HARVARD UNIVERSITY

Ernst Mayr Library

of the Museum of

Comparative Zoology

*«0*ae

ISSN 1051-3825

Hiaz, R. E., M. T. Leong, L. L. Grismer, and N. S. Yaakob. A New Species of Dibamus

(Squamata: Dibamidae) from West Malaysia

Grismer, L. L., J. L. Grismer, and T. M. Youmans. A New Species of Leptolalax( Anura: Megophryidae)

FROM PULAU TlOMAN, WEST MALAYSIA * *

Leong, T. M. and L. L. Grismer. A New Species of Kukri Snake, Oligodon (Colubridae), from Pulau

Tioman, West Malaysia 12-16

Stuart, B. L. and H. Heatwole. A New Philautus (Amphibia: Rhacophoridae) from Northern Laos 17-21

Diesmos, A. C., G. V. A. Gee, M. L. Diesmos, R. M. Brown, P. J. Widmann, and J. C. Dimalibot.

Rediscovery of the Philippine Forest Turtle, Heosemys leytensis (Chelonia; Bataguridae), from

Palawan Island, Philippines 22-27

Feldman, C. R. and J. F. Parham. Molecular Systematics of Old World Stripe-Necked Turtles

(Testudines: Mauremys ) 28-37

Hutchison, J. H„ P. A. Holroyd, and R. L. Ciochon. A Preliminary Report on Southeast Asia’s Oldest

Cenozoic Turtle Fauna from the Late Middle Eocene Pondaung Formation, Myanmar 38-52

Joyce, W. G. and C. J. Bell. A Review of the Comparative Morphology of Extant Testudinoid Turtles

(Reptilies: Testudines) 53-109

Le, Minh, T. Hoang, and D. Le. Trade Data and Some Comments On the Distribution of Mauremys

ANNAMENSIS (SlEBENROCK, 1 903) 110-113

Perala, J. and R. Bour. Neotype of Testudo terrestris ForsskAl, 1775 (Testudines, Testudinidae) 114-119

Schilde, M., D. Barth, and U. Fritz. An Ocadia sinensis x Cyclemys shanensis hybrid (Testudines:

Geomydidae) 120-125

Shi, H„ Z. Fan, F. Yin, and Z. Yuan. New Data on the Trade and Captive Breeding of Turtles in

Guangxi Province, South China 126-128

Stuart, B. L. and S. G. Platt. Recent Records of Turtles and Tortoises from Laos, Cambodia, and

Vietnam 129-150

Auffenberg, K., K. L. Krysko, and W. Auffenberg. Studies on Pakistan Lizards: Cyrtopodion stoliczkai

(Steindachner, 1867) (Gekkonidae: Gekkoninae) 151-160

Dulger, B., j. H. Ugurta§, and M. Sevinq:. Antimicrobial Activity in the Skin Secretion of Bufo viridis

(Laurenti, 1768) 161-163

Du§en, S., M. Oz, and M. R. Tunq:. Analysis of the Stomach Contents of the Lycian Salamander

Mertensiella luschani (Steindachner, 1891) (Urodela: Salamandridae), Collected from Southwest

Turkey 164-167

Ebrahimi, M., H. G. Kami, and M. Stock. First Description of Egg Sacs and Early Larval Development

in Hynobiid Salamanders (Urodela, Hynobiidae, Batrachuperus ) from North-Eastern Iran 168-175

Jarrar, B. M., and N. T. Taib. Histochemical Characterization of the Lingual Salivary Glands of the

House Gecko, Ptyodactylus hasselquistii (Squamata: Gekkonidae) 176-181

Kami, H. G. The Biology of the Persian Mountain Salamander, Batrachuperus persicus (Amphibia,

Caudata Hynobiidae) in Golestan Province, Iran 182-190

Khan, M. S. Annotated Checklist of Amphibians and Reptiles of Pakistan 191-201

(Continued on inside back cover)

1 IU

Asiatic

Herpetological

Research

Volume 11 • 2008

Chengdu Institute of Biology of the Chinese Academy of Sciences

Asiatic Herpetological Research Society at the Museum of Vertebrate Zoology,

University of California

Honorary Editor-in-Chief

Er-mi Zhao

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China

Editor-in-Chief

Yue-zhao Wang

Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, Sichuan, China

Associate and Managing Editor Associate Editor

Raul E. Diaz Theodore J. Papenfuss

University of Kansas Medical Center; University of Museum of Vertebrate Zoology, University of

Kansas Museum of Natural History, Kansas, USA California Berkeley, California, USA

Consulting Editor

James F. Parham

California Academy of Sciences

Editorial Board

Kraig Adler

Cornell University, Ithaca, New York, USA

Natalia B. Ananjeva

Zoological Institute, St. Petersburg, Russia

Steven C. Anderson

University of the Pacific, Stockton, California, USA

Aaron Bauer

Villanova University, Villanova Pennsylvania, USA

Christopher Bell

University of Texas, Austin, Texas, USA

Leo Borkin

Zoological Institute, St. Petersburg, Russia

I-Jiunn Cheng

Institute of Marine Biology, National Taiwan Ocean University,

Keelung, Taiwan, China

Wen-hao Chou

National Museum of Natural Science, Taichung, Taiwan, China

Ilya Darevsky

Zoological Institute, St. Petersburg, Russia

Indraneil Das

Madras Crocodile Bank, Vadanemmeli Perur, Madras, India

William E. Duellman

University of Kansas, Lawrence, Kansas, USA

Jinzhong Fu

Department of Integrative Biology, University of Guelph, Guelph,

Ontario, Canada

Robert F. Inger

Department of Zoology, Field Museum of Natural History, Chicago,

Illinois, USA

Xiang Ji

Hangzhou Normal College, Hangzhou, Zhejiang, China; Nanjing

Normal University, Nanjing, Jiangsu, China

Pi-peng Li

Shenyang Normal University, Shenyang, Liaoning, China

Robert W. Murphy

Royal Ontario Museum, Toronto, Ontario, Canada

Goren Nilson

University of Goteborg, Goteborg, Sweden

Nikolai Orlov

Zoological Institute, St. Petersburg, Russia

Hidetoshi Ota

Department of Biology, University of the Ryukyus, Nishihara,

Okinawa, Japan

Soheila Shafii

University of Shahid Bahonar, Kerman, Iran

Hai-tao Shi

Hainan Normal University, Haikou, Hainan, China

Xiao-ming Wang

Department of Zoology, East China Nonnal University, Shanghai,

China

Yehudah Werner

Hebrew University, Jerusalem, Israel

Xiao-mao Zeng

Chengdu Institute of Biology. Chinese Academy of Sciences,

Chengdu, Sichuan, China

Asiatic Herpetological Research (AHR) Volume 1 1 is published by the Chengdu Institute of Biology of the Chinese Academy of Sciences (CIB)

and the Asiatic Herpetological Research Society (AHRS) at the Museum of Vertebrate Zoology, University of California. The editors encourage

authors from all countries to submit articles concerning, but not limited to, Asian herpetology. All correspondence should be sent via email to the

editorial office at ahr@cib.ac.cn. Authors should consult Guidelines for Manuscript Preparation and Submission at the end of this issue and on

the web. Website For more information with regards to subscription, manuscript submission, contacts, back issues and general questions visit

AHR’s website at http://www.Asiatic-Herpetological.org.

Asiatic Herpetological Research Previous volumes were published by the Asiatic Herpetological Research Society (AHRS) and the Chinese

Society for the Study of Amphibians and Reptiles (CSSAR) as follows: Vol. 10 (2004), Vol. 9 (2001), Vol. 8 (published in 1999), Vol. 7 (1997).

Vol. 6 (1995), Vol. 5 (1993), Vol. 4 (1992), Vol. 3 (1990), and Chinese Herpetological Research Vol. 2, which was published at the Museum of

Vertebrate Zoology, 1988-1989, as the journal for the Chinese Society for the Study of Amphibians and Reptiles. Volume 2 succceeded Chinese

Herpetological Research 1987, published for the Chengdu Institute of Biology by the Chongqing Branch Scientific and Technological Literature

Press, Chongqing, Sichuan, China. Acta Herpetologica Sinica ceased publication in June, 1988.

2008

Asiatic Herpetological Research, Vol. 1 1

pp.

1-9

A Preliminary Study of the Lizard Fauna and Their

Habitats in Northwestern Iran

F. Ahmadzadeh1’*, B. H. Kiabi2, H. G. Kami3 and V. Hojjati4

MCZ

LIBRARY

1 Department of Biodiversity and Ecosystem Management, Environmental Sciences Research

Institute, Shahid Beheshti University, Evin, Tehran, Iran,

2 Department of Biology, Faculty of Sciences, Shahid Beheshti University, Tehran, Iran,

3 Department of Biology, Faculty of Sciences, Agricultural Sciences and Natural

Resources University, Gorgan, Iran,

4Damghan Islamic Azad University, Damghan, Iran.

* Corresponding author E-mail: f_ahmadzade@sbu.ac.ir

MAY 07 2008

HARVARC

UNIVERSIT

Abstract.- Northwestern Iran has unique geographical and climatic conditions that support a rich flora and fauna. In

view of the lack of in-depth studies on the lizards of the region, an investigation was started in the northern part of

Ardabil Province for an inventory of this component of the fauna and their habitats. Collections were made from

October 2003 to June 2005 and 165 specimens were collected and identified. Five families, 12 genera and 15 species

are represented, including Agamidae: Laudakia caucasia, Phrynocephalus persicus, Trapelus ruderatus; Lacertidae:

Lacerta media media, Lacerta strigata, Lacerta brandtii, Darevskia raddei raddei, Eremias strauchi strauchi,

Eremias arguta, Ophisops elegans; Scincidae: Mabuya aurata transcaucasica, Eumeces schneiderii princeps,

Abelepharus bivittatus\ Anguidae: Pseudopus apodus and Gekkonidae: Cyrtopodion caspium caspium. Comparing

this list to the data provided by Anderson (1999), it seems that most of the lizards are being reported for the Province

for the first time. The families Gekkonidae and Anguidae are newly recorded, and the gecko Cyrtopodion caspium is

first recorded from the west and northwest of Iran. With seven species represented in the area, lacertids have the high-

est species diversity among the lizard families and need further study. Habitat features also have been given for all

species.

Keywords.- Iran, Ardabil, fauna, lizard, Lacerta.

Introduction

General information about the herpetofauna of Iran has

been provided by Mertens (1957), Anderson (1966),

Tuck (1971, 1974), Latifi (1984, 1991), Balouch and

Kami (1995) and Kami and Vakilipoure (1996a, 1996b).

Furthermore, a handbook of amphibians and reptiles of

the Middle East has been published by Leviton et al.

(1992), a book on the Lizards of Iran was recently pub-

lished by Anderson (1999) and an updated checklist to

the lizards of Iran was provided by Firouz (2000).

Despite these publications, the lizards of Iran are still

poorly-known and infrequently collected, with many

new species still being discovered (Rastegar-Pouyani,

1996; Rastegar-Pouyani and Nilson, 1998). Studies on

the lizards of Ardabil Province are also very limited

(Ahmadzadeh, 2004).

The aim of this study is to determine in detail the

lizard fauna and their habitat features in the northern

part of Ardabil Province, which is of particular signifi-

cance considering the unique geography and vegetation

of the region. Moreover, this study will collect baseline

population data for future management.

Materials and Methods

The area of study is in the Northwest part of Iran, specif-

ically, the northern part of Ardabil Province (38° 15' E,

to 39° 40' E, 47° 30' N to 48° 00' N). The region is sur-

rounded by the Alborz Mountains and the Caspian Sea

to the east, Aras village is to the north. Arasbaran pro-

tected area and Gare-Dagh Mountain to the west and the

Sabalan Mountain chains to the south (Fig. 1). Altitude

ranges between 20 m in the Moghan steppe to 4,888 m

on the Sabalan Mountain. The study was carried out

between October 2003 and June 2005. All of the samples

were caught by hand and some lizards which are active

and difficult to catch, such as green lizards (e.g., Lacerta

m. media and L. strigata ), were captured by dust shot.

Locality data and their habitat features were recorded for

all species encountered during the study. However, all

have been preserved in accordance to standard methods

(Formalin 1 0%) and voucher specimens are stored in the

Biodiversity and Ecosystem Management Department

Collection (BEMD) at Shahid Beheshti University of

Iran. Specimens were identified with Leviton et al.

(1992) and Anderson (1999) using morphometric meas-

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Asiatic Herpetological Research, Vol. 1 1

2008

Figure 1. The study area, the northern part of Ardabil Province of Iran.

urements, coloration and pholidosis features (including

the number, structure and range of plates).

Results

A total of 165 samples were collected in the study area,

comprising 15 species in 12 genera and 5 families. The

species composition is given in Table 1. Distribution of

species are presented in Figure 2.

Family: Agamidae

Laudakia caucasia caucasia (Eichwald, 1831)

Laudakia c. caucasia is widely distributed in the study

area, preferring montainous habitats and eroded sand

canyons in flooded plains adjacent to mountains, rock

cliffs, old houses and stony walls near roads. At 6 km2,

210 specimens were recorded on 10 June 2004 in

Meshkinshar. This species was also collected on 24

November and 3 March in the Meshkinshahr and

Arshagh areas at elevations between 500-2,800 m.

Specimens were light olive to dark gray in ground color

with adult snout- vent length of 152 mm in males and

155 mm in females.

Phrynocephalus persicus persicus De Filippi, 1863

This species was rarely encountered in the study area.

Four specimens were captured in Arshagh, Alma village

- a semi-arid area with ephemeral plants in spring and

loamy soil. Xerophyte vegetation, both plants and bush-

es, grow in these areas. Dorsal coloration was light

brown with three dark transverse marks, within which

on the hind limbs were enlarged tubercular scales. The

largest female was 40 and 48 mm snout-vent and tail

length, respectively. During the study period, no males

were found.

Trapelus ruderatus ruderatus (Olivier, 1 804)

The small agamid lizard Trapelus r. ruderatus was found

on open stony ground and in cultivated fields with

sparse weed vegetation in autumn. On sunny summer

days, it hides under weeds such as Euphorbia spp.,

Chenopodium spp. and Chrozophora tinctoria. Its activ-

ity appears to begin in early June and extends to late

September. In total, 10 specimens were collected on the

harvested wheat and barley fields in Gooshe area at

approximately at 10:30 AM. Ground color was typically

grayish-brown with five dark transverse bars on the

trunk which were interrupted by a series of light ovoid

vertebral spots (Fig. 3). The largest male examined with

distinct callous preanal scales, had a 65 mm snout-vent

and 74 mm tail length. The largest female had measure-

ments of 63 mm and 75 mm, respectively.

Family: Anguidae

Pseudopus apodus (Pallas, 1775)

Pseudopus apodus occurs throughout the Hyrcanian for-

est of northern Iran. It has recently been collected from

the Arasbaran protected area, but there are no records for

Ardabil Province. This species was found in grassland

and shrubby vegetation near streams. On a sunny day,

four P. apodus were observed in a pond. The ground

color of the dorsum was gray with zig-zagging blackish-

brown stripes in the juvenile. The head was light yellow-

ish-brown in adults with the remainder of the body dark

brown. The longest adult had a 520 mm snout-vent

length and a 670 mm tail.

Family: Gekkonidae

Cyrtopodion caspium caspium (Eichwald, 1831)

An isolated population of Cyrtopodion caspium was

found in the Moghan Steppe for the first time, represent-

ing a new family record for northwestern Iran. One spec-

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Asiatic Herpetological Research, Vol. 11

3

Table 1. Lizard species collected from the sfudy area.

imen was collected at night on walls of an old house at

20 m elevation. After sunset they fed on various noctur-

nal insects around lights. Dorsal scales are strongly

keeled. Dorsal body coloration was light gray with five

dark transverse bars on the body and 11-12 on the tail

(Fig. 4). In Pars-Abad, one male specimen with a snout-

vent length of 75 mm and a tail length of 70 mm was

measured.

Family: Lacertidae

Darevskia raddei raddei (Boettger, 1892)

Darevskia r. raddei is common in rocky areas where

Laudakia caucasia is also frequently found. In March,

this species was seen on vertical surfaces of rocks in the

Kapas Mountains near Meshkin-Shahr. Darevskia r. rad-

dei is various shades of light brown dorsally and more

common in the rocky habitats than other lacertids.

Specimens were found below altitudes of 900 m in

Meshkinshar, but in the Salavat and Arshag Mountains,

it was collected at altitude up to 2,400 m. The relation-

ship of this subspecies to D. r. vanensis in northwestern

Iran requires further study. The largest male had a snout-

vent of 71 mm and tail length of 131 mm. One adult

female had a 70 mm snout-vent length and 130 mm tail

length (Fig. 5).

Lacerta brandtii De Filippi, 1863

Lacerta brandtii was collected under stones, on foothills

and in the burrows of other animals in open arid bushy

and stony habitats in the Razeye area. Large numbers of

this lizard were also found in Samian District, 100 km

from the study area on a foothill surrounded by cultivat-

ed land. The relationship between the two Iranian popu-

lations of this species in Esfahan Province and east

Azarbijan Province remains problematic. This species is

less active in comparison to other lacertid lizards such as

Darevskia raddei. The dorsal surface was olive-gray

with small black spots (Fig. 6) and the ventral surface

had 8 longitudinal rows of plate. The longest male spec-

imen had total length of 192 mm. The tail of this species

displays autotomy (approximately 60% of total speci-

mens).

Eremias arguta (Pallas, 1773)

Eremias arguta has a limited distribution in the study

area: one adult specimen was collected in a harvested

barley field on a sunny day near to the Ardabil-Meshkin

road and two juvenile specimens were captured in the

Ardabil Airport area in August 2004. This lizard had a

white belly and a dorsum with white spots edged with

black on a grayish background (Fig. 7) that sometimes

formed transverse bands in the adults. Our adult speci-

mens had a 95 mm snout-vent length and a 1 10 mm tail.

Eremias strauchi strauchi Kessler, 1 878

There are two subspecies of this lizard in Iran - Eremias

s. strauchi and E. s. kopetdaghica , of which only the

first was found in the study area. The specimen was col-

lected in the eastern part of the study area in the Arshag

plain under wheat straw in a dry, stony harvested field.

Eremias s. strauchi is active and hides in shrubby vege-

tation. Five eggs of this lizard were found under an

Artemisia sp. shrub on 14 June 2004 in Amir-Abad vil-

lage. In the study area one male specimen was measured

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Asiatic Herpetological Research, Vol. 1 1

2008

Figure 2. Distribution of species in the study area: (a) L. caucasia, (b) Ph. persicus, (c) T. ruderatus , (d) P. apodus , (e)

C. caspium, (f) D. raddei.

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Asiatic Herpetological Research, Vol. 11

5

Figure 2. (continued) (g) E. arguta, (h) E. strauchi, (i) L. brandtii, 0) L. media, (k) L. strigata, (I) O. elegans.

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Asiatic Herpetological Research, Vol. 1 1

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Figure 2. (continued) (m) A. bivitattus, (n) E. schneiderii, (o) M. aurata.

with a 71 mm snout-vent length and had a 128 mm tail.

It’s olive-gray color pattern does not vary greatly among

populations (Fig. 8).

Lacerta media media (Lantz and Cyren, 1 902)

Lacerta m. media was very common in the grassy and

shrubby areas along the Khyave Chaye and Garesoo

river banks. One male specimen was captured under a

stone near a bean field. Specimens were observed at an

altitude of 2,100 m and males were seen on stony walls

near the roads at the end of the winter. The dorsal sur-

face of the adult male, unlike juveniles, was green with-

out any light lines or spots. Females were dark olive-

brown with large lateral spots that disappeared with age.

The largest lacertid collected during the study was one

male specimen with a snout-vent of 117 mm and a tail

length of 272 mm. This species exists in two differently-

spotted morphs, with specimens from cultivated fields

being larger than those from other habitats.

Lacerta strigata Eichwald, 1831

Lacerta strigata was most frequently found in the

Hyrcanian Forest in northern Iran and in some bushy

and wooded streams banks associated with this forest,

such as the Arax River in the northern part of the study

area. Large numbers of this species were seen in Pars-

Abad, Bilasovar and Germi near streams with dense

Tamarix and Rubus vegetation. One specimen was cap-

tured far from the Hyrcanian Forest in an open harvested

wheat field on 25 August 2004. Most collection sites

represent new locality records. The general color of the

dorsum was light green in males and dark-olive to

brown in females; it was more strongly spotted than

Lacerta m. media. Females were also smaller with more

numerous dark spots. The largest male had a snout-vent

length of 160 mm and a tail length of 100 mm, while the

2008

Asiatic Herpetological Research, Vol. 11

7

Figure 5. Darevskia raddei raddei. Figure 6. Lacerta brandtii.

Figure 7. Eremias arguta.

Figure 9. Eumeces schneiderii princeps.

Figure 8. Eremias strauchi.

Figure 10. Mabuya aruata transcaucasica.

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Asiatic Herpetological Research, Vol. 1 1

2008

largest temale had measurements of 97 mm and 187

mm.

Ophisops elegans Menetries, 1832

Ophisops elegans is widely distributed, but is most com-

mon on the Moghan Steppe. A large isolated population

was found in Amir-Abad village. Specimens were active

and encountered almost everywhere, particularly in dry

stony habitats. Males and females both showed different

color patterns during the reproductive period. Dorsal

coloration was generally olive-green to brownish with

two light dorsolateral stripes that disappear in the adult.

An adult female from the Moghan Steppe had a snout-

vent length of 64 mm and a tail length of 1 10 mm, and a

male from Amir-Abad village had a snout-vent of 70

mm and a tail length of 120 mm.

Family: Scincidae

Ablepharus bivittatus (Menetries, 1832)

The Two-streaked Snake-eyed Skink, Ablepharus bivit-

tatus, has only been found in Amir-Abad village on

Ardabil-Germi Road on a slope with large spiny cushion

vegetation where it was sympatric with Ophisops ele-

gans and Eremias strauchi. This active lizard has a high

population density in the Neur Lake area in southern

part of Ardabil Province reaches. Body coloration on the

dorsum and tail is bronze-brown. The largest adult

female specimen reached 60 mm in snout-vent length.

Eumeces schneiderii princeps (Eichwald, 1839)

This species lives on sand dunes, stony hills and dry

river beds. We captured one male on a foothill in

Meshkinshahr on 16 June 2004 at 08:00 AM, where

Mabuya aruata transcaucasica was also found. This

lizard is very active, hides in burrows and can jump

approximately 2 m. In comparison to other scincid

lizards, Eumeces schneiderii occurs in relatively few

localities - overgrazing and destruction of habitat is

threatening extirpation of this species in the study area.

The dorsum was brownish with a narrow creamy-white

lateral line from the posterior labial through the ear

along the sides to the groin (Fig. 9). Total length (snout-

vent + tail length) of the captured male was 240 mm.

Mabuya aurata transcaucasica (Chernov, 1 926)

Mabuya aurata transcaucasica lives in sandy areas and

small hills that are covered with Astragalus and

Acantolimon vegetation. This lizard often jumps from

stone to stone for hunting insects especially grasshop-

pers. The sympatric occurrence of Mabuya aurata tran-

scaucasica, Darevskia raddei raddei and Laudakia cau-

casia caucasia has been documented on Salvat

Mountain in a rocky habitat. On the Arshagh Mountains,

juveniles with blue tails were found in cliffs, but we

could not find adult specimens at this locality. Dorsal

coloration is olive-brown with dark spots in longitudinal

rows. These spots disappear on the tail and head (Fig.

10). In the study area a specimen with a 1 15 mm snout-

vent length and a 125 mm tail was collected.

Literature Cited

Ahmadzaheh, F. 2004. Preliminary studies of the lizard's

fauna and their habitats in Meshkinshahr district.

Enviromental Sciences 1(2): 39-44.

Anderson, S. C. 1999. The lizards of Iran. Society for

the study of Amphibians and Reptiles, 442 pp.

Anderson, S. C. 1996. The turtles, lizards, and amphib-

ians of Iran. Ph.D. Thesis. Stanford University. 660

pp.

Baloutch, M. and H. G. kami. 1995. Amphibians of Iran.

Tehran University Publication, Tehran. 177 pp.

Firouz, E. 2000. A Guide to the Fauna of Iran (In Per-

sian). Iran University Press, Tehran. 491 pp.

Kami, H. G. and A. Vakilipoure. 1996a. Geographic dis-

tribution: Bufo bufo. Herpetological Review 27(3):

148.

Kami H. G. and A. Vakilipoure. 1996b. Geographic dis-

tribution: Rana camerani. Herpetological Review

27(3): 150.

Latifi, M. 1984. The snakes of Iran. Iran Department of

the Environment, Tehran. 221pp.

Latifi, M. 1991. The snakes of Iran. Society for the

Study of Amphibians and Reptiles. Contributions to

Herpetology 7.viii + 159 pp.

Leviton, A. E., S. C. Anderson, K. A. Adler and S. A.

Minton 1992. Hand book to Middle East

Amphibians and Reptiles. Oxford, Ohio. Vii + 252

pp.

Mertens, R. F. W. 1957. Weitere Unterlagen zur

Herpetofauna von Iran 1956. Jahreshefte des

Vereins fur vaterlandische Naturkunde in

Wurtemberg 112(1): 118-128.

Rastegar-Pouyani, N. 1996. A new species of Asaccus

(Sauria: Gekkonide) from the Zagros Mountain,

Kermanshahan Province, western Iran. Russian

Journal of Herpetology 3(1): 11-17.

2008

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9

Rastegar-Pouyani, N. and G. Nilson 1998. A new

species of Lacerta (Sauria: Lacertidae) from the

Zagros Mountain, Esfahan Province, west-central

Iran. Proceeding of the California Academy of

Science, ser. 4 50(10): 267-277.

Tuck, R. G. 1971. Amphibians and reptiles from Iran in

the United State National Museum Collection.

Bulletin of the Maryland Herpetological Society

7(3): 48-36.

Tuck, R. G. 1974. Some amphibians and reptiles from

Iran. Bulletin of the Maryland Herpetological

Society 10: 59-65.

Submitted: 07 August 2006

Accepted: 24 August 2007

pp. 10-12

Asiatic Herpetological Research, Vol. 11

2008

A Second Record of Ptyctolaemus gularis (Peters, 1864)

from Bangladesh

M. Farid Ahsan*, Ghazi S. M. Asm at and S. Chakma

Department of Zoology, University of Chittagong, Chittagong 4331, Bangladesh.

* Corresponding author E-mail: mfaridahsan@yahoo.com

Abstract.- Ptyctolaemus gularis (Peters, 1864), the blue-throated lizard, was collected from a hilly stream in

Rangamat District in Bangladesh in July 2003 and November 2004, representing the second recorded occurrence of

this species in Bangladesh.

Keywords.- Ptyctolaemus gularis , blue-throated lizard, occurrence, habitat, ecology, morphology, Bangladesh.

Introduction

The blue-throated lizard, Ptyctolaemus gularis (Peter,

1864), has been previously described from Meghalaya,

Assam, the Chittagong Hill Tracts, Tibet and China

(Boulenger, 1890; Hora, 1926; Smith, 1935; Zhao and

Adler, 1993). Boulenger (1890), who developed the

lizard taxonomy of the Indian Subcontinent, examined

two specimens of P. gularis, the type specimen from

Calcutta, preserved in the Berlin Museum, and a speci-

men from Sadiya, Assam, in the British Museum.

Following Boulenger (op. cit.), Hora (1926) reported ten

specimens from Assam and Nainimukh (correctly

spelled Mainimukh), Chittagong Hill Tracts, which are

presently deposited in the Zoological Survey of India.

The single specimen from Nainimukh represented the

first record of this species in Bangladesh. This record

has been subsequently overlooked by other authors,

including Ahsan, 1998; Khan, 1982; Sarker and Sarker,

1988.

Observations and Discussion

During a herpetological survey of Bangladesh, one spec-

imen of Ptyctolaemus gularis was collected from

Rangamati District (part of the Chittagong Hill Tracts)

on 18 July 2003 (Fig. 1). Two other specimens were

later collected from Rampahar about 50 km east of

Chittagong City in Kaptai National Park (22.30.425' N,

092.10.446' E), Rangamati District, on 25 November

2004 (Fig. 1). The first specimen was collected from

Rupkari Chara (23.12.126' N, 092.10.628' E), a hilly

stream of the Rupkari Union Parishad under Baghaichari

Upazila. The collection site is approximately 7 km

northwest from the Baghaichari Upazila headquarter. At

the time of collection, approximately 1300 h, the speci-

men was observed on a large stone hunting insects. This

specimen has been deposited in the departmental muse-

um of Zoology, University of Chittagong, Chittagong,

Bangladesh (Fig. 2A, B). The other two specimens, cur-

rently in the collection of S. Chakma, were collected

approximately 150 m apart between 1300 and 1400 h.

These animals were also collected while they were hunt-

ing for insects. One of us (MFA) also observed this

species in Chittagong at the Chunati Wildlife Sanctuary

in 1990.

With these new records, it is likely that

Ptyctolaemus gularis also occurs in the hills of Sherpur,

Jamalpur, Hobiganj, Moulvibazar, Sylhet (i.e., British-

Indian Assam), Khagrachari, Bandarbans (part of

Chittagong Hill Tracts) and Cox’s Bazar Districts in

Bangladesh, which share similar habitats.

Habitat and Ecology

Ptyctolaemus gularis is a terrestrial, diurnal species that

is frequently found south of the Brahmaputra River in

India (Smith, 1935; Daniel, 2002) and uncommonly

encountered in the southeastern hilly forests of

Bangladesh. It is most often observed in search of food

on land, stones and logs near streams and water-logs.

The first collection locality visited in 2003 was a narrow

stony stream, with the hills on both sides covered with

bamboo brakes (muli [Melocanna bambusoides ]),

gameri ( Gmelina arborea) and teak ( Tectona grandis)

trees. Ferns and some natural herbs grew between the

stones. The second and third specimens collected in

2004 were found on the slopes of a hilly, stony stream

close to a waterfall. The upper canopy was dominated by

garjan (Diptero carpus spp.) and gutgutia (P rotium ser-

ratum) trees, and the lower canopy and forest floor were

densely covered by shrubs and herbs.

2008

Asiatic Herpetological Research, Vol. 1 1

11

84'

28

"7~

t.

bb

V ~. ,-V

V- -O

v.

•'t

26°

24

22

NEPAL

LEGEND

1= Sherpur

2= Jamalpur

3= Sylhet

4= Hobiganj

5= Moulovibazar

6= Khagrachari

7= Rangamati

8= Kaptai \

9= Chittagong (Chunoti)

10= Cox's Bazar

?= Not Confirmed

#= Confirmed

84l 86 88

Figure 1. Map showing collection localities.

SCALE

40 80 Mi

lt r1

0 40 80 120 Km

Identification

The collected specimens can be most readily separated

from congeners by having three parallel longitudinal

folds on each side of the throat that converge posteriorly

(cf. Boulenger, 1890; Smith, 1935) (Fig. 2C). Other use-

ful characters include an olive-brown dorsum with dark

transverse bars and/or spots, two curved dark brown

cross-bars between the eyes separated by a central light

bar, a dark stripe below the eye to the angle of the

mouth, dark blue throat folds, and limbs and a tail with

dark cross-bars above and yellowish-white cross-bars

below (cf. Boulenger, 1890; Smith, 1935).

The head is also rather long and narrow with

unequally-sized upper scales that are strongly keeled.

The dorsal body scales are also unequally-sized, with

large, strongly-keeled scales and smaller feeble ones.

Several mid-dorsal rows also point backwards and

upwards and the ventral scales are strongly keeled and

mucronate. The limbs are moderate in size; the third and

fourth fingers are equal while the fourth toe is much

longer than the third. The tail is rounded, slender and

covered with sub-equal keeled scales (cf. Boulenger,

1890; Smith, 1935). Table 1 compares the lengths of the

present specimens with those collected previously.

Acknowledgments

We wish to thank Dr. M. A. G. Khan and an anonymous

reviewer(s), who kindly reviewed an earlier version of

this manuscript. Mr. M. S. Islam and Mr. M. A. W.

Chowdhury kindly helped in drawing the map.

Table 1. Comparison of recently collected specimens with those from earlier collections.

* Mean and raw data within brackets

12

Asiatic Herpetological Research, Vol. 1 1

2008

Figure 2. Dorsal (A) and ventral (B) aspects of

Ptyctolaemus gularis collected. Inset (C) shows the

gular region folds converging posteriorly, diagnostic of

this species.

Literature Cited

Ahsan, M. F. 1998. Country report for Bangladesh-

Herpetofauna of Bangladesh: present status, distri-

bution and conservation. Pp. 9-17. In: A. de Silva

(ed.), Biology and Conservation of Amphibians,

Reptiles and Their Habitats in South Asia

(Proceedings of the International Conference in

Biology and Conservation of the Amphibians and

Reptiles of South Asia, held at the Institute of

Fundamental Studies, Kandy and University of

Peradniya, Sri Lanka, August 1-5, 1996). Amphibia

and Reptile Research Organization of Sri Lanka

(ARROS).

Boulenger, G. A. 1890. The fauna of British India

including Ceylon and Burma: Reptilia and

Batrachia. Taylor and Francis, London. 541 pp.

Daniel, J. C. 2002. The book of Indian reptiles and

amphibians. Oxford University Press, Oxford. 238

pp.

Hora, S. L. 1926. Notes on lizards in the Indian

Museum: II. On the unnamed collection of lizards

of the Family Agamidae. Records of the Indian

Museum 28: 415-420 + 1 plate.

Khan, M. A. R. 1982. Wildlife of Bangladesh: a check-

list. The University of Dhaka, Dhaka. 173 pp.

Sarker, M. S. U. and N. J. Sarker. 1988. Wildlife of

Bangladesh (a systematic list with status, distribu-

tion and habitat). The Rico Printers, Dhaka. 59 pp.

Smith, M. S. 1935. Fauna of British India including

Ceylon and Burma: Reptilia and Amphibia, Vol. II-

Sauria. Taylor and Francis Ltd., London. 441 pp. +

1 map + 1 plate.

Zhao, E-M. and K. Adler. 1993. Herpetology of China.

Society for the Study of Amphibians and Reptiles,

Oxford, Ohio. 522 pp.

Submitted: 11 November 2006

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 11

pp. 13-16

Observations on the Ovipositional Behavior of the Crest-less

Lizard Calotes liocephalus (Reptilia: Agamidae) in the

Knuckles Forest Region of Sri Lanka

A. A. Thasun Amarasinghe1’2 and D. M. S. Suranjan Karunarathna1’3

[The Young Zoologists' Association of Sri Lanka, National Zoological

Gardens, Dehiwala, Sri Lanka.

Corresponding authors E-mails: 2 aathasun@gmail.com; 3 dmsameera@gmail.com

Abstract.- A mature female Calotes liocephalus lying on the ground in Pitawala in the Knuckles Forest Region of Sri

Lanka. This is the first described observation of the ovipositing of Calotes liocephalus. The ovipositional behavior

consisted of digging a hole to lay eggs, laying the eggs, scraping soil to bury the eggs, filling of the spaces between

the eggs, the tight compression of the soil and camouflaging the nest.

Keywords.- Agamidae, Calotes, egg-laying behavior, Knuckles, Sri Lanka, conservation.

Introduction

There are eighteen species of agamid lizards in Sri

Lanka, fifteen of them are endemic to the island (Bahir

and Surasinghe, 2005; Manamendra-Arachchi et al.,

2006; Samarawickrama et al., 2006). Seven species

belong to the genus Calotes. Five of them (C. ceylonen-

sis Muller, 1887; C. liocephalus Gunther, 1872; C.

liolepis Boulenger, 1885; C. nigrilabris Peters, 1860; C.

desilvai Bahir and Maduwage, 2005) are endemic. The

remaining two Calotes (C. calotes [Linnaeus, 1758); C.

versicolor (Daudin, 1802]) are probably widespread

species throughout South East Asia. According to the

published literature, Calotes liocephalus is a largely

arboreal species found only in parts of the Knuckles

Forest Region in Sri Lanka (Manamendra-Arachchi and

Liyanage, 1994). Its conservation status is Rare and

Endangered (Bahir and Surasinghe, 2005). It can be dis-

tinguished from its congeners by the presence of an

oblique fold in front of the shoulder, a lower jaw that is

rather short, a head without spines (or rarely a rudimen-

tary spine above the ear), enlarged supraocular scales

and poorly-developed dorsinuchal crests on the head and

lower neck (Manamendra-Arachchi, 1 990). Adults have

a snout to vent length of 9 1 mm, a head length of 37 mm,

a tail length of 261 mm and an axilla to groin length of

43 mm (Deraniyagala, 1953).

Location of observation.- Observations were made

approximately 1 km from Matale-Pallegama Road in

Pitawala in the Knuckles Forest Region (altitude: 783

m) in Matale District, Central Province. The habitat con-

sisted mainly of disturbed home gardens (Ekanayake

and Bambaradeniya, 2001). The ground was covered

with small amounts of wet leaf litter and the soil was

soft. There was approximately 10% canopy cover and

the undergrowth consisted primarily of grasses.

Observations of the lizard was made by the unaided eye

from 2 m away between the hours of 1420 and 1600 hrs.

The animal was not disturbed during observation. All

measurements were taken to the nearest 0.1 mm using

dial calipers.

Observations

A mature female Calotes liocephalus (snout to vent

length: 54.0 mm, head length: 19.4 mm, head width:

11.9 mm, tail length: 156.0 mm, axilla to groin length:

26.5 mm) lying on the ground, approximately 50 cm

from the road, was observed on 21 June 2006 at about

1420 hr. The temperature was 23.6°C and the humidity

93%. The weather was gloomy and the cloud cover was

8/8.

Digging the nest hole.- First, the lizard lifted the anteri-

or part of its body using its forelimbs. It then looked

around for ~10 min. During this period it repeatedly

turned its head 1 80° five times, without moving its body

(Fig. 1). The female then began digging into the ground

while scraping the soil with its forelimbs, which was

thrown backward under its body through its raised hind

limbs. This continued for approximately 5 minutes (Fig.

2). After that it stopped digging and looked around for

approximately 5 min. while repeatedly turning its head

180° three times, without moving its body (Fig. 3).

Again, it continued digging and this time the female dug

the hole continuously for approximately 10 min. It

stopped and looked around for about 5 min. while turn-

ing its head 1 80° around three times, without moving its

body as in Figure 3. After that, it continued to dig the

14

Asiatic Herpetological Research, Vol. 11

2008

Figure 1. Figure 2.

Figure 3.

Figure 4.

Figure 5.

Figure 6.

Figure 7.

Figure 8.

2008

Asiatic Herpetological Research, Vol. 1 1

15

Figure 9a.

Figure 9b.

Figure 10.

Figure 12.

hole for another half an hour, stopping three more times

for 5 min. each, to rest. The hole was dug into the ground

at a 45° angle. The final hole was 92.6 mm deep and

79.1 mm in diameter (Fig. 4). During the rest intervals

the body was coiled inside the hole with the anterior half

bent at an angle of 90° to looking around (Fig. 5). There

was a drizzle for ~15 minutes, but the female continued

digging.

Laying the eggs. - After half an hour of digging, the

female turned its body 180° clockwise, placing the pos-

terior part of its body inside the hole. It then looked

around again (Fig. 6). The significance of this egg laying

Figure 13.

behavior was that the female removed herself slowly

from the hole without lifting her limbs while it was lay-

ing its eggs (Figs. 7-8). Eight eggs were laid at a rate of

one per minute. The eggs were pure white and elliptical,

with a mean length of 14.8 mm and a mean width 8.6

mm. After the eggs were laid, the female came out of the

hole completely and started looking around (Fig. 9a, b).

Then the female crept back into the hole for 1 5 min. to

pack and place the eggs below ground level using the

anterior part of its lower jaw (Fig. 10).

Buiying the eggs and camouflaging the nest.- After

coming out of the hole, the female turned 180° clock-

16

Asiatic Herpetological Research, Vol. 1 1

2008

wise and began to drag the soil towards the hole using its

lorelimbs. The dragged soil was thrown backwards

under its body while it lifted its hind limbs (Fig. 11).

After dragging the soil for about 5 min., it turned 180°

counter-clockwise and began pressing the soil with the

anterior half of its lower jaw for half an hour. The hole

was filled up to 18.4 mm below ground level (Fig. 12).

After looking around, it dragged the surrounding Albizia

saman (Family: Fabaceae) leaves over the nest site for

camouflage (Fig. 13). It remained motionless for 2 min.

and then ran towards the forest, during which time it was

caught for measurement and then released.

Discussion

The oviposition behavior of this species varies from the

oviposition behavior of Calotes versicolor. According to

Amarasinghe and Karunarathna (2007), C. versicolor

places its cloacal aperture over the opening of the hole

while laying its eggs, but C. liocephalus places the pos-

terior part of the body inside the hole while laying eggs.

C. versicolor also lifts the anterior part of the body with

its forelimbs while turning its head to look around, but

C. liocephalus coils its entire body inside the hole while

bending the anterior part of its body to look around. C.

versicolor makes a knocking noise while packing and

placing the eggs in the hole using its lower jaw while the

C. liocephalus places them softly without making any

noise. After the observation the eggs were removed from

the hole and the hole was subsequently examined. The

bottom was conical and the soil was soft, dark and wet.

Finally the eggs were buried in a home garden to hatch.

After approximately two and half months we observed

five small holes where the hatchlings had come out.

Unfortunately we could not observe the hatchlings.

A Few diagrams, brief descriptions and notes of

Calotes liocephalus are available in popular journals,

books and magazines but almost nothing exists on the

pre and post mating behavior, egg laying behavior, cap-

tive breeding and their hatchlings. In addition Calotes

liocephalus is an endemic, rare and threatened species

and therefore it may become extinct if their population

does not increase. For such a situation to be achieved,

captive breeding methods may be needed for ex-situ

conservation of this species. In addition further observa-

tions are also needed for the conservation of Calotes lio-

cephalus.

Acknowledgments

We wish to thank Mr. Kelum Manamendra-Arachchi

(WHT - Wildlife Heritage Trust) for reviewing the man-

uscript and Mrs. Zeenia Nissam of the Department of

Zoology, Faculty of Natural Sciences of the Open

University, Sri Lanka, for her generous support for the

field visit. We also thank Mr. Niranjan Karunarathna and

Mr. Gayan Wijeytunga (YZA - The Young Zoologists’

Association) for assisting the fieldwork. Finally, Ms.

Debbie McCormick and Mr. F. S. Abeywickrama are

acknowledged for their help in preparing this paper.

Literature Cited

Amarasinghe, A. A. T. and D. M. S. S. Karunarathna.

2007. Beobachtungen zum Eiablageverhalten der

Indischen Schonechse Calotes versicolor

(Daudin, 1802) (Reptilia: Agamidae) in einem

anthropogenen Biotop in Sri Lanka. Sauria,

Berlin, 29(3): 27-30.

Bahir, M. M. and T. D. Surasinghe. 2005. A conservation

assessment of the agamid lizards of Sri Lanka. The

Raffles Bulletin of Zoology, Suppl. 12: 381-392.

Deraniyagala, R E. P. 1953. A colored atlas of some ver-

tebrates from Ceylon, Tetrapod Reptilia. National

museums of Sri Lanka.

Ekanayake, S. and C. N. B. Bambaradeniya. 2001.

Trekking in the Knuckles forest - A trekking guide

to Alugallena, Dekinda and Nitre cave nature trails.

IUCN Sri Lanka.

Manamendra-Arachchi, K. 1990. A guide to the

Agamids in Sri Lanka. Occ. Papers of the Young

Zoologist Association of Sri Lanka. 5: 1-8.

Manamendra-Arachchi, K. and S. Liyanage. 1994.

Conservation and distributions of the agamid

lizards of Sri Lanka with illustrations of the extant

species. Journal of South Asian Natural History

1(1): 77-96.

Manamendra-Arachchi, K., A. de Silva and T.

Amarasinghe. 2006. Description of a second

species of Cophotis (Reptilia:Agamidae) from the

highlands of Sri Lanka. Lyriocephalus 6(1): 1-8.

Samarawickrama, V. A. M. P. K., K. B. Ranawana, D. R.

N. S. Rajapaksha, N. B. Ananjeva, N. L. Orlov, J.

M. A. S. Ranasinghe and V. A. P. Samarawickrama.

2006. A new species of the Genus Cophotis

(Squamata: Agamidae) from Sri Lanka. Russian

Journal of Herpetology 13(3): 207-214.

Submitted: 10 October 2006

Accepted: 23 September 2007

2008

Asiatic Herpetological Research, Vol. 11

pp.

17-23

On the Status of the Chinese Pitviper Ceratrimeresurus shenlii Liang and

Liu in Liang, 2003 (Serpentes, Viperidae), with the Addition of

Protobothrops cornutus (Smith, 1930) to the Chinese Snake Fauna

Patrick David1’*, Haiyan Tong2, Gernot Vogel3 and Mingyi Tian4

1 Departement Systematique et Evolution, USM 602 Taxonomie-collection - Reptiles et Amphibiens,

CP 30, Museum National d’Histoire Naturelle, 57 rue Cuvier, F-7523J Paris Cedex 05, France,

2 16, cour du Liegat, F -7 5013 Paris, France,

dm Sand 3, D-6911 5 Fleidelberg, Germany,

4 College of Natural Resources & Environment, South China Agricultural University, Wushan,

Guangzhou 510640, People’s Republic of China.

* Corresponding author E-mail: pdavid@mnhn.fr

Abstract.- Ceratrimeresurus shenlii Liang and Liu in Liang, 2003 was described as a new genus and a new species

on the basis of the first “homed” specimen of pitviper recorded from the People’s Republic of China. This taxon has

been overlooked in the literature. The original description is here translated verbatim into English. The holotype is

compared with other homed pitvipers known from Asia. On the basis of its scalation and pattern, Ceratrimeresurus

shenlii is synonymized with Protobothrops cornutus (Smith, 1930). The range of this latter species, previously

endemic to Vietnam, is expanded northeastwards by approximately 780 airline km. A brief comment on the zoogeog-

raphy of South China is given.

Keywords.- Serpentes, Ceratrimeresurus, Ceratrimeresurus shenlii, Protobothrops cornutus, China, Vietnam, taxon-

omy, zoogeography.

Introduction

A new Chinese genus and new species of Asian pitviper,

Ceratrimeresurus shenlii Liang and Liu in Liang, 2003

has remained overlooked in the literature.

Ceratrimeresurus shenlii was not included in the latest

checklist of the Trimeresurus complex (Gumprecht et

al., 2004), nor announced in Wolfgang Wiister’s invalu-

able website “Venomous Snakes Systematic Alert”

(http://biology.bangor.ac.uk/~bssl66/update.htm).

Lastly, this taxon was not considered by Malhotra and

Thorpe (2004, 2005) in their revision on the pitvipers of

the Trimeresurus complex.

The description of Ceratrimeresurus shenlii Liang

and Liu in Liang (2003: 411; Plate 8: Fig. 13. Type local-

ity: “Working site 02 at Wuzhishan forest, Ruyuan

Xian”, Guangdong Province) appeared in a chapter of a

book on the natural history of Nanling Nature Reserve,

located in Nanling Mountains, in the north of

Guangdong Province (Pang, 2003), a fact that may

explain that the new taxon remained overlooked by the

herpetological community. However, this new species

was merely mentioned in September 2004 in the forum

of a website dedicated to venomous snakes

(http://www.venomdoc.com/fomms - last viewed on

May 30th, 2005), where this species was regarded, with-

out explanations, as a synonym of Protobothrops cornu-

tus. It was also tentatively regarded as a synonym of

Protobothrops cornutus by Vogel (2006).

Only three other taxa of Asian pitvipers with more

or less raised supraoculars were previously known from

the mainland: Trimeresurus wiroti (see David et al.,

2006), Protobothrops cornutus and Triceratolepidophis

sieversorum. In this paper, we provide a translation of

the original description. The new taxon is, according to

the re-examination of the holotype, compared with the

currently known homed species of mainland Asia and its

status is discussed.

Materials and Methods

Body and tail lengths were measured to the nearest mm.

The number of ventral scales is counted according to

Dowling (1951). The terminal scute is not included in

the number of subcaudals. The numbers of dorsal scale

rows are given at one head length behind head, at mid-

body (i.e. at the level of the ventral plate corresponding

to a half of the total number of ventrals), and at one head

length before vent, respectively. Values for symmetric

head characters are given in left/right order.

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Asiatic Herpetological Research, Vol. 1 1

2008

Abbreviations of measurements and other meristic

characters.-

Measurements and ratios: HL: head

length; SVL: snout-vent length; TaL: tail

length; TL: total length; TaL/TL: ratio tail length /total

length.

Meristic characters: DSR: formula of dorsal scale

rows; MSR: number of dorsal scale rows at midbody;

IL: infralabials; SC: subcaudals; SL: supralabials; VEN:

ventrals.

Museum abbreviations.- BMNH: Natural History

Museum, London, UK. - FMNH: Field Museum of

Natural History, Chicago, USA. - MNHN: Museum

National d’Histoire Naturelle, Paris, France. -

ZFMK: Zoologisches Forschungsmuseum Alexander

Koenig, Bonn, Germany.

Results

Translation of the original description of

Ceratrimeresurus shenlii.- The original description

appeared in Liang (2003: 411), but is credited to Liang

and Liu. It was published in Chinese with a short

English summary. A poor quality, small color photo-

graph of the holotype appears on Plate 8, Figure 13 of

the book. A verbatim translation of the original descrip-

tion should read as follows (numbers placed in square

brackets refer to our comments placed after this transla-

tion):

Jiao laotietou Ceratrimeresurus shenlii Liana

|2| °

and Liu, gen. and sp. nov. (See colour plate 13 )

The upper eyelid forms on each side a raised triangle

covered with small scales, the base of the triangle being

slightly like a three-sided pyramid; the length of the tri-

angular horn is 1 .5 times the diameter of the eye, and the

height from the tip of the triangle to the edge of the

upper eyelid is equal to the eye diameter. The tip of the

snout is blunt, the upper snout surface is broad and flat-

tened, with the lateral edge slightly upturned. The length

of the snout is about a 1/4 of head length and 1/3 of head

width. The nostril is placed near the tip of the snout,

slightly directed sideward and almost rounded in shape,

the opening being in the middle of a divided nasal scale;

there is one upper nasal scale. There are 5 small scales

between the two nostrils . The upper head surface is

covered with granular scales, which become progres-

sively larger and more keeled from the front to the back

of the head. The cephalic surface is slightly convex and

becomes progressively flat and broader backwards.

There are 7 small interorbital scales . There are 11

small scales surrounding the base of the triangular horn.

The tip of the triangular horn is one rather large scale, to

which is adjacent a smaller scale which makes the horn

looking as bifurcated. The eye is rather large and the

third of its volume is out of the head. The iris is a verti-

cally elongated oval. There is a row of small scales

around the orbit and many scales between the snout side

and temporal region. The forward sunken part of the

occiput is roughly equal to the length of the snout, with

the end of the neck plunged in it. The neck is slender, its

diameter being less than half of the head width. There

are 14 supralabial scales , among which the first and

second ones are separated from the nasal scales by a row

of 3 small scales, and the third to fifth ones do not enter

the loreal pit161, but are separated from it by a row of

small scales; the edge of the upper jaw is convex down-

wards; 14 infralabials, the fourth being the largest, the

first to the fourth ones in contact with the anterior chin

shields, and the posterior chin shields being rather large.

One large fang is located on the anterior part of the

upper jaw, followed by 2 smaller teeth. No teeth on the

palatine and pterygoid . Five small teeth are present on

the lower jaw. The loreal pit is placed antero-inferiorly

to the eye.

The head is peach-shaped and slightly flattened,

with a narrow and elongated neck; the body is rather

wide but slender and long, with a long, pointed tail. The

dorsal scales are strongly keeled, 23-23-21-17 rows, the

rows of cervical scales are more oblique than those of

the body scales. There are 187 ventral scales +77 pairs

of subcaudals. The anal scale is single. The vertebral

scales are normal.

The back of the body is of a grey color, which

becomes light grey on the sides. On the head upper sur-

face, there is a ‘X’ shaped pattern, made of a blotch

extending from the nasal scale to the front of the oppo-

site triangular, and of a blackish brown elongated blotch

extending from the posterior of the horn through the

rearward supraorbital scale (behind the horn) backwards

up to the posterior of the head. A white streak extends

from the posterior margin of the eye through the tempo-

ral up to the posterior of the head, followed by a blackish

brown stripe from the comer of the mouth to the sides of

the throat. The upper and lower lips are marked with

blackish brown square shaped spots. Diffuse and irregu-

lar spots cover the rest of the body throughout. The dor-

sal side of the head and the body is brown in color.

Dorsal blotches extend on each side from the middle of

the body to the vent, the right and the left rows being off-

set and these blotches being linked together by their

inner comer; they are merged together in the middle of

the body and linked each other again by the inner comer

on the posterior part of the body, then merged together

again from the vent to the end of the tail. Light grey-

brown spots on the lower part on the sides of the body,

roughly square in shape or almost rounded, oval or

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Asiatic Herpetological Research, Vol. 1 1

19

reduced to short crossbars. The venter is pale grayish-

brown, without other spot in the middle, so that the ven-

tral side of the body is light grey-brown color through-

out. There is a pointed scale on the tip of the tail. The

total length of the specimen: 362 + 78 = 440 mm, length

of the tail/total length = 0.564, weight = 24 g.

The specimen was collected in the thatch on the top

ot a house near a forest, coiled with the head in the mid-

dle ot its body. Not very active. It was knocked down

with a little stick on the ground and captured. The local-

ity is^the “Working site 02” at Wuzhishan forest, Ruyuan

Xian , July 1996. The specimen is deposited in the

Laboratory of Zoology of the Faculty of Biotechnology,

Jinan University'91.

Comments on the original description. - In this original

description, no mention of the sex of the specimen is

given, although it is a female according to the shape of

the base of the tail. Other comments are:

[1] : Translation: “Homed iron-head [snake]”. The ver-

nacular name “ Laotietou ” is given in China to

Protobothrops mucrosquamatus, due to the shape

of its head, looking like an ancient iron.

[2] : It should rather appear as Plate 8: fig. 13.

[3] : This value includes the two supranasals plus the

scales separating these latter ones.

[4] : If one counts the cephalic scales on a line connect-

ing the middle of the supraoculars, the value is 14

(see below).

[5] : This value is obviously erroneous; we counted 8 SL.

[6] : This is obviously a lapsus for the orbit.

[7] : this lack of teeth on the palatine bones is surprising

and may be an artifact. Teeth are very commonly

miscounted, as they are masked by the tissues and

one usually needs to peel back the gum tissues to

count the sockets. All the tooth counts in this

description may be unreliable (A. Malhotra, pers.

comm, January 2006).

[8] : Ruyuan County, in northern Guangdong Province,

close to the Guangdong-Hunan border. The

Nanling National Nature Reserve is located in the

centre of Nanling Mountains, which extend from

northeast Guangxi to southwest Fujian.

[9] : Guangzhou, Guangdong Province.

Redescription of the holotype of Ceratrimeresurus

shenlii (Figs. 1-3).- The original description includes

some imprecision or mistakes. In combining data of the

original description and our own data, the morphology is

as follows:

Body moderately stout; head subtriangular, wide at its

base, clearly distinct from the neck, thick and swollen

when seen from the side, depressed between the uplifted

areas of the supraoculars. Snout average in relative

length, about one quarter of HL, bluntly rounded when

seen from above, depressed in its center, obliquely trun-

cated when seen from the side, with a distinct canthus

rostralis. Eye relatively large. Tail long and tapering.

The holotype is now in average condition and somewhat

dessicated:

SVL: 362 mm; TaL: 78 mm; TL: 440 mm; ratio

TaL/TL: 0.177.

VEN: 187; SC: 77, paired, plus one terminal scale; anal

shield entire.

DSR: 23-21-17 scales, rhomboid, distinctly keeled.

Rostral visible from above, broader than high, trian-

gular; nasals subrectangular, divided, with a round nos-

tril opening near the tip of the snout, directed slightly

sideward and almost rounded in shape; 2 intemasals on

each side, separated by 2 small scales; 4/4 canthal

scales, slightly larger than adjacent snout scales, border-

ing the canthus rostralis ; 2 elongate upper preoculars

above the loreal pit; lower preocular forming the lower

margin of loreal pit; 3/3 small postoculars; 5/5 supraoc-

ulars on an uplifted triangular area, of which the two

central supraocular scales are strongly enlarged, triangu-

lar and strongly obliquely erected (“horn like”) and

extending out of the head margin, 1.5 times the diameter

of the eye, convergent and originating from the same

base covered with small scales; 8 or 9 slightly enlarged

scales on upper snout surface on a line between the

scales separating the intemasals and a line connecting

the anterior margins of eyes, smooth, juxtaposed, irreg-

ular in shape; 14 cephalic scales on a line between the

base of the supraoculars (including the scales covering

the uplifted basal area), smooth, flat and juxtaposed;

occipital scales larger than cephalic scales, distinctly

keeled; temporal scales small, obtusely keeled: 9 SL. 1st

SL separated from nasal; 2nd SL bordering the anterior

margin of the loreal pit, 3rd SL largest, separated from

the subocular by 1 scale; 4th SL much smaller than 3rd

SL, separated from the subocular by 1 scale; 5th and pos-

terior SL much smaller; 14 IL. first four pairs in contact

with anterior chin shields.

The coloration and pattern agree with that of the

original description. The light postocular streak is quite

conspicuous, but the overall pattern of the body is rather

faded.

Discussion

Nomenclatural considerations.- The combined descrip-

tion of the genus and species was brief and may raise

some questions about its validity. Two points need to be

discussed. The first one relates to the combined descrip-

tion of the genus Ceratrimeresurus and of its sole

20

Asiatic Herpetological Research, Vol. 1 1

2008

Figure 1. Ceratrimeresurus shenlii, holotype. General

view of body. Photograph by Tian Mingyi.

Figure 3. Ceratrimeresurus shenlii, holotype. Lateral

view of the head. Photograph by Tian Mingyi.

included species, shenlii. According to Art. 13.4 of the

International Code of Zoological Nomenclature (1999),

in the present case, such a “description of a new nominal

genus and a single included new nominal species is

deemed to confer availability on each name under

Article 13.1.1.” Art. 13.1.1 requires that every new name

published be accompanied by a description purported to

differentiate the taxon. This combined description is

hence considered to be complying with the requirements

of the Code.

The second point relates to the identification of the

holotype. No number was cited in the description, and

this specimen was still deposited in Prof. Liang Qishen’s

private collection in December 2005. Nevertheless, as

the name of the collection into which the specimen will

be eventually deposited was clearly indicated in the

original description, and as it was confirmed to us that

the specimen will eventually be deposited in this collec-

tion, we consider that this point complies with Art.

16.4.2 of the Code and makes valid the description of

Figure 2. Ceratrimeresurus shenlii, holotype. General

view of body. Photograph by Tian Mingyi.

Figure 4. Head of the second Chinese specimen of

Protobothrops cornutus. Photograph by Fu Jie.

the specific nomen.

A comparison of Ceratrimeresurus shenlii with other

horned species.- In the Asian mainland, only

Protobothrops cornutus and Triceratolepidophis siever-

sorum have erected supraoculars. Trimeresurus wiroti

has only slightly raised supraoculars (see David et al.,

2006). Another species occurring in western Indonesia,

Trimeresurus brongersmai, has erected hom-like

supraoculars. However, this latter species, related to

Trimeresurus wiroti, differs from the other homed

species by several characters, including the shape of its

snout (David et al., 2006). On the basis of Ziegler et al.

(2001), Herrmann et al. (2002), Herrmann and Ziegler

(2002), Herrmann et al. (2004) and of specimens exam-

ined by ourselves, a comparison between horned species

and Ceratrimeresurus shenlii is given in Table 1 .

Triceratolepidophis sieversorum differs by its

greater number of ventral scales and a different structure

of horns, free and strongly divergent. This species also

2008

Asiatic Herpetological Research, Vol. 1 1

21

*'*38

... MUli ASO

VII KAM

RA-wucmufXA

' mm*. •**„

' *!!«►! -

mm hx-im >,

Figure 5. Distribution map of of Protobothrops cornutus.

shows a peculiar structure of keels of dorsal scales,

which is not found in other homed pitvipers (Ziegler et

al., 2001). The dorsal keels of Ceratrimeresurus shenlii

are narrow and made of a single ridge.

In contrast, Ceratrimeresurus shenlii cannot be dis-

tinguished from Protobothrops cornutus otherwise than

by minor characters. In both species, the horns stem

from the same base and are free only in their outermost

part. All scalation characters are similar, including the

keeling of the dorsal scales (see Table 1). Other charac-

ters include the keeled occipital scales, the large 3rd SL

separated from the subocular by 1 scale row, 4th and 5th

separated from the subocular by 1 or 2 scales, the num-

ber of intemasals and of supraoculars. The sole differ-

ence bears on the numbers of pairs of infralabials in con-

tact with the anterior chin shield, first 3 pairs in P. cor-

nutus vs. first 4 pairs in Ceratrimeresurus shenlii. The

pattern is also similar, especially the dorsal blotches, the

upper head dark pattern and the postocular streak.

On the basis of the similarities in morphological

characters, we synonymize Ceratrimeresurus shenlii

Liang and Liu in Liang (2003) with Trimeresurus cornu-

tus Smith, 1930, now Protobothrops cornutus (see

Herrmann et al. [2004] for the generic position of this

species).

At the generic level, the point to be now discussed

is if the homed species Trimeresurus cornutus Smith,

1930 should be referred to a genus distinct from

Protobothrops, in which all other included species are

hornless. In this case, the generic nomen

Ceratrimeresurus is available. Pending molecular analy-

ses that should clarify the relationships between the

Chinese and the Vietnamese populations, we have to

rely only on morphology. Only two currently recognized

genera of mainland Asia include species with truly erect-

ed supraoculars (“horns”), namely Protobothrops (only

for P cornutus ) and Triceratolepidophis (T. sieverso-

nim). On the basis of the similarities between P. cornu-

tus and C. shenlii, we adopt a conservative approach in

considering that erected supraoculars have been conver-

gently evolved in several lineages. Consequently, we

synonymize the genus Ceratrimeresurus Liang and Liu

in Liang (2003) with Protobothrops Hoge and Romano

Hoge, 1983.

A second Chinese specimen of Protobothrops cor-

nutus has appeared on Internet in summer 2005 (Fig. 4),

from Shimentai Nature Reserve (24° 22'-24° 3 1 ' N, 113°

05'- 113° 31' E), Nanling Mountains, Yingde County,

Guangdong Province (see Jim and Xu [2002] for a

description of the area). The characters visible on this

picture of a freshly killed specimen are identical with

Table 1. A comparison between known specimens of Asian horned pitvipers.

List of cited specimens. Protobothrops cornutus. (1) ZFMK 75067, Phong Nha-Ke Bang National Park, Quang Binh Province, Vietnam (not seen; after

Herrmann et al., 2004); (2) BMNH 1946.1 .19.25, “Fan-si-pan Mts., Tonking", now Mt. Phang Si Pang, Lai Chau Province, Vietnam; (3) MNHN 1937.35,

“Tonkin”, northern Vietnam. - Triceratolepidophis sieversorum. (4) ZFMK 71262 (holotype), Phong Nha village, Phong Nha-Ke Bang Nature Reserve,

Quang Binh Province, Vietnam; (5) ZFMK 75066, Phong Nha-Ke Bang Nature Reserve, Quang Binh Province, Vietnam; (6) FMNH 255258, Hin Nammo

NBCA, Boualapha District, Khammouan Province, Laos (specimens (5) and (6) not seen; after Herrmann et at, 2002).

22

Asiatic Herpetological Research, Vol. 1 1

2008

those ot Ceratrimeresurus shenlii. The pattern is much

similar to that of Protobothrops cornutus depicted in

Herrmann et al. (2004). According to the data kindly

communicated to us by Mr. Fu Jie, the author of the pho-

tograph, this second specimen was seen in May 2005. It

was held in alcohol in the home of a member of the Yao

minority, to be most likely used as a medicinal beverage.

The locality of this second specimen lies in a hilly area

at approximately 7 5 airline km southeastwards from the

type locality of Ceratrimeresurus shenlii in Nanling

Nature Reserve. According to the Yao owner of this

specimen, this snake was caught by himself while he

was acting as a guide to a scientific survey of the local

herpetological fauna. Several specimens were collected,

but all died in captivity within some days. This species

is there considered very rare.

Conclusions

The occurrence of Protobothrops cornutus in China

makes a considerable northeastward range extension for

this latter species, previously considered endemic to

Vietnam (Nguyen et al., 2005) (see map on Fig. 5).

Ziegler et al. (2006) recorded a specimen of

Protobothrops cornutus from Ha Giang Province, in

extreme northern Vietnam, close to the border with

Yunnan and Guangxi Zhuang Autonomous Provinces.

The previously northernmost known locality, Mt. Phang

Si Pang (formerly Mt. Fan Si Pan) and Nanling

Mountains are separated by about 980 airline kilome-

ters. Chinese specimens are separated from the new

locality cited in Ziegler et al. (2006) by about 760 airline

km.

We also consider that the wide gap between the

Chinese population and its Vietnamese relatives may

most likely reflect a lack of appropriate collecting effort

in elevated areas of Guangxi Zhuang Autonomous and

Guangdong Provinces than a real geographical gap.

Protobothrops cornutus should be searched for in forests

of mountain or hill ranges such as Darning Shan and

Dayao Shan (Guangxi Zhuang Autonomous Province)

and in various hills of northern Guangdong located

between the Nanling Mountains and the mountain

ranges of Guangxi Autonomous Region. The herpeto-

fauna of this latter province is still quite poorly known.

Besides the provinces of Guangxi and Guangdong, this

species should be searched for in forested areas of south-

ern Yunnan (see Herrmann et al., 2004). Herrmann et al.

(2004) also showed that, in Central Vietnam, P. cornutus

also occurs in the lowlands. However, lowland areas of

southern China are quite dry (Anonymous, 1998). These

lowlands separate the hill or mountain ranges of south-

ern China which share a subtropical humid climate with

high annual amounts of rainfall (above 2000 mm), a sit-

uation which leads to the isolation of the populations of

P. cornutus in this region.

A discussion on the zoogeographical affinities

between North Vietnam and various regions of South

China are outside the scope of this paper, but a mere

comparison between the snake faunas of northern

Vietnam and the hills of Guangxi and Guangdong sug-

gests strong similarities. The occurrence of

Protobothrops cornutus in northern Guangdong, as well

as of Shinisaurus crocodilurus and Amphiesma bitaenia-

tum in northern Vietnam (David et al., 2005; respective-

ly Le and Ziegler, 2003) reinforces the zoogeographical

relationships of these areas connected by more or less

contiguous hill or mountain ranges receiving high annu-

al amounts of rainfall.

Acknowledgments

We are indebted to Q. Liang (Guangzhou, China), who

was instrumental in letting TM re-examine the holotype

of Ceratrimeresurus shenlii. We are grateful to A.

Malhotra (Bangor, United Kingdom), M. W. Lau (Hong

Kong, China) and T. Ziegler (Koln, Germany) for their

comments on the draft of this paper; we especially thank

T. Ziegler who made available and permitted us to cite

his paper in press. Lastly, we thank J. Fu (Guangzhou,

China) and N. Wu (Beijing, China) for their kind permis-

sion to use in our paper the picture of the second Chinese

specimen of Protobothrops cornutus.

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occurrence of Amphiesma bitaeniatum (Wall,

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group of Amphiesma parallelum (Boulenger, 1890)

(Serpentes, Colubridae, Natricinae). Salamandra

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Viperidae) based on morphology and molecular

data. Herpetologica 60(2): 211-221.

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Nomenclature. Fourth Edition. International Trust

of Zoological Nomenclature, London, xxix +306

pp.

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a remote mountain area in South China.

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four mitochondrial gene regions suggests a revised

taxonomy for Asian pitvipers ( Trimeresurus and

Ovophis). Molecular Phylogenetics and Evolution

32: 83-100.

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phylogeny of four mitochondrial gene regions sug-

gests a revised taxonomy for Asian pit vipers

(; Trimeresurus and Ovophis)". [Mol. Phylogen.

Evol., 32 (2004) 83-100], Molecular Phylogenetics

and Evolution 34: 680-681.

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luc ech nhai va bo sat Viet Nam. A checklist list of

Amphibians and Reptiles of Vietnam. Nha xuat ban

Nong Nghiep, Hanoi. 180 pp. (In Vietnamese).

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Guangdong Nanling National Nature Reserve.

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Huang, D. T. Yang and D. J. Li (Eds.). 1998. Fauna

Sinica. Reptilia Vol. 3. Squamata Serpentes.

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Pis. (In Chinese).

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S. G. Pauwels. 2001 [2000], Triceratolepidophis

sieversorum, a new genus and species of pitviper

(Reptilia: Serpentes: Viperidae: Crotalinae) from

Vietnam. Russian Journal of Herpetology 7(3):

199-214.

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Grubenotterarten in Vietnam. Mitteilungen

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Populationsschutze e.V. 18(2): 24—26.

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28(2): 29-40.

Submitted 13 June 2006

Accepted: 04 April 2007

pp. 24-30

Asiatic Herpetological Research, Vol. 1 1

20081

The Effects of Incubation Temperature On Hatching Success,

Embryonic Use of Energy and Hatchling Morphology in

the Stripe-tailed Ratsnake Elaphe taeniura

Wei Guo Du1’* and Xiang Ji1’2

1 Hangzhou Key Laboratory for Animal Sciences and Technology, Hangzhou Normal

University, 310036, Hangzhou, Zhejiang, R R. China

2 Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences,

Nanjing Normal University, Nanjing 210097, Jiangsu, P R. China

* Corresponding author E-mail: dwghz@126.com

Abstract .- We incubated eggs of Elaphe taeniura at 22, 24, 27, 30 and 32°C to examine the effects of incubation tem-

perature on hatching success, embryonic use of energy and hatchling morphology. Incubation temperature affected

incubation length and most hatchling traits examined in this study. Incubation length increased nonlinearly as tem-

perature decreased, with the mean incubation length being 101.7 d at 22°C, 86.0 d at 24°C, 66.3 d at 27°C, 53.9 d at

30°C, and 50.5 d at 32°C. Hatching successes were lower at the two extreme temperatures (41.2% at 32°C, and

50.0% at 22°C) than at the other three moderate temperatures (78.1-79.3%). Hatchlings from the extreme high incu-

bation temperature (32°C) were smaller in body size and wet body mass. High incubation temperatures resulted in

production of less developed hatchlings that characteristically had less developed carcasses but contained more unuti-

lized yolks. The proportion of energy transferred from the egg contents to the hatchling was 71.1% at 22°C, 80.2%

at 24°C, 81 .5% at 27°C, 82.6% at 30°C and 83.9% at 32°C. Taking the lowest hatching success at 32°C and the sub-

stantially prolonged incubation lengths at 22°C into account, we conclude that these temperatures are not suitable for

embryonic development in E. taeniura. Our data confirm the prediction that there are some thresholds over which

incubation temperatures can affect hatching success, embryonic use of energy and hatchling morphology.

Keywords.- Reptilia, Colubridae, Elaphe taeniura, egg, incubation, temperature, hatchling phenotype.

Introduction

As in other vertebrate and invertebrate taxa, temperature

may profoundly influence embryonic development in

reptiles. Compared to avian embryos, reptilian embryos

can develop under a relatively wide range of tempera-

tures (Birchard, 2004, Booth, 2004). Low temperatures

slow embryogenesis but usually have little lethal effect

on embryos; high temperatures result in faster embryon-

ic development (and thus, shortened incubation or gesta-

tion length) but often increase embryonic abnormality or

mortality (e.g. Andrews and Rose, 1994; Andrews et al.,

1997; Deeming and Ferguson, 1991; Sexton and

Marion, 1974; Shine and Harlow, 1996). Apart from the

effects on the rate of embryonic development and

embryonic abnormality or mortality, thermal environ-

ments experienced by developing embryos also affect a

number of phenotypic attributes of the hatchling, includ-

ing morphology (Du and Ji, 2002; Ji and Du, 2001a, b;

Overall, 1994), energy reserves (Du and Ji, 2001),

behavior (Burger, 1991; 1998), post-hatching growth

(Brana and Ji, 2000; Du and Ji, 2003; Rhen and Lang,

1995), and gender in species with temperature-depen-

dent sex determination (Janzen and Paukstis, 1991). It

has been repeatedly reported for oviparous reptiles that

eggs incubated at optimal temperatures not only exhibit

high hatching success but also produce good-quality

hatchlings.

The range of optimal temperatures for reptilian

embryos is often narrow and varies not only among but

also within species. For example, the optimal incubation

temperatures fall within the range from 24°C to 26°C in

Xenochrophis piscator (checkered keelback; Ji et al.,

2001) and Deinagkistrodon acutus (five-paced pit-viper;

Lin et al., 2005) but, in Elaphe carinata (king ratsnake;

Ji and Du, 2001b), Naja atra (Chinese cobra; Ji and Du,

2001a), Rhabdophis tigrinus lateralis (red-necked keel-

back; Chen and Ji, 2002), Dinodon rufozonatum (red-

banded wolf snake; Ji et al., 1999b; Zhang and Ji, 2002),

Ptyas korros (gray ratsnake; Du and Ji, 2002) and P.

mucosus (mucous ratsnake; Lin and Ji, 2004), generally

within the range from 26°C to 30°C. Pelodiscus sinensis

(Chinese soft-shelled turtle), however, has a wider range

of optimal incubation temperatures, because tempera-

tures exert no important effects on hatching success and

hatchling phenotypes within the range from 24°C to

34°C (Du and Ji, 2003; Ji et al., 2003). In Eumeces chi-

nensis (Chinese skink), eggs from a lower latitudinal

population have a narrower range of optimal incubation

2008

Asiatic Herpetological Research, Vol. 1 1

25

temperatures than do those from a higher latitudinal

population, primarily because of more stable thermal

environments in the former population (Ji et ah, 2002).

Overall, previous studies suggest that optimal tempera-

tures for developing embryos differ among reptiles dif-

fering in habitat use and/or distributional range.

The stripe-tailed ratsnake Elaphe taeniura is a large

sized (to 1800 mm SVL [snout-vent length]) oviparous

colubrid snake that ranges from the central and southern

provinces of China to Korea, Burma, Laos, Vietnam and

India (Zhao, 1993). Wild population of this snake have

declined dramatically due to habitat loss and over-har-

vesting over the past two decades (Zhao, 1998).

Fecundity, reproductive output and embryonic mobiliza-

tion of energy and material during incubation have been

reported for snakes from Zhoushan Islands (Ji et al.,

1999a; 2000). Because eggs were never incubated at

multiple temperatures, the range of incubation tempera-

tures optimal for developing embryos of E. taeniura

remains unknown. To fill this gap, we incubated eggs

produced by females from a southern population

(Guangxi, China) at five constant temperatures ranging

from 22°C to 32°C. Specifically, our aims are to (1)

examine the effects of incubation temperatures on hatch-

ing success, embryonic use of energy and hatchling mor-

phology, and (2) determine the range of optimal temper-

atures for embryos of E. taeniura.

Materials and Methods

We obtained 13 gravid E. taeniura (SVL: 1220-1690

mm; postoviposition body mass: 277.9-755.0 g) in June

1998 from a private hatchery in Guilin (Guangxi, south-

ern China), and brought them to our laboratory in

Hangzhou, where they were maintained in a 2000 x 800

X 800 (length X width X height) mm wire cage placed in

a room inside which air temperatures were never higher

than 30°C. Food (eggs of Coturnix coturnix) and water

were provided ad libitum. The snakes laid eggs between

23 June and 3 July (clutch size: 8.8±0.4, range: 7-11).

We collected the eggs within a few hours after being

laid. Each egg was measured (to the nearest 0.01 mm)

for length and width with a Mitutoyo digital caliper and

weighed (to the nearest 1 mg) on a Mettler balance. One

freshly laid egg from each clutch was dissected to deter-

mine the composition of eggs. Egg contents (yolk plus

embryo) were placed in pre-weighed glass dishes, and

weighed. Shells were briefly rinsed, dried by blotting

with paper towels and weighed. Egg contents and shells

were weighed again after oven drying to constant mass

at 65°C. The remaining eggs were incubated systemati-

cally at five constant temperatures (22, 24, 27, 30, and

32 [± 0.3]°C); such that eggs from single clutches were

distributed almost equally among the five temperature

treatments.

Eggs were individually incubated in covered plastic

jars containing known amounts of vermiculite and dis-

tilled water at approximately -12 kPa water potential

(vermiculite: water = 1:2). One-third of the egg was

buried lengthwise in the incubating substrate, with the

surface near the embryo exposed to air inside the jar.

Jars were equally assigned to five incubators (Guanzhou

medical instrument, China), with incubation tempera-

tures set at 22, 24, 27, 30, and 32 (± 0.3)°C, respectively.

We moved jars among the shelves in the incubator daily

according to a predetermined schedule to minimize any

effects of thermal gradients inside the incubator. Jars

were weighed on alternate days, and distilled water was

added evenly into substrates when necessary to compen-

sate for evaporative losses and water absorbed by eggs,

thereby maintaining the substrate water potential con-

stant.

The duration of incubation, measured as the number

of days to pipping, was recorded for each egg.

Hatchlings were collected, measured (for SVL and tail

length), and weighed a few hours after hatching, and

then euthanized by freezing to -15°C for determination

of composition and sex. The killed hatchlings were sep-

arated into carcass, residual yolk and fat bodies. The

Table 1. The effect of temperature on incubation duration, hatching success, and sex ratio in Elaphe taeniura. Data on

incubation duration are expressed as mean±1 SE.

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Figure 1. Adjusted means (±1 standard error) for body

mass, SVL and tail length of hatchlings from different

incubation temperatures, with initial egg mass being set

at 23.5 g. Adjusted means with different letters differ sig-

nificantly. Numbers under the error bars in the upper plot

are sample sizes, and are applicable to the other two

plots.

three components of the hatchling were dried in an oven

(65°C) to constant mass, weighed and preserved frozen

for later analyses. We determined the sex of hatchlings

by pressing on both sides of the ventral tail base with

forceps to record the presence or absence of hemipenes;

hatchlings with everted hemipenes were recorded as

males.

We extracted non-polar lipids from dried samples of

egg contents and hatchlings in a Soxhlet apparatus for a

minimum of 5.5 h using absolute ether as solvent. The

amount of lipids in each sample was determined by sub-

Temperature (°C)

Figure 2. Carcass, yolk sac and fat bodies of hatchling

Elaphe taeniura incubated at different temperatures.

Graphs show adjusted mean (± standard error), with ini-

tial egg mass as the covariate. Adjusted means with dif-

ferent letters above the error bars differ significantly.

Numbers under the error bars in the upper graph are

sample sizes, and apply to all graphs within this figure.

tracting the lipid-free dry mass from the total sample dry

mass. We determined energy density of each dried sam-

ple using a GR-3500 adiabatic bomb calorimeter

(Changsha Instruments, China).

All data were tested for normality using

Kolmogorov-Smimov test and for homogeneity of vari-

ance using Bartlett’s test. Parametric analyses were used

to analyze data when the assumptions for these analyses

were met; otherwise, nonparametric analyses were used.

Values are presented as mean ± 1 standard error, and the

significance level is set at a = 0.05.

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Table 2. Lipids and energy in egg contents and shell dry mass of hatched eggs and freshly laid eggs in Elaphe taeniura

Data are expressed as adjusted mean ±1 SE, with initial egg mass as the covariate.

Results

The mean values for incubation length varied consider-

ably among the five temperature treatments (ANOVA;

F475 = 687.2, p < 0.0001). Incubation length decreased

as incubation temperature increased, but not in a linear

pattern. The mean incubation length was shortened by

3.5 d from 30°C to 32°C, 12.4 d from 27°C to 30°C,

19.7 d from 24°C to 27°C, and 15.7 d from 22°C to 24°C

(Table 1). Incubation temperature affected hatching suc-

cess (G-test, G =11.51, df— A, p < 0.05), but not the

sexual phenotype of hatchlings (G = 4.73, df = 4,

p > 0.25). Hatching successes were apparently lower at

the two extreme temperatures (22°C and 32°C) than at

the other three moderate temperatures (24, 27, and

30°C) (Table 1).

There were no between-sex differences in all exam-

ined hatchling variables (all p > 0.05), so we pooled data

for both sexes. All examined hatchling variables, except

yolk sac, were positively correlated with initial egg

mass). ANCOVAs with initial egg mass as the covariate

showed that incubation temperatures significantly

affected wet body mass (F474 = 3.67, p <0.01; Fig. la),

SVL (F4 j4 = 4.21, p < 0.01; Fig. lc) and tail length ( F474

= 4.31,/? < 0.01; Fig. Id) of hatchlings, but not hatchling

dry body mass ( F 474 = 0.38, p > 0.05; Fig. lb). Mean

values for wet body mass, SVL and tail length were all

smaller in hatchlings incubated at 32°C than in hatch-

lings incubated at the other four temperatures (Fig. la, c,

d). Hatchlings from different incubation temperatures

differed in carcass mass (ANCOVA with the initial egg

mass as the covariate: F474 = 6.36, p < 0.001; Fig. 2a)

and yolk sac dry mass (ANOVA: F475 - 4.79, p < 0.01;

Fig. 2b), but not fatbody dry mass (F474 = 0.37,

p > 0.05; Fig. 2c). Hatchlings from 32°C had lighter car-

cass dry mass but larger residual yolk sac, whereas

hatchlings from 22°C had heavier carcass but smaller

residual yolk sac (Fig. 2a, b).

Energy contents (F4 74 = 2.78, p < 0.05) and non-

polar lipids (F474 = 2.80, p < 0.05) differed among

hatchlings from different incubation temperatures, with

hatchlings incubated at 22°C containing lower quantities

of energy and non-polar lipid than did those incubated at

other four temperatures (Table 2). Conversion efficiency

of energy during incubation at 22°C (71.1%) was thus

lower than those at 32, 30, 27, and 24°C (83.9%,

82.6%, 81.5%, and 80.2%). Similarly, conversion effi-

ciency of lipid at 22°C (55.3%) was lower than those at

32, 30, 27, and 24°C (70.9%, 69. %, 67.3% and 64.9 %).

Additionally, shells of hatched eggs were significantly

lighter than shells of freshly laid eggs (F5 84 = 3.19, p <

0.05; Table 2).

Discussion

As in numerous other reptiles, thermal environments

experienced by developing embryos affect hatching suc-

cess, incubation length, embryonic expenditure of ener-

gy, and linear dimensions (SVL and tail length) and

body composition of hatchlings in Elaphe taeniura. Our

results provide support for the previous conclusion that

reptilian embryos developing at relatively low or moder-

ate temperatures produce well developed and thus, larg-

er hatchlings (e.g. Deeming and Ferguson, 1991; Du and

Ji, 2002; Ji and Du, 2001a, b). The larger hatchling size

has an association with the greater carcass dry mass

(Chen and Ji, 2002; Du and Ji, 2002; Ji et al., 1997; Ji

and Sun, 2000). Data from the current study proved that

this conclusion is also true in E. taeniura (Fig. 1; Fig. 2).

The finding that more yolks remain unutilized at hatch-

ing when eggs are incubated at high temperatures has

been reported for nearly all reptiles studied to-date (e.g.,

Beuchat, 1988; Du and Ji, 2002; Ji and Du, 2001a, b; Lin

et al., 2005; Phillips et al., 1990; Phillips and Packard,

1994). In E. carinata (Ji et al., 1997), E. taeniura (Ji et

al., 1999a), D. rufozonatum (Ji et al., 1999b) and P. kor-

ros (Ji and Sun, 2000), hatchlings exhibit a substantial

increase in size (SVL) during the first post-hatching

days due to the transfer of resources in the residual yolk

into carcass.

The proportion of lipids transferred from the egg

contents into the hatchling was noticeably lower than

those of energy. Given that mass-specific energy density

is much higher in lipids than in proteins and carbohy-

drates, this result provides evidence that lipids are the

main source of energy for embryonic development.

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2008

Hatchling size and mass were primarily determined by

the embryonic expenditure of energy during incubation

after removing the influence of variation in initial egg

mass (Du and Ji, 2001, 2003; Ji and Du, 2001a, b).

Given that the total energy invested in an egg is fixed,

any increase in embryonic expenditure of energy during

incubation may inevitably result in production of small

sized or lighter hatchlings. Incubating eggs at low tem-

peratures or high temperatures may increase energy

expenditure for embryonic development due to the

increased incubation length and/or embryonic metabo-

lism (Booth, 1998; Booth and Astill, 2001; Packard and

Packard, 1988). Consequently, eggs incubated at moder-

ate temperatures usually produce larger and heavier

hatchlings than did those at low or high temperatures.

A prolonged exposure of eggs of E. taeniura to tem-

peratures lower than 24°C or higher than 30°C may have

a detrimental effect on embryonic development, as indi-

cated by the fact that hatching success decreases dramat-

ically at temperatures outside this temperature range

(Table 1). The mean incubation length 30°C is 53.9

days, approximately 3.4 days longer than that at 32°C,

so the ecological disadvantage of the increased incuba-

tion length (and thus, decreased growth period prior to

the onset of the first winter) due to a decrease in incuba-

tion temperature from 32°C to 30°C is less pronounced.

Considering that less developed hatchlings are produced

at 32°C and that hatching success is low at this temper-

ature, we conclude that the temperature of 32°C is out-

side the range of optimal temperatures for incubating

eggs of E. taeniura. Eggs incubated at 24°C exhibit high

hatching success and produce well developed hatch-

lings. However, the majority of hatchlings from eggs

incubated at 24°C appear between late September and

mid-October, so the growth prior to the onset (late

November) of the first winter for these hatchlings is

about 1.5-2 months. As incubation length increasingly

increases as temperature decreases in E. taeniura (Table

1 ), we expect that the disadvantage of incubating eggs of

E. taeniura at temperatures lower than 24°C can be very

pronounced due to the increasingly prolonged incuba-

tion length. For eggs of reptiles incubated under natural

conditions, the prolonged incubation length at low tem-

peratures also increases exposure of eggs to the effects

of adverse biotic (increased microbial contamination)

and abiotic factors(extreme thermal and hydric condi-

tions) in the incubation environment of the eggs, which

potentially reduces hatching success. Thus, the tempera-

ture of 24°C is suitable but not optimal for incubating

eggs of E. taeniura. Taking the energy expenditure dur-

ing incubation, the rate of embryonic development and

hatchling phenotypes into account, we consider that

temperatures around 27°C are optimal for incubating

eggs of E. taeniura.

Acknowledgments

This work was supported by a grant from Zhejiang

Provincial Natural Science Foundation. Many thanks

were given to X.-Z. Hu, W.-Q. Xu, and J.-Q. Du for their

assistant in the laboratory.

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Submitted: 1 7 November 2006

Accepted: 22 September 2007

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pp.31-38

Behavioral Observations and Descriptions of the Endangered Knobby

Newt Tylototriton wenxianensis and Their Application in Conservation

Da-Jie Gong* and Mai Mu

Department of Biology, College of Life Science, Northwest Normal University

Lanzhou, Gansu, China.

* Corresponding author E-mail: gongdj@nwnu.edu.cn

Abstract.- Tylototriton wenxianensis is an endangered species at the status of VU. This paper introduces observations

and studies including breeding, territorial, communication and antipredatory behaviors. The rules and mechanisms of

the behaviors are recorded separately and described in the paper. Relationships between the behaviors and environ-

ment are also explained tor the purpose of describing how the environment acted on the behaviors and how the behav-

iors adapted to the environment. The paper ends with conservation plans in connection with behavioral ecology: A)

protect the particular habitat and avoid anthropogenic threats, B) artificial construction for natural migration and gene

communication and C) artificial breeding and re-introduction into nature.

Keywords.- Observations, Tylototriton wenxianensis , behaviors, habitats, conservation.

Introduction

Tylototriton wenxianensis (Caudata: Salamandridae) is

peculiar in China and poorly known. It is only found

along the boundary between Gansu and Sichuan

Province, narrowly distributed in Wenxian, Qingchuan

and Pingwu (details see Table 1 and Fig. 1). It was

defined as a threatened species with the status of VU

(IUCN, 2006).

A representative of the Family Salamandridae, T.

wenxianensis has rough and toxic skin with seasonal

colors. The bilateral warts stay longitudinal and clus-

tered in the same size and the boundaries between them

are not clear enough. It is dark all over apart from the

red-orange fingers, toes and the venter of the tail (Fei,

1993).

The newt lives in the heavily forested mountains at

approximately 940 m. The adult is terricolous and usu-

ally wanders about the pool. It stays hidden under wet

gravel or small muddy caves covered by fallen leaves in

the daytime and appears at night for preying (Gong and

Mou, 2006).

Behavioral studies on tailed amphibians have

recently been reported. The studies were mainly focused

in the following fields: breeding behaviors, including

sexual recognition and sexual selection (Dawley, 1984),

modes of courtship and mating (Arnold et al., 1972,

1977; Salthe, 1967, 1974), sperm competition (Arnold,

1977; Halliday, 1998; Massey, 1988; Sparreboom, 1995;

Verrel et al., 1998), reproductive behavior (Fang, 1984;

Harris et al., 2002; Park et al., 2000), parental care

(Cramp, 1995; Nussbaum, 1985; Peterson, 2000), evolu-

tion of reproduction (Arnold, 1977; Veith, 1998; Verrell

and Krenz, 1998,); migratory behavior (Amtzen, 2002;

Douglas, 1979; Griffiths, 1996; Serbiolova, 1995;

Twitty, 1966); territorial behavior (Mathis, 1991; Mathis

et al.,1998; Mathis et al.,2000; Simons et al., 1997);

communications (Holliday, 1997; Houck, 1988; Houck

and Sever, 1994; Rollmann, 1999; Verrell, 1989,1989a;);

antipredatory behavior (Brodie, 1990; Graves and

Quinn, 2000; Maerz et al.,2001; Sih et al., 2000, 2003;

Storfer and Sih, 1998; Sullivan, 2002; Woody and

Mathis, 1997). All the above provide a source of refer-

ence for the behavioral study on T. wenxianensis.

Methods

Migratory behavior.- We chose the habitat in Qingchuan

as the site to observe, where a large population was

found (Gong and Mou, 2006). The newts are not active

year round except for the breeding season, so their

migratory behaviours seem quite obvious and clear. We

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2008

Date

Figure 2. Reproductive migration of Tylototriton wenxianensis. The polyline represents temperature, columns repre-

sent number of the migratory newts and “ * ” rainy days.

made continuous observations when they migrated for

breeding towards the pools nearby. We marked each

migratory group with varisized loops and observed by

radio tracking and GPS. Finally we made records of the

time, route length, size of groups and sex ratio in the

migration.

Reproductive behavior.- Reproductive behaviors

included mating and spawning. We observed the course

and took down the data of sex ratio, time, envioronment

and quantity of the spawns etc.

Conmmunication.- Communication happens along with

courtship. We tried to understand how the newts com-

municate with each other and why. The communications

included chemical signals and body-contacts (Jiang,

2004). Mechanisms of chemical signals from glands are

expected to be understood through dissection. Types of

the body-contacts and their effects on courtship were

studied.

Territorial behaviors - We measured the size of the ter-

ritory occupied by 6 female newts and size of the tails

and bodies in contrast. Aggressive behaviors related to

the available prey in the environment were also

observed. Relationships between the data and phenome-

non were discussed and concluded.

Antipredatory behavior.- The newts were less aggres-

sive without structures for aggressivity (Jiang, 2004).

They moved too slowly to escape attacks from enemies

and to defend themselves. We made some model ene-

mies and demonstrated the predation and antipredatory

behaviors artificially.

Results and Analysis

Behaviors.- We made continuous observations on the

migratory behaviors and found 186 adults including 170

females and only 16 males, the ratio was 170:16 ($:$)

= 10.625:1. Breeding lasted from early April to late May

with a peak from April 8th to May 6th. The average tem-

perature was 15.5±4°C (Fig. 2).

Like many other newts, T .wenxianensis does not

seem to have significant secondary sexual characters. It

distinguishes and selects the opposite sex primarily by

sight and sense of smell. The male seemed more active.

It courted the female by wagging its tail and dancing

round. Meanwhile, the female was secreting chemicals

and emitting a strong smell to attract the male. Soon the

male bit the female on the tail (or hind limb), then they

circled around both with their tails wagging (Fig. 3).

Experiments show that the male prefers to choose a big-

ger one with more ovums and a stronger smell.

Mating did not accompany spawning in the mean-

time. It was the female that selected where to spawn.

The male moved to the pool and settled down earlier

than the female. However, the female left soon after

spawning, while the male stayed untill the end of the

Figure 3. Model of mating behavior. Shows the male (black) wagging its tail and dancing round.

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33

Table 1 . Distribution and flora of T. wenxianensis along the boundary between Gansu and Sichuan Province.

Figure 4. The sperm transmission. Swells show the col-

loidal secretion.

Figure 6. Rigid body: the reaction to anti-predator. Shows

ribs jutting through the side warts.

Figure 5. The courtship behavior. Shows the transmis-

sion of pheromones in the fantail.

Figure 7 The raised tail and warning color on the ventral

surface of the tail.

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Figure 8. The protuberant glands and warning color on

the back. Shows harmful and toxic skin secretion, glan-

diform warts, spine glands, caudal glands, glandiform

warts, spine glands, caudal glands, tergal glands, etc.

Figure 10. Mimicry: newt playing dead, with venter

upward and body rigid.

v

Figure 9. Playing dead. Shows the newts played dead

with upward bottom and rigid body.

Figure 11. The breeding pool (after spawning). Vicinity of

Wuxing Village, Sichuan Province, southwest China.

Table 2. Relation between spawning territory, food and size of six female newts.

Table 3. The antipredatory behaviors of T. wenxianertsis, grouped by different types. The observations were made in

the field and in artificial conditions, a. harmful skin secretion; b. toxic skin secretion; c. parotids; d. glandiform warts; e.

spine glands; f. caudal glands; g. tergal glands; h. tergal colors; i. ventral colors; j. rigid body; k. roll of body; I. exposed

abdomen; m. raised tail; n. raised jaw; o. arched body; p. larger head; q. swagging tail; r. curved forehead; s. extended

nerve.

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35

Figure 12. The artificial breeding box made of glass, 110

x 40 x 60 cm (lateral view). Shows the constitution and

features of the breeding ecotope under artificial control.

breeding season. The analysis from Douglas (1979):

First, to choose the optimum condition for spawning and

to reduce risks in breeding migration; Second, to control

the male genotype effectively through sexual selection.

Mating behavior happened on land, while spawning

underwater. We analyzed the reason: the adult newt

mainly lives a terricolous life. Sometimes it approaches

the pools or streams for food. The observations on the

behaviors in captivity indicate that the adults needed

almost no water unless they felt too dry. They had adapt-

ed to the climate overland. In addition, in reproductive

seasons, inorganic conditions overland near the pools

may help them to secrete pheromones and sex hormones

which could be significantly reduced underwater.

The newt took no attack on its kin, but became quite

different towards non-related individuals. The newt rec-

ognized its kin through chemical signals. In the hot sum-

mer, the adult admitted the posterity into its territory

sometimes and food became insufficient. Occupying ter-

ritory usually accompanied attacks and fights. That

depended on food quantity, size of body and environ-

ment (Table 2). The newt with longer tail and body

occupied larger territory, closely related to the food

quantity.

Communications were drived mainly by chemical

signals and body contact. Chemical signals are neces-

sary for the accurate and regular courtship behavior. The

newts distinguished the sex and attracted each other by

transmission of pheromones (Fig. 4) and secretion of

hormones (Fig. 5), which later brought about the mating

behavior.

Body contact was also important, particularly in

breeding behavior. Tactile signals were transmitted

through touch on the mouth, collisions and friction

between bodies.

In the long course of evolution, series of antipreda-

tory behaviors had formed against captures, such as

escapes, cryptic coloration, rigid body (Fig. 6), apose-

matic coloration (Fig. 7, Fig. 8), camouflage (Fig. 9),

warning postures (Fig. 7), imitative toxicants, particular

skeletal adaptations (Fig. 6), chemical defenses and

playing dead (Fig. 10) etc. (Table 3).

Relationship between the behaviors and environment-

al female finished spawning in a short time without

male involved. Spawning lasted 2-3 days smoothly in

rainy seasons without interference. They got to the

spawning pools no sooner than they selected the proper

sites. Spawning began one day later. The female

spawned one a time lasting half a hour, and the whole

course 5-6 hours. The course maintained a even pace

slowly without a peak.

In Qingchuan, three pools were found with female

newts and a mass of spawn. The three conditions for

spawning sites were: A) ground covered with plants (the

plants may be divided into three layers: the upper con-

sisting of tall sparse laurisilvae, bushes in the middle and

wet weeds at the bottom), B) spawning sites with semi-

permanent pools, and the water soaked out after rain, C)

it chose a mesa with loose soil as the spawning site on

the hillside, covered by fallen leaves (Fig. 11).

The newt preyed mainly on insects, earthworms and

snails. Like other amphibians, it stayed hidden in the day

and preyed in the night. The behaviors changed sensi-

tively with the temperature and sunlight.

In summers, plants flourished and numerous insects

appeared, when the newt acts frequently. After thunder-

storms, earthworms come out from the soil and supply

food to the newts. In winter, the newts have to hibernate

in response to the lack of food and low temperature.

Conservation plans.-

(1) Protect the particular habitat and avoid anthro-

pogenic threats.

•Figure 10 shows the particular breeding habitat. The

newts enter the pools and ground nearby only in

Table 4. Relationship between the conditions and hatching rate under artificial control.

*Huang (2007) reported that pH 5.2-6.0 was most suitable for the development of larvae.

36

Asiatic Herpetological Research, Vol. 11

2008

reproductive seasons, so it is of great significance to

protect the similar pools. The pools were more or less

50 cm deep, 10 m2 in size and at an elevation of 1000

m. Pieces of rocks piled around. Soil and mud around

the pool was covered densely by rotted leaves. The

vegetation nearby were mainly made up of shrubs and

arbors (see Table 1).

Human activities, especially farming and poaching

severely threatens breeding. Recommendations about

plans to avoid these threats: A) build up fences around

the mating sites and breeding pools to prevent the

natural enemies and poachers from breaking in, B)

reduce farming and grazing by livestock, especially

around irrigation water from the pools, C) make (and

enforce) laws to punish poachers.

(2) Artificial construction for natural migration and

gene communication.

•Protect the whole habitat and ensure the natural

migratory behavior. The migratory routes are uaually

blocked by farmland etc., so it may help keep the

proper migration to build up artificial passages across

the farmland.

Connect the adjacent pools by building canals as

much as possible in order to ensure the communica-

tion of gene from different populations, especially

from breeding groups. The measure may help avoid

inbreeding depression and loss of genetic diversity in

a small population and also help with evolution of the

species.

(3) Artificial breeding and re-introduction into nature

•In view of the high sex ratio ($:$ = 10.625:1 on

average, n = 186) in the breeding season and the low

rate of hatchability (46.54%; n = 1272) in nature, it is

quite feasible to increase the hatching rate and reduce

the sperm competition between the males in artificial

conditions. We directly gathered the females and

eugenic males with longer bodies (longer tails and

bodies seem dominant in the sex competition apt to

survive under natural selection (Jiang, 2004) and

higher sperm density, and also keep the sex ratio at

1:1 in a artificial glass box (Fig. 12) to avoid compe-

tition and injury as well as to ensure high-quality

inheritance.

Studies showed that factors influencing the hatching

rate were natural enemies (such as snakes), climate

changes and pollution (Tian et al., 1997). It is an

effective approach to raising the survival rate and

enlarging the population by hatching out the larvae in

artificial conditions and re-introducing them into nature.

The conditions include temperature, humidity, sunlight

period and pH of water etc. (Table 4). Re-introduce the

juveniles into nature when they complete metamorpho-

sis and get ready to land.

Acknowledgments

We wish to thank K. R. Zhang for assistance and valu-

able help with our investigation of distribution of T.

wenxianensis and J. R. Teng who provided us the speci-

mens of T. wenxianensis.

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Accepted: 22 September 2007

2008

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pp.39-44

Observations on the influence of Seasonality, Lunar Cycles,

and Weather Condition on Freshwater Turtle Activity in Sarawak,

East Malaysia (Borneo)

Karen A. Jensen1’* and Indraneil Das1

1 Institute of Biodiversity and Environmental Conservation

Universiti Malaysia Sarawak,

94300, Kota Samara han, Sarawak, Malaysia.

Corresponding author E-mail: kitti Jensen@yahoo.com

Abstract.- Freshwater turtles were surveyed at two sites in Sarawak: Loagan Bunut National Park and Balai Ringin.

Capture results were tested against environmental factors such as lunar phase, weather and seasonality to examine

differences in activity level. Proportionally, soft-shelled turtles were most active during the full moon (29.0%) and

the last quarter lunar phase (29.4%). Hard-shelled turtles were active during the full moon 50.0% of the time. Both

soft-shelled and hard-shelled turtles were more active during overcast periods (53.0% and 66.0%, respectively).

Seasonality did not seem to affect soft-shelled turtle activity, while hard-shelled turtles were active 50.0% of the time

during the dry South-west Monsoon from June to September.

Keywords.- Testudines, Malaysia, Borneo, capture success, environmental factors.

Introduction

Southeast Asia has a highly diverse freshwater turtle

fauna due to a combination of factors, including the

presence of major mountain massifs, some of the largest

archipelago systems in the world, large tracts of lowland

forests, streams and rivers, high precipitation and tropi-

cal climate (Iverson, 1992; Lovich, 1994). Due to their

cryptic nature and the presence of intense hunting in the

recent past, however, these enigmatic species have been

difficult to study. Consequently, there is a paucity of

published information regarding the current status and

basic ecology of the Southeast Asian fauna, including

those populations endemic to Borneo. As part of a larger

study on the ecology of Amyda cartilaginea (described

in Jensen, 2006), the activity of freshwater turtles in

Borneo in relation to environmental factors, such as

lunar phase, precipitation, and seasonality, were

assessed.

Materials and Methods

The primary study area was Loagan Bunut National

Park (03° 44-03° 00' N, 114° 09-114° 17' E) in north-

ern Sarawak, which is within a three hour drive to the

town of Miri. Field work was concentrated at the Park,

but two visits were also made to Balai Ringin (01° 03'

00" N, 110° 45' 00" E), a fishing village about two hours

driving distance from Kuching. Both sites are located

within peat swamp forests (Fig. 1).

Loagan Bunut National Park contains the only

freshwater floodplain lake in Sarawak (Sayer, 1991),

encompassing 650 ha2 at its maximum diameter. The

lake is completely dry during prolonged droughts.

Annually, the lake completely dries between three and

six times, typically in February, May, and June.

A variety of techniques were attempted to assess the

most effective method for capturing Amyda carti-

laginea. One method employed was the use of hoop

traps according to techniques described by Frazer et al.

(1990), Legler (1960), and Vogt (1980). Native hoop

traps called 'bubu' were ineffective in trapping turtles.

Another local fishing device called a 'selambau' caught

a single turtle in Balai Ringin.

Figure 1. Locations of Balai Ringin and Loagan Bunut

National Park in Sarawak, East Malaysia.

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 1. Total number of individuals of freshwater turtle

species caught during the present study. The asterisk

refers to an unsexed carcass. Sites include: 1 = Loagan

Bunut National Park; 2 = Balai Ringin; 3 = Matang

Wildlife Centre; and 4 = Vicinity of Mulu National Park.

Manual capture, otherwise known as 'muddling'

(Cagle, 1943), was an effective, albeit labor-intensive,

method of collecting turtles in the surrounding forests

and streams, although it was only effective during low

water periods. The technique consists of wading through

streams and probing areas of sand or mud and among

roots with a stick, hands, or feet. Comparisons with

other studies were not possible as there are no compara-

tive studies of Amyda cartilaginea available.

Hard-shelled turtles were searched for in forested

areas by walking 100 m transects (2 m wide) through the

forest, looking under leaves, tree roots, and debris. The

turtles were most frequently located as they crossed

trails or other open areas.

At Loagan Bunut National Park, 51 field days were

spent during five sampling trips. Over the course of 33

evenings, two hoop nets and 60 baited hand lines were

set, totaling 2,046 trap nights. At Balai Ringin, 16 field

days were spent during two sampling trips; in total, 45

baited hand lines were set over 16 evenings, totaling 720

trap nights. There were 2,766 trap nights in total.

With the exception of the Balai Ringin trips, traps

were set for a minimum of seven days. The baits used,

chosen on the basis of availability, consisted of parts of

chicken, pork, or local fishes such as Ikan Kali ( Clarius

nieuhofii), Ikan Toman ( Channa micropeltes) or Ikan

Haruan ( Channa striatus). If habitat conditions changed

noticeably, the traps were re-located to a nearby site with

at least one meter of water depth.

Lunar phase was recorded at the time of capture to

test for possible differences in turtle activity level during

the different phases of the lunar cycle. Turtles that were

captured while physically active, as opposed to resting,

were used in the present study. “Active” animals were

qualified as those specimens caught with traps or lines

because they must have swam or walked to the area of

capture. Three Amyda cartilaginea were found by mud-

dling and one Cyclemys dentata was found buried near

the trunk of a tree. These animals were inactive at the

time of capture and were not included in subsequent

analyses. One A. cartilaginea was caught by baited line

on a trip near Gunung Mulu National Park, and was

included in subsequent analyses, as were any active

hard-shelled turtles found in localities other than the

study sites.

Lunar phase was divided into four categories; new

moon, first quarter, full moon, and last quarter. New

moon was defined as when the non-illuminated side is

facing the Earth; at this time, the moon is not visible,

except during a solar eclipse. First quarter moon was

defined as the phase when one half appears to be illumi-

nated by direct sunlight; during this phase, the illuminat-

ed fraction of the moon's disk increases. Full moon was

the phase when the moon was completely illuminated by

direct sunlight. Last quarter moon was defined as the

phase when one half of the moon appears to be illumi-

nated by direct sunlight; during this phase, the illuminat-

ed fraction of the moon's disk decreases. Moon phases

were obtained from the U.S. Navy Astronomical

Applications Department website (U.S. Navy, 2003). In

the analyses, hard-shelled and soft-shelled species were

pooled separately due to their presumed different behav-

ior, capture method, and habitat use.

To determine if weather affected capture success,

weather conditions at the time of collection was record-

ed, being divided into three categories: clear, overcast,

and raining. Overcast was defined as times when the sky

was completely cloudy and grey, and clear weather was

defined as entirely clear to having some white cumulus

clouds.

Seasonality was divided into the North-east

Monsoon (wet season), the South-west Monsoon (dry

season) and non-monsoonal times, which occurred dur-

ing April, May and October. The North-east Monsoon

prevails from November to March and the South-west

Monsoon occurs from June to September. The North-

east Monsoon brings the majority of precipitation to

Sarawak, while the South-west Monsoon season is typi-

cally characterized by dry weather.

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Asiatic Herpetological Research, Vol. 1 1

41

Last Quarter,

29.4

New, 1 1.8

1st Quarter,

29.4

Full, 29.4

Figure 2. Percentage of Amyda cartilaginea collected

while physically active during various lunar phases.

Last Quarter,

25

New, 16.7

1st Quarter,

8.3

Full, 50

Figure 3. Percentage of hard-shelled turtles collected

while physically active during various lunar phases.

Twenty-two days were spent searching for turtles

during the North-east Monsoon, 15 days were spent

searching during the South-west Monsoon and 30 days

were spent searching during non-monsoonal times.

Results

Species richness - A total of 34 individual turtles from

four species were found at the sites examined (Table 1).

Loagan Bunut National Park.- A total of five freshwa-

ter turtle species were recorded over a period of 5 1 field

days and 2,046 trap nights. In all, 14 Amyda cartilaginea

were captured: six males, six females and two juveniles.

Six Cyclemys dentata were collected: one female, four

juveniles and one unsexed carcass. Three juvenile Cuora

amboinensis were collected.

Of the 14 Amyda cartilaginea captured at Loagan

Bunut National Park, three individuals were found by

muddling, and 11 were caught during the 2,046 trap

nights. Of the 1 1 trapped animals, ten were caught on

handlines with Ikan Kali and one was caught on a hand-

line baited with Ikan Haruan. No more than a single tur-

tle was collected per night, representing a 0.54% trap

success, with only 1 1 out of 2,046 trap nights being suc-

cessful.

Balai Ringin.- Three freshwater turtle species were col-

lected over a period of 1 6 days and 720 trap nights: one

female Cyclemys dentata , one female Heosemys spinosa

and five Amyda cartilaginea (four females and one juve-

nile).

Of the turtles captured at Balai Ringin, three were

caught using handlines baited with Ikan Kali, one was

found in a selambau and one was caught in a bubu. The

trapping success rate was 0.69%.

Additional species.- An adult Amyda cartilaginea was

caught in the vicinity of Mulu National Park (4° 1' 15

N, 114° 54' 2" E). Three Heosemys spinosa (one male,

one female, and one juvenile) were also found by vari-

ous Universiti Malaysia Sarawak personnel while per-

forming wildlife surveys at Matang Wildlife Centre (01°

36’ 398" N, 110° If 33" E).

Effects of lunar phase on capture success.- During the

new moon phase, traps and lines were set for 8 days; two

adult Amyda cartilaginea (one male, one female) were

captured. During the first quarter moon phase, traps and

lines were set for 18 days; five adult A cartilaginea (one

male, four females) were captured.

During the full moon phase, traps and lines were set

for 14 days; five A. cartilaginea were captured (four

females, one juvenile). During the last quarter phase,

traps and lines were set for nine days; five A. carti-

laginea were captured (two males, one female, two juve-

niles). These data are shown in Figure 2. The active

hard-shelled turtle species captured were Heosemys

spinosa (four individuals), Cuora amboinensis (three

individuals) and Cyclemys dentata (five individuals, one

of which was inactive). During the new moon lunar

phase, one juvenile Cuora amboinensis and one juvenile

Cyclemys dentata were collected over a period of three

days. During the first quarter lunar phase, 10 days were

spent searching for turtles with only one female

Heosemys spinosa (8.3% of the total) captured. During

the full moon lunar phase, collections over 1 1 days pro-

duced one female and one juvenile Heosemys spinosa ,

one juvenile Cuora amboinensis , and one female and

two juvenile Cyclemys dentata. During the last quarter

lunar phase, collections over five days yielded one male

Heosemys spinosa , one juvenile Cuora amboinensis and

one juvenile Cyclemys dentata (Fig. 3).

Results for both soft-shelled and hard-shelled tur-

tles indicate that lunar phase may not have an influence

on their activity patterns. A larger sample size with at

least one radio-tagged species would provide more sig-

nificant results.

Effects of weather on capture success.- During clear

weather, three Amyda cartilaginea (one male, two juve-

42

Asiatic Herpetological Research, Vol. 1 1

2008

Clear, 0

Rain, 34

Overcast, 53

Figure 4. Percentages of Amyda cartilaginea collected Figure 5. Percentages of hard-shelled turtles collected

during different weather conditions. during different weather conditions.

Non-Monsoon,

35

Northeast

Monsoon, 30

Southwest

Monsoon, 35

Non-Monsoon,

31

Southwest

Northeast

Monsoon, 46

Monsoon, 23

Figure 6. Percentages of all Amyda cartilaginea collected

based on seasonality.

niles) were collected, nine individuals were captured

during overcast weather (three males, six females), and

six individuals were captured when it was raining (four

females and one juvenile) (Fig. 4).

For hard-shelled turtles, 12 individuals were found

on the forest floor or on forest trails when it was clear,

while only four were found when it was raining (one

female, one male and one juvenile Heosemys spinosa

and one female Cyclemys dentata). When it was over-

cast, eight turtles were found (one female Heosemys

spinosa, three juvenile Cuora amboinensis, and four

juvenile Cyclemys dentata ) (Fig. 5).

Effects of seasonality on capture success.- Amyda car-

tilaginea capture success was examined between sea-

sons. As might be expected, the water levels of both the

lake and its tributaries at Loagan Bunut National Park

and the riparian habitats in Balai Ringin were lower in

the dry season. Consequently, three of the 20 individuals

captured were found buried in the mud. During the wet

season, six female Amyda cartilaginea were captured,

while in the dry season, only seven soft-shelled turtles

(three males, two females, and two juveniles) were cap-

tured. During the non-monsoon seasons, seven soft-

shelled turtles (four males, two females, one juvenile)

were captured, representing 41% of the total (Fig. 6).

Thirteen hard-shelled turtles were collected. One

female Cyclemys dentata was found buried under the

hollowed trunk of a tree, and although inactive at the

Figure 7. Percentages of all hard-shelled turtles collected

based on seasonality.

time of capture, was included in this component of the

analysis since we were looking at the overall effects of

capture and seasonality. Six of these turtles were collect-

ed during the wet season (one female Heosemys spinosa ,

two juvenile Cuora amboinensis, and three juvenile

Cyclemys dentata). Three of the turtles were found dur-

ing the dry season (one juvenile Heosemys spinosa, one

juvenile Cuora amboinensis, and one juvenile Cyclemys

dentata). Four turtles were captured during non-mon-

soon times (one male and one female Heosemys spinosa,

and two female Cyclemys dentata) (Fig. 7).

All four turtles found buried in mud or hidden in a

tree hollow were collected in the dry season. During the

North-east Monsoon, these localities would have been

covered by at least two meters of water.

Discussion

Capture rates for both Amyda cartilaginea and hard-

shelled turtles were low, (0.54% and 0.69%, respective-

ly), indicating that the populations of these species may

be at critically low levels, although this is difficult to

substantiate considering the paucity of historical data for

southeast Asia.

Turtle capture rates were tested against three envi-

ronmental factors: lunar phase, weather, and season. In

the lunar phase analysis, it appeared that a new moon

may have some influence on the movements of Amyda

cartilaginea. At a capture rate of 11.8% (compared to

2008

Asiatic Herpetological Research, Vol. 1 1

43

29.4% for all other phases), the darkness of the sky may

have an effect on the foraging capabilities of this

species. Other predatory species have also been noted as

having increased foraging activity with increased moon-

light (Brigham and Barclay, 1992). A capture rate of

50.0% during the full moon phase indicated that hard-

shelled turtles may need lunar illumination for foraging

activity.

The effects of lunar phase on changes in animal

behavior are well known. Tigar and Osborne (1999)

hypothesized that fewer predaceous arthropods were

active during full moons than new moons, possibly

because of the increased risk of vertebrate predation.

Alvarez-Castaneda et al. (2004) concluded that fewer

rodent remains were present in bam owl ( Tyto alba) pel-

lets during full moons, indicating that rodent activity

may be decreased during this phase, which is supported

by other studies that have found rodent activity to be

linked to lunar phase. O'Farrell (1974) found that the

most important factors affecting rodent activity was the

amount of time between sunset and sunrise, as well as

lunar phase. Price et al. (1984) reported that bright

moonlight reduces the overall activity of nocturnal

rodents. Church (1960a), concluded that ovulation of the

common Asian toad ( Duttaphrynus melanostictus) was

correlated with the lunar cycle in Java. Church (1960b)

also found this to be the case with the crab-eating or

mangrove frog ( Fejervarya cancrivora ) in Java.

In the present study, 53.0% Amyda cartilaginea

collections occurred during overcast weather, 29.0%

ocurred during rain events, and 18% occurred when the

skies were clear. A total of 66.0% of hard-shelled turtles

captured during overcast conditions, 34.0% were cap-

tured during rainy conditions and none were captured

during clear weather, indicating that turtles may favor

overcast weather for moving and foraging. Seasons did

not have a dramatic affect on the capture rate of turtles,

however, more information is necessary to make this

determination with any confidence. Clearly, a large

amount of effort is required to examine the behavior of

turtles, as well as other animals, especially when con-

ducted across multiple seasons, lunar phases, weather

conditions, or even years. This paper thus presents pre-

liminary information on the influences of environmental

factors on turtle behavior in Borneo.

Acknowledgments

This project was made possible through financial sup-

port from Chelonian Research Foundation's (CRF)

Linnaeus Fund, British Chelonian Group (BCG)

Conservation Grant, Idea Wild, Universiti Malaysia

Sarawak ('The herpetofauna of Loagan Bunut' ; funda-

mental grant number: 1/94/441/2004 [179] and 'Studies

on the natural history and systematics of the herpetofau-

na of peat swamp forest of Sarawak, East Malaysia';

fundamental grant number: l/26/303/2002[40], the

UNDP-GEF ('Conservation and Sustainable Use of

Tropical Swamp Forests and Associated Wetland

Ecosystems', UNDP-GEF Funded Project

MAL/99/G31). Thanks are due to the Sarawak

Biodiversity Centre for providing a research permit

(Research Agreement Number: SBC-RA-0073-KAJ

and Research Permit Number: SBC-RP-0085-KAJ). We

also thank the Sarawak Forestry Department for provid-

ing permission to survey freshwater turtles in Sarawak

(Permit number: 03697). Special thanks are due to the

Sarawak Forestry Corporation and the staff at Loagan

Bunut National Park for permission and use of equip-

ment.

Literature Cited

Alvarez-Castaneda, S. T. S., N. Cardenas and L.

Mendez. 2004. Analysis of mammal remains from

owl pellets (Tyto alba), in a suburban area in Baja

California. Journal of Arid Environments 59(1):

59-69.

Brigham, R. M. and R. M. R. Barclay, 1992. Lunar

influence on foraging and nesting activity of com-

mon poorwills ( Phalaenoptilus nuttallii). The Auk

109(2): 315-320.

Cagle, F. R. 1943. Turtle populations in southern

Illinois. Copeia 1942(3): 155-162.

Church, G. 1960a. Annual and lunar periodicity in the

sexual cycle of the Javanese toad, Bufo melanostic-

tus Schneider. Zoologica 45(13): 181-189.

Church, G. 1960b. The effects of seasonal and lunar

changes on the breeding pattern of the edible

Javanese frog, Rana cancrivora Gravenhorst.

Treubia 25(2): 215-233.

Frazer, N. B., J. W. Gibbons, and T. J. Owens. 1990.

Turtle trapping: preliminary tests of conventional

wisdom. Copeia 1990(4): 1150-1152.

Iverson, J. B. 1992. Global correlates of species richness

in turtles. Herpetological Journal 2: 77-81.

Jensen, K. A. 2006. Ecology and use of the Asian soft-

shell turtle (Amyda cartilaginea), with notes on

other species. Unpublished MSc Thesis, University

Malaysia Sarawak, Kota Samarahan. xxii+140pp.

44

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2008

Legler, J. M. 1960. A simple and inexpensive device for

trapping aquatic turtles. Utah Academy

Proceedings 37: 63-66.

Lovich J. E. 1994. Biodiversity and zoogeography of

non-marine turtles in Southeast Asia. Pp. 380-

391. In: S.K. Majumdar, F.J. Brenner, J.E. Lovich,

J.F. Schalles and E.W. Miller (eds.) Biological

Diversity Problems and Challenges. Pennsylvania

Academy of Science, Easton, Pennsylvania.

O'Farrell, M. J. 1974. Seasonal activity patterns of

rodents in a sagebrush community. Journal of

Mammalogy 55(4): 809-823.

Price, M. V., N. M. Waser. and T.A. Bass. 1984. Effects

of moonlight on microhabitat use by desert

rodents. Journal of Mammalogy 65(2): 353-356.

Sayer, J. 1991. Rainforest buffer zones. Guidelines for

protected area managers. IUCN Forest

Conservation Programme, Cambridge, UK. 94 pp.

Tigar, B. J. and P. E. Osborne. 1999. The influence of the

lunar cycle on ground-dwelling invertebrates in an

Arabian desert. Journal of Arid Environments

43(2): 171-182.

U.S. Navy. 2003.

http://aa.usno.navy.mil/faq/docs/moon_phases.ht

ml.

Vogt, R.C. 1980. New methods for trapping aquatic tur-

tles. Copeia 1980(2): 368-371.

Submitted: 05 January 2007

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 45-49

Effect of Stocking Density on the Energy Budget of Juvenile

Soft-Shelled Turtles (Pelodiscus sinensis)

Runzhen Jing1 and Cuijuan NlU1’*

1 Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering,

College of Life Sciences, Beijing Normal University, Beijing, 100875, People’s Republic of China.

* Corresponding author E-mail: cjniu@bnu.edu.cn

Abstract.- The present work investigates the effect of stocking density on the energy budget of juvenile soft-shelled

turtles ( Pelodiscus sinensis ). Turtles (body weight: 16.22±0.28 g) were stocked at densities of SD1 (8 animals/m-,

0.14 kg/m2), SD2 (48 animals/m2, 0.81 kg/m2) and SD3 (96 animals/m2, 1.62 kg/m2) in aquaria in triplicate for each

treatment. The experiment lasted for 35 days. Survival rate, coefficient of size variation, productivity, and apparent

digestibility coefficient were not significantly different at the three stocking densities. While there were no significant

differences between treatments SD2 and SD3, turtles in group SD1 showed a lower excretion rate and significantly

higher food intake and growth rate. Turtles in group SD1 also showed higher crude lipid content and lower crude ash

content. No significant differences were found among the treatments in body moisture and crude protein.

Keywords.- Survival, growth, food consumption, stress, body composition.

Introduction

Stocking density is one of the most important biotic fac-

tors in aquaculture because it directly influences sur-

vival, growth, behavior, health, feeding, and production.

High densities may interfere with intra-population inter-

actions and eventually affect biomass gain. The relation-

ship between stocking density and growth for fish has

been shown to be positive (Papst et al., 1992), negative

(Hengsawat et al., 1997; Irwin et al., 1999) or density-

independent (Fairchild and Howell, 2001; Rowland et

al., 2004; Rowland et al., 2006), depending on different

experimental density ranges. In the fish farming indus-

try, it is very important for the farmer to know the opti-

mum stocking density of the animals being reared to

maximize production and profitability.

The soft-shelled turtle ( Pelodiscus sinensis ) is a

commonly cultured aquatic reptile species in China with

a yield of more than 140,000 tons in 2004 (Shen et al.,

2006; Zhang, 2005). Despite the fact that the aquacul-

ture of this species is widespread, scientific studies con-

cerning the effects of stocking density on biological

characters are limited (Mayeaux et al., 1996). The objec-

tive of the present study is to evaluate the effect of stock-

ing density on the energy budget of juvenile P. sinensis.

Materials and Methods

Turtles and rearing conditions.- Juvenile Pelodiscus

sinensis (body weight: 16.22±0.28 g) were obtained

from a commercial turtle farm in Beijing. Turtles were

reared in rectangular aquaria (80 length [L] x 35 width

[W] x 30 cm height [H]), with 1 1 individuals per aquar-

ium, at a water depth of 15 cm. Water temperature was

maintained at 29.5±0.5°C by a thermo-controlled heater.

Aquaria were supplied with dechlorinated water. The

dissolved oxygen level was over 5 mg/L and the pH was

7.95. Natural photoperiod was followed. Turtles were

fed to satiation once daily at 1500 h. Commercial turtle

food was used with 0.5% Cr203 added for the apparent

digestibility coefficient assay. Proximate dry matter

composition of the diet was as follows: moisture 3.97%;

crude protein 40.27%; crude lipid 7.04%; and crude ash

15.73%. Energy content was 16.06 kJ/g. Turtles were

allowed to acclimate to the laboratory conditions for

three weeks before the experiments began.

Experimental process.- Healthy turtles were randomly

stocked at initial densities of SD1 (8 animals/m2, 0.14

kg/m2), SD2 (48 animals/m2, 0.81 kg/m2) and SD3 (96

animals/m2, 1 .62 kg/m2) in aquaria (40 L x30 W x30 cm

H) in triplicate for each treatment. There were no signif-

icant differences in initial average body weight or coef-

ficient of size variation within each aquarium among the

treatments. The experiment lasted for 35 days. The final

densities were 0.34 kg/m2, 1.20 kg/m2 and 2.26 kg/m2,

respectively. To maintain a constant numbers of animals,

an alternative turtle with approximately the same body

weight was added when an initial turtle died. All the

water in the tanks was replaced by an equal amount of

fresh water daily after surplus food was removed. The

aquaria were inspected once daily for mortalities and

dead turtles were removed immediately after detection.

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 1. Survival, specific growth rate, food consumption, apparent digestibility coefficient, and excretion of juvenile

soft-shelled turtles ( Pelodiscus sinensis ) held at different stocking densities (Mean ± S. E.)1.

'Values in each row with different superscript letters are significantly different (p < 0.05).

2Means are significantly different among treatments (p < 0.01).

3Means are extremely different among treatments (p < 0.001).

Turtles were weighed to an accuracy of 0.1 g before and

after the experiment following three days starvation. Six

turtles at the beginning of the experiment and all turtles

remaining at the end of the experiment were sacrificed

and dried at 65°C to constant weight for analysis of body

biochemical composition. Crude protein was determined

by the Kjeldahl method, crude lipid was extracted by

ether, and crude ash was determined after 12 h of burn-

ing at 550° in a muffle furnace. Energy contents were

measured using a calorimeter (CA-4P, Shimadzu,

Japan). All samples were analyzed in triplicate.

Measurements of various components of the energy

budget.- A weighed excess of feed pellets was fed to the

turtles once daily (at 1500 h) with a fraction of feed

retained for determination of dry matter content.

Uneaten food was collected an hour later and dried.

Food intake was determined as the difference between

the food supplied and the food left uneaten.

Fresh complete feces were collected once daily.

Cr->03 content in the diet and feces were determined by

the method described in detail by Bolin et al. (1952).

The apparent digestibility coefficient (ADC) and the

energy lost via feces (F) were calculated by the follow-

ing expressions:

ADC (%) = 100 x (1- Cr203 content in diet/Cr203

content in feces)

productivity were calculated by the following formulae:

CV (%) = 1 00 x Standard deviation/ Average body

weight

SGR (%/day) = 100x(lnW2-lnW1)/(t2- ft)

Productivity (g/daydn2) = (Wt2-Wtl)/S/( t2- ft)

where W2 and Wft express final average weight at time

t2 and initial average weight at time ft in days, respec-

tively. Wt2, Wtl and S express biomass at day t2, biomass

at day ft, and aquarium surface area, respectively.

Energy allocated to growth was calculated from weight

gain (g) and energy content (kJ/g) of the whole body.

Energy lost via excretion was calculated from the

ammonia and urea excreted using energy equivalents for

ammonia (24.83 J/mg N) and urea (23.03 J/mg N)

(Elliott, 1976). Ammonia and urea concentrations inside

the water were measured by firstly catalyzing urea to

ammonia using urease, and then assaying the total

ammonia via standard Nessler’s colorimetric technique.

The turtles were kept in a given amount of renewed

experimental water for 48 h and fasted during the meas-

urement. Water samples were taken before and after this

period.

The energy budget for juvenile animals can be

described as:

F = I x (100- ADC) x Ef/1 00

where I and Ef are food consumption in dry weight and

feces energy content, respectively.

The coefficient of variation in body weight (CV)

within each aquarium, specific growth rate (SGR), and

C=F+U+R+G

where C is the energy in the food consumed, F is the

energy lost in fecal production, U is the energy lost in

nitrogenous excretory products, R is the energy spent in

metabolism, and G is growth energy. In this study, C, F,

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Asiatic Herpetological Research, Vol. 1 1

47

Table 2. Chemical composition of juvenile soft-shelled turtles ( Pelodiscus sinensis) held at different stocking densities

in 35-day experimental period (Mean ± S. E.)1.

‘Crude protein, crude lipid, and crude ash based on the contents in the dry matter.

'Values in each column with different superscript letters are significantly different p < 0.05).

2Means were extremely different among treatments (p < 0.001).

U, and G were determined directly, and R was calculated

by the equation:

R = C- F- U-G.

Statistical analysis.- All data were analyzed with SPSS

for Windows, Version 1 1 .0. A one-way ANOVA was used

to test the differences among treatment means when

assumptions of normality and homogeneity were met.

When a significant treatment effect was found, the

Least-Significant-Difference (LSD) test was applied to

determine which specific pairs differed. The nonpara-

metric Kruskal-Wallis test was applied when the

required homogeneity of variance and normality were

not satisfied. A regression analysis was carried out to

estimate the relationship between stocking density and

growth rate. The significant level was set at p < 0.05.

Results

All turtles in one aquarium of treatment SD2 died due to

a malfunction of the thermo-controlled heater. This

replicate was not taken into account for any statistical

comparisons.

The effects of stocking density on survival, specific

growth rate, food consumption, apparent digestibility

coefficient, and excretion.- Survival rates were not sig-

nificantly different among the three treatments (Table 1 ),

but all showed a negative relationship with increased

stocking density (r = -0.708,/? = 0.050). Stocking densi-

ty showed a clear influence on final body weight, specif-

ic growth rate, food consumption, and excretion, as

identified by the statistical significance (Table 1).

Turtles in group SD1 showed significantly higher food

intake and growth rate than those held at the other two

densities, which did not differ significantly from each

other. Lower excretion rate was observed in group SD1

compared to groups SD2 and SD3. The apparent

digestibility coefficients of juvenile turtles ranged from

74.29% to 78.14%. The relationship of SGR and stock-

ing density (SD, animals/m2) can be described as the lin-

ear model or the quadratic model:

SGR = -0.0171 x SD + 2.4360

o < 0.01, R2= 0.769)

SGR - 0.0003 x SD2 - 0.053 1 x SD + 2.8805

o < 0.01, R2 = 0.915).

The effect of stocking density on body composition. -

Body composition for each treatment group is shown in

Table 2. There were significant differences in lipid and

ash contents between treatments (F25 = 11.520,

p = 0.013; F2 5 = 64.577, p = 0.000). Crude lipid con-

tents of group SD1 were much higher than those of

groups SD2 and SD3 while crude ash contents were

lower. No significant differences were found among

treatments in body moisture and crude protein

(F2 5 = 0.155, /?= 0.860; F2 5 = 3.412,/? = 0.116).

The effect of stocking density on energy ; budget.- No

marked differences were found among treatments in F/C

and R JC (Table 3; F2 5 = 0.058,/? = 0.945; F25 = 2.561,

p = 0.171). Stocking density significantly influenced

U/C and G/C (F2 5 = 27.151, p = 0.002; F2 5 = 6.243,

p = 0.044). Energy budgets for the different treatments

can be described as:

100C = 10. OF + 0.3U + 73.3R + 16.5G; SD1

100C = 10.3F + 0.7U + 78.3R + 10.8G; SD2

100C = 10. OF + 0.8U + 79. 1R + 10.2G; SD3

Discussion

Stocking density has been considered to be chronically

stressful to reared animals (Vijayan and Leatherland,

1988). Several studies have also demonstrated that

increased stocking density has a negative effect on sur-

vival and growth (Penha-Lopes et al., 2006; Schram et

al., 2006), except in some fish species that exhibit

schooling behavior (Jorgensen et al., 1993;

Papoutsoglou et al., 1998). This impaired growth by

stocking density may be attributed to reduced food con-

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Asiatic Herpetological Research, Vol. 11

2008

Table 3. Energy budget of juvenile soft-shelled turtles ( Pelodiscus sinensis) held at different stocking densities in 35

day experimental period (Mean ± S. E.)1.

C: food intake in energy; F: energy lost in fecal production; U: energy lost in nitrogenous excretion; R: energy lost in metabolism; G: energy allocated to

growth.

'Values in each column with different superscript letters are significantly different (p < 0.05).

2Means are significantly different among treatments (p < 0.01).

3Means are extremely different among treatments (p < 0.001).

sumption, lowed food conversion rate or increased

metabolic cost (Jorgensen et al., 1993; Li and Brocksen,

1977; Vijayan and Leatherland, 1988).

In the present study, an obvious trend of decreased

survivorship of juvenile soft-shelled turtle with elevated

stocking density was observed. This agrees with the

results of Mayeaux et al. (1996), who reported that com-

mon snapping turtles ( Chelydra serpentine ) stocked at

58 animals/m2 exhibited greater mortality, lower weight

gain, and higher food consumption compared to those

stocked at 29 animals/m2. Food consumption also

reduced with increasing stocking density in the present

study, however, conflicting with the above snapping tur-

tle results. This discrepancy may be caused by differ-

ences in life habit or varying experimental conditions.

Knights (1985) observed that more aggressive (and usu-

ally larger) eels of Anguilla anguilla ate more, while the

feeding of smaller subordinates was inhibited, even

when food was offered to excess. In the present experi-

ment, despite food being divided between at several

spots in each tank, a similar phenomenon was observed

during the feeding process. It is likely that the appetite

of the subordinate turtles was suppressed and their

growth inhibited when the turtles were grouped at high

density.

The lower feed intake in treatments SD2 and SD3

appear to explain the lower growth in the same treat-

ments, because reduced food ingestion reduces the

amount of energy available for growth (Table 1).

Moreover, the proportions of food energy spent in

growth (indicated by the gross energy efficiency, Table

3) in treatments SD2 and SD3 were obviously lower in

relation to that in treatment SD1 (10.8% and 10.2%,

compared to 16.5%). These results may suggest that the

ingested energy was not efficiently converted to body

reserves, especially at high stocking density.

Juvenile soft-shelled turtles tend to grab each other

with their sharp claws, and grabbing activities often

result in injuries of the toes and neck, and may even

result in death. Agonistic interactions among individuals

and elevated swimming activity also lead to increased

metabolic expenditure. In the present experiment, turtles

of groups SD2 and SD3 exhibited hyperactivity com-

pared to group SD1, and had reduced lipid and higher

ash contents. The difference in chemical composition in

these turtles suggest that elevated stocking density may

induce extra energy expenditure, subsequently allocat-

ing less energy to storage.

In conclusion, the pattern of energy allocation of the

turtles in the present experiment was significantly influ-

enced by different stocking densities. Turtles cultured at

lower density had a relatively higher survival rate, dis-

tinctly higher growth rate and transfer more consumed

energy to growth. The lower energy input and lower

gross energy efficiency in treatments SD2 and SD3 may

have contributed to their reduced growth rate.

Furthermore, the excretion of nitrogenous wastes to the

environment was relatively lower with reduced stocking

density. Conversely, higher stocking density could result

in higher productivity to some degree, since there were

no significant differences in productivity among the

treatments. We suggest that the turtle farmer pursue an

optimal stocking density based on profitability, consid-

ering that lower stock densities are shown to be related

to increased survivorship, growth rate and feed utiliza-

tion, while also being associated with a reduction in

nitrogenous wastes.

Acknowledgments

We are thankful to Ms. C. Huang and our other col-

leagues at the animal physiological ecology lab, college

of life science, Beijing normal university, for their valu-

able advice and assistance to the experiment. This work

was funded by the National Natural Science Foundation

of China (No. 30271014, No. 30671598).

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Submitted: 26 October 2006

Accepted: 29 May 2007

pp. 50-56

Asiatic Herpetological Research, Vol. 1 1

2008

Genetic Variation and Trans-species Polymorphism of

MHC Class II B Genes in Reptiles

En Li1, Xiao Bing Wu1’* and Peng Yan1

1 Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological

Resources, College of Life Science, Anhui Normal University, 241000 Wuhu, China.

* Corresponding author E-mail: wuxb@mail.ahnu.edu.cn.

Abstract.- Trans-species polymorphism has been extensively documented for the major histocompatibility complex

(MHC) in mammals, fishes and birds, but not for non-avian reptiles. Our study has addressed this by focusing on three

species of the reptiles: Chinemys reevesii, Plestiodon chinensis and Alligator sinensis. Using polymerase chain reac-

tion (PCR) and nucleotide sequence analyses, we examined a total of twenty-five sequences of exon 2 of the MHC

class II B genes in these species. High allelic variability was observed among sequences within each of these species,

indicating extensive MHC polymorphism. Nonsynonymous substitution rates (< dN ) exceeded synonymous substitution

rates ( ds ) greatly within the antigen-binding sites (ABS), suggesting the effect of balancing selection. Phylogenetic

analysis of these reptile sequences clearly supports the hypothesis of trans-species polymorphism. We therefore con-

fidently conclude that trans-species polymorphism in the MHC is now known for reptiles, as well as mammals, fishes

and birds. This suggests that the main function of the MHC (presentation of peptides to T lymphocytes) has remained

largely unchanged despite of long periods of evolution.

Keywords.- Major Histocompatibility Complex (MHC), reptiles, trans-species polymorphism.

Introduction

The genes of the major histocompatibility complex

(MHC) code for polymorphic membrane glycoproteins

that play a key role in the T-cell mediated immune

response (Klein, 1986). There are two distinct classes of

MHC molecules, class I and class II, which are encoded

by separate but tightly linked loci. The diverse, but

always specific, antigen-binding properties of the MHC

class I and II molecules determine which foreign pep-

tides can be identified to trigger an immune response.

Such MHC-dependent recognition of certain antigens

has been considered as an important contributing factor

in susceptibility to disease (Klein, 1986). In many verte-

brate species, the MHC class I and class II loci exhibit

an extraordinarily high degree of polymorphism, partic-

ularly in exon 2 of the beta genes. This variation is prob-

ably maintained through some kind of balancing selec-

tion related to interactions between the immune system

and pathogens (Parham and Ohta, 1996), although it has

not been resolved as to whether the selection is over-

dominant (heterozygote advantage hypothesis), frequen-

cy dependent (rare-allele advantage hypothesis) or a

combination of these factors (Hughes and Hughes,

1995; Hughes and Yeager, 1998; Hill et al., 1992;

Hughes, 2000; Thurz et ah, 1997).

A characteristic feature of the MHC genes is trans-

species polymorphism, i.e. the existence of allelic line-

ages shared by related species, supporting the theory that

the divergence of MHC allelic lineages predate specia-

tion (Graser et ah, 1996; Klein, 1987; Ottova’ et ah,

2005). For MHC genes, this kind of polymorphism has

been well-documented in mammals, but for other verte-

brate classes, the data on trans-species polymorphism

are either fragmentary or unavailable. Only in fish

(Klein et ah, 1998; Ottova et ah, 2005) and birds (Hess

and Edwards, 2002; Richardson and Westerdahl, 2003)

is there clear evidence for the interspecific sharing of

MHC alleles, but these species are of recent origin and

do not provide information about long-term persistence

of allelic lineages. In an attempt to obtain such informa-

tion, we decided to compare the polymorphism in three

reptiles (. Alligator sinensis, Chinemys reevesii and

Plestiodon chinensis) with that in two other closely-

related reptiles {Alligator mississippiensis and Caiman

crocodilus ).

In this study, we investigate genetic variation at

exon 2 of the MHC class II B genes, including part of

the putative antigen-binding sites, in three reptiles. We

choose this particular exon because it is known to be

highly polymorphic in primates and a variety of other

terrestrial species. Our purposes in this study are: first,

to analyze the variability of MHC class II B genes

among the species listed above; second, to test for the

influence of selection on amino-acid polymorphism, i.e.

a positive (balancing) selection in exon 2; and finally, to

document whether the trans-species polymorphism in

MHC class II B genes also exits in reptiles.

2008

Asiatic Herpetological Research, Vol. 1 1

51

Table 1 . The genetic parameters of the sequences within

each analysed species.

Note: N: number of sequences; L: sequence length (pb); S: variable sites;

x: nucleotide diversity; p: amino acid diversity.

Materials and Methods

Isolation of genomic DNA.- Total genomic DNA was

isolated from 20-50 pL of blood using standard phenol-

chloroform extraction methods (Sambrook and Russell,

2001). The sampled individuals (without existing

sequence data on GenBank) included a total of four

Chinemys reevesii (terrapin) and two Plestiodon chinen-

sis (saurian) from natural populations.

Polymerase chain reaction (PCR).- A 166 bp fragment

of exon 2, from the class II B genes coding for part of

the peptide-binding region, was amplified by PCR using

the following degenerate primers reported by Shi et al.

(2004): the forward (sense) primer MHC-UP 5’-

AAGG(T/G/C)C(C/G)AGTG(T/C)TACT(T/A)(C/T)A(

T/G/C)(T/G/C)AACGG-3’; the reverse (anti-sense)

primer MHC-DP 5’-

TAGTTGTG(C/G)C(G/T)GCAG(A/T)A(C/G)GTGTC-

3’. PCR reaction were performed in 30 pL of reaction

mixture containing 10 mM Tris-HCl (pH 8.3), 1.5 mM

MgCl2, 150 pM dNTP, 1 pM of each primer, 20-100 ng

of isolated genomic DNA and 1 unit of Taq DNA

Polymerase (Promega). Thermocycler conditions were

as follows: an initial denaturation for 5 min at 94°C, fol-

lowed by 35 cycles, each consisting of 30 s at 94°C, 40

s at 52°C, and 40 s at 72°C. The final extension at 72°C

was for 10 min. PCR products were separated in a 2%

agarose gel containing ethidium bromide (0.5 pg mL1).

Separated PCR products were visualized under UV light

and photographed to examine the banding patterns.

Cloning and sequencing.- Following agarose gel elec-

trophoresis, PCR products of appropriate size were

recovered, purified and concentrated using the DNA Gel

Extraction Kit (V-gene Biotechnology Limited). The

purified PCR products was ligated into the pGElVD-T

Vector using the TA cloning kit (Promega); Competent

Escherichia coli DH5a cells were transformed in a liga-

tion reaction, and positive clones were identified by

blue/white selection, as described in the manufacturer’s

protocol. Twenty to thirty positive clones were selected

for each individual. Insert size was verified by PCR

using Ml 3 universal forward and reverse primer.

Different inserts were screened by single-strand confor-

mation polymorphism (SSCP) analysis and sequenced

using the dideoxy nucleotide chain termination method

(Sanger et al., 1977) on an Applied Biosystems 377

automated sequencer.

Data analysis.- Nucleotide and inferred protein

sequences were aligned using the CLUSTAL X software

(Jeanmougin et al., 1998). MHC sequences from close-

ly-related species were acquired using the GenBank

BLAST program (Altschul et al., 1990). Genetic dis-

tances were measured using the two-parameter method

(Kimura, 1980). The computer package MEGA 2.1

(Kumar et al., 2001) was used to estimate the rate of

nonsynonymous (dN) and synonymous (ds) substitutions

according to Nei and Gojobori (1986), applying the

Jukes and Cantor (1969) correction for multiple hits.

The differences between these rates was evaluated with

a /-test with infinite degrees of freedom according to the

test statistic t = d!s{d)\ s(d) is the standard error of d and

Table 2. Numbers (mean±standard error) and relative rate (d^/ds) of nonsynonymous (dN) and synonymous (ds) sub-

stitutions per nucleotide in exon 2 sequences given for all sites and for pABS and non-pABS for comparison of three

species.

Asterisks indicated the significance of two-tailed t-test in the order: * p < 0.01 , ** p < 0.001 .

Figure 1. Amino acid sequences translated from nucleotide sequences of exon 2 from MHC class II B genes of C. reevesii, P. chinensis and A. sinensis. The Alsi

sequences are from our laboratory work (Shi et al. 2004). Asterisks (*) indicate the putative antigen-binding sites correspond with those for human class II

sequences; dots (.) indicate identity with the consensus sequence at the top; dashes (-) gaps introduces to achieve optimal sequence alignment.

52

Asiatic Herpetological Research, Vol. 1 1

2008

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Asiatic Herpetological Research, Vol. 1 1

53

0.05

Figure 2. Phylogenetic tree of MHC class II B genes

nucleotide sequences in different reptiles constructed by

neighbor-joining method in MEGA. Bootstrap values

from 1000 replications were indicated above the branch-

es, values less than 50% are not shown.

is given by s(d) = [Var^) + Var^)]1 2 (Kumar et al.,

1993). These analyses were performed on the 24 codons

comprising the ABS sites (as defined in the crystal struc-

ture of a human class II molecule, DRB1 (Brown et al.,

1993)) and on all remaining non-ABS codons in the

amplified segment. The phylogenetic tree was construct-

ed using MEGA 2.1 program for distance-based meth-

ods, applying the neighbour-joining (NJ) algorithm.

Bootstrap analysis (1000 replications) was performed to

determine the reliability of the branching pattern in the

phylogenetic tree. To further quantify polymorphism,

nucleotide diversity (jt) was also calculated using the

computer application DnaSP (Rozas et al., 1999).

Results

Amount and extent of variation.- In total, fifteen

sequences were obtained from Chinemys reevesii and

Plestiodon chinensis (see Table 1); sequence identity

was confirmed by sequencing multiple clones in both

directions. These sequences are designated Chre for

Chinemys reevesii and Plch for Plestiodon chinensis , in

accordance with the proposed nomenclature (Klein et

al., 1990), and have been deposited in GenBank under

the accession numbers AY937200-AY937207 (for

Chre-\~Chre-&), AY772946-AY77295 1 and AY764032

(for Plch-\~Plch-l). Ten sequences (. Alsi-\~Alsi-\0\

GenBank accession numbers AY49 1421 -AY49 1430) of

exon 2 of the class II B genes from \hrze Alligator sinen-

sis, used for the subsequent data analysis, were also sub-

mitted. All sequences except for one from A. sinensis

(Alsi-S), which had six nucleotides deletions, were

166bp in length. Published sequences were aligned with

those derived here, illustrating numerous variable sites

in Chinemys reevesii (84 [=50.6%]), P. chinensis (12

[=7.2%]) and A. sinensis (38 [=22.9%]); these numbers

are consistent with those seen for this fragment in other

species. Nucleotide diversity was also calculated within

species, with all three species exhibiting a mean pair-

wise nucleotide diversity of 0.22203, 0.02180 and

0.09236, respectively. Furthermore, Chre-5 was also

found to be identical to Plch- 3 in P. chinensis, and Alsi-

1 in A. sinensis. Sharing of these same alleles in different

species was also reported in Hedrick et al. (2002).

Amino acid variation within Chinemys reevesii,

Plestiodon chinensis and Alligator sinensis was 35.8%,

5.2% and 18.4%, respectively (Table 1). Aligned amino

acid sequences are presented in Figure 1. The putative

antigen-binding sites (pABS), corresponding to those in

the human class II sequences (Brown et al., 1993), are

indicated by an asterisk. The term “putative” has used

here because the actual antigen-binding sites for reptiles

have not yet been verified. Among the Chinemys

reevesii sequences, 92.9% (13 out of 14 codons) of

pABS are variable and 63.4% (26 out of 41 codons) of

the nonbinding sites (non-pABS) are variable. Within A.

sinensis, 71.4% of pABS and 31.7% of the non-pABS

are variable, and in P chinensis, 21.4% of pABS (3 of

14) are variable, while 12.2% (5 of 41 positions) of non-

pABS are variable. The numbers of synonymous substi-

tutions (ds) and nonsynonymous substitutions (dN) per

nucleotide in exon 2 sequences are given in Table 2. The

ratio of dN to ds tended to be greater than 1 .0, particular-

ly for pABS, which has a ratio consistent with that seen

in other MHCs, suggesting that there is selection for

amino acid replacements in the antigen-binding region.

Meanwhile, dN/ds for pABS and non-pABS in Chinemys

reevesii and A. sinensis were all larger than 1.0.

Phylogenetic analysis.- BLAST searches in genome

sequence databases have revealed that a number of alle-

les from other reptiles exhibit a high degree of similarity

to the sequences derived here. Of these, three Caiman

crocodilus alleles {Cacr-\~Cacr-2>\ Accession Numbers

AF256651, AF256652 and AF277661) and three

Alligator mississippiensis alleles (Almi-\~Almi- 3;

Accession Numbers U24402-U24404) were chosen for

54

Asiatic Herpetological Research, Vol. 1 1

2008

phylogenetic analysis. A neighbor-joining tree showing

the relationships among these nucleotide sequences is

presented in Figure 2. The sequences of Chinemys

reevesii and Plestiodon chinensis tend to cluster togeth-

er. Interestingly, Caiman crocodilus sequences were

widely dispersed in the tree (supported by high bootstrap

values), being more similar to the crocodile sequences.

A cluster of four Alsi sequences (Alsi- 2, Alsi- 3, Alsi- 5,

Alsi-6) showed a higher degree of similarity to Pick

sequences than to other lineages. The phylogenetic tree

supports the hypothesis of trans-species polymorphism,

as indicated by the clustering of lineages from different

species and the presence of sequences from different

species in the same allelic lineage. This trans-species

allelic similarity is not unusual for MHC genes, as it has

been proposed that MHC allelic lineages are maintained

by selection and are often older than the species them-

selves.

Discussion

In the present study, we investigated exon 2 (of MHC

class II B genes) sequences from three reptiles and

examined within species polymorphism. The results

revealed relatively high amounts of variability in both

nucleotide and amino acid sequences (Table 1), as well

as a pattern of evolution consistent with those seen in a

variety of mammalian species, including humans.

However, the level of genetic variation within each of

these species differed, possibly reflecting different pat-

terns of evolution and population genetic structure.

Levels of polymorphism are higher in Chinemys reevesii

dead Alligator sinensis compared to Plestiodon chinensis ,

which may prove to be of value in future studies on pop-

ulation genetics and conservation biology.

As an important genetic component of the verte-

brate immune system, variation in the MHC is signifi-

cant to consider selective pressure due to parasitic or

pathogen resistance. It has been suggested that species

or populations with low MHC diversity might be partic-

ularly susceptible to infectious disease and parasites

(Hedrick and Kim, 2000; O’Brien and Evermann, 1988).

Furthermore, in this and other studies (Hedrick et al.,

2002; Ottova et al., 2005), identical MHC alleles have

been found amongst different species; the sharing of

these likely homologous sequences may be due to expo-

sure to similar (or the same) antigens present throughout

the evolution of each species.

Balancing selection appears to play a determinant

role in MHC evolution (Bematchez and Landry, 2003),

evidence of which is the presence of more nonsynony-

mous (< dN ) than synonymous ( ds ) substitutions in anti-

gen-binding sites (Binz et al., 2001; David and Helena,

2003; Hedrick et al., 2002). In the present study, the

observed excess of nonsynonymous substitutions, par-

ticularly at putative antigen-binding sites, indicates that

nonsynonymous sites evolve faster than synonymous

sites. This implies the presence of balancing selection

(or positive Darwinian selection), which favors new

variants and increases MHC diversity, which has been

observed in a number of species (Hughes and Nei,

1989). In the case of Chinemys reevesii and Alligator

sinensis, however, a significant excess of nonsynony-

mous substitutions was also found in non-pABS sites,

possibly suggesting that reptile ABS sites may not exact-

ly correspond to those in humans as originally defined

by Brown et al. (1993). Similar findings were reported

for the Pacific salmon (Miller and Withler, 1996) and

Sonoran topminnow (Hedrick et al., 2001).

The phylogenetic tree suggests that some Alsi

sequences are more similar to Plch sequences than to

other Alsi sequences, with species sequences intermin-

gling to form several significantly-supported clusters

(Fig. 2). This intermixing suggests a trans-species per-

sistence of MHC class II exon 2 sequences, with some

allelic lineages predating species cladogenesis. The pro-

longed maintenance of MHC alleles is contrary to what

would be expected from neutral loci, supporting the idea

that long-term balancing selection on the MHC alleles

has occurred (Figueroa et al., 1988). Our results are con-

sistent with the theory of trans-species evolution in

MHC alleles (Klein, 1987), which has been previously

supported by studies on mammals and fish (reviewed in

Hedrick, 2001), suggesting that MHC polymorphism is

widespread in the Vertebrata.

In conclusion, we are presenting strong evidence

for trans-species polymorphism at exon 2 of class II

gene in reptiles. The polymorphism is putatively main-

tained by balancing selection and is restricted to what

are apparently functional loci on (primarily) the ABS

sites. These observations suggest that the MHC carries

out the same function in reptiles as it does in mammals,

but additional research needs to be conducted, particu-

larly with regards to trans-species polymorphism and

specific binding loci across taxa.

Acknowledgments

We greatly thank Dr. R. Hu from the Division of Biology

at the California Institute of Technology for correcting

the English in this paper. This study was supported by

project grants (No.30270213 and 30470244) from the

National Natural Science Foundation of China, the

Foundations for Excellent Youth in Anhui Province

(04043049) and a Special Foundation for Anhui Key

Laboratory of Biotic Environment and Ecological

Security, Anhui Normal University.

2008

Asiatic Herpetological Research, Vol. 1 1

55

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Submitted: 25 October 2006

Accepted: 23 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp.57-61

The Biology and Taxonomic Status of the

Sunken Ear Frog (Rana tormotus Wu, 1977)

PlPENG Li* AND YUYAN Lu

Liaoning Key Laboratory of Biodiversity and Evolution, Shenyang

Normal University, Shenyang 110034, P. R. China.

* Corresponding author E-mail: lipipeng@yahoo.com

Abstract.- The biology and taxonomic status of the sunken ear frog, Rana tormotus Wu, 1977, are reviewed and

briefly discussed. This is a rare species restricted the mountain streams and rivers of Anhui and Zhejiang provinces

in East China, and is characterized by a sunken tympanic membrane that forms an external ear canal, similar to that

seen in birds. The male, which is known to produce ultrasonic sounds when calling, has a more deeply sunken mem-

brane. The karyotype of this frog is 2n = 26, having five pairs of large, eight pairs of small and seven pairs of sub-

metacentric chromosomes. The frog is active during the night and females are uncommonly encountered. Specimens

are often found in the same habitat as Megophrys boettgeri, Bufo gargarizans, Rana limnocharis, R. schmackeri, Paa

spinosa, Amolops wuyiensis and A. ricketti. This frog was first described within Rana , but it was recombined in

Amolops because its tadpole was of the “ Amolops type”, even though the tadpole was unknown at the time. The

recently-discovered tadpole has no abdominal sucker and the poison glands, smaller than and similar to those of R.

schmackeri with LTRF (1:4-4/111:1-1), making it distinct from the “Amolops- type” tadpole. Adult and larval morphol-

ogy, as well as developmental characters, support the placement of this species in Wurana, a new genus setup recent-

ly-

Keywords.- Amphibia, Ranidae, Rana , tormotus, Wurana, Amolops.

Introduction

Rana tormotus Wu, 1977 is a characteristic frog with a

sunken ear membrane that is particularly distinct in the

male, giving this species the common name of “sunken

eared frog” or “concave-eared torrent Frog,” the former

of which translates to “Ao Er Wa” or “Wa Er Wa ” in

Chinese. In 1972 and 1974, Ermi Zhao, Guanfu Wu and

two assistants collected one female and 18 male frogs

with sunken tympana at Taohua Creek on Mt. Huanshan.

These frogs were subsequently described as members of

the new species Rana tormotus (Sichuan Institute of

Biology [Wu, G. F.]). During the next 23 years, only a

few reports on this frog’s taxonomy and karyotype were

published (Chen, 1991; Fei et al., 1991; Guo and Dong,

1986; Huang et al., 1990). The species was subsequently

moved to Amolops by Fei et al. (1991), because its tad-

pole might be of the “ Amolops type”, even though the

tadpole was unknown at the time. The correct taxonomic

placement of this species is currently ambiguous (Global

Amphibian Assessment, 2005; Zhao and Adler, 1993;

Zhao and Zhao, 1994; Zhao et al., 2000;).

Recently, the ecology, bioacoustics and evolution-

ary history of this species have been explored by Liu and

Hua (2001), Wu and Wu (2002) and Feng et al. (2002,

2006). We have also completed surveys on its distribu-

tion and habitat (at Jiande, Zhejiang and its type locality

at Huanshan, Anhui), which are presented below. We

further placed this species in the new genus Wurana on

the basis of developmental characters and adult and lar-

val morphology (Li et al., 2006).

Distribution and habitat - This sunken ear frog is a rare

endemic species restricted to the mountain streams and

rivers of East China at elevations between 150 and 750

m. It is currently known only from the type locality

(Taohua Creek, Hotspring Creek, Fu Creek and Xiang

Creek of Huanshan, Anhui Province) and two locations

in Zhejiang province (Huang, et al, 1990, personal com-

munication with Prof. Qinghui Gu): two small creeks in

the Jiangde Forestry Centre and the creeks of Anji

County. The latter creeks are filled with large rocks and

are surrounded by trees, brushes and grass (Figs. 1-3).

All frogs were collected at night since they could

not be found during the day. Adult males were found on

rocks in the river and in the surrounding trees and

shrubs, and were located by their calls. No females were

observed; in Liu and Hua (2001), it was reported that

females were collected only after midnight following the

appearance of males. It was suggested that the females

were not commonly found because they occupied higher

tree branches (Wu and Wu, 2002). Neither sex was

observed following the breeding season.

Wu and Wu (2002) reported that the frogs were only

found in shrubs along the flat parts of the river. In com-

parison, at the bases of mountains we found that trees

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Asiatic Herpetological Research, Vol. 1 1

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115° 120°

Figure 1. Distribution of Rana tormotus (red square) in

Anhui and Zhejiang province.

were preferentially chosen (57.32% at Taohua Creek and

51.9% at Fu Creek), followed by shrubs (31.71% and

40.51%) and then rocks in the water (10.98% and

7.95%). Liu and Hua (2001) observed 11 of 53 frogs on

branches, four on sand near vegetation along the river

bank and the remainder on exposed sand and rock in the

river far from any vegetation. In the present study at

Taohua Creek, 20% of frogs were found on tree branch-

es, 40% on shrubs, 20% on grass leaves and 20% on

rocks; specimens were never found along sandy river

banks, where they may have been excluded by Rana

species. The differences in observations between sur-

veys may be due to climatic variation.

Males were kept in an aquatic box simulating the

natural environment. During the day, the frogs hid in

gaps between stones and vegetation, but at night they

emerged on the surrounding leaves and rocks despite

variation in weather (including rain).

The other frogs found in the same habitat as Rana

tormotus at the type locality were Megophrys boettgeri,

Bufo gargarizans, Rana limnocharis , Rana schmackeri

(Boettger, 1892), Paa spinosa (David, 1875) and

Amolops wuyiensis (Liu and Hu, 1975) (Liu and Hua,

2001; Li, Lu and Lii, 2006). Amolops ricketti was found

instead of A. wuyiensis at Jiande. Frogs observed in the

Figure 2. Type locality of Rana tormotus, Taohua Creek

of Mt. Huanshan in Anhui province, China.

surrounding areas included Rana nigromaculata ,

Microhyla heymonsi, R. livida and R. japonica (Wu and

Wu, 2002). Tadpoles of Rana schmackeri, Paa spinosa

and Amolops wuyiensi were also collected from Taohua

Creek.

Karyotype and Ag-banding pattern.- Guo and Dong

(1986) reported the karyotype and Ag-banding pattern of

Rana tormotus. The karyotype is 2n = 26, consisting of

five pairs of large and eight pairs of small chromosomes.

A secondary constriction is present near the centromere

on the long arms of chromosome 6 and 10. No hetero-

morphic chromosomes were present. One homologous

pair of NORs were found in the secondary constriction

of chromosome 10 using Ag-AS staining techniques.

There were also seven pairs of submetacentric chromo-

somes, the largest among frogs with a 2n = 26 karyotype

in the Raninae (Guo and Dong, 1986; Pan et al., 2002).

Calling and related morphological characters. -

Recently, Feng et al. (2002, 2006) detailed the extraordi-

narily rich vocal repertoire of the sunken ear frog. These

frogs produce countless vocalizations, some of which

share features of bird songs or primate calls - e.g., ultra-

sonic frequency, multiple upward and downward FM

sweeps and sudden the onset and offset of selective har-

monic components within a call note. Most frog calls go

either up or down, and no others are known to extend

into the ultrasonic range. Frame-by-frame video analysis

of the frog's calling behavior suggests the presence of

two pairs of vocal sacs that may contribute to its remark-

able call-note complexity. Electrophysiological studies

of the frog’s auditory midbrain confirmed that its audi-

ble range extends into the ultrasonic (Xu et al., 2005).

This characteristic can likely be explained by the fact

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Asiatic Herpetological Research, Vol. 11

59

Figure 3. Another locality of Rana tormotus at Jiande

county of Zhejiang province, China.

that the frog lives by noisy streams that produce acoustic

signals with significant ultrasonic harmonics that would

mask normal calls; selective pressure on this species

could eventually produce a call that would not be

masked by the wideband noise produced by the river

(Peter et al., 2003).

With regard to sound reception, the sunken ear frog

has a unique structural autapomorphy not seen in other

Anura - a sunken tympanic membrane that forms an

external ear canal like that seen in birds. The tympanic

membrane is more deeply sunken in the male, suggest-

ing that it receives the airborne sound in a manner some-

what different from that in the female (Feng et al., 2006;

Xu et al., 2005).

Tadpoles and taxonomic status.- Several different

species of tadpoles from the frog’s type locality were

reared to metamorphosis in the lab. The first morpho-

type was identified as Amolops wuyiensis, which had an

abdominal sucker and poison glands; the second to

fourth morphotypes were without either of these struc-

tures. Among latter, one identified as Paa spinosa had a

relatively larger body and body-tail length, and froglets

that very closely resembled the adult in general mor-

phology. The remaining two morphotypes were signifi-

cantly smaller and had the same LTRF (1:4-4/111:1-1); of

these, one was externally similar to the adult of Rana

schmackeri and one was similar to the adult of R. tormo-

tus (Fig. 4); the characters used to aid in the separation

of these two species were those listed in Li et al. (2006)

and another paper discussing this species in the present

journal issue (Li et al., 2007).

These characters are enough to verify that Rana

tormotus does not have an Amolops- type larva, and

Figure 4. Tadpole, froglets and adult male of Rana tormo-

tus (Wu, 1977). (A) Adult male and froglets, (B) tadpoles,

(C) froglet just after metamorphosis, (D) froglet in ventral

view.

should be placed elsewhere. This conclusion was veri-

fied by examining and comparing the skeletons of Rana

tormotus to the ranine genera Amolops , Pseudoamolops,

Rana and Staurior (Li et al., 2006).

Li et al. (2006) designated Rana tormotus as the

type species of the new monotypic genus Wurana (ety-

mologically, the specific epithet honors Wu Guanfu for

the research on this and other frogs), which is known

from Anhui and Zhejiang Provinces, China. Wurana is

diagnosed as follows:

Adult.- Dorsolateral folds relatively thick and

wide; tympanum deep, forming an external audito-

ry canal that is pronounced in the male; no temporal

folds; male without humeral glands; tarsal folds

absent; tips of fingers and toes expanded into small

disks with circummarginal grooves on outer three

digits; width of crossbar on terminal phalanx much

less than 0.3 times phalanx length.

Larva.- Small type tadpole with weak homy beak;

two rows of lower labial papillae with bases origi-

nating in same line; oral disc emarginate laterally,

with single row of truncate marginal papillae in

posterolateral margin of upper lip and with wide

rostral gap; labial tooth row formula (LTRF) usual-

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Asiatic Herpetological Research, Vol. 1 1

2008

ly 1:4-4/111: 1-1 (sometimes 1:3-3/111:1-1); without

external gland groups; spiraculum on the left with

free tube.

From the general external morphological and skele-

tal characters, Wurana is closer to Rana, especially to

some of the odorous frogs, than Amolops.

The surveys done by Qinghui Gu, Pipeng Li and

others have shown that Wurana tormota is likely either

a threatened or “rapidly declining” species (Global

Amphibian Assessment, 2005; Liu and Hua, 2001). It

has a restricted distribution in a region that is heavily-

impacted by human activities such as sight-seeing,

therefore continued biological research and regional

conservation are strongly recommended.

Acknowledgments

This study was supported by Natural Science

Foundation of China (grant no. 30470206 to Pipeng Li).

We thank Prof. Ermi Zhao, Qinghui Gu, Guangfu Wu,

Hui Zhao, Xisheng Tang, Song Huang and Shunqing Lb

for their help in field surveys and research.

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1-24.

Feng, A. S, P. M. Narins and C.H. Xu. 2002. Vocal acro-

batics in a Chinese frog, Amolops tormotus.

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Chongqing, China. Pp 124-126.

Fei, L, C. Y. Ye, J. P. Jiang, F. Xie and Y. Z. Huang.

2005. An illustrated key to Chinese amphibians.

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Frost, D. 2006. Amphibian Species of the World 3.0, an

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Li, P. P., Y. Y. Lu, A. Li and F. L. Yu. 2008. Tadpole of a

little-known frog, Wurana tormotus. Asiatic

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Li, P. P, Y. Y. Lu and S. Q. Lb. 2006. Taxonomic status

of Rana tormotus Wu, 1977 with description of a

new genus of subfamily Raninae. Sichuan Journal

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Liu, B. R. and T. M. Hua. 2001. Ecological research of

Rana tormotus. Journal of Biology 18(3): 27-28.

Pang, Q. P, Y. Ye. and Y. T. Wen. 2003. Chromosome

data of Chinese amphibians. Chinese Journal of

Zoology 37(4): 49-62.

Peter, M. N, A. S. Feng and W. Y. Lin. 2004. Old World

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910-913.

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tormotus Wu. Acta Zoologica Sinica 23(1):

113-115.

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Chengdu, China.

Submitted: 16 October 2006

Accepted: 25 November 2007

pp. 62-70

Asiatic Herpetological Research, Vol. 1 1

2008

A New Species of Brown Frog from Bohai, China

Pipeng Li1’*, Yuyan Lu1 and Ang Li1

1 Liaoning Key Laboratory of Biodiversity and Evolution,

Shenyang Normal University, Shenyang 110034, P. R. China.

* Corresponding author E-mail: lipipeng@yahoo.com

Abstract.- A new species of brown frog is described from Mt. Culai in Shandong province, China. The new species

differs from other Chinese members of the R. longicrus group (R. zhenhaiensis, R. chaochiaoensis and R. omeirnontis)

in that the head is wider than long and female leg is longer than that of the male. Furthermore, the body is larger the

web on the inner side of the male fifth toe nearly extends to the toe tip, the dorsal color is reddish-brown, there are

no gray or dark bars across the eyes or spots on the back, the labial tooth row formula is frequently 3(2-3)/3, the male

tibia is slightly longer than the foot, the dorsal masculine line is absent and the ventral masculine line is weakly devel-

oped.

Keywords.- Ranidae, Rana, brown frog, new species, China.

Introduction

Brown frogs, also known as wood frogs (Liu, 1946), are

a widespread, complex and diverse group in the genus

Rana. Thirteen brown frogs are known from China, five

of which were previously recognized as R. japonica

(Pope and Boring, 1940). These five species, together

called the southern Chinese Brown Frog (2n = 26), occur

south of the Yangtse River and in Taiwan. The brown

frog in Taiwan was revived as R. longicrus Stejneger,

1898 and the species on the mainland were subsequently

considered to be R. chaochiaoensis Liu, 1945, R.

chevronta Hu and Ye, 1981, R. omeirnontis Ye and Fei,

1993, R. zhenhaiensis Ye, Fei and Matsui, 1995 and R.

japonica (Fei et al., 1993; Fei et al., 2005; Liu and Hu,

1961; Ye et al., 1995).

The species of southern Chinese brown frog do not

overlap in their distributions. Rana chaochiaoensis and

R. omeirnontis are more western in distribution, with the

former found in Yunnan, Guizhou and Sichuan

Provinces, and the latter found in Sichuan and Gansu

Provinces, as well as some counties in Guizhou, Hunan

and Hubei Provinces (Fei et al., 2005, Li et al., 2005). R.

zhenhaiensis occurs in eastern and southeastern China,

in Anhui, Jiangsu, Zhejiang, Jiangxi, Hunan, Fujian,

Guandong and Guangxi Provinces (Fei et al., 2005; Li et

al., 2005). R. chevronta is also western in distribution,

found only on Mt. Omei (Fei, 1999; Fei et al., 2005; Li

et al., 2005). In northern China, R. japonica, now

replaced by R. zhenhaiensis and no R. japonica in China

(Ye et al., 1995), was recorded formerly at Mt. Culai in

Shandong Province and Jixian county in Tianjin (Wang

et al., 1997; Wang et al., 1995). The two other species of

brown frog found in northern China are R. chensinensis

and R. kunyuensis, found in Shandong peninsula (Li et

al., 2006; Lu and Li, 2002), 500 km away from Mt.

Culai.

The present study, part of a project funded by the

National Natural Science Foundation of China, was con-

ducted in a region near the Bohai Sea, where the authors

collected three species of brown frogs (Li et al., 2005; Li

et al., 2006; Lu and Li, 2002). Several specimens resem-

bling Rana zhenhaiensis and R. omeirnontis were col-

lected on Mt. Culai, and were subsequently described as

a “species belong to Rana longicrus species group in

Shandong Province” (Lu et al., 2005). However, after

comparing these specimens with representatives of R.

zhenhaiensis and R. omeirnontis collected from their

type localities and the original descriptions of these

species, the frog from Mt. Culai appears to be a distinct

and separate species. This new species is here placed

within Rana as part of the R. longicrus group and its

relationship to other Chinese members of the group is

discussed.

Materials and Methods

From May 2005 to March 2006, surveys were conducted

at Mt. Culai, Shandong Province (Fig. 1), and the type

localities of Rana zhenhaiensis (Caiqiao of Beilun (for-

merly Zhenhai County), Zhejiang Province) and R.

omeirnontis (Longdong of Mt. Omei, Sichuan Province),

where adults, juveniles and tadpoles were collected.

Some tadpoles were reared through metamorphosis to

confirm their identification, as well as to describe and

compare their coloration with respect to the adults, or at

least until stages 36-38 for proper description.

Specimens were raised in captivity in plastic boxes (260

x 175 x 160 mm) filled with 1.5 L of water. Egg yolk and

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Asiatic Herpetological Research, Vol. 1 1

63

Figure 1. Collection Area in Shandong province, China.

A: Culai Mountain.

vegetable leaves were regularly provided. Frogs and tad-

poles were preserved in 10% formalin and deposited in

the collections of Shenyang Normal University.

Measurements were made with digital calipers to

the nearest 0.01 mm. Abbreviations are as follows: SVL

= snout-vent length; HDL = head length, from tip of

snout to rear of jaws; HDW = maximum head width;

SNT = snout length, from tip of snout to anterior comer

of eye; EYE = diameter of exposed portion of eyeball;

IND = intemasal distance; IOD = interorbital distance at

narrowest point; TMP = horizontal diameter of tympa-

num; TEY = tympanum-eye distance, from anterior edge

of tympanum to posterior comer of eye; FAHL = fore-

arm and hand length; FAW = forearm width; TLL = total

length of leg; TIB = tibia length; TFL = tarsus and foot

length; FL = foot length, from proximal edge of inner

metatarsal tubercle to tip of fourth toe.

All tadpoles were staged according to Gosner

(1960). Tadpoles in stage 33, including both reared spec-

imens and those preserved immediately after capture,

were measured and used in descriptions. Measurements

and terminology follow McDiarmid and Altig (1999).

The labial tooth row formula follows those outlined by

McDiarmid and Altig (1999) and Dubois (Li, 2006).

All measurements were taken with a digital caliper

(to the nearest 0.01 mm) under a stereomicroscope,

except for total length, which was measured with the

caliper directly. Photographs were taken with Nikon

D100 and Sony 7 1 7digital cameras.

Data from Rana zhenhaiensis and R. omeimontis

were taken from their original descriptions and from

additional specimens collected from their respective

type localities and other localities stored in Chengdu

Institute of Biology (CIB) and Museum of Natural

History of Shenyang Normal University (SYNU).

Taxonomy

Rana culaiensis, new species

(Figs. 2-3)

Holotype and type locality.- An adult male, Field num-

ber YT050526007, collected by Lu Yuyan on 27 May

2005 from Mt. Culai (1 17° 18' E, 36° 02' N), Taian City,

Shandong province, China (Fig. 1), at 900 m elevation.

Paratypes.- An adult female, Field number

YT050526005, other information as forthe holotype.

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Figure 2. Holotype of Rana culaiensis, sp. nov. and its

allied species from type localities. (A) Dorsal, (B) Lateral

and (C) Ventral view of R. culaiensis, (D) Lateral view of

R. zhenhaiensis, (E) Lateral view of R. omeimontis, (F)

Dorsal and (G) Lateral view of tadpole of R. culaiensis at

stage 33, (H) Palmar view of hand in R. culaiensis, (I)

Palmar view of hand in R. zhenhaiensis.

Five males, Field number YT050526001-004,

YT050526006, collected by Lu Yuyan on 27 May 2005

from ML Culai (36° 02-03' N, 117° 17-18' E, Taian

City, Shandong province, China, at 690-900 m eleva-

tion.

Tadpoles - Other information as for the holotype.

Diagnosis and comparisons.- This new species is super-

ficially similar to Rana zhenhaiensis, but it can be dis-

tinguished by the following characters: 1) average

snout-vent length larger in adult males (53.6 mm) and

females (62.0 mm); 2) head length slightly less than

head width; 3) toes 3/4 webbed, with web on inner side

of male fifth toe nearly extending to tip; 4) dorsal color

reddish brown, without gray or dark bar across eyes or

spots on back; 5) dorsal masculine line absent and ven-

tral masculine line weakly developed; 6) male tibia

slightly longer than foot; 7) labial tooth row formula fre-

quently 3(2-3)/3. Furthermore, with respect to similar

brown frogs in China, the female leg of R. culaiensis is

longer than the male leg.

On the other hand, this new species can be separated

from all other southern Chinese brown frogs on the basis

Figure 3. Flolotype of Rana culaiensis, sp. nov. (A)

Dorsal view, (B) Ventral view, (C) Lateral view of head,

(D) Palmar view of hand, (E) Tarsal view of Foot. Scale

bar = 10 mm.

of a head that is wider than long and a female leg that is

longer than that of the male. When comparing Rana

culaiensis to other southern Chinese brown frogs in the

R. longicrus group, it appears to be a very well-defined

species with conspicuous diagnostic features (Fig. 4;

Table 2) that easily separate it from the superficially

similar R. zhenhaiensis, R. omeimontis and R.

chaochiaoensis .

From R. zhenhaiensis in having larger males and

females, longer female legs, a more well-developed web

on inner side of male toe 5 (ill developed in R. zhen-

haiensis), an indistinct ventral masculine line (on both

sides in R. zhenhaiensis ) and a different breeding season

(from March to April in R. culaiensis and January to

March in R. zhenhaiensis).

From R. omeimontis in having slightly curved dor-

solateral fold (straight in R. omeimontis), relatively

longer female legs, an indistinct ventral masculine line

(on both sides in R. omeimontis), a larger TMP:EYE

ratio (0.66) and different breeding seasons (July to

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65

R. culaiensis R. zhenhaiensis R. omeimontis

Figure 4. Rana culaiensis, sp. nov. and its allied species from their type localities. (A) dorsal view of male and (B)

ventral view of male (scale bar = 10 mm).

October in R. omeimontis).

From R. chaochiaoensis in having slightly curved

dorsolateral fold, relatively longer female legs (legs of

subequal length in both sexes of R. chaochiaoensis), an

indistinct ventral masculine line, a yellowish-white

female venter (reddish-orange in R. chaochiaoensis ),

three rows of teeth on the tadpole lower lip (four rows in

R. chaochiaoensis ) and a different breeding season

(April to May in R. chaochiaoensis , with some breeding

seen as late as August).

In distribution, Rana culaiensis is allopatric to its

related species. It is a common case in the brown frogs

in China, such as Rana chevronta which only found

located at a narrow area in Mt. Omei (Fei et al., 2005)

and R. kunyuensis, which is closed to R. amurensis and

located in Mt. Kunyu only (Che et al., 2007; Li et al.,

2005).

Description of holotype.- An adult female with SVL

62.0 mm; HDW slightly wider than HDL and head

strongly depressed; snout rounded (more so on projec-

tion beyond lower jaw) and SNT slightly longer than

EYE; canthus rostralis distinct, loreal region slightly

oblique; nostril slightly closer to tip of snout; IND wider

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Table 1. Measurements (mm) of allotype and paratypes of Rana culaiensis, sp. nov.

Note: * size range, ** mean ± SD (n = 5), *** % SVL.

than IOD and IOD wider than upper eyelid width; tym-

panum large, round, TMP two-thirds diameter of EYE

and separated from eye (TEY) by one-third of TMP;

vomerine teeth developed in slightly oblique groups

between and behind choanae, with groups narrowly sep-

arated in “\ /” shape; tongue deeply notched and with

papillae.

FAHL less than half of SVL; fingers obtuse with

relative length of fingers II < IV < I < III; subarticular

tubercles prominent; three metacarpal tubercles distinct

(inner one large and outer two separated at bases). TLL

relatively long; tibio-tarsal joint reaching nostril, mak-

ing TIB about 57.8% of SVL; heels overlapping when

limbs folded at right angles to body; TIB slightly longer

than FL; toes also obtuse with tips similar to those of

fingers, toes 3/4 webbed with subarticular tubercles well

developed; web of inner side of male fifth toe nearly

extending to tip of toe; inner metatarsal tubercle oval,

outer metatarsal tubercle weakly developed.

Skin rather smooth above, with few warts; glandu-

lar dorsolateral fold running along each side of body

behind eye to insertion of hind leg and slightly curved to

temporal fold above tympanum; temporal fold distinctly

curving posteriorly from above tympanum to large trian-

gular gray patch behind eye; ventral surface of body

smooth except for posterior and median surfaces of

femora, which are covered with coarse granular glands

(small granules).

Measurement of holotype.- SVL 56.8 mm; HDL 17.15

mm; HDW 17.9 mm; SNT 7.8 mm; IND 5.32 mm; IOD

3.51 mm; UED 3.97 mm; EYE 6.26 mm; TMP 3.69 mm;

FAHL 23.63 mm; FAW 7.6 mm; TLL 106.17 mm; TIB

34.2 mm; TFL 47.91 mm; FL 33.7 mm.

Coloration of holotype in life (in preservative). -

Dorsum evenly reddish or yellowish-brown (gray),

without gray or dark interorbital bars; stripe on upper lip

dark brown with white blotches, extending from tip of

snout to venter of eye, joining with dark reddish-brown

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67

(yellow) stripe running to behind arm insertion; lower

lip with brown speckles; rictal glands brown (yellow);

limbs gray dorsally with four and five dark cross-bars on

thigh and tibia, respectively; sides of body light yellow.

Dorsolateral told pale brown (yellow); throat and belly

creamy-yellow (white with pale gray nebulous marks);

triangular patch gray to somewhat black (gray with tym-

panum dark brown); metacarpal tubercles and nuptial

pad dark brown (black).

Description of paratypes.- The paratypes, six adult

specimens, one female (YT050526005) and five males

(YT05052600 1-004, YT050526006) approximate the

holotype in almost all pertinent details. The measure-

ments of paratypes summarized in Table 1. However,

there are some minor differences between paratypes and

the holotype as follows:

Male paratypes.- One male specimen with small

tubercles on sides of body covered with black. And

the following characters of male paratypes as sec-

ondary sexual characters: smaller than female with

FAW much thickened; male with strong nuptial

pads on the inner-dorsal side of first fingers, extend-

ed to figure tip and separated at metacarpus; no

vocal sacs; lineae musculinae indistinct ventrally

and absent dorsally; TLL is shorter than the female

TLL.

Female paratype.- The female specimen with light

jacinth spots ventrally in life (pale gay flecks in pre-

servative).

Tadpoles.- The body ovoid in dorsal profile, pear-like in

lateral profile; darkly colored in life (tail more grey in

preservative). In stage 33, body length 16 mm, tail

length 34 mm, length of hind limb 9 mm; snout rounded,

eye dorsolateral, nostril slightly closer to snout tip;

spiraculum small, on left side of body and with no free

tube; vent dextral, tube of vent continuous with ventral

caudal fin; dorsal fin rising from base of tail. Tail height

about half of body length with apex obtuse; musculation

weakly developed on pointed tip; mouth anteroventral,

with row of labial papillae on lower lip and mouth cor-

ner (papillae of lower lip regularly arranged); comer of

mouth with several additional papillae; labial tooth row

formula frequently 3(2-3)/3, length of tooth row long,

homy beak weak and narrow.

Habitat.- During field work at Mt. Culai of Shandong

province in 2005 and 2006, we surveyed the Mt. Culai

and the nearby mountains and collected the trogs

described here as a new species in a forest brook (alt.

630-900m) covered with gramineous grass following

the breeding season. The tadpoles in stage 28-34 were

collected on 27 May 2005. No eggs were found at that

time, suggesting that the breeding season for this species

was in March and April.

Etymology.- Rana culaiensis is so named as it is appar-

ently restricted to Mt. Culai of Shandong procince, East

China.

Taxonomic account.- As Liu (1946) indicated, “among

the Chinese amphibian the woodfrog group presents a

problem most difficult to solve. Great confusion exists

in the literature, as there has been no satisfactory com-

parative study of preserved museum specimens of differ-

ent species, and no careful investigation in the fields.” In

some species groups, the frogs are quite conservative in

their morphology and very difficult to separate (Liu and

Hu, 1961; Lu and Li, 2002; Tanaka et ah, 1996; Xie et

al., 2000), as is also the case for the many species of

Eurasian brown frogs (Kim et al., 2002). Although much

progress has been made in the systematics of these

species, many brown frogs are still difficult to identify

and some species may yet remain undescribed (Che,

2007; Lu and Li, 2005).

The brown frogs of the Rana longicrus group (for-

merly treated as the Rana japonica group) included four

species ( Rana zhenhaiensis, R. chaochiaoensis , R. omei-

montis, and R. chevronta) in the mainland of the south-

ern China and one species {Rana longicrus ) in Taiwan

(Fei et al., 2005; Xie et al., 2000). These frogs once clas-

sified as R. japonica by Pope and Boring (1940), The

species-level status of R. chaochiaoensis , R. chevronta

and R. longicrus has never been questioned, but some

researchers still treat R. omeimontis and R. zhenhaiensis

as R. japonica (Zhao et al., 2000), even though Xie et al.

(2000) provided significant morphological, ecological,

cytological and morpholometrical support to verify the

status of all five species. Well-supported phylogenetic

analyses have also been provided (Che, 2007; Jiang and

Zhou, 2001; Yang et al., 2001). The cluster tree provided

by Xie et al. (2000) illustrates a close relationship

between R. omeimontis and R. chaochiaoensis , as well

as a close relationship between R. zhenhaiensis and R.

chevronta.

The Culai frogs once reported as new province

record under Rana japonica (Wang et al. 1997), but R.

japonica was replaced by R. zhenhaiensis by Ye, Fei and

Matsui (1995). Here we treated it as a new species with

some difference from other related species in the group

and it maybe close to R. zhenhaiensis and omeimontis

(Fig. 4; Table 2). As allopatric to its congeners, it shows

the same case of as Rana chevronta and R. kunyuensis.

Material examined. -

Rana chaochoensis from CIB (n = 70). (males)

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 2. Comparisons between Rana culaiensis and its allies.*

* data of Rana chaochiaoensis , R. omeimontis and R. zhenhaiensis cited from Xie et al. (2000).

CIB37841,

CIB3785 1,

CIB37859,

CIB37873,

CIB37860,

CIB37876

CIB37842,

CIB37852,

CIB37866,

CIB37877;

CIB37862,

id CIB84495

CIB37847, CIB37850,

CIB37855, CIB37857,

CIB37867, CIB37870,

(females) CIB378853,

CIB37869, CIB37874,

from China, Sichuan

Province, Zhaojue County (as Chaocho formerly, type

locality), (males) CIB37643, CIB37683, CIB37703;

(females) CIB37641, CIB37642, CIB37644, CIB

37681, CIB37699, CIB37700 and CIB37707 from

China, Sichuan Province, Yuexi, Mianning, and

Yanyuan Counties, (males) CIB37712, CIB37713,

CIB37715, CIB37723, CIB37731, CIB37734,

CIB37735, CIB377747, CIB37748, CIB37754,

CIB37755, CIB37756, CIB37758, CIB37760; (females)

CIB37720, CIB37721, CIB37724, CIB37725,

CIB37773 and CIB37777 from China, Guizhou

Province, Weining County, (males) CIB37796,

CIB37799, CIB37798, CIB37801, CIB37802,

CIB37803, CIB37804, CIB37805, CIB37806, (females)

CIB37786, CIB37787, CIB37788, CIB37790,

CIB37792, CIB37793 and CIB37830 from China,

Yunnan Province, Kunming City, (males): CIB37840,

CIB37839 and (female) CIB37838 from China, Yunnan

Province, Lijiang County.

Rana chevronta from CIB (n = 1): (male) CIB65I0028

from China, Sichuan Province, Mt. Omei (type locality).

Rana omeimontis from SYNU (n = 8): (males)

SYNU050274, SYNU050275, SYNU050278,

SYNU06080522, SYNU06080523; (females)

SYNU050276, SYNU050277 and SYNU050279 from

China, Sichuan Province, Omei Mt (type locality).

Rana zhenhaiensis from SYNU (n = 18): (males)

SYNU050267, SYNU050268, SYNU05027 1 ,

SYNU06020126, SYNU06040129, SYNU06040130,

SYNU06040131, SYNU06040132, SYNU06040133’,

SYNU06040134, SYNU06040135; (females)

SYNU050269, SYNU050270, SYNU050272,

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Asiatic Herpetological Research, Vol. 1 1

69

SYNU050273, SYNU06020127, SYNU06020128 and

SYNU06040136 . China, Zhejiang Province, Beilun

region (formaerly Zhenhai county, type locality).

Acknowledgments

This study was supported by the Natural Science

Foundation ot China (grant no. 30470206 to Pipeng Li).

We wish to thanks G. F. Wu and H. Zhao, as well as to

my graduated students W. Wang., P. W. Wang and X. Z.

Cui for their help in Field surveys.

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Lu, Y. Y, P. P. Li, W. Wang, P. W. Wang and X. Z. Cui.

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1997. A new record of Ranidae from Shandong

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Submitted: 13 September 2006

Accepted: 25 November 2007

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pp.71-75

The Tadpole of A Little-known Frog, Rana tormotus Wu, 1977

Pipeng Li*, Yuyan Lu, Ang Li and Lina Yu

Herpetodiversity Research Group, Shenyang Normal University, P. R. China 110034.

* Corresponding author E-mail: lipipeng@yahoo.com

Abstract.- The tadpoles of a Chinese endemic and rare frog, Rana tormotus were collected from the type locality and

reared with other coexisting tadpoles of Paa spinosa, Rana schmackeri and Amolops wuyiensis in comparison. The

tadpole of Rana tormotus is small (about 27 mm in total length) and brown to slightly olive in color. Its body is ovoid

in dorsal view, widest at about midpoint, depressed and elliptical in lateral view. Body length is nearly one-third ol

total length. Lateral line pores are visible on the body and tail. No glands (such as ventral and dorsal glands in tadpoles

of genus Amolops) are visible. The snout is round, slightly flatted in dorsal profile, and rounded in lateral and ventral

profiles. Eyes directed dorsolaterally with diameter 31% of body height, closer to tip of snout than eye. Spiracle is

short and sinistral posteriorly. Tail approximately 2.1 times body length. Tail fins convex and approximately fusiform.

Tail tip is V-shaped or narrowly rounded. Vent tube is dextral and attached to ventral fin.

Oral disc is large and anteroventral in position. Labial tooth row formula 5(2-5)/4(l). Upper jaw sheath is finely

serrate and narrow, lower jaw sheath is finely serrate and shallowly V-shaped. No abdominal sucker was observed

behind the oral disc. It is similar to those of Rana andersonii and R. schmackeri in shape and oral disc characters.

From the characters of the tadpole of Rana tormotus, it does not belong to the Amolops type. It should be placed

in the genus Rana {sensu lato ) as Rana tormotus firstly or placed in a new genus (as Wurana by Li et al. [2006]) by

further analysis.

Keywords.- Amolops tormotus, Rana tormotus , Wurana tormota, tadpole, sunken ear frog, concave-eared torrent frog.

Introduction

Rana tormotus Wu, 1977 is an arboreal frog in the fam-

ily Ranidae found in the mountain streams of the Anhui

and Zhejing Provinces of Eastern China (Sichuan

Institute of Biology (Wu, G. F.), 1977; Zhao and Adler,

1993). This frog has an interesting characteristic: the

males warble melodies like a bird in order to attract

females (Feng et al., 2002), calling nightly from the low

vegetation along the banks of rivers and streams. Their

vocal repertoire is extraordinarily rich; individual calls

exhibit multiple upward and downward frequency

sweeps, rapid frequency “steps,” and sudden onset and

offset of selective harmonic components within a note

(Feng et al., 2002; Peter et al., 2004). This is the first

species of frog known to use diverse rising and falling

modulations - most frog calls only go either up or down.

These calls are also the first terrestrial frog noises

known to extend into the ultrasonic range (Feng et al.,

2002; Peter et al, 2004). Both of these phenomena are

related to the frog’s unique ear.

This frog, which is called the “sunken ear frog” or

“concave-eared torrent frog” in Chinese, has a conspic-

uous character that makes it different from most other

frogs: the males have visible ear canals leading to

eardrums within the skull, similar to Amolops cavitym-

panum Boulenger (Sichuan Institute of Biology [Wu G.

F.], 1977; Feng et al., 2002; Fei et al., 1991, 2005). Fei

et al. (1991, 2005) and Dubois (1992) placed this species

in Amolops because its tadpole might “belong to [the]

Amolops type” (Fei et al., 1990 (1991); Zhao and Zhao,

1994). Because the tadpoles have never been recorded,

however, it has been argued that this species should

instead be assigned to "Rana" (Zhao and Zhao, 1994;

Zhao et al., 2000; Global Amphibian Assessment, 2005).

Here, for the first time, we describe the tadpole of

this little-known species of frog and provide information

on its natural history. The importance of these findings

lies in the necessity for larval characters in anuran clas-

sification (Chou and Lin 1997), and may allow for clar-

ification of the uncertain position of Rana tormotus in

relation to Rana and Amolops.

Materials and Methods

Field work was conducted in a small stream of

Longjiang Forest, Zhejiang Province (China), and the

type locality of Taohuaxi stream in the Huangshan Mts,

Anhui province. Field studies were done from June to

August 2005.

The tadpoles of Rana tormotus Wu, 1977, Paa spin-

osa (Daivd, 1875), Rana schmackeri Boettger, 1892 and

A. wuyiensis (Liu and Hu, 1975) were observed and

sampled in Taohuaxi, a permanent stream. Some tad-

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Asiatic Herpetological Research, Vol. 1 1

2008

poles ol R. tormotus and R. schmackeri were reared until

stages 36-38 (Gosner, 1960) for description, or through

metamorphosis to confirm their identification and to

describe and compare adult coloration. Tadpoles were

raised in captivity in a plastic box (260 x 175 x 160 mm)

with 1.5 L ot water. Egg yolk and vegetable leaves were

provided regularly. The tadpoles were preserved in 5%

formalin. This material, together with adult voucher

specimens, is deposited in the collections of Shenyang

Normal University.

Tadpoles were staged according to Gosner (1960).

Tadpoles in stage 38, included both reared and freshly

captured specimens, were used in the descriptions and

measured. No changes were observed in the oral mor-

phology or general shape of reared tadpoles.

Measurements, terminology, and labial tooth row formu-

la follow Altig and McDiarmid (1999); labial tooth row

formula also follows Dubois (1995).

All measurements were taken with a digital caliper

(0.01 mm) and a stereomicroscope, except for total

length, which was measured directly with a hand caliper.

Drawings were made with the aid of a camera lucida

attached to a stereomicroscope. The photographs were

taken with a Nikon D100 camera.

Measurements are abbreviated as follows: BL

(body length), TL (total length), TaL (tail length), BW

(maximum body width), BH (maximum body height),

TH (height of tail), DFH (dorsal fin height), VFH (ven-

Figure 1. Standard morphometric measurements for tad-

poles used in this study.

tral fin height), SO (snout-ocular axis distance), SN

(snout-nasal axis distance), E (eye diameter), IN

(intemarial distance), IO (interorbital distance), SS

(snout-spiracle distance), ODW (oral disc width).

Standard measurements are shown in Figure 1.

Decription of external morphology at stage 38.- The

mean measurements and standard deviations of eight

tadpoles in stage 38 are shown in Table 1. Mean total

length at stage 38: 27±1.07 mm (n = 8; Table 1). Body

ovoid in dorsal view, widest at midpoint, depressed and

elliptical in lateral view; lateral part of marginal papillae

of oral disc slightly visible; Body length nearly one-third

(32%) of total length, body 1.5 times longer than wide,

2.1 times longer than high, 1.4 times wider than high.

Lateral line pores (neuromasts of caudal, dorsal,

supranaso-orbital, infranaso-orbital, lateral, mental,

postgular, postspiracle and pregular lines) visible on

body and tail. No glands visible. Snout rounded, slightly

flatted in dorsal profile, rounded in lateral and ventral

profiles; eyes moderate, not part of dorsal profile, direct-

ed dorsolaterally, diameter 31% of body height, separat-

ed by distance about 2.8 times eye diameter; interorbital

distance 88% of body width; nostrils directed dorsolat-

erally, closer to tip of snout than eye, intemarial distance

60% of interorbital distance.

Spiracle sinistral, short, posterior, opening slightly

above midline at about 5/7 of body length, directed pos-

terodorsally at about 15°, lateral wall longer than medial

wall, inner wall confluent with body, wall forming

around aperture.

Tail approximately 2.1 times body length and 3.0

times body width, maximum height 28% of tail length,

maximum tail height at end of first third of tail length.

Tail musculature highest at base, slightly higher than

dorsal and ventral fins, gradually tapering to pointed tip,

weakly developed. Tail fins convex and approximately

fusiform. Dorsal fin originates from tail muscle (the pos-

terior edge of the first section) near tail-body junction,

tallest just past to midpoint; ventral fin of nearly equal

height throughout its length; dorsal fin height E3 times

ventral fin height at highest point. Tail tip V-shaped or

narrowly rounded; vent tube dextral, short, attached to

ventral fin.

Oral disc large, anteroventral in position, width

about 0.73 times distance between eyes and approxi-

mately 45% of body width, emarginate laterally, with

single row of truncate marginal papillae in lateral poste-

rior margin of upper lip and wide rostral gap, two rows

of completely marginal papillae on lower lip but bases of

papillae originate in same line; rostral gap equal in

length of A-l. No lateral submarginal papillae. Labial

tooth row formula 5(2-5)/4(l) and l:4+4/l+l:3 (follow-

ing Dubois [1995]); A-l and A-2 longest, slightly longer

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Asiatic Herpetological Research, Vol. 11

73

Table 1. Mean measurements and standard deviation

(mm) of eight tadpoles in stage 38 of Gosner (1960).

than all other rows and similar in length, A-2 gap nar-

row, length of row becoming progressively shorter from

A-3 to A-5 (A-5 22% length of A-2); P-2 and P-3 equal

in length and longer than P-1 and P-4 (80% P-2); A-2

gap narrower than P-1. Labial teeth small, blunt and

devoid of cusps; longest at middle of row, teeth becom-

ing progressively smaller from PI to P4, but equal in P2

and P3, teeth of A i smaller than A2 and those of A2

equal. Jaw weakly developed. Upper jaw sheath finely

serrate, narrow, width slightly less than width of lower

jaw; lower jaw sheath finely serrate, shallowly V-

shaped.

From stage 26 onward, labial tooth row formula sta-

bilizes with very small variation in some specimens.

Larval denticles disappear after disappearance of vent

tube. Tadpole matching Orton’s Type-IV category

(Orton, 1953): oral disc elaborate and spiracle sinistral.

In preserved specimens, dorsal surface of body dark

brown; gut and heart visible ventrally, not visible later-

ally; anterior half of ventral part of body pale brown

with dark brown spots on sides, and abdominal region

whitish without dark brown spots. Coloration of muscu-

lar part of tail similar to that of body in reticulated pat-

tern. Tail fins with small brown spots.

In life, tadpole body coloration dark-brown with

head pale brown. Pupil of eye round, black, and

enclosed by narrow sliver ring.

Natural history notes.- Relative to the size of the adult

(male 35 mm, female 48 mm), the tadpole is small. If the

tadpole had not been reared to adulthood, it would have

been difficult to believe that the tadpole and frog were of

the same species.

The tadpoles gathered in groups in the small stream,

and were found to swim freely among small stones

where there was no swift water current or side pools that

were poorly connected with the main permanent stream

at night. During the daytime, the tadpoles hid under

stones and were rarely seen. The tadpoles were noctur-

nal and used their sites for grazing, avoiding areas with

silty sediment and fast water flow. The large oral disc

and numerous blunt rows of teeth suggested a greater

capacity for grazing than for suspension feeding.

The color of the tadpoles camouflaged them against

the small stones and sand on the bottom of the stream

where they spent the day. Although collected from a

mountain stream, the tadpoles were able to live in their

jars for a long time, sometimes in good condition after

three day’s travel. After 40 days of captivity, the larvae

completed metamorphosis and became froglets similar

to the adult in body shape and coloration. The biggest of

the tadpoles was less than 30 mm in total length.

Froglets were 11.4 mm (10-11.6 mm, n = 5, SD = 0.73)

from snout to vent just after metamorphosis.

The frogs and larvae that were found coexisting

with Rana tormotus in the stream were Paa spinosa

Figure 2. Rana tormotus tadpole at stage 38 (Gosner,

I960), (A) Dorsal view, (B) Lateral view, (C) Ventral View',

(D) Oral disc.

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Asiatic Herpetological Research, Vol. 1 1

2008

(Daivd, 1875), Rana schmackeri and A. wuyiensis (Liu

and Hu, 1975). While the tadpole of R. schmackeri was

similar to R. tormotus in shape, coloration and general

oral disc characters, the other two appeared conspicu-

ously different.

Although Rana schmackeri also had a small tadpole

belonging to Orton’s Type-IV category (Orton, 1953), it

was larger than the tadpole of Rana tormotus. The total

length of R. schmackeri tadpole was more than 30 mm

from stage 35, while the maximum total length of the R.

tormotus tadpole was less than 30 mm at any later stage.

At stage 36, the body and tail length was 11.1 mm and

20.25 mm for R. schmackeri, and 8.36 mm and

18.83mm for R. tormotus. At stage 44, the back of R.

schmackeri became greenish, and after metamorphosis,

yellow patches appeared on the back.

The tadpole of Amolops wuyiensis was also of the

small type, but the ventral suctorial disc easily identified

it. This species was found at the edge of side water-bod-

ies of the stream with little current. The tadpole used the

suctorial ventral disc to adhere to the rocky substrata to

overcome the stream’s water current.

The tadpole of Paa spinosa is of the big type, and is

found alone or in groups of a few individuals at the bot-

tom of pools beside or below cascades and gently flow-

ing parts of the Taohuaxi stream. The oral disc of this

species is emarginate, with two rows of marginal papil-

lae, lateral submarginal papillae and a strong beak. The

most conspicuous character of the tadpole is a gray or

black band at the base of tail. The labial tooth row for-

mula is 5(2-5)/3(l).

Discussion of taxonomic status.- Examination of the

tadpole of Rana tormotus reveals that it is not of the

“ Amolops type”, because there is no abdominal sucker

and no ventral or dorsal glands (Yang, 1991). The tad-

pole of this species is more similar to that of Rana

andersonii (Liu, 1940) and O. schmackeri in shape and

oral disc morphology, but the tadpole and froglet just

after metamorphosis is smaller, differently shaped, and

without green or yellow back patches.

The body and tail length of the Rana andersonii tad-

pole averaged 12.27 mm (12-12.5 mm, n = 3) and 26.27

mm, and the froglet 12.7 mm (Liu, 1940). Four days

after metamorphosis, uneven green patches appeared on

the back (Liu, 1940).

While the larvae are similar in appearance, the

adults of odorous frogs and Rana tormotus are remark-

ably different in morphology, with the sunken ear and

absence of odor gland cells in the skin of R. tormotus

being most the notable characters.

Based on the above evidence, this species should

either be left to Rana as Rana tormotus, or placed in the

new genus as Wurana tormota, although further study is

needed.

Acknowledgments

We are grateful to Prof. Gu H. Q., Dr. Lu S. Q., Mr. Tang

X. S. and Ms. Zhao H. for their field working help. This

research was supported by China National Nature

Science Foundation and Special Invited Professor Grand

of Shenyang Normal University to Dr. Pipeng Li.

Literature Cited

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vae: relationships among developmental modes,

morphologies and habitats. Herpetological

Monographs 3: 81-109.

Altig, R. and R. W. McDiarmid. 1999. Diversity: famil-

ial and generic characterizations. In R. W.

McDiarmid and R. Altig (eds). Tadpoles: The

Biology of Anuran Larvae. Pp 259-337. Univ. of

Chicago Press, Chicago.

Chou, W. H. and J. Y. Lin. 1997. Tadpoles of Taiwan.

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Science 7: 1-98.

Dubois, A. 1992 Notes Sur la classification des Ranidae

(Amphibiens Anoures). Bull. Mens Soc. Linn. Lyon

61(10): 305-352.

Dubois, A. 1995. Keratodont formula in anuran tad-

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33: 1-15.

Fei, L, C. Y. Ye. and Y. Z. Huang. 1990 (1991). Key to

Chinese Amphibia. Pp 124-126, Chongqing Branch,

Science and Techinology Literature Publishing

House, Chongqing.

Fei, L., C. Y. Ye., J. P. Jiang., F. Xie. and Y. Z. Huang.

2005. An illustrated Key to Chinese amphibians.

Sichuan Publishing Group-Sichuan Publishing

House of Science and Technology, Chengdu.

Feng, A. S., P. M. Narins and C. H. Xu. 2002. Vocal

acrobatics in a Chinese frog, Amolops tormotus.

Naturwissenschaften. 89(8): 352-6. Epub 2002 Jun

22.

Global Amphibian Assessment. 2005. Global

Amphibian Assessment - Detailed Report: Amolops

tormotus. In: http://www.globalamphibians.org/ser-

vlet/GAA?searchName=Amolops+tormotus

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Gosner, K. L. 1960. A simplified table for staging anu-

ran embryos and larvae with notes on identification.

Herpetologica 16:183-190.

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Narins, P. M., A. S. Feng, W. Y. Lin, et al. 2004. Old

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115(2): 910-913.

Sichuan Institute of Biology (Wu, G. F.) 1977. A new

species of frogs from Huang-Shan, Anhui - Rana

tormutos Wu. Acta Zoologica Sinica, Beijing,

23(1): 113-115.

Yang, D. T. 1991. Phylogenetic systematics of the

Amolops group of ranid frogs of Southeastern Asia

and the Greater Sunda Islands. Fieldiana: Zoology,

Chicago (USA), new ser., No. 63: 1^12.

Zhao, E. M., and K. Adler. 1993. Herpetology of China.

Published by the Society for the Study of

Amphibians and Reptiles in cooperation with the

Chinese Society for Study of Amphbians and

Reptiles. Oxford, Ohio, USA.

Zhao, E. M., H. W. Chang, and X. L. Zhao. 2000.

Taxonomic bibliography of Chinese amphibia and

reptilia, including karyological literature.

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Literature - Catalogue and Indices. Chengdu

Science and Technology University Press,

Chengdu.

Submitted: 03 March 2006

Accepted: 25 November 2007

pp. 76-79

Asiatic Herpetological Research, Vol. 1 1

2008

A Brief Report on the Life History of Batrachuperus taibaiensis

at Ping He Liang of Tsinling Mts.

Pipeng Li*, Yuyan Lu and Ang Li

Liaoning Key Laboratory of Biodiversity and Evolution, Shenyang

Normal University, Shenyang 110034, R R. China.

* Corresponding author E-mail: lipipeng@yahoo.com

Abstract.- Batrachuperus taibaiensis is a high-mountain, stream salamander endemic to China, and compared to con-

geners, is more northeastern in distribution and found at lower elevations. The distribution, life history and measure-

ments of the larva and adult of this species were recorded from 2005 to 2006 at Ping He Liang of Tsinling Mts. The

life history of the genus is discussed in the context of refining species definitions for those Batrachuperus found in

the Tsinling Mts. Past surveys carried out in Tsinling Mts found several different Batrachuperus species and there is

a need to clarify which species really exist. The distribution of Batrachuperus taibaiensis is discussed in the paper.

Keywords .- Life history, stream salamander, Batrachuperus taibaiensis.

Introduction Materials and Methods

Much of the taxonomy and phylogeny of the Chinese

Amphibia remain unresolved, as is much of the knowl-

edge on their basic natural history, generally because

observations on natural history are not considered to be

worthy of publication and the gathering observations is

often time intensive (Greene, 1993). Nevertheless, these

observations may contribute to phylogenetically inform-

ative characters (de Queiroz and Wimberger, 1993) and

data critical for developing conservation and manage-

ment strategies (Mendelson et al., 1999).

The high-mountain stream salamanders, genus

Batrachuperus Boulenger, 1878, contains seven species

that occur in Western China and adjacent Myanmar

(Frost, 2007). The natural history of the species in this

genus remains largely unknown or unpublished because

individuals generally hide under stones in small moun-

tain streams at high altitude, although reports on the

common species B. pinchonii (David) have been made

by Liu (1945).

Recently, one new species ( Batrachuperus

taibaiensis) was found in the upper Heihe River in the

Tsinling Mts. of Shaanxi Province, China. Compared to

congeners, this salamander is more northeastern distri-

bution and found at lower elevations (Song et al., 2001).

During collecting trips (1987-2005) along the rivers and

streams of Ping He Liang, the senior author collected

several B. taibaiensis and collected data on their natural

history. The following is a brief excerpt from the results

of a series of herpetological surveys made from 2005 to

2006.

Amphibian surveys were made on the south side of the

Tsinling Mts. (33° 36' N, 108° 28' E, 1800-2000 m)

between Huo Di Tang and Xun Yang Ba of Ningshan

County, Shaanxi province, China, from April 2005 to

June 2006. Ping He Liang reaches an altitude of 2160 m

at the National Way. Batrachuperus taibaiensis was also

surveyed at its type locality on the north side of the

Tsinling Mts in April 2005.

Adult specimens were stored in 10% formalin.

Larvae and egg cases were observed live in the lab.

Results

Identification.- Batrachuperus taibaiensis (Fig. 1) can

be separated from congeners by its relatively large body

size and lack of homy covers on the palms and tarsa.

Distribution.- The distribution of Batrachuperus

taibaiensis is concentrated in the upper part of two rivers

and their associated streams on the northern and south-

ern sides of Ping He Liang (Fig. 1). The pH of the water

was somewhat acidic (pH = 5. 5-6.0). Specimens were

found under rocks in river headwaters and streams in the

study area 2-3 km from the top of Ping He Liang (above

1800 m).

In the streams and rivers where Batrachuperus

taibaiensis was found, adults of a number of other

amphibians occurred: Ranodon tsinpaensis Liu and Hu,

Bufo gargarizans Cantor, Ran a chensinensis David, Paa

quadrana (Liu, Hu and Yang), Bufo andrewsi Schmidt.

Tadpoles of Bufo andrewsi and S. ningshanensis Fang

2008

Asiatic Herpetological Research, Vol. 1 1

77

Figure 1. (A) Habitat of Batrachuperus taibaiensis. (B) Adult and larvae of B. taibaiensis from Ping He Liang. (C) and

(D) are egg cases of B. taibaiensis.

were also observed, the latter of which has its type local-

ity in the same region. Hyla tsinlingensis Liu and Hu,

another endemic Chinese amphibian, was found in near-

by ponds. Bufo gargarizans Cantor, Bufo andrewsi

Schmidt, H. tsinlingensis Liu and Hu, Rana chensinen-

sis David P. quadrana (Liu, Hu and Yang) and

Batrachuperus pinchonii were more widespread at

lower elevations near the study area.

The study area encompassed approximately 1 5 km2

of Subalpine conifer habitat covered with well-devel-

oped vegetation. The canopy often arched over the

rivers and streams.

Reproduction and growth.- The breeding season most

likely occurs from April to July, as the youngest larvae

with external gills and the most number of young sala-

manders in different stages of development were collect-

ed on August 3rd, 2005; young salamanders were not col-

lected after April 16th, 2005.

Egg-cases, previously unknown for this species,

were found adhered to the under-side of rocks in the

river in April 1 6lh, 2005. The body of the egg-case was a

curled columnar tube with tapered ends; the case was

smooth and almost entirely transparent with thin longi-

tudinal striations; fresh cases were the color of milk; for-

78

Asiatic Herpetological Research, Vol. 1 1

2008

Table 1. Measurements of Batrachuperus taibaiensis

(n=3)

A: larva just after hatching; B: developed larva; C: larva with fill shrivel-

ing

malin-fixed cases became brittle. The cup-like cap at the

free end of the case was more soft and delicate than the

remainder of the case (Fig. 1). Cases were 15.0-17.0

mm in length with a diameter of 18.0-20.0 mm. There

were 27-29 eggs or embryos in each egg case. Egg

diameter was 5. 0-5. 5 mm.

Free larvae appeared at stream edges under small

stones at the beginning of August. The early larvae,

compared to developed larvae, juveniles and adults, in

that the color was lighter gray with the dorsum yellow-

ish-green and the venter yellowish; the dorsal pigment

faded after fixation. The forelimb of the early larva was

fully developed with four formed fingers; the fourth toe

bud appeared after formation of the first three toes. The

fore limbs developed earlier than the hind limbs. The

labial folds were well-developed and the pores of the lat-

eral line organ were visible on the head. Eyes were

small, black and covered with a transparent membrane;

eyelids were absent. Balancers were absent from the side

of head. There were four pairs of external gills (decreas-

ing in size posteriorly); the filaments of the last gill were

very short, white and hidden beneath the third gill; the

first to third gills resemble those of Batrachuperus pin-

chonii (Liu, 1945). The vertebral groove was distinct

along the length of the trunk. The tail was much higher

and shorter than that of the adult.

The developed larva was blacker than the younger

larva, and larvae with gill regeneration were nearly

black. Pores of the lateral line organs were more con-

spicuous in the head and shoulder regions. Eyelids were

well developed (as in Batrachuperus pinchonii [Liu,

1945]). Gill filaments were blacker and longer than

those seen in earlier larva, but shorter than larva with gill

regeneration. Fingers and toes were well-developed.

Fifteen specimens from various developmental

stages were measured (Table 1). Larval head length was

26.3%, 32.58% and 34.96% of total body length, while

adults head length was 23.91% in the male and 24.69%

in the female. Larval eye length was slightly larger than

that of adults.

Discussion

Need for clarification of Batrachuperus species in

Tsinling Mts.- Song (1983) and Yuan (1984) reported

Batrachuperus pinchonii from the Tsinling Mts. in Mao

Tai Zi, Liuba County, and Huo Di Tang Forest, Ningshan

County. Later, from 1985 to 1989, the senior author and

a colleague from Shaanxi Normal University collected

two species of Batrachuperus from Huo Di Tang and

Xun Yang Ba Forests, Ningshan County, which are on

the northern and southern slopes of the same Mountain:

B. pinchonii was collected at lower elevations while B.

tibetanus was collected at higher elevations (Li and

Fang, 1993 and unpublished data). At the same time,

Song collected a new species of Batrachuperus from

Zhouzi County - B. taibaiensis - which was supported

by sequence data from cytochrome b (Song et al., 2001).

Unfortunately, these species were either excluded (Fei et

al., 2005) or treated under incorrect names (Han and Lu,

2003; Zhang and Jia, 2002) in subsequent publications.

Batrachuperus occurs throughout the Tsinling Mts.,

extending to Foping, Zhouzi, Zashui and Ningshan

Counties. Batrachuperus taibaiensis has also been

found in Kangxian, Wenxian and Fenxian in Gansu

Province, as well as Liuba and Nanjiang in northern

Sichuan Province (Zeng, 2004); these identifications

were made using molecular techniques because it is

often difficult to identity species of Batrachuperus using

traditional methods (Zeng, 2004).

Based on what is now known about the biology,

morphology and distribution of Batrachuperus taibaien-

sis, we consider that all specimens of B. tibetanus col-

lected from the Tsinling Mts south to Shaanxi,

Southeastern Gansu and the North Sichuan Provinces

(such as Micanshan and Dabashan) are actually repre-

sentatives of B. taibaiensis. Considering this, the full

range of B. taibaiensis may actually extend as far as

southeastern Shaanxi Province and the Shenlongjia Mts

in northwestern Hubei Province. To corroborate these

assumptions, it will be necessary to re-examine previ-

ously collected specimens from these localities to verify

their identity. Further fieldwork and molecular analyses

will likely be necessary.

The need to focus on Batrachuperus life history.- The

high-mountain stream salamander Batrachuperus cur-

rently was considered to contain seven species that

occur in Western China and adjacent Myanmar (Frost,

2008

Asiatic Herpetological Research, Vol. 11

79

2007). Most of these species are restricted to China, and

B. tibetanus and B. pinchonii are significant in their use

as experimental animals in embryological, morphologi-

cal and ecological investigations (Li and Fang, 1993;

Xu, 1992, 1993; Zhang and Jia, 2002; Han and Lu,

2003; Fei et ah, 2005).

Excluding one report by Liu (1945) on

Batrachuperus pinchonii, little data on life history has

previously been available for species in this genus.

Limited notes on B. longdongensis Liu and Tian, 1978,

B. cochranae Liu, 1950 and B. yenyuanensis Liu, 1950

have also been published for populations in White-drag-

on-pool at Chin-ting (Jinding) of Mt. Omei, Pao-hsing-

hsien (Baoxing County), Yen-yuan-hsien (Yanyuan

County) and Tien-shui (Tianshui) and Sia-ho (Xiahe)

Counties of Gansu Province. Data from Fei et al. (2005)

are also useful for understanding the biology of these

native animals.

Here we have reported additional life history data

for a new species in the genus, Batrachuperus taibaien-

sis, one of the largest stream salamanders in China. The

tube-like egg case is longer and thicker than those in

other species, it is strongly coiled and contains more

(27-29) eggs. In comparison, the egg case of B. pin-

chonii is cylindrical and cayenne-shaped with 7-12 eggs

(Liu, 1945); the case of B. yenyuanensis is linear and has

6-13 eggs (Zhao and Yang, 1997). The larvae of this

species are also unique.

Acknowledgments

This research was supported by National Nature Science

Foundation of China (No:30470206) and Special Invited

Professor Grand of Shenyang Normal University to

Pipeng Li. We are grateful to Dr. Zeng Xiaomao sending

her unpublished Ph. D. dissertation, Prof. Wu G. F. and

Zhao H. for field help, and three anonymous reviewers

for their reviews and corrections of the manuscript.

Literature Cited

De Queiroz, A. and P. H. Wimberger. 1993. The useful-

ness of behavior for phylogeney estimation : levels

of homoplasy in behavioral and morphological

characters. Evolution , 47(1): 46-60.

Fei, L., C. Y. Ye., Y. Z. Huang., J. P. Jiang and F. Xie.

2005. An illustrated key to Chinese amphibians.

Sichuan Publishing Group, Sichuan Publishing

House of Science and Technology, Chengdu.

Frost, D. R. 2007. Amphibian species of the World. 5.0,

an Online Reference, http://research.amnh.org/her-

petology/amphibia/references.php?id-25385

Greene, H. W. 1993. What’s good about natural history.

Herpetological Natural History 1: 3.

Han, Y. P. and X. Y. Lu. 2003. Population Structure and

Conservation of Batrachuperus tibetanus in the

Tsinling Mountains. Chinese Journal of Zoology

38(2): 68-70.

Li, P. P. and R. S. Fang. 1993. Microstructural study of

skin and its gland on Bactrachuperus pichonii. Acta

Herpetologica Sinia, Guiyang 1-2: 15-18.

Liu, C. C. 1945. Natural history studies of West China

amphibian IX: Life history of Batrachuperus pin-

chonii (David). Journal of West China Border

Research Society 15(B): 44-55.

Mendelson, J. R., III., Ustach, P. C. and A. Nieto-

Montes de Oca. 1999. Description of the tadpole of

Bufo tutelarius, natural history notes on the Bufo

valiiceps group , and a key to the tadpoles of the

group. Journal of Herpetology 33 (2): 324-328.

Song, M. T. 1983. Batrachuperus pinchonii in Mt

Tsinling. Chinese Journal of Zoology 1 8(4): 1 3—

141.

Song, M. T., X. M. Zheng., G. F. Wu., Z. J. Liu and J. Z.

Fu. 2001. A new species of Batrachuperus from

Northwestern China. Asiatic Herpetological

Research 9: 6-8.

Xu, J. and J. C. Chen. 1992. A preliminary on the repro-

ductive ecology of the Batrachuperus tibetanus.

Chinese Journal of Zoology 27(5): 33-36.

Yuang, H. 1984. A herpetological survey in the

Huoditang forest of Tsinling Mountains. Acta

Herpetologica Sinica 2(1): 70-71.

Zhang, Y. H. and L. Z. Jia. 2002. Microstructure and

ultrastructure of vitellogenesis in oocytes of the

stream salamander ( Batrachuperus pinchonii). Acta

Zoologica Sinica 48(4): 534-542.

Zeng, X. M. 2004. The phylogenetic study of Western

hynobiids in China. Ph.D. Dissertation, East China

Normal University 2004 (2): 13 1-1 34.

Zhao, E. M and D. T. Yang. 1997. Amphibians and

Reptiles of The Hengduan Mountains region.

Beijing: Science Press.

Submitted: 19 September 2006

Accepted: 25 November 2007

pp. 80-84

Asiatic Herpetological Research, Vol. 1 1

2008 1

An Investigation of the Morphometric Characteristics of Eggs of the

Chinese Alligator (Alligator sinensis)

Wei Zhi Meng1, Xiao Bing Wu1’* and Lu Sheng Wu2

1 Anhui Province Key Laboratory for Conservation and Exploitation of Biological Resource, College

of Life Science , Anhui Normal University, 1 East Beijing Road, Wuhu 241000, China,

~ Division of Science and Technology, Jinan University, 601 Huangpu Avenue, Guangzhou 5 10632, China.

* Corresponding author E-mail: wuxb@mail.ahnu.edu.cn

Abstract.- In this study we analyzed morphometric intraspacific differences in the eggs of the Chinese alligator,

Alligator sinensis, (egg mass, egg width and egg length). Data were collected for 1460 eggs from 40 clutches at the

Anhui Research Center for Chinese Alligator Reproduction (ARCCAR). Our results found that the mean clutch size

was 36.5 eggs, mean egg mass per clutch was 41.9 g, and clutch mass was 1519.7 g. We generated three regression

equations relating to relationships among egg width, length and mass. The average of clutch size in 2004 was much

higher than it was in 1985 as indicated by a two-sample t-test.

Keywords.- Egg shape index, egg length, egg width, egg mass, clutch size.

Introduction

Studies of the reproductive ecology of Alligator sinensis

are important for our understanding of its conservation

biology and status. Several studies about the reproduc-

tive ecology of Alligator sinensis are available and

include information on egg incubation in captivity (Gu

and Zheng, 1983; Liang and Pan, 1990), nest excavation

(Huang and Watanabe, 1986), the relationships between

egg hatching and environmental factors (Wang et al.,

2000) and captive breeding (Cheng and Wang, 1984;

Wang and Zhang, 2000; Xu et al., 1989). Egg character-

istics are important when examining reproductive ecol-

ogy, as has been illustrated in previous studies (Cariello

et al., 2004; Du, 2003; Huang et al., 2003; Reese, 2000),

but little of this research has been devoted to Alligator

sinensis. We herein record data on egg characteristics of

the Chinese alligator in the ARCCAR, and analyze these

characteristics to investigate intraspecific differences,

providing fundamental information for further study of

the influence of egg shape on hatching rate and quality

of young alligators.

Materials and methods

Measurements of eggs - A total 1460 eggs from 40

clutches were collected between 5 and 16 July 2004 at

the artificial breeding area of the ARCCAR. Eggs were

collected within 12 hours of laying and taken to a hatch-

ing room where their length and width were measured

with digital calipers (precision 0.01 mm). Egg mass was

taken using a scale (precision 0.1 g).

Statistical analysis.- We analyzed the morphological

characteristics of the eggs using the statistical software

SPSS (Version 10.0). Data on the distribution of egg

characteristics (including egg length, width, mass, egg

shape index [length/width], clutch size, clutch mass and

clutch mean egg mass) were analyzed using descriptive

statistics. The coefficient of variation (SD/Mean) was

used to study variation in egg characteristics. Regression

analyses were used to examine the correlation among

egg width, length and mass. Hoyt (1979) put forward an

empirical formula:

W = KWXY2

where Kw is the coefficient of mass, W is egg mass, X is

egg length, and Y is egg width.

Results

Descriptive statistics of the morphological characteris-

tics of the eggs of the Chinese alligator.- Table 1 pro-

vides descriptive statistics for the morphological charac-

teristics of the eggs examined. From the frequency dis-

tributions of these morphological characteristics

(Fig. 1), it is apparent that in most cases, egg length

ranges from 51.42 mm to 60.00 mm, egg width from

32.49 mm to 37.51 mm, egg mass from 33.34 mm to

48.33 mm, clutch mass from 1200 g to 1800 g, clutch

size from 30 to 45, and clutch mean egg mass from 35.0

g to 47.5 g.

The egg of the Chinese alligator usually has the

form of an elongate ellipse, although some eggs deviated

from this shape. From the frequency distribution of ego

2008

Asiatic Herpetological Research, Vol. 1 1

81

Table 1. Descriptive statistics of the morphological characteristics of the eggs of Alligator sinensis.

shape index, it was evident that in most cases, egg shape

falls between 1.50 and 1.72.

Intraspecific difference of egg morphological charac-

teristics.- Since there were a high proportion of mal-

formed eggs in four of the 40 clutches, only the 36

remaining clutches were analyzed. Figure 2 shows the

CV (coefficient of variation) of egg morphological char-

acteristics, indicating morphological variation among

eggs of the same clutch.

There were differences in variation among the mor-

phological characteristics of the eggs laid by different

females. The CV of egg morphological characteristics

were calculated from Table 1 , revealing that variation in

egg mass (CV = 0.18) and clutch size (CV = 0.17) were

the greatest.

The correlations among the CV of egg morpholog-

ical characteristics were analyzed using a Pearson corre-

lation analysis (Table 2). Table 2 indicates that the CV of

the egg shape index had a significant positive correlation

with the CV of egg width. The CV of egg mass had a

significant positive correlation with the CV of egg

length.

Correlations of egg length, egg width and egg mass.-

Data (including egg length, width and mass) collected

from 1445 eggs (malformed and broken eggs not includ-

ed) were used to generate scatter plots. These plots indi-

cated that both egg width and length had a positive cor-

relation with mass, while length was negatively correlat-

Table 2. Pearson Correlations of CV of four egg morpho-

logical characteristics.

** Correlation is significant at p < 0.01 (2-tailed).

ed with width. In order to obtain the exact correlation

between the two parameters, the influence of other

parameters was eliminated by using a partial correlation

analysis. The results showed that egg length had a sig-

nificant positive linear relationship with egg mass

(r = 0.875, p < 0.00 1 ); egg width had a significant posi-

tive linear relationship with egg mass

(r = 0.856, p < 0.001); and egg width had a significant

negative linear relationship with egg length (r = -0.638,

p< 0.001).

Linear regression analysis was used to analyze the

relationships between egg width (Fig. 4), length, and

mass (Fig. 3). Two regression equations were generated

(1) W = -29.855 + 1.265 XL

R2 = 0.681, p < 0.001; df= 1443

(2) W = -53.078 + 2.699 Xw

R2= 0.637, p< 0.001; df= 1443.

Where W is egg mass (g), XL is egg length (mm), Xw is

egg width (mm).

We were able to approximate the original egg mass

from data about egg width and length using the two

equations.

In order to estimate egg mass (W) more exactly, we

used the empirical formula given by Hoyt (1979) to

derive another regression equation. From the scatter

plots of egg mass and the volume (V) of the cube

approximately the volume of the egg (egg width X egg

width X egg length), it is evident that egg mass had a sig-

nificant positively correlation with the volume of this

cube. Linear regression analysis (Fig. 5) yielded the fol-

lowing equation expressing the relationship between egg

mass and volume:

(3) W= 1.656 + 0.00057V

R2 = 0.928, p < 0.001; df= 1443,

where V = XLXW2, so the final equation is:

82

Asiatic Herpetological Research, Vol. 1 1

2008

(a) Egg length (mm)

(b) Egg width (mm)

(c) Egg mass (g) (d) Egg shape index

(e) Clutch mass (g) (f) Clutch mean egg mass (g)

Figure 1. Frequency distributions of egg morphological

characters of Alligator sinensis.

(4) W = 1 .656 + 0.00057XlXw2,

p < 0.05). The data collected in 1985 were from the orig-

inal parent generation captured from the wild, which is

no longer breeding, leaving the FI generation to consti-

tute the dominant portion of the breeding population

(Wu et al., 1999, 2005). Many reproductive characteris-

tics of squamate reptiles are fictile, clutch size being one

of them. In the wild, adult alligators must face pressures

relating to natural selection potentially reducing their

full reproductive potential, but in the ARCCAR, the

nutrition of the alligators is regulated by artificial diets,

maximizing their reproductive potential. One way to

quantify this potential is to examine egg morphological

characteristics, which are directly influenced by nutri-

tion.

Egg width and length data can be collected easily,

but data on egg mass are more difficult to obtain because

some eggs might break while the female alligator pro-

tects her clutch, or if it is usurped by other females.

Furthermore, there is variation in egg mass during incu-

bation (Wang and Zhang, 2000). In bird species exam-

ined in the wild, investigators have correlated egg

weight, width and length (Hoyt, 1979; Zhao and Ma,

1997; Zhou, 1994) in order to calculate egg mass. The

regression equations (1), (2), and (4) established in this

study have been found to accurately estimate the origi-

nal egg mass from data on egg width and length, provid-

ing an efficient means of measurement that can also be

applied to the mass of recently-hatched young alligators

if only the eggshell is available. This information subse-

quently be used to estimate the constitution of the young

alligators, gain basic information on wild populations,

and develop a sound basis for investigation of wild pop-

ulations. We conducted an analysis of variance test

(ANOVA) to determine whether the egg masses predict-

ed by the three equations differed from actual observa-

tions. The results showed no significant differences

(F = 0.891,/? > Poos), illustrating that all three equations

can be used to calculate egg mass, although the accuracy

of equation (3) (R2 = 0.928) is higher than that of the

other two [(1) (R2= 0.681); (2) (R2 = 0.637)].

where W is egg mass (g), XL is egg length (mm) and Xw

is egg width (mm).

Discussion

In 1985 it was recorded that the clutch size of Chinese

Alligators at the ARCCAR, based on 29 clutches, was

26.2 (SD = 3.9; n = 29) (Xu et al, 1989). Means of

clutch sizes in 1985 and 2004 were compared by a two-

sample t-test, revealing that the average clutch size in

2004 was much higher than it was in 1985 (t = 7.77,

0.60-

c

o

+5 0.50 H

2

> 0.40 H

■ Egg shape index

■ Egg length

□ Egg width

□ Egg mass

..... , -MMMWMUUy

Case Number

Figure 2. Coefficient of variation of egg morphological

characteristics.

2008

Asiatic Herpetological Research, Vol. 1 1

83

60.00-

3

^ 50.00-

</>

f3

E

240.00

G)

LU

30.00

45 .00 50 .00 55 .00 60 .00 65 .00 70 .00 75 .00

R Sq Linear = 0.681 '

Egg length (mm)

Figure 3. The relationship between egg mass and length.

Egg width (mm)

Figure 4. The relationship between egg mass and width.

Figure 3. The relationship between egg mass and length.

Though selection females produce progeny of a cer-

tain size that are able to escape natural enemies and

obtain food efficiently. Compared to progeny size, the

number of progeny should show larger variation within

a single brood (Lin and Ji, 2004). The results of this

study suggest that the CV of egg mass (0.12) is smaller

than the CV of clutch size (0.17), meaning that clutch

size in the Chinese alligator exhibits greater variation

than does egg mass.

Egg shape index can be used to describe the shape

of eggs. From Table 2 we conclude that the CV of egg

shape index has a significant positive correlation with

the CV of egg width. This suggests that, compared to

egg length, egg width has a greater influence on egg

shape. According to studies on the hatching rate of some

birds and reptiles (Fang et ah, 2004; Fang et al., 2001;

Zhu, 2002), egg shape had been found to be an impor-

tant variable in hatching rate, however, little research on

the relationship between egg shape and hatching rate has

been made, and is in need of further investigation.

Acknowledgments

This work was supported by National Natural Science

Foundation of China (NSFC, No. 30270213, 30470244),

the Foundation for Excellent Youth in Anhui Province

(04043409) and Key Laboratory of Biotic Environment

and Ecological safety in Anhui Province.

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effects of incubation thermal environments on

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the relationship of Chinese alligator egg hatching

and environmental factors], Sichuan Journal of

zoology 19(2): 82-83. (In Chinese)

Wang, R. P. and X. F. Zhang. 2000. [Studies on changes

in egg mass and embryonic metabolic rate of the

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temperatures]. Journal of Economic Animal 4(4):

49-54. (In Chinese)

Wu, X. B., Y. Q. Wang, K. Y. Zhou, J. S. Nie, C. L. Wang

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Submitted: 01 November 2006

Accepted: 22 April 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 85-92

A New Species of Rhacophorus (Anura: Ranidae) from China

Yun Ming Mo1, Jian Ping Jiang2’*, Feng Xie1 and Annemarie Ohler3

1 Natural History Museum ofGuangxi, Nanning 530012, China,

2Chengdu Institute of Biology, The Chinese Academy of Science, Chengdu 610041, China,

}Departement Evolution et Systematique Museum national d'Histoire naturelle, Paris 75005 France.

* Corresponding author E-mail: jiangjp@cib.ac.cn

Abstract.- A new brown tree frog species, genus Rhacophorus , is described on the basis of seven adult male specimens

collected from Cenwanglaoshan Nature Reserve, Guangxi, in southern China. This frog can be distinguished from all

other Asian Rhacophorus Kuhl and van Hasselt, 1822 by the combination of: skin brown and smooth; Y-shaped car-

tilage visible dorsally on tips of fingers and toes; outer fingers one-third webbed; distinct dermal ridges present on

forearms, above vent, and calcars present on heels; anterior and posterior surface of thighs tangerine in color without

distinct dark or light spots; tympanum distinct and large, about 6.6% of SVL; dorsum brown with wide dark cross-

shaped mark.

Keywords.- Amphibia, Rhacophoridae, Rhacophorus , new

Introduction

The genus Rhacophorus Kuhl and van Hasselt, 1 822, a

member of the family Rhacophoridae, contains approxi-

mately 60 species ( sensu stricto ) (Frost, 2007) that are

distributed in the tropical and temperate zones of East,

South, and Southeast Asia. Liem (1970) outlined a con-

servative definition of the genus (. Rhacophorus sensu

stricto ), and while some authors recognize the genus in

this sense (Jiang et al., 1987; Fei et al., 1990; Zhao and

Adler, 1993; Inger et al., 1999; Malkmus et al., 2002;

Frost, 2007; Frost et al., 2006), others have adopted a

broader definition ( Rhacophorus sensu lato) that

includes the genus Polypedates (Tian and Jiang, 1986;

Dubois, 1987, 1992; Fei, 1999; Fei et al., 2005). In a

recent review of the Rhacophrinae, a new generic classi-

fication was proposed for the Rhacophorinae (Delorme

et al., 2005), where some members of Rhacophorus

were transferred to the new genus Aquixalus. This clas-

sification was adopted by Frost (2007) and Frost et al.

(2006) with some modification. In view of the various

interpretations of the Rhacophoridae, the classification

of the treefrogs should still be considered unstable

(Wilkinson et al., 2002; Matsui and Orlov, 2004), and as

stressed by Frost et al. (2006), the boundaries of

Rhacophorus should be considered tentative.

All Chinese Rhacophorus {sensu lato ) have been

found in southern region north to Qinling, with most

species distributed in the tropical and subtropical

regions. Some new species were recently added to the

genus from the regions in and around southern China: R.

hainanus Zhao et al., 2005 from Hainan, R. minimus

Rao et al., 2006 from Guangxi, R. yinggelingensis Chou

et al., 2007 from Hainan, and R. jarujini Matsui and

species.

Panha, 2006 from eastern Thailand.

During the survey of the herpetofauna of

Cenwanglaoshan Nature Reserve, Guangxi, China, in

May 2004 and 2005, seven specimens of a small brown

Rhacophorus were collected (Fig. 1), which appeared to

be distinct from other congeners hitherto known from

China (Fei et al., 2005) and nearby countries (Bourret,

1942), including Vietnam (Inger et al., 1999; Orlov et

al., 2001), Thailand (Taylor, 1962) , Laos (Stuart, 1999),

India (Inger and Dutta, 1987), and Burma (Zug et al.,

2003). These specimens, which resemble some south-

eastern Asia members of the genus, are described below

as a new species.

Materials and Methods

Morphological data - The seven specimens included in

the new species were collected by hand in

Cenwanglaoshan Nature Reserve, Guangxi, China, in

May 2004 and 2005. Five were preserved in 10%

buffered formalin, and two were preserved in 70%

ethanol. Morphological information of related species

used for comparison was obtained from the literature

listed below.

Measurements - Sixteen body measurements were

made using dial callipers to the nearest 0.1 mm: SVL =

snout-vent length; HL = head length from tips of snout

to the commissure of the jaws; HW = head width at the

commissure of the jaws; SL = snout length from tip of

snout to the anterior comer of the eye; INS = intemarial

space; IOS = interorbital space, i.e., the smallest space

between the inner edge of upper eyelid; UEW = width of

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Asiatic Herpetological Research, Vol. 1 1

2008

O R. maxim us

0 R. translineatus

A R. verrucopus

■ R. bipunctcittis

&R. kio

▲ R. hounglienensis

* R. luoshan

Figure 1. Localities recorded for the six species belonging to the Rhacophorus reinwardtii group in China, with the

locality recorded for R. hoanglinensis in Vietnam.

upper eyelid; ED = diameter of eye; TD = horizontal

diameter of tympanum; LAHL = length of lower arm

and hand; HAL = hand length; HLL = hindlimb length;

TL = tibia length; FTL = length of foot and tarsus; FL =

foot length; and TFDD = third-finger disc transverse

diameter.

Analyses of advertisement calls - We recorded the

advertisement calls of this new species using a

Panasonic SV-MP21V recorder (parameter set as

22050Hz, 16 bit, monophone, wav file). Calls were ana-

lyzed using Cool Edit Pro V2.1 and BatSound V3.0.

Environmental parameters recorded during collection

(2010-2340 h) were as follows: air temperature 22°C;

moisture 92%.

Museum acronyms.- CIB, Chengdu Institute of

Biology, Chinese Academy of Sciences; GXNM,

Natural History Museum of Guangxi, Nanning, China.

Taxonomy

Rhacophorus laoshan, new species

Fig. 2

Holotype- Catalog number GXNM 2005081. Adult

male collected in Cenwanglaoshan Nature Reserve

(Guangxi, China) on 19 May 2005 (106° 24' 8.22” E,

24° 29' 1 .98” N) at 1389 m altitude by Yunming Mo.

Paratypes - Six adult males, all from the same site as the

holotype: CIB 2831k collected on 19 May 2004 by

Jianping Jiang, Annemarie Ohler, Yunming Mo, and

Feng Xie; GXNM2005079, GXNM2005082,

GXNM2005095, CIB2005080, and CIB2005094 col-

lected on 19-20 May 2005 by Yunming Mo.

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Asiatic Herpetological Research, Vol. 11

87

Figure 2. (A-D) Holotype of Rhacophorus laoshan sp. nov.; adult male GXNM2005081.

Diagnosis - This new species Rhacophorus laoshan is a

small brown treefrog (SVL 35. 1±1 .3 mm; n = 7) that can

be distinguished from all other Asian Rhacophoridae by

the combination of the following characters: skin brown

and smooth; Y-shaped cartilage visible dorsally on tips

of fingers and toes; outer two fingers one-third webbed;

dermal ridges present on forearm, above vent, and on

heel calcars; anterior and posterior surfaces of thigh tan-

gerine in color and without distinct dark or light spots;

tympanum large, about 6.6% of SVL; dorsum brown

with wide dark cross.

Description of holotype.- Body size small (SVL = 35.4

mm) and moderately elongate. Head moderately com-

pressed, dorsally flat, and wider (HW = 13.9 mm) than

long (HL = 13.8 mm). Snout bluntly pointed, projecting,

with length (SL = 6.4 mm) longer than horizontal diam-

eter of eye (ED = 4.6 mm). Canthus rostralis distinct

with loreal region slightly concave. Nostril oval and

closer to tip of snout than eye; interorbital space almost

flat, and larger (IOS = 4.5 mm) than upper eyelid (UEW

= 3.9 mm) and intemarial space (INS = 4.0 mm).

Tympanum rounded with diameter (TD = 2.3 mm) half

that of eye; tympanum-eye distance about half tympa-

num diameter. Supratympanic fold distinct, present from

posterior comer of eye to above and behind insertion of

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2008

arm. Vomerine ridges oblique, at an angle of 45° to body

axis, and closer to choanae than to each other. Vomerine

teeth on tongue pear-shaped, with deep notch on poste-

rior end.

Forearm plus hand about half snout-vent length,

with forearm (6.7 mm) shorter than hand (10.9 mm);

torearm with distinct dermal ridge from elbow to wrist

(Fig. 2); relative length of fingers: 3 > 4 > 2 > 1; fingers

with dermal fringes (Fig. 2); third and fourth fingers

with web on basal one-third; tips of all fingers with well-

developed disks with distinct circummarginal grooves;

discs relatively wide compared to fingers, except for

first finger; Y-shaped cartilage easily observable on

backs of fingers; subarticular tubercle developed; super-

numerary tubercles below base of fingers distinct; inner

metacarpal distinct, large, flat, and oval.

Hind limbs rather long, with tibiotarsal articulation

reaching middle of eye when leg stretched forward;

heels strongly overlapping when limbs folded at right

angles to body; tibia (TL 18.0 mm) about half SVL and

longer than thigh (12.8 mm) and foot (FL 14.9 mm); rel-

ative length of toes: 4>3 = 5>2> 1; tips of all toes with

moderately-sized disks (slightly smaller than those of

fingers except that of first finger); discs with distinct cir-

cummarginal grooves that are relatively wide compared

to toe width; Y-shaped cartilage easily observable on

backs of toes; web present, half developed, I 1 - 2 1/2 II 1

- 2 1/2 III 1 - 2 1/2 IV 2 - 1 V dermal fringe along toe V

distinct; subarticular tubercles prominent on all toes;

inner metatarsal tubercle distinct and oval; outer

metatarsal tubercle absent.

Dorsum smooth with granules scattered along sides

of body and head, along lower mandible, and back of

forearm; venter, head, and limbs covered with flat gran-

ules; outer edge of forearm and tarsus-metatarsus with

granulose ridge; dermal calcars present on heels; gran-

ules above vent forming transverse skin fold.

Color in life.- Dorsum chocolate brown; broad trans-

verse strip present medially on upper lids and interor-

bital space; back with broad cross-shaped marking (Fig.

2); limbs with broad transverse stripes: 2 on forearms,

2-3 on carpals and fingers, 3 on thighs and tibiae, and

4—5 on feet and toes; anterior and posterior surfaces of

thighs tangerine in colour and usually without distinct

dark or light spots; inner surface of tarsus and foot tan-

gerine; belly light gray-brown and without dark spots.

Male secondary sexual characters - Adult males with

nuptial pad on the base of first finger; internal subgular

vocal sacs present with two elongate openings; linea

masculina absent.

Variation.- Most variation was found in the appearance

of the wide cross-shaped mark on the dorsum. Usually,

this mark was visible except for when the dorsum was

more darkly-colored. The snout of some specimens was

green, and some specimens had a green spot on the

shoulder; ovate yellow spots on the sides of the body

sometimes formed a line.

Etymology - The species is named after the locality, i.e.,

Cenwanglaoshan Natural Preserve in northwestern

Guangxi, China, where it was found.

Measurements.- Sixteen body measurements are pro-

vided in Table 1 .

Habitat and ecological notes.- This new species was

found in a secondary broad-leaf forest with bamboo

undergrowth. Dense grass and deciduous leaves covered

the ground (Fig. 3). There were almost no perennial

streams in the region. During April and May, especially

at night after rainfall, males were heard calling loudly in

the forest, with six to nine calls making up a chorus. The

holotype and paratypes were found on branches and

leaves of trees one to three meters from the ground.

Advertisement calls.- The analyzed results of the calls

by Cool Edit Pro 2.1 and BatSound indicated that calls

were emitted every 17-25 seconds and lasted about

3.6-A.6 seconds. The calls had 19-26 notes (6 individu-

als, 12 calls) (Fig. 4a), with a note interval of

0.171-0.267 seconds (mean ± SD: 0.189±0.0184, 6 indi-

viduals, 12 calls, 44 notes). The dominant frequency was

2000 Hz and the second dominant frequency was 4000

Hz (Fig. 4b).

Comparisons.- The new species Rhacophorus laoshan

is most similar in appearance to R. hoanglienensis

(Orlov et al., 2001) and R. verrucopus Huang, 1983. It

species can be distinguished from R. hoanglienensis by

a combination of the following characters: (1) anterior

and posterior surfaces of the thighs are orange-red and

without distinct dark or light spots (black and white ver-

miculation is present in R. hoanglienensis ); (2) body

size smaller (SVL 35.1±1.3 mm; n = 7) than in R. hoan-

glienensis (SVL 43.2 mm; n = 1); (3) tympanum more

distinct and larger (TD = 2.3 mm, about 6.6% of SVL)

than that of R. hoanglienensis (TD = 2.1 mm, about

4.86% of SVL); (4) wide dark cross-shaped mark pres-

ent on dorsum; (5) venter light gray-brown and without

spots (not creamy-white with small dark spots that

merge in the distal parts of the fore and hind limbs).

Furthermore, the new species emits a call every 17-25

sec. (see above), while R. hoanglienensis calls every 3-5

min (Orlov et al., 2001), and white lines running from

the supratympanic fold to tip of snout through eyelid and

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Asiatic Herpetological Research, Vol. 1 1

89

Table 1 . Measurements (in mm) of Rhacopnorus laoshan sp. nov. Variations (mean ± SD) are shown for paratypes and

ratios to SVL (%). Abbreviations as used in text.

canthal ridge of female of R. hoanglienensis (Bain and

Truong, 2004). This new species can be distinguished

from R. verrucopus by the latter lacking vocal sac, head

longer than width, outer fingers 1/2 webbed, the dermal

ridge on forearm and above vent more weak, and the

dorsum without the wide dark cross mark present

(Huang, 1983). Rhacophorus laoshan can be separated

from related species that also have dermal flaps on the

forearms, tarsus, vent, or heel as follows. Rhacophorus

laoshan shows reduced toe webbing, which is more

extensive in R. annamensis, R. bipunctatus, R. exe-

chopygus, and R. reinwardtii (Inger et al., 1999); com-

plete webbing on the feet is seen in R. kio , R.

nigropalmatus, R. reinwardtii (Ohler and Delorme,

2006), R. maximus (Liu and Hu, 1961; Fei, 1999), R.

pardalis (Brown and Alcala, 1998; Inger, 1954, 1966;

Inger and Stuebing, 1997; Malkmus et ah, 2002), R.

prominanus (Taylor, 1962; Inger, 1966), and R. robin-

soni (Taylor, 1962; Inger, 1954) have. The absence of a

dark spot in temporal region distinguishes the new

species from R. cyanopunctatus (Malkmus et ah, 2002);

and R. bipunctatus (Inger et ah, 1999).

Rhacophorus baluensis can be separated from the

new species by being larger (male SVL = 50-55 mm)

(Malkmus et ah, 2002) and by having dark transverse

bars and irregular light or dark blotches on the dorsum

(Inger and Stuebing, 1997; Malkmus et ah, 2002).

Rhacophorus bimaculatus (Peters, 1867) is also larger in

size (SVL = 65 mm) and has fingers that are almost fully

webbed (Fei, 1999). Almost fully-webbed fingers are

also seen in R. dulitensis (Taylor, 1962; Inger and

Stuebing, 1997). Rhacophorus gauni can be separated

from the new species by having a conspicuous white

spot below the eye (Inger and Stuebing, 1997). The skin

of R. kajau is leafy green dorsally and usually has

minute white spots scattered on the back, head, and

exposed surface of the limbs (Inger and Stuebing, 1997;

Das, 2007). Rhacophorus rhodopus has more developed

finger and toe webbing, which is also red in color (Liu

and Hu, 1959; Fei, 1999), not grey-brown. Rhacophorus

translineatus has dark transverse bars on dorsum

(Sichuan Institute of Biology [Hu et ah, 1977; Fei,

1999]) while R. laoshan has a dark wide cross-shaped

mark.

The new species can be distinguished from

Rhacophorus appendiculatus by having irregular low

ridges on the back, as well as a narrow flap on the heel

(Inger and Stuebing, 1997; Malkmus et ah, 2002).

Rhacophorus calcaneus differs by having oval or round

dark brown spots on the dorsum, small enameled white

spots, or pinkish dorsolateral bands and a thin black net-

work enclosing large white spots ventrolaterally (Inger

et ah, 1999). Rhacophorus verrucosus has the vomerine

teeth absent (not present) and a low conical tubercle on

the heel (Inger et ah, 1999). Rhacophorus bisacculus has

no prominent projections in the infra-anal area, but it

may have several short, pointed tubercles (Inger et ah,

1999). Rhacophorus naso has a dorsolateral fold and a

skin projection on the tip of the snout (Fei, 1999).

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Asiatic Herpetological Research, Vol. 1 1

2008

Figure 3. Habitat of Rhacophorus laoshan sp. nov.

Remarks.- Those Rhacophorus with dermal flaps are

distributed in the tropical region of southeastern Asia,

extending north to the southern slopes of the eastern

Himalayas, the Hengduan Mountains, the Yunnan-

Guizhou Plateau, and the Nanling Mountains. Within

this area, most species are distributed north of 20° N. Of

these taxa, R. bipunctatus, R. maximus, R. kio, R. hoan-

glienensis, R. laoshan , R, translineatus, and R. verruco-

pus, are distributed along the southern border of China

(Fig. 1).

Rhacophorus bipunctatus (recorded as R. bimacu-

latus by Fei (1999) and Fei et al. (2005) in their list of

Chinese amphibians) has the widest Chinese distribution

of these seven species (Fig. 1), and is also known from

Northeastern India, Myanmar, Thailand, Laos and

Vietnam. In China this species occupies the tropical

region of the southern slopes of the Yunnan-Guizhou

Plateau, the Nanling Mountains, and southern Medog.

Rhacophorus maximus has and R. bipunctatus are

known from Yunnan and Tibet. The northern extent of R.

kio in China is the southern border regions of Yunnan

and Guangxi (Ohler and Delorme, 2006; Fei, 1999).

Rhacophorus translineatus and R. verrucopus are found

along the southern slope of the eastern Himalayas in

Medog, while R. hoanglienensis and R. laoshan are

known from the southern slope of the Yunnan-Guizhou

Plateau.

In summary, there are only five Rhacophorus

species with dermal flaps along the southern slopes of

the Yunnan-Guizhou Plateau and the Nanling

Mountains: R. maximus, R. kio , R. hoanglienensis, R.

laoshan, and R. bipunctatus. Of these, the new species

A.

0 1 2 3 4 5 6

Time (seconds)

Figure 4. Audio-spectrogram with FFT size 512 (A), and

power spectrum (B) of advertisement calls of the new

species Rhacophorus laoshan (Holotype)

R. laoshan is the most northern, R. bipunctatus the most

eastern and R. kio and R. hoanglienensis the most south-

ern.

Lastly, only four rhacophorids were recorded in

Cenwanglaoshan Nature Reserve (Mo and Xie, 2005),

including the new species, suggesting that further sur-

veys of the herpetofauna along the southern slopes of

Yunnan-Guizhou Plateau and the Nanling Mountains

should be carried out to further explore the little-known

biodiversity of this region. Such efforts may elucidate

more species or populations of Asian treefrogs or other

new species.

Acknowledgments

We are grateful to Prof. Fei Liang, Ye Changyaun (CIB),

and Prof. Masafumi Matsui (Kyoto University) and Dr.

R. Steven Wagner (Central Washington University) for

comments on the manuscript, Mr. Jian Li (CIB) for the

line drawing, and Mr. Zhiming Xie (GXNM) for field

assistance. This work was supported partially by NSFC

(No. 30000018, 30670245) and Life Science Special

Fund of Chinese Academy of Sciences (CAS) Supported

by the Ministry of Finance (STZ-01-19) to Jian-ping

Jiang.

2008

Asiatic Herpetological Research, Vol. 1 1

91

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C. F. B. Haddad, R. O. de Sa', A. Channing, M.

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Submitted: 28 November 2006

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 93-97

The Life History of Triturus vittatus vittatus (Urodela)

in Various Habitats

Oren Pearlson1’2’3 and Gad Degani1’2’*

{MIGAL-Galilee Technology Center, P.O. Box 831, Kiryat Shmona 11016, Israel,

2 School of Science and Technology, Tel ITai Academic College, Galilee, Israel,

3 Institute of Evolution, Faculty of Sciences and Science Education, University of Haifa, Israel.

* Corresponding author E-mail: gad@migal.co.il

Abstract.- The life cycle of Triturus v. vittatus at localities of various altitudes in Israel, ranging from 212 to 740 m

above sea level (ASL), were studied. Mature newts were observed only around winter rain pools, where they arrived

before the pools filled with water. The males left the ponds after spawning, while females left after eggs were

oviposited on plants and other substrata, according to the conditions of the ponds. Males (9-1 1 cm long, weighing

4. 3-5. 3 g) were slightly bigger than females (8.5-10 cm long, weighing 3. 1-4.3 g) Females laid 18-68 eggs each.

Fifteen to 30 days after oviposition, larvae hatched and from April to July, remained in the ponds to develop. Various

anuran larvae were found in the same breeding sites, including Hyla savignyi, Bufo viridis, Rana bedriagae and

Pelobates syriacus. Larval and adult Salamandra infraimmaculata were found to inhabit several of the rain pools

simultaneously, although the period during which both stages existed together was brief. Although temperature and

oxygen levels in the pools were not significantly different between breeding sites in the various habitats, development

took longer to complete at the more elevated sites.

Keywords.- Newt, larvae, Triturus v. vittatus , winter pool, Israel, life cycle.

Introduction

The life cycle and ecological conditions necessary for

the banded newt ( Triturus vittatus) have not been well

studied, although some aspects of the biology and life

cycle of the subspecies T. v. vittatus have been docu-

mented in Israel, Europe and the Mediterranean region

(Raxworthy, 1989; Olgun et al., 1997). Three subspecies

of the banded newt are currently recognized: T. v. vitta-

tus, found along the eastern edge of the Mediterranean,

ranging from Turkey to Israel; T. v. cilicensis, found in

areas bordering the east and northeast of the

Mediterranean; and T. v. ophryticus, located in the

Caucasus to the east and south of the Black Sea.

In Israel, Triturus v. vittatus is found from the north

to the central coastal plains, where conditions are most

extreme. The biology and life cycle of the populations in

northern Israel and Upper Galilee have been previously

described (Degani, 1986; Degani and Mendelssohn,

1983). Throughout their adult aquatic phase and larval

periods, T. v. vittatus mainly inhabits winter pools that

sometimes disappear at the beginning of summer

(Degani and Kaplan, 1999). The terrestrial adults reach

the pond at the beginning of the rainy season before the

ponds fill with water, and when the ponds are filled,

enter them for their aquatic phase.

Materials and Methods

In order to locate Triturus v. vittatus, all of the main

types of aquatic habitats inhabited by amphibian larvae

in northern Israel were investigated. The procedure fol-

lowed was that described by Degani and Kaplan (1999).

The various Triturus v. vittatus populations around

winter rain ponds and rock pools in northern Israel were

studied during four consecutive years (2001-2005) (Fig.

1). The elevations of these habitats ranged from 212 to

740 m ASL and represented a number of extreme eco-

logical and physical conditions. Water parameters were

measured every two weeks during the time the pools

were filled. In situ temperature and dissolved oxygen

data were obtained by a hand-held oxygen meter (WTW,

Oxi330 set, Germany). Water parameters were analyzed

by one-way analysis of variance (ANOVA), followed by

the Student-Newman-Keuls (SNK) test, for which

Graph-Pad Prism software (Graph Pad, San Diego, CA)

was used. The level of significance between groups was

set at p < 0.05.

Larvae were collected with a hand net (Degani and

Mendelssohn, 1983), identified to species and grouped

by specific water body.

94

Asiatic Herpetological Research, Vol. 1 1

2008

Golan

Heights

Mount

Hermon

LEBANON

T. v. cilisensis

T. v. vittatus

Naharia #

Mount

Meiron

Haifa

Lake

Kinneret

Mount

Carmel

Nazareth

SYRIA

Figure 1. Various ponds in Israel colonized with newts examined in the study.

Results

The life cycle of Triturus v. vittatus is presented in

Figure 2. Males and females arrived at the dried ponds

before the beginning of the heavy rains in October and

November and entered them when they were filled.

Male newts (9-11 cm long and weighing 4. 3-5. 3 g)

were found to be slightly bigger than females (8.5-10

cm long and weighing 3. 1-4.3 g), with no significant

difference in size detected for either sex between ponds

(Fig. 3).

After mating, the males left the ponds while the

females remained in the water to deposit between 18-68

eggs on plants or rock surfaces. The larvae hatched 19-

29 days later and remained in the rain pool for 30 to 75

days. Hatching time and duration spent in the pool was

dependent on water temperature (Fig. 4), with develop-

ment being slower at higher altitudes.

Various anuran larvae were found in the same

breeding sites, including Hyla savignyi, Bufo viridis,

Rana bedriagae, and Pelobates syriacus. Larval and

adult Salamandra infraimmaculata were found to inhab-

2008

Asiatic Herpetological Research, Vol. 1 1

95

Figure 2. Life cycle of T. v. vittatus: Terrestrial (A) and

aquatic females (B), eggs on plants in the pond few days

after spawning (C), larvae one day before hatching (D)

and 2 days after hatching (E), and developing larvae (F).

it several of the rain pools simultaneously, although the

period during which both stages existed together was

brief. The eggs and larvae developed in the ponds only

when temperatures rose above 1 8°C, the threshold tem-

perature also necessary for metamorphosis in S. infraim-

maculata.

From winter to spring temperatures rose from 5°C

to 30°C (Fig. 4). No significant differences in tempera-

ture were observed between the various ponds [p > 0.05,

F-value = 0.1766-1.186) during the periods when the

newt larvae were present, although temperatures in

Dovev pond were lower at the beginning of the growth

period during the years 2001-2002, 2003-2004 and

most of the growth period in 2004-2005. Dissolved oxy-

gen concentrations did not vary between sites {p > 0.05,

F-value = 0.3489-2.326), ranging between 2-27 mg/L;

concentrations stayed between 5-10 mg/L for most of

the growth period. High oxygen concentrations were

detected during the larval growth period and during

completion of metamorphosis (Fig. 4).

Discussion

Since newt larval development is dependent on an

aquatic habitat, the locations of breeding sites changed

from year to year depending on water availability. This

site flexibility is an important environmental adaptation

in a semi-arid country such as Israel. The data obtained

from the five different newt populations examined in

this study were consistent with those data obtained in

previous studies on the same subspecies in Upper

Galilee (Degani and Kaplan, 1999; Degani and

Mendelssohn, 1983) and the coastal plains of Israel

(Geffen et al., 1987), supporting observations that T. v.

vittatus is present in water in Israel between December

and April. Adults of T. v. ophryticus in northern Turkey

differ in that they are usually found in the water from

%

§

i-i

3

"S3

£

n Matityahu Q. pond a Dovev pond a Pharaa pond

m Amiad waterholes ^Nahalit pond

Figure 3. The measurements of mature females (N = 27) and males (N =15) from various populations.

96

Asiatic Herpetological Research, Vol. 1 1

2008

2001-2002

Temperature

2003-2004

T em perature

Oxygen

Amiad

Pharaa

Matityahu

Nahalit

Dovev

Q.

2004-2005

Temperature

Oxygen

■Amiad

■ Pharaa

Figure 4. Water temperatures and oxygen concentration of various breeding sites where T. v. vittatus newts were pres-

ent during winter and spring 2001 to 2005.

2008

Asiatic Herpetological Research, Vol. 11

97

early March to late Octoberor November, depending on

the climate and altitude (Kutrup, 2005b). We suggest

that the differences between the two subspecies are due

to regional climate differences.

Kutrup et al. (2005a) studied the food of the banded

newt, Triturus v. ophryticus, at different sites in Trabzon

in northern Turkey and discovered that the newts con-

sume a wide variety of invertebrates during their aquatic

phase. In Israel, the Salamandra infraimmaculata and T.

v. vittatus have a very similar diet, composed of various

invertebrates (Degani and Mendelssohn, 1978; Geffen et

al, 1987).

In summary, the present study examined the life

cycle of Triturus v. vittatus in northern Israel, which was

found to vary depending on the unpredictable presence

of rain pools necessary for juvenile development.

Among the different ponds, large variations were found

in the length of the larval growth period, as well as in the

time required for completion of metamorphosis. In con-

trast, no differences were observed in the ecological

parameters and water quality of the ponds during the lar-

val growth period.

Literature Cited

Degani, G. 1986. Growth and behavior of six species of

amphibian larvae in winter ponds in Israel.

Hydrobiologia. 140: 5-10.

Degani, G. and D. Kaplan. 1999. Distribution of

amphibian larvae in Israeli habitats with change-

able water availability. Hydrobiologia 405: 49-56.

Degani, G. and H. Mendelssohn. 1978. The food of

Salamandra salamandra (L.) tadpoles in Israel in

different habitats. Israel Journal of Ecology

C.19-C.45.

Kutrup, B., U. Bulbul and N. Yilmaz. 2005b. Age struc-

ture in two populations of Triturus vittatus ophryti-

cus at different altitudes. Amphibia-Reptilia 26:

49-54.

Olgun, K., V. Tok, J. Amtzen and O. Turkozan. 1997.

The taxonomic status of the banded newt ( Triturus

vittatus) in southern Turkey. The Herpetological

Journal 7: 169-171.

Raxworthy, C. 1989. Courtship, fighting and sexual

dimorphism of the banded newt, Triturus vittatus

ophryticus. Ethology 81: 148-170.

Degani, G. and H. Mendelssohn. 1983. The habitats,

distribution and life history of Triturus vittatus vit-

tatus (Jenyns) in the Mount Meron area (Upper

Galilee, Israel). British Journal of Herpetology 6:

317-319.

Geffen, E., S. Gafny and A. Gasith. 1987. Contribution

to the knowledge of the biology of the banded newt,

Triturus vittatus vittatus , in rainpools in Israel.

Israel Journal of Zoology 34: 213-223.

Kutrup, B., E. Akir and N. Yilmaz. 2005a. Food of the

banded newt, Triturus vittatus ophryticus

(Berthold, 1846), at different sites in Trabzon.

Turkish Journal of Zoology 29: 83-89.

Submitted: 08 Januaty 2007

Accepted: 22 September 2007

pp. 98-104

Asiatic Herpetological Research, Vol. 11

2008

Sexual Dimorphism and Female Reproduction in

Lacerta vivipara in Northeast China

Peng Liu, Wen Ge Zhao*, Zhi Tao Liu, Bing Jun Dong and Hui Chen

Department of Biology, Institute of Life and Environment Sciences,

Harbin Normal University, Harbin, 150025, China.

* Corresponding author E-mail: zhaowenge311@126.com

Abstract.- Lacerta vivipara is a small lacertid lizard that inhabits much of Europe and northern Asia. From the end

ot May to the beginning of October in 2003, these common lizards were collected from a population in Heilongjiang

Province (northeast China) in order to study sexual dimorphism and female reproductive traits. Through the exami-

nation of external morphological traits, such as snout-vent length, head length, head width, head height, tail length,

body weight, rows of ventral and mid-dorsals scales, ventral color, tail base and femoral pores, analyses revealed the

presence of a distinct sexual dimorphism. Males possessed a bulging tail base, a salmon-pink venter and a thorn in

the femoral pore. Females had significantly more rows of ventral scales and fewer mid-dorsal scales than males. Adult

males were larger in head size and had a longer tail, whereas adult females were larger in body size and weight. Male

juveniles and neonates were larger in head size than females of the same age and female neonates were larger in body

size than male neonates. The rates at which head length, head width and head height increased with increasing SVL

(snout-vent length) was allometric in females.

Females produced a single clutch every breeding season, with 3-12 young per clutch. While clutch size and

neonate mass were not positively correlated with maternal SVF, clutch mass was, suggesting that sexual dimorphism

in this species is due (in part) to differences in reproductive investment between the sexes. The larger head of males

is likely an adaptation for male-male combat while the larger relative body length of females is a result of selection

for higher fecundity.

Keywords.- Sexual dimorphism, female reproductive adaptations, allometry, Facertidae, Lacerta vivipara.

Introduction

Sexual dimorphism in body size, body shape, and col-

oration is widespread in many Chinese lizards, includ-

ing Takydromus septentrionalis, Sphenomorphus indi-

cus, Eremias argus, Gekko japonicus, Plestiodon ele-

gans , Plestiodon chinensis, Erendas brenchleyi,

Phrynocephalus vlangalii and Eremias multiocellata

(Du and Ji, 2001; Ji and Du, 2000; Fin and Ji, 2000; Fi

et al., 2006; Xu and Ji, 2003; Zhang et ah, 2005).

Previous studies strongly suggest that sexual dimor-

phism results from a balance between numerous selec-

tive pressures differing in influence between the sexes

(Shine, 1989; Schoener et ah, 1982; Vitt and Cooper,

1985). Consequently, various hypotheses have been pro-

posed to explain sexual dimorphism, including, female

choice in mate selection, male aggressive behavior

(Andersson, 1994; Cooper and Vitt, 1993), fecundity

selection (a selection leading to larger body-cavity size

in females) (Griffith, 1990), differential mortality due to

differences in longevity (Shine et ah, 2002), and food-

niche divergence (Fin and Ji, 2000). Because reproduc-

tive output is associated with numerous morphological

traits in lizards, data on female physiology and repro-

duction are crucial to understanding the origin of sexual

dimorphism in the group (Du and Ji, 2001; Ji and Du,

2000; Fin and Ji, 2000; Fi et ah, 2006; Zhang et ah,

2005).

The common lizard, Lacerta vivipara Jacquin,

1787, has the largest geographic range of any terrestrial

squamate reptile, extending across Eurasia from western

Europe to Japan. In China, it is found in Heilongjiang

Province, Xijiang Province and Inner Mongolia. It is a

small (approximately 4-5 g), diurnal, non-territorial

lizard typically found in open spaces surrounded by

pine-broadleaf mixed forest (Zhao et ah, 2006).

Due to both its abundance in nature and unique dual

oviparous and ovoviviparous reproductive modes,

Lacerta vivipara has been the focus of numerous mor-

phological studies (Guillaume, 2006; Smajda and

Majlath, 1999; Fecomte et ah, 1992; Wermuth, 1955).

Despite this abundance of detailed quantitative exami-

nation (Dong, et ah 2004; Fang and Tang, 1983; Zhao et

ah, 2006), however, details on Chinese populations of

the species and the relationship between its reproductive

ecology and morphometry remains poorly understood .

To examine the relationships between sexually dimor-

phic, morphometric traits in males and females (from

adults to neonates), and their relationships to offspring

2008

Asiatic Herpetological Research, Vol. 1 1

99

Figure 1. The tail base and ventral color of a male (top)

and female (bottom) Lacerta vivipara.

number and mass, we studied a population of L. vivipara

in Sunwu County, Heilongjiang Province, in northeast

China (49° 39' 19.2" N, 127° 34' 10.1" E; elevation 304

m). Morphological measurements were taken from

lizards collected in the field. Females gave birth to

young under simulated field conditions. Particular atten-

tion was paid to examining (1) sexual dimorphism in

ecologically important morphological traits and (2) the

relationship between female size and offspring size and

number. The results demonstrate that increased male

head size is an adaptation for combat and increased

female body length is an adaptation for higher fecundity.

Materials and Methods

Specimen collection and housing.- From the end of

May to the beginning of October in 2003, 183 lizards

(121 females, 62 males) were collected and analyzed. It

was assumed that the lizards were collected randomly,

thereby making the sample representative of the popula-

tion as a whole. Most of the males sampled were used

only for the collection of morphological data and were

released immediately following measurement; all

females were retained for subsequent analysis. The

retained lizards were transported to a nearby field station

and housed in a 7.5 x 1.8 x 1.0 (length x width x height)

m3 enclosure on the ground. The bottom of the enclosure

was covered with grass, branches and stones to simulate

the lizards’ natural habitat. Food (insects and spiders)

and water in small dishes were provided ad libitum. A

humid environment was maintained by spraying the sub-

strate with water daily. The lizards were marked by toe

clipping and back-painting.

Morphometry.- For each lizard collected, the following

five variables were measured with digital calipers to the

nearest 0.01 mm: snout-vent length (SVF; from the tip

Sid. Dev = 1.03

Mean = 23

N =62.00

o

<-

o

Zj

cr

o

s-

U.

24 25 26 27 28 29

Ventral s

Std Dev = 1.17

Mean = 26

N =121.00

Figure 2. Frequency distribution of rows of ventral scales

in males and females.

of the snout to the anterior margin of the cloacal lips);

head length (HF; from the tip of the snout to the poste-

rior margin of the skull); head width (HW; the largest

width of the head); head height (HH; the largest height

of the head ); tail length (TL; from the anterior margin of

the cloacal lips to the tip of the tail; specimens with

regenerated tails were excluded); body weight (BW),

number of ventral and mid-dorsals scale rows. Femoral

pores, venter color and tail base width were used to sex

individuals. Specimens with a SVL of 47 mm or more

were considered to be sexually mature adults (139 spec-

imens total); specimens with a SVF of 36-47 mm were

considered juveniles (30 specimens); specimens with a

SVF of less than 36 mm were considered neonates (14

specimens). .

Female reproduction.- Gravid females were separated

from each other in 30 x 25 x 25 (length x width x depth)

cm3 cages in order to accurately associate newborns with

their mothers. Enclosures were checked at least once a

day for neonates, which were immediately measured

and weighed after birth. Postpartum females were indi-

vidually weighed and measured for SVL. Clutches

Asiatic Herpetological Research, Vol. 1 1

2008

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2008

Asiatic Herpetological Research, Vol. 11

101

Snout-vent length (mm)

Figure 3. Linear regressions of head length, head width

and head height with SVL in Lacerta vivipara. The

regression equation is indicated in the figure. See text for

statistical analyses. Solid dots and lines: females; open

dots and dashed lines: males.

Snout-vent length (mm)

Figure 4. Linear regression of litter mass on female SVL

in Lacerta vivipara. The regression equation is indicated

in the figure. See text for statistical analyses.

including dead young, stillboms, or unfertilized eggs

were excluded from statistical analyses. Clutch mass

(RLM) was calculated by dividing litter mass by post-

partum female mass (Shine, 1992). Relative fecundity

was calculated by using the residuals derived from the

regression of litter size on maternal SVL (Olsson and

Shine, 1997).

Statistical analysis.- Whenever parametric statistics

were applied, a normal distribution was verified using

the Kolmogorov-Smimov test. Homoscedasticity was

verified using Levene's Test for Equality of Variances.

For significant departures from normality or

homoscedasticity, data were loge-transformed before

analysis. To test for sexual dimorphism in the data,

absolute values of morphometric measurements were

compared between sexes using a linear regression analy-

sis, one-way analysis of variance (ANOVA) and one-

way analysis of covariance (ANCOVA) with SVL as the

co variate.

All statistical analyses were performed using SPSS

(Statistical Package for the Social Science) vll.5 for

Windows. Homogeneity of slopes was checked prior to

testing for differences between adjusted means. Values

are presented as mean ± standard error and the signifi-

cance level in set at p < 0.05 for all statistical tests.

Table 2. Slope (b), intercept (a) and adjusted R square (r2) estimated from reduced major axis regressions for each

trait against SVL in female neonates, juveniles, and adults.

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 3. Descriptive statistics of female reproductive

traits and snout-vent length of Lacerta vivipara (n = 26).

Results

Sexual dimorphism.- In males, the base of the tail

bulged because of the presence of the hemipenes and the

venter of the tail was salmon pink. In females, the base

of the tail was slender and the venter had a saffron-yel-

low to off-white tint (Fig. 1). Femoral pores (8-12) were

small and black in females and neonates while it was

accompanied by a thorn in adult male. There were more

vertical scale rows in females (24—29) than in males

(21-25) (Mann- Whitney Test, Z = -10.377, p < 0.01;

Fig. 2), and males had more mid-dorsal scales

(31.63±1.46) than females (30.6 l=tl .48) (Mann-

Whitney Test, Z = -3.514 , /? < 0.01).

The largest male and female were 59.07 and 71.40

mm SVL, respectively. The mean SVL was larger in

adult females (58.69±5.44 mm) than in adult males

(51.86±3.13 mm) (t = -9.304, p < 0.001). Body weight

was greater in adult females (t = -3.710, p < 0.001) and

tail length was larger in adult males (^ = 2.519,/? < 0.01).

An ANCOVA test controlling for SVL found that adult

males had a larger head size (head length, head width

and head height) compared to adult females of the same

SVL (ANCOVA; HL, F = 140.145, p < 0.001; HW,

F= 48.800,/? < 0.001; HH, F= 50.035, P< 0.001). Head

length and head width were larger in juvenile males than

in juvenile females (ANCOVA; HL, F = 11.380,

p < 0.0 1 ; HW, F= 12.134,/? < 0.01) and head length was

larger in neonate males than in neonate females (ANCO-

VA; HL, F= 18.515,/? < 0.01). Body length was larger

in neonate females than in neonate males (t = -3.073,

p < 0.01) (Table 1).

The rates at which head length, head width, and

head height increased with increasing SVL were all

greater in males than in females (Fig. 3). Although the

rates of increase were the same in adult males as they

were for juvenile and neonate males (ANCOVA; HL,

F = 2.972, p = 0.059 > 0.05; HW, F = 0.476,

p = 0.624 > 0.05; HH, F = 5.091, p = 0.09 > 0.05), this

was not the case for females (ANCOVA; HL, F 8. 1 75,

p < 0.001; HW, F = 5.586, p = 0.005 < 0.01; HH,

F = 6.143, p = 0.03 < 0.05). In female neonates, head

length and width did not increase proportionally to SVL

(b < 0) and rate of head width was greater in female

juveniles than in female adults (b = 0.163 vs. b = 0.051)

(Table 2).

Female reproductive traits.- Female Lacerta vivipara

produced a single clutch of 3-12 young every breeding

season (Table 3). Clutch mass was positively correlated

with maternal SVL (r = 0.55, F = 5.43, p < 0.0 1 ; Fig. 4),

whereas clutch size (r = 0.38, F = 3.75, p = 0.06) and

neonate mass (r = 0.37, F = 3.38, p = 0.06) were not.

Neonate mass was independent of relative fecundity

(r = 0.15, F= 0.56,/?= 0.46).

Discussion

Consistent with previous studies of European popula-

tions of Lacerta vivipara (Gvozdik and Damme, 2003;

Kratochvil et al., 2003; Smajda and Majlath, 1999;

Wermuth, 1955), the present study found that sexual

dimorphism in head size, abdomen length, and tail

length was widespread in Chinese populations, suggest-

ing that these sexually dimorphic traits evolved a very

long time ago and has remained in the species as it dis-

persed across Asia. Lacerta vivipara is similar to other

lizards (e.g., Plestiodon laticeps, Plestiodon elegans,

Phrynocephalus vlangalii, Takydromus septentrionalis,

Tropidurus torquatus) (Du and Ji, 2001; Vitt and

Cooper, 1985; Zhang and Ji, 2000; Zhang et al., 2005) in

that the males have a larger head and longer tail while

females have a longer snout-vent length, increased body

weight a longer abdomen, and more rows of ventral

scales.

Sexual differences in head size are common within

the Lacertidae (Huang, 1998; Molina-Boija et al., 1998).

Since long periods of evolutionary time are often

required to manifest these differences (Kratochvil et al.,

2003), proximate environmental factors can be less

important determinants of sexual dimorphism in head

size than ultimate ones, such as phylogenetic history.

Sexual dimorphism may simply be the result of phylo-

genetic history and is maintained through competition

over mates (intra- and inter-sexual selection)

(Kratochvil et al., 2003; Shine, 1989).

According to most speculation, variations in allom-

etry in Lacerta vivipara are adaptive responses related to

differences in both the ecology and reproductive behav-

ior of the two sexes (Kratochvil et al., 2003). Although

it has been reported that a larger head is a male adapta-

tion to feeding on larger prey (Schoener et al., 1982),

there is little intersexual dietary divergence in L. vivipa-

2008

Asiatic Herpetological Research, Vol. 1 1

103

ra (Zhao et al., 2006). In contrast, the present study sup-

ports the conclusion that larger male heads are an adap-

tation for intersexual combat (Gvozdik and Damme,

2003). There is also evidence to support the possibility

that a longer male tail provides armament in combat and

improves the male’s ability to escape (Barbadilloo and

Bauwens, 1997; Barbadilloo et al., 1995; Brana, 1996;

Herrel et al., 2001). Color dimorphism is hormonal in

origin, becoming noticeable at the onset of sexual matu-

rity; this dimorphism apparently aids in sexual identifi-

cation and maintaining social hierarchy (Adriana, 2005).

Females have a considerably larger number of

transverse rows of scales covering the venter of the

abdomen and have a relatively large abdomen compared

to males of the same size. The present data showed that

maternal size is the main determinant of reproductive

output in Lacerta vivipara, with larger females produc-

ing heavier clutches. This offers strong evidence to sup-

port the hypothesis that selection for higher fecundity

results in the evolution of a longer trunk.

In other species of lizards such as Takydromus

septentrionalis, Podarcis muralis, Gekko japonicus,

Plestiodon chinensis and Sphenomorphus indicus, sexu-

al dimorphism in head size occurs at earlier ontogenetic

stages (Zhang et al., 2005). Our results reveal a similar

pattern in Lacerta vivipara, in that changes in allometry

vary at different ontogenetic stages between the sexes,

resulting in a distinct dimorphism. The neonates have

larger heads to obtain more foods to increase the trunk,

so that the sex individuals have no significant difference

in the size. With the growth of the body, the rate growth

of the head slows in female and head length and head

width is decreased, and quickly increased in juveniles

till the adults. Adult females of L. vivipara sacrifice head

and tail growth for increased abdomen (and body cavity)

length in order to achieve a greater reproductive output.

In conclusion, Lacerta vivipara exhibits a sexual dimor-

phism in size, color, and shape that can be linked to sex-

ual selection. In females, characteristics allowing for

higher reproductive output are selected for in females,

resulting in larger bodies and energy allocation directed

to early reproduction instead of growth. In males, char-

acteristics selecting for increased numbers of copula-

tions are selected for.

Acknowledgments

We are deeply indebted to Yu-guo Xia, Feng Zhao and

Zhi-gang Xie for collecting, housing and measuring

specimens in the field. Thanks also are given to Chun-

zhu Xu, Lin-lin Liu, Dian-wei Li and Zhi-ying Zhang

for dealing with the data. The Heilongjiang Provincial

Bureau of Education for scientific research funded this

work.

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Submitted: 31 October 2006

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 105-109

Sperm Morphology of Five Rhacophorus (Amphibia: Anura:

Rhacophoridae) Species from China

Li Mei Qin1’2, Zhong Hua Zheng1’*, Jian Ping Jiang1, Feng Xie1 and Yun Ming Mo3

1 Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, 61 0041, China,

2 Postgraduate university of Chinese academy of sciences, Beijing, 100049, China,

3 Natural History Museum of Guangxi, Nanning, 530023, China.

* Corresponding author E-mail: zhengzh@cih.ac.cn

Abstract.- Sperm shape and size of five species of the genus Rhacophorus from China were investigated in the present

study. Our results reveal the presence of two possible monophyletic lineages: the first is composed of R. chenfui, R.

dugritei and R. omeimontis, has relatively small spermatozoa with a with a coiled head and a thin tail, while the sec-

ond, composed of R. mutus and R. megacephalus, have spermatozoa that are longer with a straight head and filiform

shape.

Keywords.- Amphibian, Rhacophoridae, Rhacophorus , Polypedates, spermatozoa, morphology.

Introduction

The genus Rhacophorus {sensu lato) is a member of the

family Rhacophoridae and contains approximately 80

species worldwide, 24 of which are found in China (Fei

et al., 2005). Rhacophorids are predominantly arboreal,

sharing with basal ranids expanded digital pads and

mantellids the intercalary phalangeal elements (Frost et

al., 2006). These frogs are distributed in the tropical and

sub-tropical regions of eastern and southern Asia; in

China, they inhabit southern areas north to Qinling.

Phylogenetic and taxonomic relationships within this

genus are still conjectural and controversial. For

instance, the genus Polypedates is valid according to

Frost et al. (2007), but it is absent in Fei et al. (2005).

Furthermore, Frost et al. (2007) placed R. chenfui , R.

dugritei , R. omeimontis in Rhacohporus , while R. mutus

and R. megacephalus were placed in the genus

Polypedates by Fei et al. (2005).

Previous studies have revealed that some morpho-

logical characters of the spermatozoa were unique to

given taxa in the Anura (Kuramoto and Joshy, 2000;

Kuramoto, 1996; Zheng et al., 2000a, 2000b, 2002). For

example, the spermatozoa of most Rana species are

characterized by a cylindrical head and a thin tail, while

spiral and corkscrew-shaped sperm head and waved tail

are typical of spermatozoa in the family Megophryidae

(Zheng et al., 2002), and fusiform-shaped spermatozoa

are unique to the family Bombinatoridae (Zheng et al.,

2000a). These studies make it evident that sperm mor-

phology is not only variable between taxa, but can be

useful for elucidating taxonomic relationships

(Kuramoto and Joshy, 2000). In this study, the shape and

size of spermatozoa in Rhacophorus chenfui, R.

dugritei, R. omeimontis, R. megacephalus and R. mutus

were examined for the purpose of resolving their cryptic

phylogenetic relationships.

Materials and Methods

Collecting localities are listed in Table 1 . All specimens

were collected during their breeding seasons, (i.e., May

to July) from 1998 to 2005.

Frogs were euthanized by inserting a medical nee-

dle through the occipital ostium to destroy the spinal

cord. Next, the testes were removed and immediately

fixed with 10% formaldehyde, squashed and macerated

with a clean toothpick; sperm were suspended on slides,

air-dried and stained with acid carmine for 40 seconds.

Slides were examined on a ZEISS Axioplan2 light

microscope (LM). Other testes were fixed with 3% glu-

taraldehyde for about two hours and centrifuged at 3000

rpm for 30 s; the supernatant was discarded and the pel-

let rinsed with double distilled water. A drop of the

resulting sperm suspension was placed on a cover slip,

air-dried, coated with gold, and observed on a JEOL

JSM-5900LV scanning electron microscope (SEM).

Spermatozoan pictures were shot by ZEISS

AxioVision 4.0. Sperm length was measured by soft-

ware Arc View GIS 3.2. Sperm length data were ana-

lyzed with SPSS 11.5. The length of sperm head of

Rhacophorus chenfui , R. dugritei, R. omeimontis were

calculated by the formulate L=Nl7t (L: the length of

sperm head, tt = 3. 14, 1 = the diameter of helix, N = the

number of turns in the helix).

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Asiatic Herpetological Research, Vol. 1 1

2008

Figure 1. Spermatozoa of five tree frogs, a: Rhacophorus chenfui ; b: R. dugritei; c: R. omeimontis ; d: R. mutus ; e: R.

megacephalus; f: detail of the sperm head of R. mutus. A: acrosome; H: head; T: tail.

Figure 2. The total length of spermatozoa in different pop-

ulations of Rhacophorus mutus and R. megacephalus.

Units: pm.

Results

The spermatozoa of all five species consisted of two por-

tions, the head and the tail. The sperm head of

Rhacophorus chenfui (Fig. la) was shaped like a coiled

spiral, the tail was thin and wavy (shared with character-

istic shared with R. dugritei (Fig. lb) and R. omeimontis

(Fig. lc)), and the slightly-coiled apical section was

likely the acrosome. The shape of the sperm in R. mutus

(Fig. Id) was filiform, consisting of a straight, thick

head and a thin wavy tail. A thinner section (Fig. If, see

arrow) at the tip of sperm head was likely the acrosome

(Fig. If, a). Bends and waves could usually be observed

from the midpoint of the sperm.

Spermatozoa measurements are shown in Table 1.

Total sperm length in Rhacophorus chenfui and R.

dugritei is much shorter than those measured for R.

megacephalus and R. mutus; the sperm of R. omeimontis

was of intermediate length.

The sperm head of Rhacophorus dugritei was the

shortest and thinnest, and the head of R. omeimontis was

longer than that of R. dugritei , and thinner than those of

all other four species. Head length in R. chenfui was also

relatively short, and it was also slightly thicker than that

of R. dugritei. R. megacephalus had the largest sperm

head among all five species, with the head of R. mutus

being only slightly smaller.

The coiled head of Rhacophorus chenfui , R. omei-

montis and R. dugritei were largely similar. However,

the sperm head of R. chenfui was longer than the tail ,

with the ratio of sperm head to total sperm length being

1:0.86.

Sperm size of Rhacophorus megacephalus and R.

mutus differed remarkably between populations. The

spermatozoa of R. megacephalus in Sichuan population

were longer than those in the Guangxi population, and

those of R. mutus were longer than those in the Yunnan

population compared to the Guangxi population (Table

1; Fig. 2).

2008

Asiatic Herpetological Research, Vol. 1 1

107

Table 1 . Sperm measurements of Rhacophorus species.

Unit: pm. N: number of spermatozoa;

Discussion

Spermatozoa morphology.- The morphology of the

sperm observed here for Rhacophorus chenfui, R.

dugritei and R. omeimontis appeared to be very similar

to that reported by Mizuhira et al. (1986) for R. arboreus

and R. schlegelii, as well as that reported by Kuramoto

(1996) for R. viridis amamiensis, R. owstoni and R.

moltrechti. The sperm heads of latter species, however,

while coiled, did not appear to have the two twisted sub-

coils characteristic of the former three species.

Conversely, the shape of the spermatozoa in R. mutus

and R. megacephalus were very similar to those seen in

Polypedcites leucomystax, P. megacephalus (Kuramoto,

1996) and P. maculatus (Kuramoto and Joshy, 2000),

where the head is linear (not coiled) and no fibers in the

tail. Based on the variation of sperm morphology

observed here, the five species examined can be divided

into two groups: tthe first group (consisting of R. chen-

fui, R. dugritei and R. omeimontis) is characterized by a

helical sperm head and a thin sperm tail, and the second

group (consisting of R. mutus and R. megacephalus) is

characterized by a thread-like sperm head and a thin tail.

Sperm size differ remarkably between species, also

between populations. The average length of spermato-

zoa in Rhacophorus chenfui was 123.45±8.60pm, while

that of R. megacephalus was 230. 25±1 9.81 pm, a differ-

ence of statistical significance (P < 0.01, n = 20, one-

way analysis of variance). In the different populations of

R. megacephalus , total spermatozoa length in the

Sichuan, Yunnan, Guangxi and Taipei populations was

241.16 pm, 230.65 pm, 222.04 pm, 213.1 pm

(Kuramoto, 1996), respectively, illustrating a reduction

in length towards the Pacific Ocean (Fig. 3). This phe-

nomenon was also observed in R. mutus (average length

228.84 pm in the Yunnan population and 202.9 pm in

the Guangxi population).

The implications for taxonomy from sperm atological

data.- On the basis of skeletal characters, Liem (1970)

placed Rhacophorus dugritei and R. omeimontis into the

genus Polypedates. Uncertain as to the limits of these

two genera, Fei (1999) and Fei et al. (2005) tentatively

treated all Polypedates as Rhacophorus. Based on

molecular evidence, however. Frost et al. (2007) placed

R. chenfui, R. dugritei and R. omeimontis in

Rhacohporus, and placed R. mutus and R. megacephalus

in Polypedates. In view of the spermatological data pre-

sented here, the latter hypothesis presented by Frost et

al. (2007) appears to be best supported.

108

Asiatic Herpetological Research, Vol. 1 1

2008

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Figure 3. Map of collection localities.

Acknowledgments

This research was funded by grants from National

Natural Science Foundation of China, No.30570195

Literature Cited

Fei L. 1999. Pp. 161-175. In: Atlas of amphibians of

China. Henan publishing house of science and tech-

nology, Zhengzhou. (In Chinese).

Fei, L., C. Y. Ye, J. P. Jiang, F. Xie and Y. Z. Huang.

2005. Pp. 169-175. In: [An illustrated key to

Chinese amphibians]. Sichuan publishing group,

Sichuan publishing house of science and technolo-

gy, Chengdu. (In Chinese)

Frost, D. R., T. Grant, J. Faivovich, R. H. Bain, A. Haas,

C. B. Haddad, R.O. De Sa, A. Channing, M.

Wilkingson, S. C. Donnellan, C. J. Raxworthy, J. A.

Campbell, B. L. Blotto, P. Moler, P. C. Drewes, R.

A. Nussbaum, J. D. Lynch, D. M. Green, and W.C.

Wheeler 2006. Pp. 243-247. in: The amphibian tree

of life. American Museum of Natural History,

Library-Scientific publication, New York.

Kuramoto, M. 1996. Generic differentiation of sperm

morphology in tree frogs from Japan and Taiwan.

Journal of Herpetology 30(3): 437^143.

Kuramoto, M. and S. H. Joshy. 2000. Sperm morpholo-

gy of some Indian frogs as revealed by SEM.

Current Herpetology 19: 63-70.

Liem, S. S. 1970. The morphology, systematics, and

evolution of the Old World treefrogs

(Rhacophoridae and Hyperoliidae). Fieldiana:

Zoology 57: 1-145.

Mizuhira, V., Y. Futaesaku, M. Ono, M. Ueno, J.

Yokofujita and T. Oka. 1986. The fine structure of

the spermatozoa of two species of Rhacophorus

( arboreus , schlegelii ). I. Phase-contrast microscope,

scanning electron microscope, and cytochemical

observations of the head piece. Journal of

Ultrastructure and Molecular Research 96: 41-53.

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Zheng, Z. H, L. Fei, and C. Y. Ye. 2002a. [Study of the

morphology of spermatozoa of the genus Bombina ].

Cultum Herpetologica Sinica 8: 222-227 . (In

Chinese)

Zheng, Z. H., L. Fei, and C. Y. Ye. 2000b. [Study on the

morphology of the spermatozoa of Megophrys

(Amphibia: Pelobatidae) from China]. Journal of

Applied and Environmental Biology 6(2): 161-165.

(In Chinese)

Zheng, Z. H., L. Fei, C.Y. Ye, F. Xie and J. P. Jiang.

2002. [Comparative study of the sperm morphology

of Chinese Megophryinae and its taxonomical

implications (Amphibia: Pelobatidae)]. Acta

Zootaxonomica Sinica 27(1): 167-172. (In

Chinese)

Submitted: 07 December 2006

Accepted: 13 March 2007

pp.110-131

Asiatic Herpetological Research, Vol. 1 1

2008

The Herpetofauna of Nallamala Hills, Eastern Ghats, India: An

Annotated Checklist, With Remarks on Nomenclature, Taxonomy,

Habitat Use, Adaptive Types and Biogeography

C. Srinivasulu1 and Indraneil Das2’*

1 Wildlife Biology Section, Department of Zoology,

Osmania University, Hyderabad 500 007, Andhra Pradesh, India,

2 Institute of Biodiversity and Environmental Conservation,

Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia.

* Corresponding author E-mail: idas@ibec.unimas.my; hamadryad2004@hotmail.com

Abstract- We present an inventory of the herpetofauna of the Nallamala Hills, Eastern Ghats, south-eastern India. The

fauna, as currently known, includes 20 species of amphibians belonging to 14 genera in six families and 64 species

of reptiles belonging to 42 genera in 15 families. Divided in habitat types, the herpetofauna can be classified into

species tolerant of disturbed habitats; exclusively scrub species (and for reptiles, from rocky biotopes); scrub and bor-

dering agricultural fields; and exclusively mesic forest species. For one species, lack of ecological information pre-

cludes its allocation to a specific habitat category. Significant diversity of squamates (including gekkonids, scincids,

and colubrids) are known from these ranges, several of which endemic or largely restricted to scrub forests of

Peninsular India. Mesic forests remain poorly explored, and support hitherto undescribed species among the herpeto-

fauna. Adaptations seen amongst the herpetofauna of the Nallamala Hills include a diversity of dietary and habitat

types, including, among amphibians, ant specialists; predators of small vertebrates; folivores; fossorial; terrestrial;

aquatic or aquatic-margin; and arboreal forms. Amongst reptiles, adaptive types includes ant- and worm-eaters; pred-

ator of crop pests; predator of small or medium-sized vertebrate prey; egg-predators; fish-eaters; frog- and toad-

eaters; and one near-exclusive snake-eater. In terms of habitat usage, reptiles exceed amphibians in species richness,

on account of their greater capacity of surviving in relatively arid regions.

The Eastern Ghats contributes significantly to both species richness and endemicity of the Indian region, includ-

ing representatives of endemic genera and species. Nonetheless, these hills continue to receive less attention for con-

servation compared to the relatively better-known Western Ghats.

Keywords.- Amphibians, reptiles, biodiversity, ecology, Nallamala Hills, Eastern Ghats, India.

Introduction

Nallamala Hills (14° 26' - 16° 31' N and 78° 30' - 80° 10'

E) are a group of low hill ranges with an average altitude

of ca. 500 m in the central Eastern Ghats complex in the

state of Andhra Pradesh, south-eastern India (Fig. 1).

From the Palnad Basin in the north to the Tirupati Basin

in the south, the Nallamala Hills runs for a distance of

ca. 430 km with an average width of 30 km (Anon,

1965; Srinivasulu and Nagulu, 2002). An unbroken

chain of rugged hills with precipitous cliffs covering an

area of ca. 7,640 km2, it encompasses six districts

(Nalgonda, Mahbubnagar, Kurnool, Cuddapah,

Prakasam and Guntur) in Andhra Pradesh State.

Running parallel to it in the south-eastern side is the

Balapalli and Palakonda Ranges, while on the western

side, towards the north, are the Erramala Range. The

vegetation is typically of the southern tropical dry decid-

uous and southern tropical moist deciduous forest types

intermingled with scrub (Champion and Seth, 1968),

although the Nallamalas show representatives of many

vegetation types known from the Eastern Ghats, includ-

ing dry deciduous, moist deciduous, dry evergreen,

riverine and scrub forest (see R. K. Rao, 1998; R. S.

Rao, 1998). Dry deciduous forests are dominant.

Common species found here include Antidesma acidum,

Canthium parviflorum , Cerisoides turgida, Cissus palli-

da, Cochlospermum religiosum , Colebrookea oppositi-

folia, Dalbergia lanceolaris , Dalbergia paniculatum,

Diospyros melanoxylon, Ehretai laevis, Lagerstroemia

parviflora, Pterocarpus marsupium, Syzygium alterni-

folium, and Tamilnadia uliginosa. A forest type with

Boswellia serrata and Chloroxylon swietenia as the

dominant species occurs near Chalama, a Terminalia

coriacea and Anogeisus latifolia type occur in eastern

Nallamala, a Phoenix type with Phoenix loureivie as the

dominant species forming a pure stand on rocky substra-

ta occur between Ramannapenta and Gundla

Brahmeshwaram Metta Wildlife Sanctuary (GBM).

Moist deciduous forests are restricted to sheltered sites

with high rainfall such as GBM, upper Ahobilam and

Iskagundam; common species include: Careya arborea ,

2008

Asiatic Herpetological Research, Vol. 1 1

17

16

- 15

14'

78'

79

80°

Figure 1. Maps showing the location of the Nallamala Hills, Andhra Pradesh, south-eastern India. On top left, map of

India, showing Andhra Pradesh State; on bottom left, map of Andhra Pradesh, showing location of Nallamala Hills; and

on right, the Nallamala Hills, with localities mentioned in the text.

Dillenia pentagyna. Ficus hispida, Barleria strigosa,

Adiantum lunulatum , Oroxylona indicum , Trema orien-

talis and Pimpinella wallichiana. The Nallamalas are

home to many endemic species of Eastern Ghats includ-

ing: Andrographis nallamalayana, Ericaulon lushing-

tonii, Dicliptera beddomei , Premna hamiltonii,

Euphorbia linearifolia var. nallamalayana ,

Rostellularia vahlii, Andrographis beddomei,

Rostellularia vahlii var. rupicola, Boswellia ovalifoliata,

Cycas beddomei, Chaemaesyce linearifolia,

Chaemaesyce senguptae, Crotalaria madurensis,

Cro talar ia paniculata nagarjunakondensis, Indigofer a

barberi, Pterocarpus santalinus, Albizia sikharamensis ,

and Eriolaena lushingtonii. Pterocarpus marsupium and

Cycas beddomii are well known endemics. Another

interesting feature of the flora is the exhibition of gigan-

tism as exemplified by the shrubby climber Marsdenia

tenacissima, the leaves of which measure up to 32 cm.

Other climbers such as Bauhinia vahlii and Enteda

pursetha are dominant over other vegetation. The cli-

mate is generally hot and dry with temperatures rising

up to 43-45°C during May and dropping to 8-12°C in

December. The Nallamala Hills receive on an average

900-1,000 mm rainfall annually.

The Nallamala Hill Range has been conveniently

divided into three zones (Fig. 1): i.) the Northern

Nallamala Hills (the expanse of hill ranges between the

Palnad Basin and the River Krishna that flows approxi-

mately 130 km through the hills); ii.) the Central

Nallamala Hills (the expanse of hill ranges between the

River Krishna and the British railway track between

Nandyal and Guntur passing through Chalama, Bogada.

and Diguvametta); and iii.) the Southern Nallamala Hills

(the expanse of hills between the British railway track

and the Tirupati basin near Rajampet (14 1 T N and 79°

10’ E). Two contiguous protected areas, the

Nagarjunasagar Srisailam Tiger Reserve and the Gundla

Brahmeshwaram Metta Wildlife Sanctuary (with a col-

lective area of 4,762 km2) have been set aside to con-

serve the rich biodiversity of this tract.

The first of the faunal surveys conducted in the

Nallamala Hills dates back to 1930 when the ornitholo-

gist, Salim Ali (1896-1987), of the Bombay Natural

History Society, collected bird specimens from

Mannanur and Farahabad on the Amrabad Plateau in the

Northern Nallamala Hills during the Hyderabad State

Ornithological Survey (see Lozupone et al., 2004, for a

gazetteer of localities; Srinivasulu and Nagulu, 2002).

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Asiatic Herpetological Research, Vol. 1 1

2008

Subsequently, the Zoological Survey of India conducted

two faunistic surveys to collect vertebrate fauna in the

vicinity of the area in the Northern Nallamala Hills that

was to be submerged due to the construction of the

Nagarjunasagar Dam on River Krishna. Between 1980

to present date many surveys and other studies (Agrawal

and Bhattacharyya, 1976; Bhushan 1986, 1994;Murthy,

1968, 1986; Nagulu et al., 1998; Rao et ah, 1997; Rao et

al. 1999; Rao et ah, 2005; Rao et al., 2004a, b,c,d; Reddy

et al., 2004; Sharma, 1969, 1971, 1976; Srinivasulu and

Nagulu, 2002; Srinivasulu and Rao, 1999; Srinivasulu

and Rao, 2000; Srinivasulu, 2001b, 2002, 2003) have

been conducted documenting the faunal elements found

in the Nallamala Hills.

History of Herpetofaunal Studies

The earliest zoological collections from these hill ranges

were made by Thomas Claverhill Jerdon (1811-1872), a

member of the Asiatic Society, and also an important

contributor to mammalogy, ornithology, and herpetol-

ogy (see Das, 2004, for a brief biographic account).

Jerdon’s papers were published in the Journal of the

Asiatic Society of Bengal. As Civil Surgeon of Nellore

in 1842, Jerdon collected extensively in the then poorly-

known region between Madras and Nellore, discovering

many novelties amongst the vertebrate fauna, and most

famously, the Jerdon's courser, Rhinoptilus bitorquatus

(see an account in Bhushan, 2003). As a result of his col-

lections during the time, the following now familiar her-

petological species were described by Jerdon himself:

Microhyla rubra (Jerdon, 1854), Hoplobatrachus cras-

sus (Jerdon, 1854), Hemidactylus subtriedrus Jerdon,

1853, and Oligodon taeniolatus (Jerdon, 1853).

The Zoological Survey of India undertook the first

herpetological survey of the Nallamala Hills, between

1962 and 1963 (reported by Murthy, 1968; Sharma,

1969; 1971; 1976). As part of the Eastern Ghats

Herpetological Survey, Dr. Hem Singh Pruthi (7-1953),

Plant Protection Adviser to the Government of India and

entomologist with the ZSI (see Lai, 1954, for an obitu-

ary), collected herpetofauna from the Nallamala Hills in

1929 which were identified by Dr. Malcolm Arthur

Smith. Under the State Faunal Diversity Documentation

Project, initiated by the Survey, additional specimens

were collected from localities in the Nallamala Hills

(Murthy, 1986; Sanyal et al., 1993; Sarkar et al., 1993).

The first author of the present report made observa-

tions on the herpetofauna of Northern and Central

Nallamala Hills between late 1995 and early 2000. A

research team from the Department of Zoology,

Osmania University, Hyderabad, also documented the

herpetofaunal diversity while executing an Andhra

Pradesh Forest Department-sponsored project on the

effects of man-made barriers on wildlife in Gundla

Brahmeshwaram Metta Wildlife Sanctuary in the

Central Nallamala Hills, between 1998 and 2000.

Observations on the herpetofaunal diversity made dur-

ing these two studies between 1995 and 2000 have been

listed in an unpublished document (Srinivasulu, 2001a).

The Andhra Pradesh Forest Department, in collaboration

with Department of Botany, Sri Krishnadevaraya

University, Anantapur (for flora) and Department of

Zoology, Osmania University, Hyderabad (for fauna)

initiated All Taxa Biodiversity Inventorization Project in

2001 (Rao et al., 2004e) in which the first author was

involved. Voucher specimens of amphibians and reptiles

collected during this project have been deposited in the

State Forest Department’s Eco-Resource Monitoring

Lab, located in Sunnipenta, Kumool District. Between 3

- 16 June 2003, CS along with a research scholar from

Zoological Survey of India, Hyderabad, and other vol-

unteers visited Nagarjunasagar Srisailam Tiger Reserve

to study the voucher specimens of the herpetofauna in

Eco-Resource Monitoring Lab, Sunnipenta and collect

fresh voucher specimens to be deposited in the National

Zoological Collection housed at the Freshwater

Biological Station of the Zoological Survey of India,

Hyderabad, India (Srinivasulu et al., 2006).

Materials and Methods

Literature review and faunistic surveys by the first

author for acquiring voucher specimens, both for the

Andhra Pradesh Forest Department (January 2001 to

June 2003) and the Zoological Survey of India (June

2003), and records of observations made by the first

author between December 1995 to December 2004 in

the Nallamala Hills form the basis of the diversity of

herpetofauna reported here. Voucher specimens collect-

ed following techniques detailed in Heyer et al. (1994),

including collections along 100-200 m transects and

sampling within 50 sq m quadrats, at elevations between

150-570 m ASL. Vegetation in the area of sampling is

dry deciduous and scrub forest types. Several moist

decidous forest patches were also surveyed, including

along seasonal streams, particularly for amphibians.

Specimens were preserved and were deposited both at

the State Forest Department Collection housed at ERM

Labs, Sunnipenta, Kumool District and the National

Zoological Collection at the Freshwater Biological

Station, Zoological Survey of India, Hyderabad. Certain

large-growing (and threatened/protected) species con-

sidered easily-identifiable in the field (e.g., Crocodylus

palustris and Python molurus) were not collected.

Photographic vouchers were deposited in the Natural

History Museum at the Department of Zoology,

Osmania University, Hyderabad. All records from the

southern Nallamala Hills are based on sight records.

2008

Asiatic Herpetological Research, Vol. 1 1

113

The annotated lists of amphibians and reptiles pro-

vided below include information on their distribution in

the Nallamala Hills, their habitat and qualitative impres-

sions of abundance. Details of the vouchers are also pro-

vided. It the voucher specimen/s and/or photographic

voucher are present, they are indicated by [S] or [P] fol-

lowed by abbreviation of the place where housed.

Abbreviations used include: ZSIK (National Zoological

Collection, Zoological Survey of India, Kolkata), ZSIH

(National Zoological Collection, Freshwater Biological

Station, Zoological Survey of India, Hyderabad), ERM

(Eco-Resource Monitoring Lab, Andhra Pradesh Forest

Department, Sunnipenta), and NHMOU (Natural

History Museum, Department of Zoology, Osmania

University, Hyderabad). Nomenclatural remarks con-

cerning species are for those names that are different

from that generally prevailing in the literature in Indian

herpetology, especially the Fauna of British India vol-

umes by Smith (1931-43).

Results

The herpetofauna of the Nallamala Hills, as currently

known, includes 20 species of amphibians belonging to

12 genera in four families and 64 species of reptiles

belonging to 42 genera in 1 5 families. Recently, Rao et

al. (2005) published an account of herpetofauna of the

Nallamala Hills putting on record about 66 species of

herpetofauna (including 1 8 species of amphibians in 1 1

genera in 4 families and 48 species of reptiles in 34 gen-

era in 12 families) based on collections made from 16

locations between 15° 35'N (Isukagundam) to 16° 37' N

(Vijayapuri) and 78° 39'E (Saileshwaram) to 79° 17' E

(Vijayapuri) between November 2001 and September

2004. Rao et al.’s (2005) paper suffers from numerous

problems (including misidentifications and erroneous

nomenclature, in addition to dubious first record

claims), and grossly under-represents the herpetofauna

of the region, while ignoring to emphasize the endemic

reptiles of the Nagaijunasagar area.

Of the herpetofaunal species listed in this work,

voucher specimens of 76 species are either at the

National Zoological Collection of the Zoological Survey

of India, at Kolkata (48 species) and Hyderabad (20

species) or in the Eco-Resource Monitoring Laboratory,

Sunnipenta (62 species). Vouchers of 1 1 species are at

Kolkata, and 14 are at Sunnipenta. Eight taxa listed in

this report are either based on literature reports or on

sightings.

Annotated List of Amphibians

Order: Anura

Family: Bufonidae

1. Bufo stomaticus Liitken, 1862

Bufo stomaticus C. F. Liitken. 1862. Vidensk. Meddr.

Danske Naturh. Foren. 1862: 305.

Northern (Sarkar et al., 1993; Srinivasulu et al., 2006),

Central (Srinivasulu et al., 2006) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (A1844, Al 141), ZSIH

(ZSI/FBS/N/1 148, 1150-1153).

2. Bufo scaber Schneider, 1799

Bufo scaber J. G. Schneider. 1799. Hist. Nat. Amph.:

222.

Northern (Srinivasulu et al., 2006), Central (Rao et al.,

2005; Srinivasulu et al., 2006) and Southern Nallamala

Hills. Scrub, open forests and near agricultural fields.

Uncommon. [S] ERM (ERMA-5a); [P] NHMOU

(NHMOU. Amph. P. 1-03).

Remarks: First record claim from the region by Rao et

al. (2005) is erroneous as it has been already reported

from the Nallamala Hills by Subba Rao et al. (1994).

Dubois and Ohler (1999) showed that Bufo scaber

Schneider, 1799 has priority over Bufo fergusonii

Boulenger, 1892.

3. Duttaphrynus melanostictus (Schneider, 1799):

Bufo melanostictus J. G. Schneider. 1799. Hist. Nat.

Amph.: 216.

Northern (Rao et al., 2005; Sarkar et al., 1993;

Srinivasulu et al., 2006), Central (Rao et al., 2005;

Sarkar et al., 1993; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Abundant. [S] ZSIK (A 1144, A1145,

Al 845, A1997-98, A8373-75, A8377-81), ERM

(ERMA-1 a).

Family: Dicroglossidae

4. Euphylyctis cyanophlyctis (Schneider, 1799)

Rana cyanophlyctis J. G. Schneider. 1799. Hist. Nat.

Amph.: 137.

Northern (Rao et al., 2005; Sarkar et al., 1993;

Srinivasulu et al., 2006), Central (Rao et al., 2005;

Sarkar et al., 1993; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Abundant. [S] ZSIK (A6941, A6943,

A 1948, A8424-26, Al 108-13, A1130, A1989, A7810-

11, A6947, Al 131-32, A1830-34, A1838, A1941-47),

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Asiatic Herpetological Research, Vol. 1 1

2008

ERM (ERMA-3a); [P] NHMOU (NHMOU.Amph.P.7-

03).

Remarks: Euphlyctis Fitzinger, 1843 was revived from

synonymy of Rana Linnaeus, 1758 by Dubois (1992).

5. Euphylyctis hexadactylus (Lesson, 1834)

Rana hexadactyla R. P. Lesson. 1834. Voyage Indes-

Orient.: 331.

Northern (Rao et al., 2005; Srinivasulu et al., 2006),

Central (Rao et al., 2005; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Abundant. [S] ERM (ERMA-1 0a);

[P] NHMOU (NHMOU. Amph. P.8-03).

6. Fejervarya cf. limnocharis (Gravenhorst, 1829)

Rana limnocharis J. L. C. Gravenhorst. 1829. Rept.

Mus. Zool. Vratis. Delic. Mus. Zool: 42.

Northern (Sarkar et al., 1993; Srinivasulu et al., 2006),

Central (Rao et al., 2005; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Common. [S] ZSIK (A8438-40,

A7813-14, A1990, A6946), ERM (ERMA-1 7a); [P]

NHMOU (NHMOU. Amph. P.9-03).

Remarks: Fejervarya Bolkay, 1915 was recognized as a

subgenus of Rana Linnaeus, 1758 by Dubois (1992),

and as a genus by Iskandar (1998).

7. Hoplobatrachus crassus (Jerdon, 1854) Jerdon’s

Bull Frog

Rana crassa T. C. Jerdon. 1854. J. Asiatic Soc. Bengal

22(5): 531.

Northern (Sanyal et al., 1993; Srinivasulu et al., 2006),

Central (Rao et al., 2005) and Southern Nallamala Hills.

Scrub, open forests and near agricultural fields.

Abundant. [S] ZSIK (A1843, A6945, A8409-14), ERM

(ERMA-1 3a); [P] NHMOU (NHMOU. Amph.P. 10-03).

Remarks: Hoplobatrachus Peters, 1863 was revived

from synonymy of Rana Linnaeus, 1758 by Dubois

(1992). See also Grosjean et al. (2004).

8. Hoplobatrachus tigerinus (Daudin, 1803)

Rana tigerina F.-M. Daudin. 1803. Hist. Nat.: 64; PI.

XX.

Northern (Sanyal et al., 1993; Srinivasulu et al., 2006),

Central (Rao et al., 2005; Sanyal et al., 1993;

Srinivasulu et al., 2006) and Southern Nallamala Hills.

Scrub, open forests and near agricultural fields.

Abundant. [S] ZSIK (A6944, A8443^5, Al 138-39,

A 1995), ZSIH (ZSI/FBS/N/1 145), ERM (ERMA-1 5a).

9. Sphaerotheca breviceps (Schneider, 1799)

Rana breviceps J. G. Schneider. 1799. Hist. Nat. Amph.:

140.

Northern (Srinivasulu et al., 2006), Central (Rao et al.,

2005; Sanyal et al., 1993; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Abundant. [S] ZSIK (A8400, A8448,

A 1940), ZSIH (ZSI/FBS/N/1 143, 1144, 11461, 1147,

1155-58), ERM (ERMA-12a); [P] NHMOU

(NHMOU.Amph.P. 11-03).

Remarks: Rao et al. (2005) included an erroneously

identified photograph (image 13 at www.zoosprint.org/),

which is, in fact, that of Sphaerotheca dobsoni, a taxon

that also is present in the Nallamala Hills (see below). In

support of long-separated evolutionary lineages, repre-

senting distinct monophyletic radiations of the Africa,

Madagascar and southern Asia, Vences et al. (2000)

argued for the partition of Tomopterna into three lineag-

es. Thus, the earliest available name for the Asian

species is Sphaerotheca.

10. Sphaerotheca dobsonii (Boulenger, 1882)

Rana dobsonii G. A. Boulenger. 1882. Cat. Bat. British

Mus.: 32; PI. 3, Fig. 1.

Northern (Srinivasulu et al., 2006), Central (Srinivasulu

et al., 2006) and Southern Nallamala Hills. Scrub, open

forests and near agricultural fields. Uncommon. [P]

NHMOU (NHMOU.Amph.P. 12-03).

11. Sphaerotheca rolandae (Dubois, 1983)

Rana ( Tomopterna ) rolandae A. Dubois. 1983. Alytes:

2(4): 163.

Northern (Srinivasulu et al., 2006), Nallamala Hills.

Open forests. Rare. [P] NHMOU

(NHMOU.Amph.P. 1 3-03).

Remarks: Rao et al.’s (2005) claim of this taxon (as

Tomopterna rolandae) as the first record from Andhra

Pradesh is based on erroneous identification. The vouch-

er specimen and the photograph included in the report

are that of Sphaerotheca breviceps (image 14 at

www.zocsprint.org /).

Family: Microhylidae

12. Kaloula taprobanica Parker, 1934

Kaloula pulchra taprobanica H. W. Parker. 1934.

Monogr. Frogs. Microhylidae: 86.

Northern (Rao et al., 2005) and Central (Rao et al.,

2005; Srinivasulu et al., 2006) Nallamala Hills. Open

forests and scrub areas. Uncommon. [S] ZSIH

(ZSI/FBS/N/1 159), ERM (ERMA^la); [P] NHMOU

(NHMOU. Amph. P.2-03).

Remarks: The first record of its occurrence in Andhra

Pradesh reported by Rao et al. (2005) is erroneous, as

Sivakumar et al. (2003) had reported its occurrence in

the State from Sriharikota Island Nellore District. Rao et

al. (in review, a) puts on record for its occurrence in the

Nallamala Hills.

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115

13. Microhyla ornata (Dumeril and Bibron, 1841)

Engystoma ornatum A.-M.-C. Dumeril & G. Bibron.

1841. Erp. Gen. 8: 745.

Northern (Sarkar et al., 1993; Srinivasulu et al., 2006),

Central (Rao et al., 2005; Sarkar et al., 1993; Srinivasulu

et al., 2006) and Southern Nallamala Hills. Scrub, open

lorests and near agricultural fields. Common. [S] ZSIK

(Al 846-M-8, A7817, A8451, A1140, A1996), ZSIH

(ZSI/FBS/N/1 141, 1160), ERM (ERMA-8a); [P]

NHMOU (NHMOU.Amph. P.3-03).

14. Microhyla rubra (Jerdon, 1854)

Engystoma rubrum T. C. Jerdon. 1854. J. Asiatic Soc.

Bengal 22(2): 534.

Northern (Srinivasulu et al., 2006), Central (Rao et al.,

2005; Sarkar et al., 1993; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Uncommon. [S] ZSIK (A8463-64),

ERM (ERMA-6a); [P] NHMOU (NHMOU.Amph.P.4-

03).

15. Ramanella variegata (Stoliczka, 1872)

Callula variegata F. Stoliczka. 1872. Proc. Asiatic Soc.

Bengal 1872(6): 111.

Central (Srinivasulu et al., 2006) and Southern

Nallamala Hills. Scrub and open to close forests.

Uncommon. No vouchers, based on sightings.

16. Uperodon globulosus (Gunther, 1864)

Cacopus globulosus A. C. L. G. Gunther. 1864. Reptiles

British India: 416.

Central (Rao et al., 2005; Srinivasulu et al., 2006;

Srinivasulu et al., 2006) Nallamala Hills. Scrub and

open forests. Rare. [S] ZSIH (ZSI/FBS/N/1 138), ERM

(ERMA-7a); [P] NHMOU (NHMOU.Amph.P.5-03).

17. Uperodon systoma (Schneider, 1799)

Rana systoma J. G. Schneider. 1799. Hist. Nat. Amph.:

144.

Northern (Rao et al., 2005; Srinivasulu et al., 2006),

Central (Rao et al., 2005; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Common. [S] ZSIH

(ZSI/FBS/N/1 142, 1149, 1154), ERM (ERMA-2a); [P]

NHMOU (NHMOU. Amph. P.6-03).

Family: Petropedetidae

18. Indirana leithii (Boulenger, 1888)

Rana leithii G. A. Boulenger. 1888. Ann. & Mag. nat.

Hist. Ser. 6, 2: 506.

Northern (Srinivasulu et al., 2006) and Central

(Srinivasulu et al., 2006) Nallamala Hills. Scrub forests.

Rare. [S] ERM (ERM/A24).

Remarks: Indirana Laurent, 1986 was revived from syn-

onymy of Rana Linnaeus, 1758 by Dubois (1992). This

species had been sighted on two occasions near

Rollapenta in Central Nallamalla Hills and on one occa-

sion near Ahobilam in Southern Nallamala Hills by the

first author (Srinivasulu et al., 2006), who has also stud-

ied a single specimen in the collection of ERM Labs

(ERM/A24), Sunnipenta that had been identified by

Varad Giri of the BNHS.

Family: Ranidae

19. Hylarana sp.

Central (Rao et al., 2005; Rao et al., in review, b)

Nallamala Hills. Riparian forest. Rare. [S] ERM

(ERMA-14a).

Remarks: The systematic status of the population,

referred to Rana temporalis (Gunther, 1864) by previous

workers, is under study by the second author, who

assigns it to Dubois' (1992) subgenus Sylvirana, elevat-

ed to generic rank in Frost et al. (2006). Currently this

generic name is a synonym of Hylarana (See Frost et al.,

2007).

Family: Rhacophoridae

20. Polypedates maculatus (Gray, 1834)

Hyla maculata J. E. Gray. 1834. 111. Indian Zook: PI.

LXXXII; Fig. 1.

Northern (Rao et al., 2005; Sanyal et al., 1993;

Srinivasulu et al., 2006), Central (Rao et al., 2005;

Sanyal et al., 1993; Srinivasulu et al., 2006) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Abundant. [S] ZSIK (A8403), ZSIH

(ZSI/FBS/N/1 140, 1161), ERM (ERMA-lla); [P]

NHMOU (NHMOU.Amph.P. 14-03).

Annotated List of Reptiles

Order: Crocodilia

Family: Crocodylidae

1. Crocodylus palustris Lesson, 1831

Crocodylus palustris R. P. Lesson. 1831. Bull. Sci. Nat.

Geol. 25: 121.

Northern Nallamala Hills. Under the Central

Government sponsored crocodile rehabilitation pro-

gramme, some crocodiles were reintroduced both at

backwaters of Nagarjunasagar Reservoir in Vijaypuri

vicinity, Srisailam Reservoir and Ethipothala (described

by Srinivas et al., 1999). Their numbers have dwindled

due to poaching, but some crocodiles do survive in both

these areas. No vouchers, based on the literature and

indirect evidence.

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2008

Order: Chelonia

Family: Geoemydidae

2. Melanochelys trijuga (Schweigger, 1812)

Emys trijuga A. F. Schweigger. 1812. Prod. Monogr.

Chel.: 310.

Northern, Central and Southern Nallamala Hills.

Waterbodies, streams and rivers. Uncommon. No vouch-

ers, based on sightings.

Remarks: Assumed, on the basis of locality, to belong to

the nominotypical form.

3. Pangshura tentoria (Gray, 1834)

Emys tentoria J. E. Gray. 1834. Proc. Zool. Soc. London

1834(2): 54.

Northern, Central and Southern Nallamala Hills.

Waterbodies, streams and rivers. Uncommon. No vouch-

ers, based on sightings.

Remarks: Allocated to Pangshura Gray, 1869 rather

than Kachuga Gray, 1856 by Spinks et al. (2004).

Family: Testudinidae: Tortoises

4. Geochelone elegans (Schoepff, 1795)

Testudo elegans J. D. Schoepff. 1795. Hist. Test. 3: 111;

PI. XXV.

Northern (Rao et al., 2005), Central (Rao et al., 2005)

and Southern Nallamala Hills. Scrub forests and near

agricultural fields. Common. [S] ERM (ERMR-5a).

Remarks: First reported from the Nallamala Hills by

Subba Rao et al. (1994).

Family: Trionychidae

5. Nilssonia gangetica (Cuvier, 1825)

Trionyx gangeticus G. L. C. F. D. Cuvier. 1825.

Recherch Ossemens Foss. 5: 203.

Northern Nallamala Hills (Sharma, 1971). Waterbodies,

streams and rivers. Uncommon. [S] ZSIK (R21238).

Remarks: The generic nomen Aspideretes Hay, 1904,

was revived from the synonymy of Trionyx Geoffroy

Saint-Hillaire, 1809 by Meylan (1987). Praschag et al.

(2007) placed Aspideretes in the synonymy of Nilssonia,

but provided an incorrect (feminine) termination of the

species name.

6. Nilssonia leithii (Gray, 1872)

Trionyx Leithii J. E. Gray. 1872. Ann. & Mag. nat. Hist,

ser. 4 10: 334.

Northern (Sharma, 1971; Sanyal et al., 1993) and

Southern Nallamala Hills. Waterbodies, streams and

rivers. Uncommon. [S] ZSIK (R21403).

7. Lissemys punctata (Bonnaterre, 1789)

Testudo punctata M. Bonnaterre. 1789. Tableau Encycl.

Method. Nat.: 30.

Northern (Sanyal et al., 1993), Central and Southern

Nallamala Hills. Waterbodies, streams and rivers.

Common. [S] ZSIK (Specimen not traceable).

Order: Squamata

Family: Agamidae

8. Calotes rouxii (Dumeril & Bibron, 1837)

Calotes rouxii A.-M.-C. Dumeril & G. Bibron. 1837.

Erp. Gen. 4: 407.

Northern (Rao et al., 2005), Central (Rao et al., 2005)

and Southern Nallamala Hills. Rocky outcrops in open

and scrub forests, and agricultural fields. Common. [S]

ZSIH (ZSI/FBS/N/1 172), ERM (ERMR-lOa); [P]

NHMOU (NHMOU. Rep. P. 1-03).

9. Calotes versicolor (Daudin, 1802)

Agama versicolor F.-M. Daudin. 1802. Hist. nat. Rept.

3: 395; PI. XLIV.

Northern (Rao et al., 2005; Sharma, 1971; Sanyal et al.,

1993), Central (Rao et al., 2005) and Southern

Nallamala Hills. Rocky outcrops in open and scrub

forests, and agricultural fields. Abundant. [S] ZSIK

(R20249-60, R20181-85, R21286-88, R21289,

R21431-33, R21367, R21275, R21434-35, R21276,

R2 1422-25, R21277-80, R21368-70, R21281,

R21283-85, R24456, R20293, R20214-15), ZSIH

(ZSI/FBS/N/1 165, 1169), ERM (ERMR-12a).

10. Psammophilus blanfordanus (Stoliczka, 1871)

Charasia blanfordana F. Stoliczka. 1871. Proc. Asiatic

Soc. Bengal 1871(9): 194.

Northern (Sanyal et al., 1993; Sharma, 1971), Central

and Southern Nallamala Hills. Rocky outcrops in open

and scrub forests. Common. [S] ZSIK (R21436,

R24685, R24659).

11. Psammophilus dorsalis (Gray in Griffith &

Pidgeon, 1831)

Agama Dorsalis J. E. Gray in: E. Griffith & E. Pidgeon.

1831. Class Reptilia 9: 56.

Northern (Rao et al., 2005; Sharma, 1971), Central (Rao

et al., 2005) and Southern Nallamala Hills. Rocky out-

crops in open and scrub forests. Common. [S] ZSIK

(R21291, R20295), ERM (ERMR-lla).

12. Sitana ponticeriana Cuvier, 1829

Sit. (= Sitana ) ponticeriana G. J.-L.-N.-F. D. Cuvier.

1829. Reg. Anim. 2: 43.

Northern (Rao et al., 2005; Shanna, 1971; Sanyal et al..

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Asiatic Herpetological Research, Vol. 11

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1993), Central (Rao et al., 2005; Sanyal et al., 1993) and

Southern (Sanyal et al., 1993) Nallamala Hills. Rocky

outcrops in open and scrub forests and agricultural

fields. Abundant. [S] ZSIK (R20284, R21256, R21270,

R21413-15, R2 127-72, R21348-50, R21267, R21257,

R2 1 4 1 9-2 1 , R2 1258-61, R21253-56, R21269,

R21357-64, R24436, R24439, R20178, R20216,

R2 1262-68, R21352, R21416, R21357-62, R24668,

R24455, R24462, R20223, R20294, R24684), ZSIH

(ZSI/FBS/N/1 166, 1170, 1171), ERM (ERMR-9a); [P]

NHMOU (NHMOU. Rep. P.2-03).

Family: Chamaeleonidae

13. Chamaeleo zeylanicus Laurenti, 1768

Chamaeleo zeylanicus J. N. Laurenti. 1768. Syn. Rept.:

46.

Northern (Rao et al., 2005), Central (Rao et al., 2005)

and Southern Nallamala Hills. Open and scrub forests,

and agricultural fields. Common. [S] ERM (ERMR-

13a); [P] NHMOU (NHMOU.Rep.P.1-01).

Family: Gekkonidae

14. Cnemaspis sp.

Remarks: An unidentified species of Cnemaspis was

encountered in Central and Southern Nallamala Hills.

Three specimens that were collected by the first author,

deposited in the Eco-Resources Monitoring Labs,

Sunnipenta in March 2002, were lost due to attack by

ants. Specimens were collected from the leaf litter in a

dry stream near Chinnarutla. Rare. [S] ERM (Lost).

Cnemaspis otai Das and Bauer (2000) is known from

Vellore region, in extreme northern Tamil Nadu State,

adjacent to Andhra Pradesh, and the Nallamala speci-

mens may be either this nominal species, or an unde-

scribed species.

15. Hemidactylus bowringii (Gray, 1845)

Doryura bowringii J. E. Gray. 1845. Cat. Lizards British

Mus.: 156.

Northern (Sanyal et al., 1993) Nallamala Hills. Inhabits

human-altered habitats and other dilapidated man-made

structure. Rare. [S] ZSIK (R24465).

16. Hemidactylus brookii (Gray, 1845)

Hemidactylus brookii J. E. Gray. 1845. Cat. Lizards

British Mus.: 153.

Northern (Rao et al., 2005; Sharma, 1971; Sanyal et al.,

1993), Central (Rao et al., 2005) and Southern

Nallamala Hills. Open forests, old temples, also human

commensal, found in houses and other dilapidated man-

made structures. Abundant. [S] ZSIK (R2 1240-44,

R24669, R2 1404-05, R23237, R24435, R20179,

R23687, R23699), ZSIH (ZSI/FBS/N/1 174), ERM

(ERMR-2a).

17. Hemidactylus flaviviridis Riippell, 1835

Hemidactylus flaviviridis E. Riippell. 1835. Neue

Wirbelth. -Fauna Abyss., Amph. 18: PI. 6; Fig. 2.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Human commensal, found in houses

and other man-made structures. Uncommon. [S] ZSIH

(ZSI/FBS/N/1 173), ERM (ERMR-3a).

18. Hemidactylus frenatus Dumeril & Bibron, 1836

Hemidactylus frenatus A.-M.-C. Dumeril & G. Bibron.

1836. Erp. Gen. 3: 366.

Northern (Sanyal et al., 1993), Central (Rao et al., 2005)

and Southern Nallamala Hills. Open forests, old tem-

ples, also human commensal, found in houses and other

dilapidated man-made structures. Common. [S] ZSIK

(R23700), ERM (ERMR-30a).

19. Hemidactylus giganteus Stoliczka, 1871

Hemidactylus giganteus F. Stoliczka. 1871. Proc. Asiatic

Soc. Bengal 1871(9): 193.

Northern (Rao et al., 2005; Sanyal et al., 1993), Central

(Rao et al., 2005) and Southern Nallamala Hills. Open

forests, old temples, and other dilapidated man-made

structures. Uncommon. [S] ZSIK (R21411-12), ZSIH

(ZSI/FBS/N/1 167, 1168), ERM (ERMR-la).

20. Hemidactylus leschenaultii Dumeril & Bibron,

1836

Hemidactylus leschenaultii A.-M.-C. Dumeril & G.

Bibron. 1836. Erp. Gen. 3: 364.

Northern (Rao et al., 2005; Sanyal et al., 1993), Central

(Rao et al., 2005) and Southern Nallamala Hills. Open

forests, old temples, also human commensal, found in

houses and other dilapidated man-made structures.

Common. [S] ZSIK (R20180, R23693, R24458,

R24466, R24660, R24510, R24513), ERM (ERMR— 4a).

21. Hemidactylus reticulatus Beddome, 1870

Hemidactylus reticulatus R. H. Beddome. 1870. Madras

Monthly J. Med. Sci. 1: 33.

Northern (Sanyal et al., 1993), Central (Rao et al., 2005)

and Southern Nallamala Hills. Rocky outcrops in open

and scrub forests. Uncommon. [S] ZSIK (R2 1245-46,

R2 1247-53, RR21346, R21343-45, R21406-10,

R23216), ERM (ERMR-17a).

22. Hemidactylus triedrus (Daudin, 1802)

Gecko triedrus F.-M. Daudin. 1802. Hist. nat. Rept. 4:

155.

Northern (Rao et al., 2005; Sanyal et al., 1993), Central

(Rao et al., 2005) and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Common. [S]

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ZSIK (21239, R24509, R24512), ERM (ERMR-7a).

Family: Lacertidae

23. Ophisops jerdoni (Blyth, 1853)

Ophisops jerdoni E. Blyth. 1853. J. Asiatic Soc. Bengal

22: 653.

Northern (Rao et al., 2005; Sanyal et al., 1993; Sharma,

1971), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Abundant. [S] ZSIK (R21302-14, R21381-92,

R21440, R24661, R24670), ZSIH (ZSI/FBS/N/1 176-

1178), ERM (ERMR-18a).

24. Ophisops leschenaultii (Milne-Edwards, 1829)

Lacerta leschenaultii H. Milne-Edwards. 1829. Ann.

Sci. nat. 16: 86; PI. VI; Fig. 9.

Northern (Sanyal et al., 1993; Sharma, 1971), Central

(Murthy, 1986) and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Uncommon.

[S] ZSIK (R2 1296-97, R21438-39).

25. Ophisops minor (Deraniyagala, 1971)

Cabrita jerdoni minor P. E. P. Deraniyagala. 1971.

Ceylon J. Sci. 32(1): 104; Fig. 1.

Northern (Rao et al., 2005; Sanyal et al., 1993; Sharma,

1971), Central (Rao et al., 2005; Sanyal, et al., 1993)

and Southern Nallamala Hills. Scrub, open forests and

near agricultural fields. Common. [S] ZSIK (R21298-

301, R2 1377-80, R24440, R24459, R24464), ERM

(ERMR-8a).

Remarks: Reviewed by Bohme and Bischoff (1991). See

also nomenclatural remarks in Arnold (1989).

Family: Scincidae

26. Lygosoma ashwamedhi Sharma, 1969

Riopa ashwamedhi R. C. Sharma. 1969. Bull. Syst.

Zook, Calcutta 1(2): 73; Fig. 2.

Endemic to Andhra Pradesh, known only from type

locality. Northern (Sharma, 1969, 1971; Sanyal et al.,

1993) Nallamala Hills. Rocky scrub forests. Rare. [S]

ZSIK (R2 11 73-77, R21179).

27. Lygosoma guentheri (Peters, 1879)

Eumeces guntheri W. C. H. Peters. 1879. S.-Ber. Ges.

Naturf. Freunde Berlin 1879(3): 36.

Central (Rao et al., 2005) Nallamala Hills. Scrub forest.

Rare. [S] ERM (ERMR^13a).

Remarks: Hitherto known only from the Western Ghats,

from Gujarat to Kerala States (Daniel and Shull,

"1963"; 1964; Daniel, 1962; Smith, 1935: 322), this is

the first record of the species from the Eastern Ghats,

28. Lygosoma punctata (Linnaeus, 1758)

Scincus punctatus C. Linnaeus. 1758. Syst. Nat. 10th ed.

1: 209.

Northern (Sanyal et al., 1993; Sharma, 1971), Central

and Southern Nallamala Hills. Scrub, open forests and

near agricultural fields. Common. [S] ZSIK(21294,

R21376, R20327, R20318), ZSIH (ZSI/FBS/N/1175);

[P] NHMOU (NHMOU. Rep. P.3-03).

29. Eutropis carinata (Schneider, 1801)

Scincus carinatus J. G. Schneider. 1801. Hist. Amphib.:

183.

Northern (Rao et al., 2005; Sanyal et al., 1993; Sharma,

1971), Central (Rao et al., 2005; Sanyal et al., 1993) and

Southern Nallamala Hills. Scrub, open forests and near

agricultural fields. Common. [S] ZSIK (R2 1292-93,

R21373-75, R21437, R20313, R21437, R24438,

R24457, R24463, R23694, R24511), ZSIH

(ZSI/FBS/N/1 179, 1180), ERM (ERMR-15a); [P]

NHMOU (NHMOU. Rep. P.4-03).

Remarks: Mausfeld et al. (2002) suggested partitioning

the genus Mabuya Fitzinger, 1826 into several genera,

allocating the Asian species to Eutropis Fitzinger, 1843.

30. Eutropis macularia (Blyth, 1853)

Euprepes macularius E. Blyth. 1853. J. Asiatic Soc.

Bengal 22: 652.

Northern (Sanyal et al., 1993; Rao et al., 2005), Central

(Sanyal et al., 1993; Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R24437, R23701), ERM

(ERMR-16a).

31. Eutropis nagarjuni Sharma, 1969

Mabuya nagarjuni R. C. Sharma. 1969. Bull. Syst.

Zook, Calcutta 1(2): 71; Fig. 1.

Endemic to Andhra Pradesh. Northern (Sanyal et al,

1993; Sharma, 1969, 1971) Nallamala Hills. Rocky

scrub forests. Rare. [S] ZSIK (R2 11 70-72), ZSIH

(ZSI/FBS/N/1 164); [P] NHMOU (NHMOU. Rep. P.5-

03).

Remarks: The photo purported to be of Mabuya bed-

domei (Jerdon, 1870) in Rao et al. (2005; image 30 at

www.zoosprint.org/) is that of Eutropis nagarjuni

(Sharma, 1969), as shown by Srinivasulu et al. (2005).

Family: Varanidae

32. Varanus bengalensis (Daudin, 1802)

Tupinambis bengalensis F.-M. Daudin. 1802. Hist. nat.

Rept. 3: 67.

Northern (Rao et ah, 2005; Sanyal et ah, 1993; Sharma,

1971), Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

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Asiatic Herpetological Research, Vol. 11

119

al fields. Common. [S] ZSIK (R21315, R21441-42),

ERM (ERMR-26a).

Remarks: For a history of the name Varanus monitor

Linnaeus, 1758, a junior synonym of Tupinambis ben-

galensis Daudin, 1802, see Mertens ( 1946; 1956; 1957)

and Sprackland (1982).

Family: Boidae

33. Eryx conicus (Schneider, 1801)

Boa conica J. G. Schneider. 1801. Hist. Amphib. 2: 268.

Northern (Rao et al., 2005; Sanyal et al., 1993; Sharma,

1971), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21331), ERM (ERMR-

14a).

34. Eryx johnii (Russell, 1801)

Boa Johnii R Russell. 1801. Continuation Account

Indian Serpents: 18; PI. xvi-xvii.

Northern (Rao et al., 2005; Sanyal et al., 1993; Sharma,

1971), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R2 1332-33), ERM

(ERMR-22a).

Family: Pythonidae

35. Python molurus (Linnaeus, 1758)

Coluber molurus C. Linnaeus. 1758. Syst. Nat. 10th ed.

1: 225.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Rocky scrub, open forests and near

agricultural fields. Uncommon. No vouchers collected,

based on sightings.

Family: Colubridae

36. Ahaetulla nasuta (Lacepede, 1789)

Coluber nasuta B.-G.-E. de L. V.-S.-I. Lacepede. 1789.

Hist. Nat. Serp. 1: 100.

Northern, Central and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Common. [S]

ERM (ERMR-6a).

Remarks: Rao et al., (2005) did not include this taxon in

their catalogue, but provided its picture (image 42 at

www.zoosprint.org/). In their list they included the sub-

species, Ahaetulla nasutus isabellinus (Wall). The single

specimen based on which the presence is reported by

Rao et al. (2005) requires further studies to confirm the

validity of the so-called subspecies, whose correct termi-

nation of subspecies nomen should be rendered isabelli-

na, to match the gender of the genus.

37. Amphiesma stolatum (Linnaeus, 1758)

Coluber stolatus C. Linnaeus. 1758. Syst. Zool. 10th

ed.: 219.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Uncommon. [S] ERM (ERMR-27a).

Remarks: The gender of Amphiesma Dumeril, Bibron

and Dumeril, 1 854 has been treated erroneously treated

as feminine since it was resurrection by Malnate (1960).

Toriba ( 1 994) showed that the genus is neuter, and the

termination of the species name should therefore be sto-

latum (see also David et al., 1998).

38 . Argyrogena fasciolata (Shaw, 1802)

Coluber fasciolatus G. Shaw. 1802. Gen. Zool.: 528.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ERM (ERMR-28a).

Remarks: Revived from the synonymy of Coluber for C.

fasciolata Shaw, 1802, by Wilson (1967).

39. Atretium schistosum (Daudin, 1803)

Coluber schistosus F.-M. Daudin. 1803. Hist. Nat. Rept.

6: 132.

Northern, Central and Southern Nallamala Hills. Near

waterbodies and paddy fields. Common. No vouchers,

based on sightings.

40. Boiga forsteni (Dumeril, Bibron & Dumeril, 1854)

Triglyphodon forsteni A.-M.-C. Dumeril, G. Bibron &

A.-H.-A. Dumeril. 1854. Erp. Gen. 7: 1077.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Uncommon. [S] ERM (ERMR-20a).

41. Boiga trigonata (Schneider in Bechstein, 1802)

Coluber trigonatus J. G. Schneider in: J. M. Bechstein.

1802. La Cepede’s Nat. Amphib.: 256; PI. 40; Fig. 1.

Northern (Sanyal et al., 1993; Sharma, 1971), Central

(Rao et al., 2005) and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Common. [S]

ZSIK (R21457), ERM (ERMR-37a).

42. Coelognathus Helena (Daudin, 1803)

Coluber helena F.-M. Daudin. 1803. Hist. nat. Rept. 6:

277; PI. LXXVI.

Northern (Rao et al., 2005; Sharma, 1971; Sanyal et al.,

1993), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21334), ZSIH

(ZSI/FBS/N/ 1181), ERM (ERMR-32a); [P] NHMOU

(NHMOU. Rep. P.6-03).

Remarks: Coelognathus Fitzinger, 1843, was revived

from the synonymy of Elaphe Fitzinger in: Wagler,

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2008

1833, by Heltenberger (2001a; b), based on visceral and

vertebrae morphology and allozyme variations.

43. Coluber bholanathi Sharma, 1976

Coluber bholanathi R. C. Sharma. 1976. Comp. Physiol.

Ecol. 1(3): 106; Fig. 1.

Endemic to Andhra Pradesh, known only from type

locality. Northern (Sanyal et al., 1993; Sharma, 1976)

Nallamala Hills. Scrub and open forests. Rare. [S] ZSIK

(R2 1335-37).

44. Dendrelaphis tristis (Daudin, 1803)

Coluber tristis F.-M. Daudin. 1803. Hist. nat. Rept. 6:

430.

Northern (Rao et al., 2005), Central (Rao et al., 2005)

and Southern Nallamala Hills. Scrub, open forests and

near agricultural fields. Common. [S] ERM (ERMR-

25a); [P] NHMOU (NHMOU.Rep.P.2-01).

45. Enhydris enhydris (Schneider, 1799)

Hydrus enhydris J. G. Schneider. 1799. Hist. Amphib. 1:

245.

Northern and Central (Rao et al., 2005) Nallamala Hills.

Scrub, open forests and near agricultural fields.

Uncommon. No vouchers collected, based on sightings.

46. Liopeltis calamaria (Gunther, 1858)

Cyclophis calamaria A. C. L. G. Gunther. 1858. Cat.

Colubrine Snakes British Mus.: 250.

Central (Rao et al., 2005) Nallamala Hills. Scrub and

open forests. Rare. [S] ERM (ERMR-29a).

47. Ly codon aulicus (Linnaeus, 1758)

Coluber aulicus C. Linnaeus. 1758. Syst. Nat. 10th ed 1:

220.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ERM (ERMR-35a).

48. Lycodon striatus (Shaw, 1802)

Coluber striatus G. Shaw. 1802. Gen. Zool.: 527.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Uncommon. [S] ERM (ERMR-44a).

Remarks: First record claim from the region by Rao et

al. (2005) is erroneous as it has been already reported

from the Nallamala Hills by Subba Rao et al. (1994).

49. Lycodon travancoricus (Beddome, 1870)

Cercaspis travancoricus R. H. Beddome. 1870a. Madras

Monthly J. Med. Sci. 1: 169.

Central (Rao et al., 2005) Nallamala Hills. Scrub and

open forests. Rare. [S] ERM (ERMR-38a).

50. Macropisthodon plumbicolor (Cantor, 1839)

Tropidonotus plumbicolor T. Cantor. 1839. Proc. Zool.

Soc. London 1829(7): 54.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ERM (ERMR-36a).

51. Oligodon arnensis (Shaw, 1802)

Coluber arnensis G. Shaw. 1802. Gen. Zool.: 526.

Northern, Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ERM (ERMR-41a).

52. Oligodon taeniolatus (Jerdon, 1853)

Coronella taeniolata T. C. Jerdon. 1853. J. Asiatic Soc.

Bengal 22(6): 528.

Northern (Rao et al., 2005; Sharma, 1971; Sanyal et al.,

1993), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Uncommon. [S] ZSIK (R2 1450-51, R24460),

ERM (ERMR^42a).

53. Oligodon travancoricus (Beddome, 1877)

Oligodon travancoricum R. H. Beddome. 1877. Proc.

Zool. Soc. London 1877(4): 685.

Northern (Sanyal et al., 1993), Central and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Uncommon. [S] ZSIK (R21338).

54. Ptyas mucosa (Linnaeus, 1758)

Coluber mucosus C. Linnaeus. 1758. Syst. Nat. 10th ed

1: 216.

Northern (Rao et al., 2005; Sharma, 1971; Sanyal et al.,

1993), Central (Rao et al., 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R2 1446-47, R21449),

ERM (ERMR-39a); [P] NHMOU (NHMOU.Rep.P.3-

01).

Remarks: David and Das (2004) showed that the correct

termination of the species nomen should be mucosa ,

rather than mucosus , to match the gender of the generic

nomen.

55. Sibynophis subpunctatus (Dumeril, Bibron &

Dumeril, 1854)

Oligodon subpunctatum A.-M.-C. Dumeril, G. Bibron &

A.-H.-A. Dumeril. 1854. Erp. Gen. 7: 58.

Central Nallamala Hills. Scrub and open forests. Rare.

[S] ERM (ERMR^lOa).

Remarks: The taxon Sibynophis subpunctatus (Dumeril,

Bibron & Dumeril, 1854), has been recently been resur-

rected from the synonymy of Sibynophis sagittaria

(Cantor, 1839) by Captain et al. (2004).

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Asiatic Herpetological Research, Vol. 11

121

56. Xenochropis piscator (Schneider, 1799)

Hydrus piscator J. G. Schneider. 1799. Hist. Amphib. 1:

247.

Northern (Sharma, 1971; Rao et al., 2005; Sanyal et ah,

1993), Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21339, R21452,

R21454-55).

Family: Elapidae

57. Bungarus caeruleus (Schneider, 1801)

Pseudoboa caerulea J. G. Schneider. 1801. Hist.

Amphib. 2: 284.

Northern (Rao et ah, 2005; Sanyal et ah, 1993; Sharma,

1971), Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21458-59), ERM

(ERMR -24a).

58. Calliophis melanurus (Shaw, 1802)

Coluber melanurus G. Shaw. 1802. Gen. Zooh: 552.

Northern (Sanyal et ah, 1993; Sharma, 1971) Nallamala

Hills. Scrub forests. Rare. [S] ZSIK (R21460).

59. Naja naja (Linnaeus, 1758)

Coluber naja C. Linnaeus. 1758. Syst. Nat. 10th ed. 1:

221.

Northern (Rao et ah, 2005; Sanyal et ah, 1993; Sharma,

1971), Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21461), ERM (ERMR-

33a); [P] NHMOU.

Family: Typhlopidae

60. Grypotyphlops acutus (Dumeril, Bibron &

Dumeril, 1844)

Onychocephalus acutus A.-M.-C. Dumeril, G. Bibron &

A.-H.-A. Dumeril. 1844. Erp. Gen. 6: 333.

Northern (Sanyal et ah, 1993; Sharma, 1971), Central

and Southern Nallamala Hills. Scrub, open forests and

near agricultural fields. Common. [S] ZSIK (R21330).

Remarks: Wallach (2003) revived Gyptotyphlops Peters,

1881 from the synonymy of Rhinotyphlops Fitzinger,

1832, to accommodate the present species.

61. Ramphotyphlops braminus (Daudin, 1803)

Eryx braminus F.-M. Daudin. 1803. Hist. Nat. Gen.

Rept. 7: 279.

Northern (Sanyal et ah, 1993; Sharma, 1971), Central

(Rao et ah, 2005) and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Common. [S]

ZSIK (R2 13 16-20, R2 1327-29, R2 1394-99), ERM

(ERMR- 19a).

Family: Viperidae

62. Daboia russelii (Shaw & Nodder, 1797)

Coluber russelii G. Shaw & F. P. Nodder. 1797. Nat.

Misc. 8: PI. 108.

Northern (Rao et ah, 2005; Sanyal et ah, 1993; Sharma,

1971), Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIK (R21341), ERM (ERMR-

34a).

Remarks: Although Obst (1983) revived Daboia Gray,

1842 from the synonymy of Vipera Laurenti, 1768, his

concept of the genus included Daboia Gray, 1842,

Macrovipera Reuss, 1927, Pseudocerastes Boulenger,

1896 and the Vipera xanthina (Gray, 1849) complex.

Hermann et ah (1992) separated these genera from each

other, and from Vipera Laurenti, 1768, restricting

Daboia to Vipera russelii Shaw & Nodder, 1797. For

comments on the spelling of the specific name, see

Dowling (1993) and Adler et ah (2000).

63. Echis carinatus (Schneider, 1801)

Pseudoboa carinatus J. G. Schneider. 1801. Hist.

Amphib. 2: 285.

Northern (Sanyal et ah, 1993; Sharma, 1971), Central

(Rao et ah, 2005) and Southern Nallamala Hills. Scrub,

open forests and near agricultural fields. Common. [S]

ZSIK (R21342, R21401-02, R24461) ERM (ERMR-

23a).

64. Trimeresurus gramineus (Shaw, 1802)

Coluber gramineus G. Shaw. 1802. Gen. Zooh: 420.

Northern, Central (Rao et ah, 2005) and Southern

Nallamala Hills. Scrub, open forests and near agricultur-

al fields. Common. [S] ZSIH (ZSI/FBS/N/1182, 1183),

ERM (ERMR-31a).

Erroneous or Doubtful Records

In addition to the records presented in the preceding

pages, the following species have been recorded from

the Nallamala Hills in the literature. These have been

shown to be in error, stemming from the use of incorrect

names or from misidentifications.

Bufo hololius Gunther, 1876, which had been

reportedly collected by Pillai and Murthy (1983) from

Nagarjunasagar area (also cited by Sarkar et ah, 1993),

has not been included in this list as this taxon is known

only from type specimen and all other specimens

assigned to this nomen need reevaluation, according to

Dubois and Older (1999).

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2008

Polypedates leucomystax (Gravenhorst, 1829), has

been recorded from the Nallamala Hills by Rao et al.

(2005), and earlier, from the Eastern Ghats, by Pillai and

Murthy (1983). The records possibly refers to either P

maculatus or another member of this complex, since

Gravenhorst's species (type locality: Java) is a mesic

area frog, approaching the present study area only in the

northeast of the country (see Dutta, 1997).

In a series of papers dealing with the ecology and

physiology of squamate reptiles, Subba Rao (1970;

1972) and Subba Rao and Rajabai (1972a; 1972b; 1974)

recorded Calotes nemoricola from Tirupati, and Subba

Rao (1994) recorded this species from the Nallamala

Hills. Whitaker and Das (1990) showed this to be erro-

neous identifications for the widespread Calotes versi-

color (Dmdin, 1802).

Subba Rao et al. (1994) reported Eutropis beddomei

(Jerdon, 1870) from all districts encompassing the

Nallamala Hills range. Recently Rao et al. (2005) also

reported it from Vijayapuri and Mallelatheertham in

Northern Nallamala Hills. This nominal species is

restricted to the Western Ghats of south-western India

and south-central Sri Lanka (Smith, 1943). As men-

tioned earlier, Rao's (2005) image 30 represents

Eutropis nagarjuni Sharma, 1969.

Rao et al. (2005) listed Cerberus rynchops

(Schneider, 1799) from Sundipenta/Sikharam, within

the Nallamala Range. This is an estuarine/coastal

species (see Das, 2002b; Whitaker and Captain, 2004),

and its record in the literature from the Eastern Ghats

Complex (e.g., Pillai and Murthy, 1983) may be from

the plains.

Sibynophis Sagittarius (Cantor, 1839) was reported

by Rao et al. (2005), based on a specimen collected from

Sunnipenta. This taxon was previously reported from the

area southeast of the Nallamala Hills, from Sriharikota

Island, Nellore District, by Rao and Sekar (1993).

Sibynophis subpunctatus (Dumeril, Bibron & Dumeril,

1 854) was recently resurrected from the synonymy of S.

Sagittarius (Cantor, 1839) by Captain et al. (2004) for

this population. A record of this species from East

Godavari District by Sanyal et al. (1993) is erroneous, as

the specimen is from Godaveri (27° 34' N and 85° 22'

E), 10 km southeast of Kathmandu, central Nepal.

Remarks on Habitat Use

Divided in habitat types, the amphibian fauna of

Nallamala Hills can be classified into: 1. Human com-

mensals or otherwise tolerant of disturbed habitats (14

species: Bufo stomaticus, Duttaphrynus melanostictus,

Kaloula taprobanica, Microhyla ornata, M. rubra,

Uperodon globulosus, U. systoma, Ramanella variega-

ta, Euphylyctis cyanophlyctis, E. hexadactylus,

Fejervarya cf. limnocharis, Hoplobatrachus crassus, H.

tigerinus, and Polypedates maculatus)', 2. Exclusively

scrub forest species (four species: Bufo scaber,

Sphaerotheca breviceps, S. dobsonii, and S. rolandae );

and 3. Exclusively mesic forest species (two species:

Hylarana sp. and Indirana leithii ). Corresponding clas-

sification for reptiles include: 1. Human commensals or

taxa otherwise tolerant of disturbed habitats (seven

species: Calotes versicolor, Hemidactylus bowringii, H.

brookii, H . flaviviridis , H . frenatus , H. leschenaultii, and

Ptyas mucosa ); 2. Exclusively scrub forest species or

from rocky biotope (21 species: Geochelone elegans,

Psammophilus blanfordanus, P dorsalis, Sitana pon-

ticeriana, Hemidactylus reticulatus, H. triedrus,

Ophisops jerdoni, O. leschenaultii, O. minor, Lygosoma

ashwamedi, L. guentheri, L. punctata, Eutropis macu-

laria, Varanus bengalensis, Eryx conicus, E. johnii,

Python molurus. Coluber bholanathi, Liopeltis cala-

maria, Sibynophis subpunctatus, and Calliophis mela-

nurus); 3. Scrub and bordering agricultural fields (22

species: Hemidactylus giganteus, Chamaeleo zeylani-

cus, Eutropis carinata, Ahaetulla nasuta, Amphiesma

stolatum, Argyrogena fasciolata, Boiga forsteni, B. trig-

onata, Coelognathus helena, Dendrelaphis tristis,

Lycodon aulicus, L. striatus, Oligodon arnensis, O. tae-

niolatus, O. travancoricus, Bungarus caeruleus, Naja

naja, Grypotyphlops acutus, Ramphotyphlops braminus,

Daboia russelii, Echis carinatus, and Trimeresurus

gramineus ); 4. Exclusively mesic forest species (three

species: Calotes rouxii, Cnemaspis sp., and Lycodon tra-

vancoricus); 5. Wetland species (10 species: Crocodylus

palustris, Melanochelys trijuga, Pangshura tentoria,

Nilssonia gangetica, N. leithii, Lissemys punctata,

Atretium schistosum, Enhydris enhydris,

Macropisthodon plumbicolor, and Xenochropis

piscator)', and 6. Habitat category unknown (one

species: Eutropis nagarjuni).

In summary, all six microhylids, two bufonids, five

ranids, one rhacophorid, in addition to one agamid, five

gekkonids and one colubrid are human commensals.

Human activities may promote creation or maintenance

of certain habitats conducive for these species (e.g.,

perennial water sources, in the form of wells, drainage

areas, etc.). Low amphibian diversity characterize scrub

forests, where community members are such as Bufo

scaber, Sphaerotheca breviceps, S. dobsonii and S.

rolandae show xeric-region and/or fossorial adaptations

(e.g., thickened skins and burrowing adaptations, such

as enlarged metatarsal tubercles on pes) and adaptations

for retaining moisture.

All turtles and crocodilians reported from the

Nallamalla Range are associated with wetlands. The sole

non-aquatic species ( Geochelone elegans) is a scrub for-

est dweller. Significant diversity of gekkonids

( Hemidactylus reticulatus and H. giganteus ), scincids

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Asiatic Herpetological Research, Vol. 1 1

123

( Lygosoma ashwamedhi, L. guentheri, L. punctata, and

Eutropis macularia ) and colubrids ( Coluber bholanathi,

Liopeltis calamaria, and Sibynophis sagittaria) are

known from the ranges, several of these endemic ( L .

ashwamedhi and C. bholanathi ) or largely restricted to

scrub forests of Peninsular India. All lacertids ( Ophisops

jerdoni; O. leschenaultii, and O. minor) and all boids

(Eryx conicus, E. johnii, and Python molurus) reported

from the Nallamalla Hills are restricted to this biotope.

Nonetheless, mesic forests remain poorly explored, per-

haps for which reason both unidentified species from the

Nallamala Ranges of amphibian ( Hylarana ) and reptile

( Cnemaspis ) may represent taxa undescribed by science.

Remarks on Adaptive Types

Adaptations seen amongst the amphibians of the

Nallamala Hills include a diversity of dietary and habitat

types. Representatives of ant specialists include all the

microhylid and most bufonid species locally represent-

ed. Additional categories include: predators of small ver-

tebrates ( Polypedates maculatus) and folivores

(Euphlyctis hexadactylus and some E. cyanophlyctis). In

terms of gross habitat usage are the fossorial (Kaloula

taprobanica, Micro hy la ornata, M. rubra, Uperodon

globulosus, and U. systoma), terrestrial ( Duttaphrynus

melanostictus, B. scaber, and B. stomaticus ), aquatic or

aquatic-margin (. Euphlyctis cyanophlyctis, E. hexadacty-

lus, Fejervarya cf. limnocharis, Hoplobatrachus cras-

sus, and H. tigerinus) and arboreal (Polypedates macu-

latus and sometimes Ramanella variegata ) species. At

least three species enter bathrooms of human dwellings

(Ramanella variegata, Kaloula taprobanica, and

Polypedates maculatus) and one (Polypedates macula-

tus) is known to apply a coat of protein on the surface of

its body prior to emerging for foraging, to prevent evap-

orative water loss. Skittering on the water surface is

known for two species (E. cyanophlyctis and juvenile E.

hexadactylus).

Adaptive types among the reptiles, when classified

by diet, include eaters of soft-bodied (e.g., ant- and

worm) prey (Grypotyphlops acutus and

Ramphotyphlops braminus)', predators of crop pests,

such as rodents (Argyrogena fasciolata, Ptyas mucosa,

and Varanus bengalensis)', predator of small or medium-

sized vertebrate prey (Python molurus, Crocodylus

palustris, Ptyas mucosa, Daboia russelii, Trimeresurus

gramineus, and Echis carinata)', egg-predators

(Oligodon arnensis, O. taeniolatus and O. travancori-

cus); primarily fish-eaters (Crocodylus palustris ,

Nils sonia gangetica, N. leithii , Atretium schistosum,

Enhydris enhydris, and Xenochrophis piscator); frog-

and toad-eaters (Macropisthodon plumbicolor and

Dendrelaphis tristis) and near-exclusive snake-eaters

(Bungarus caeruleus and Calliophis melanurus).

In terms of habitat usage, reptiles exceed amphib-

ians in species richness, on account of their greater

capacity of surviving in relatively arid regions. The

regional gekkonid diversity, within the genus

Hemidactylus includes arboreal (H. bowringii, H.

brookii, H. flaviviridis, H. frenatus, and H.

leschenaultii), terrestrial (H. triedrus) and semi-fossori-

al (H. reticulatus) types. Usage of specific habitat types

include walls of houses (Hemidactylus bowringii, H.

brookii, H. flaviviridis, H. frenatus, and H.

leschenaultii), rupicolous habitats such as rocky boul-

ders (Psammophilus blanfordanus and P. dorsalis)', fos-

sorial habits in terms of usage of soft substratum for bur-

rowing (Grypotyphlops acutus and Ramphotyphlops

braminus)', and arboreal species, utilizing trees or some

sort of vegetation (Ahaetulla nasuta, Boiga forsteni, B.

trigonata, Dendrelaphis tristis, Lycodon aulicus,

Calotes rouxii, Chamaeleo zeylanicus, and Lycodon tra-

vancoricus; the typhlopid Ramphotyphlops braminus is

also known to occasionally ascend trees in search of

prey). Other adaptive types shown by the fauna include

vegetation mimics (Chamaeleo zeylanicus and

Ahaetulla nasuta)', Batesian mimicry is shown by

Sibynophis subpunctatus (for which the model presum-

ably is Calliophis melanurus)', bipedal locomotion

(Sitana ponticeriana)', and side-winding (Echis carina-

tus, when moving on sand or other loose substrate).

Biogeography of the Eastern Ghats

The Eastern Ghats remain the poor sister of the more

well-known Western Ghats, a recognized global hotspot

of biological species diversity (e.g., Ward, 2002). Inger

(1999) lamented about the low species richness of the

amphibian fauna of the Eastern Ghats (21 species),

while Das (1996) reported 84 species of reptiles, both

significantly different from the known diversity of the

Western Ghats, which has seen an explosion of new as

well as spectacular species discoveries in recent years

(see Biju, 2001; Biju and Bossuyt, 2003). Nonetheless,

enough documentation exists to reveal a highly diverse

biota of the hill ranges that run approximately parallel to

the east coast of India.

The range itself is a weathered relict of the peninsu-

lar plateau, characterized by a series of low hills that

extend from the Khondmal Hills of Orissa State, south to

the Shevaroys of central Tamil Nadu, where they meet

the Western Ghats in the Nilgiris region (descriptions in

Das, 1996; Mani, 1995). The northern and southern sec-

tions of the Eastern Ghats are separated by the delta of

the River Godavari, which is approximately 130 km in

width. Other important breaks are formed by the

drainages of the rivers Mahanadi and Krishna. The

Biligirirangan Hills, at 1,750 m, is the highest range in

the Eastern Ghats. Moisture regimes show a general gra-

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2008

dient, Irom a relatively mesic northern range, with dry

and moist deciduous forests, to a relatively dry southern

subzone, with dry deciduous and thorn scrub (vegeta-

tional analysis in Legris and Meher-Homji, 1983).

Detailed analysis of faunal relationships along these hill

ranges, including comparative diversity of lineages as an

effect of breaks in the continuity of the ranges, humidity,

and elevational effects remain to be conducted.

We adopt Wikramanayake et al.'s (2002) ecoregion-

al approach to interpreting the distribution of the region-

al herpetofauna. These workers have classified the Indo-

Pacific Region (stretching from Afghanistan in the west

to New Guinea and the Solomons to the east), recogniz-

ing 129 ecoregions on the basis of vegetation, geology

and geological history. Within this framework, the

Nallamala Range falls within the Deccan Thom Scrub

zone (Ecoregion 23), abutting (and being influenced by)

Ecoregions 21 (Central Deccan Plateau Dry Deciduous

Forest); Ecoregion 22 (South Deccan Plateau Dry

Deciduous Forest); and Ecoregion 6 (East Deccan Dry-

Evergreen Forest). Although the Nallamala Hills also are

adjacent to Ecoregion 34 (the Godavari-Krishna man-

groves), herpetofaunal influences are absent, on account

of geological-vegetational differences.

The herpetofauna of the Eastern Ghats has a long

history of exploration, commencing with Patrick Russell

(1727-1805), the first Western herpetologist in India,

and medical doctor and naturalist with the British East

India Company, based at Vizagapatam (at present

Visakhapatnam). Russell explored the herpetofauna, pri-

marily snakes, of that region and produced a two volume

folio of water colors of snakes (also including Barkudia

melanosticta (Schneider, 1801), which was published in

1796 and 1801-1802.

Collections for faunistic inventories within the

Eastern Ghats complex have also been made by McCann

(1945), Pillai and Murthy (1983), Daniels and Ishwar

(1993), besides the contributions of the Zoological

Survey of India in the Nallamala Hills referred to earlier.

Rao and Rao (1998) studied the ecology of Barkudia

melanosticta (as B. insularis)\ Bauer and Das (2000)

studied the ecology of Calodactylodes aureus in Vellore;

Das and Chanda (1998) described a new species of

Philautus from the Visakhapatnam region; Dutta (2003)

described a new Philautus from Simlipal Hills; and Das

and Bauer (2000) described two new species of

gekkonid lizards of the genus Cnemaspis from the

Eastern Ghats.

Although less species rich than the more mesic adja-

cent regions, Ecoregion 23 supports a distinctive her-

petofauna, including arid region representatives whose

relatives are Eurasian and Afro-Ethiopian (e.g.,

Chamaeleo, Ophisops, Eryx, and Echis) and the region

also supports lineages that may be termed distinctly

autochthonous (i.e., Indian lineages, such as the genera

Uperodon, Ramanella, Indirana, Sphaerotheca,

Melanochelys, Pangshura, Nilssonia, Psammophilus,

Sitana, Argyrogena, Atretium, and Grypotyphlops ). The

presence of representatives of Indo-Malayan elements

represented here (e.g., Kaloula, Hylarana, Calliophis,

and Trimeresurus ) are explainable using Hora's (1949)

Satpura Hypothesis model, of emigration of the biota of

the Indo-Malayan region westwards. Alternative models

are available to explain the presence of these taxa in the

Eastern Ghats, including a more mesic climate in the

Indian Subcontinent up to the Eocene (van der Hammen,

1983). The climatic changes were perhaps accelerated

by widespread agriculture, specifically through the cul-

tivation of graminaceous crops (Misra, 1983), helping

further in the conversion of what was once tropical sub-

humid and dry deciduous forests into savannas.

Within the Eastern Ghats herpetofauna, endemic

genera include the limbless skinks, Barkudia (with two

species, B. insularis and B. melanostictus; see Das,

2000) and Sepsophis (a monotypic genus, containing S.

punctatus). A number of species hitherto considered

endemic to the Western Ghats have in recent years been

found within the Eastern Ghats complex, including

Indirana leithii (this report), Hylarana malabarica

(Tschudi, 1838) by Daniel and Selukar (1963), a mem-

ber of the genus Hylarana (this report) and Lygosoma

guentheri (Peters, 1879) (this report). Balachandran and

Pittie (2000) reported the occurrence of Draco from

these hills, that they allocated to D. dussumieri Dumeril

& Bibron, 1837, a Western Ghats species. Eastern Ghat

endemics found in the Nallamala Range include

Hemidactylus reticulatus, Eutropis nagarjuni,

Lygosoma ashwamedhi, and Coluber bholanathi. New

species have been described from these ranges in recent

years, including the geckos Cnemaspis otai and C. yer-

caudensis (see Das and Bauer, 2000).

Several species known from the Eastern Ghats have

not (yet) been recorded from the Nallamala Ranges.

Some may be regional endemics or appropriate habitats

may be missing on the site under study, although the

absence of some (e.g., Calodactylodes aureus), that are

known from both north and south of the range here rein-

force the argument for more sampling of the fauna.

Other Eastern Ghat endemics (e.g., Barkudia, with two

species, the monotypic Sepsophis, and

Hemiphyllodactylus aurantiacus ) among the reptiles,

and Philautus terebrans and Ichthyophis peninsularis

occur in adjacent ranges of the Ghats (see Das and

Chanda, 1998; Pillai and Murthy, 1983), and with fur-

ther collection, may prove their presence here, or be rep-

resented by hitherto unknown sister species.

2008

Asiatic Herpetological Research, Vol. 1 1

125

Conservation and Management

Parts of the Nallamala Range are within the Protected

Areas System, the levels of protection for each compo-

nent varying trom Forest Reserves, that lie within the

jurisdiction of the Andhra Pradesh Forest Department, to

National Park, that are gazetted and their protection

implemented by the Central (= Federal) Government.

The most well-known of the protected areas is the

Nagarjunasagar Srisailam Tiger Reserve and the recent-

ly gazetted Gundla Brahmeshwaram Metta Wildlife

Sanctuary.

Conservation of amphibians and reptiles represent

special challenges, for which reason, arguments have

been made to move away from species-based conserva-

tion strategies, to that addresses entire landscapes. Given

the large number of known components of the biodiver-

sity of these Protected Areas, especially non-

homoeothermous members (or non-mammal and bird

species), and the general lack of expertise to identify, let

alone understand, conservation requirements, this is

apparently a safer approach to the conservation of biodi-

versity. The situation is not unique to the Eastern Ghats:

in the Indo-Pacific region, centinelan extinction (or

species loss even before their formal description) is

known for both amphibians and reptiles (Das, 2002a;

Erdelen, 2002).

A handful of the recorded species from the

Nallamala Range are human commensals, or so-called

'weed-species', including, amongst amphibians:

Duttaphrynus melanostictus, B. stomaticus, Kaloula

taprobanica, Micro hy la ornata, M. rubra, Uperodon

globulosus, U. systoma, Ramanella variegata,

Euphylyctis cyanophlyctis, E. hexadactylus, Fejervarya

cf. limnocharis, Hoplobatrachus crassus, H. tigerinus,

and Polypedates maculatus. Scrub species of amphib-

ians include: Bufo scaber, Sphaerotheca breviceps, S.

dobsonii, and S. rolandae). Human-commensals among

the reptiles include: Melanochelys trijuga, Lissemys

punctata, Calotes versicolor, Hemidactylus bowringii,

H. brookii, H. flaviviridis, H. frenatus, H. giganteus, H.

leschenaultii , Lygosoma punctata, Eutropis carinata,

Amphiesma stolatum, Dendrelaphis tristis, Lycodon

aulicus, Ptyas mucosa , Naja naja, and Ramphotyphlops

braminus. Scrub species of reptiles include: Geochelone

elegans , Sitana ponticeriana, Chamaeleo zeylanicus,

Hemidactylus reticulatus , H. triedrus, Ophisops jerdoni,

O. leschenaultii, O. minor, Lygosoma ashwamedhi, L.

guentheri, Eutropis nagarjuni, Coluber bholanathi,

Liopeltis calamaria, Calliophis melanurus, Daboia rus-

selii, and Echis carinatus. Two species are exclusively

rupicolous ( Psammophilus blanfordanus and P dor-

salis). And only two species are considered mesic region

taxa, in that their respective congeners are exclusively

distributed in such areas (e.g., Hylarana and

Cnemaspis ).

Human-commensals generally refer to species tol-

erant of environments altered by humans. However,

many still have life histories intimately dependent on

certain habitat features, such as ponds or other standing

bodies of water, substrates that serve as burrowing refu-

gia, etc. Changes from rural to urban environments are

known to cause local extinction of amphibian species

(including Sphaerotheca ), through the removal of such

habitats, as observed in the Chennai region (Das,

unpubl.).

Three herpetofaunal species from the Nallamala

Hills are recognised as threatened, under the Red List

categories of the IUCN (World Conservation Union; see

Hilton-Taylor, 2000). These include the turtles,

Nilssonia gangetica and N. leithii, and the crocodilian,

Crocodylus palustris (all in the 'Vulnerable' category).

In the end, species protection in countries such as

India, where the pressure on land and water are large,

can only be assured in areas within protected areas. It is

therefore imperative to bring additional areas of these

hills with high diversity and/or faunal endemicity into

the country's protected areas system.

Acknowledgments

The first author thanks the successive Heads of the

Department of Zoology, Osmania University for facili-

ties, and a Research Grant (Award No. 9/132(626)/2002

EMR-I) by Council for Scientific and Industrial

Research, New Delhi. We acknowledge Andhra Pradesh

Forest Department for research and collection permits,

and logistics. The second author is grateful to the

Institute of Biodiversity, Universiti Malaysia Sarawak,

for supporting his researches on the herpetology of Asia.

We also thank M. S. Ravichandran, ZSI Kolkata, for

curatorial assistance, and Genevieve V. A. Gee,

Bhargavi Srinivasulu and an anonymous reviewer for

comments on a draft manuscript.

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pp. 132-135

Asiatic Herpetological Research, Vol. 1 1

2008

Spermatogenesis Timing in a Population Ophisops elegans

(Sauria: Lacertidae), Western Iran

Farhang Torki1’* and Ahmad Gharzi2

xFarhang Torki Ecology and Herpetology Center for Research (FTEHCR), 68319-16589,

P. O. Box: 68315-139 Nourabad City, Lorestan Province, Iran,

: Department of Biology, Faculty of Science, Lorestan University, 68135 Khorramabad, Iran.

* Corresponding author E-mail: torkifarhang@yahoo.com

Abstract.- During biological activity, specimens of Ophisops elegans were collected in western Iran, from March to

November. Testis were removed and H&E techniques were used for histological study. The results show three phases

in spermatogenesis timing as follows: (a) active phase, spermatogenesis in all specimens is active, (b) transitional

phase, spermatogenesis in many specimens are active an in other is inactive, and finally (c) inactive phase, spermato-

genesis in all specimens is inactive.

Keywords.- Spermatogenesis timing, testicular cycle, Ophisops elegans , Zagros mountains, western Iran.

Introduction

Lizards show two type of spermatogenesis; continuous

and alternate (Torki, in press a, b). In the continues type,

spermatogenesis is year-round and spermatozoa are

foudn in the lumen of the seminiferous all year (e.g.,

Hemandez-Gallegos et al., 2002; Sherbrooke, 1975;

Vieira et al., 2001). In contrast, the alternate type of

spermatogenesis occurred during a well defined period

in which spermatozoa were not found in the lumen of

seminiferous (Castilla and Bauwens, 1990; Fitch, 1970;

Torki, 2006, in press a, b, c). Continuous spermatogen-

esis occurs in tropical regions (Fitch, 1970; Hemandez-

Gallegos et al., 2002; Vieira et al., 2001), this region

limited by author into ITCZ region (Torki, 2006).

Alternate spermatogenesis occurred in non-tropical

regions, especially in temperate zones (Castilla and

Bauwens, 1 990; Torki, 2006). In the temperate-zone, the

male testicular cycle is divided into two well-defined

phases as follows: (a) the regenerative phase that occurs

in the spring and is characterized by sustained sperm

production, and (b) the degenerative phase, that begins

in late summer, where a break in spermatogenesis is

observed (Castilla and Bauwens, 1990; Fitch, 1970;

Lofts, 1987; Torki, in press b). Likewise, tropical species

in seasonal habitats also display, if less pronounced, a

regenerative phase during the wet (reproductive) season

2008

Asiatic Herpetological Research, Vol. 1 1

133

DF 1

Figure 1. Shows phase significant during degeneration

period based on DF analysis.

and a degenerative phase during the dry (non-reproduc-

tive) season (Marion and Sexton, 1971; Wilhoft and

Reiter, 1965).

Spermatogenesis of some lizards in Iranian plateau

especially in Zagros Mountains described by author

(Torki, 2006, in press a, b, c), and shows alternation

spermatogenesis in Zagros Mountains. In this study, my

purpose is determination spermatogenesis timing in

Ophisops elegans in Zagros Mountains.

Materials and Methods

Seventy-five mature male specimens of O. elegans were

collected by hand north of Lorestan province. The size

of males O. elegans is between 38.9 < SVL < 47.2 mm.

O. elegans go to hibernation period from Oct. to Feb.

(Torki, 2005). Testis were removed from each individual

by dissection, during each month from after hibernation

to before hibernation. Snout-vent lengths (to the nearest

Figure 2. Show annual period and phase in spermatoge-

nesis of O. elegans in western Iran, central Zagros.

0.5 mm) were measured for each lizard. In each lizard

maximum length and width of the left testis was meas-

ured (with electronic calipers to the nearest 0.01 mm)

and Testis Volume (TV) and estimated TV (0.1 mm3)

using the ellipsoid formula; v = 4/3n abc, where v is vol-

ume, a and c are equal to half testicular height, and b is

half testicular length (Vieira et al., 2001; Torki, 2006).

For histological analysis, testes were fixed and the epi-

didymis was fixed in 3.7% formalin, dehydrated in a

graded series of ethanol, cleared in xylem, and embed-

ded them in paraffin. Sections were stained with hema-

toxylin-eosin (H&E) and were observed with an Zeiss

Axiophoto microscope. For each individual, two char-

acters (pm) were measured: the Lumen of Seminiferous

(LS) diameter, Germinative Seminiferous (GS) diame-

ter. For data collecting, using the mean of twenty trans-

versally oriented tubules at the same section, next to the

core of the testes. Measurements were taken with an

ocular micrometer, to the nearest 1 pm. Same as author

study (Torki, 2006, in press a, b, c), Tukey HSD test and

Canonical Discriminant Functions Analysis (DFA) to

show phases significance were used.

Table 2. Shows Tukey FISD test (a = 0.05) for determina-

134

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2008

Figure 3. Light microscopy shows active phase, histological section of (a) seminiferous, (b) epididymis. LE, Lumen of

Epididymis, E: Epididymis layer, LS: Lumen of Seminiferous, GS: Germinative layer of Seminiferous, S: Spermatozoa,

SI: primary Spermatocytes, S2: secondary Spermatocytes, AM: Amorphous Material, 1C: Interstitial tissue Cell.

follows: phase (1) from Mar. to Jul., phase (2) during

Jun. to Jul., and phase (3) from Aug. to Oct.

Discussion

In this study, there was no significant relationship

between SVL*Month (p > 0.05), because adult speci-

mens were collected. Based on statistical and histologi-

cal study, I presented three phases (Fig. 2) during the

degeneration period in O. elegans; active, transitional

and inactive phase, (a) Active phase: because spermato-

zoa in the lumen of seminiferous and epididymis are

found (Fig. 3), this phase occurred from Mar. to May. (b)

Transitional phase: because spermatozoa in many speci-

mens found in lumen of seminiferous and epididymis

and in other specimens not found, this phase occurred

from Jun. to Jul. (c) Inactive phase: because in all spec-

imens lumen of seminiferous and epididymis is without

spermatozoa, this phase occurred during pre-hibernation

or from Aug to Oct. Same as O. elegans, Trapelus

lessonae show three phases (Torki, 2006, In Press c).

Two species ( O . elegans and T. lessonae) show synchro-

nism in three phases during degeneration period. In both

species (T. lessonae and O. elegans ) spermatogenesis

activity occurred during post-hibernation. In contrast, in

the agama, Laudakia nupta spermatogenesis occurred

after post hibernation in late spring and early summer

(Torki, In Press b). Body length of T. helenae is lowest

than other taxa and body length in L. nupta is biggest

other taxa and O. elegans with T. lessonae is between to

other. Based on timing of spermatogenesis activity and

body length, three types of activities of spermatogenesis

timing in lizards inhabitant Zagros Mountains as fol-

lows: (a) early active spermatogenesis, that occurred in

lowest body length, (b) late spermatogenesis that

occurred in highest body length, and finally normal

active spermatogenesis that occurred in T. lessonae and

O. elegans. Three taxa ( L . nupta, T. lessonae and O. ele-

gans) are sympatric; therefore, divergeny in timing sper-

matogenesis occurred due to body length. This is pro-

nounced confirmed by T. helenae as a lowest body

length. Torki and Rastegar-Pouyani briefly report affects

of body size to timing of spermatogenesis (2006).

However timing of spermatogenesis is many lizards is

different, but histological structure in these lizards is

similar (Gharzi and Torki, 2006 a). Nevertheless, in

many lizards timing of spermatogenesis activity

occurred due to climate condition (e.g., Duvall et ah,

1982; Fitch, 1970; Whittier et al., 1987). Nevertheless,

climate condition and geographic position are two main

factors that strongly regulated spermatogenesis timing in

lizards (Gharzi et al., 2006; Torki, 2005 b, in press a). On

the other hand, geographic variation in timing spermato-

genesis activity occurred due to climate gradient or lati-

tude gradient (Gharzi and Torki, 2006 b; Torki, in press

a). In many lizards such as genus Trapelus divergeny in

spermatogenesis activity occurred in T. lessonae and T.

agilis, is related to climate condition. In additional, spe-

ciation process due to dispersal or vicariance evidence in

two taxon of genus Trapelus are important factors for

the divergeny in timing of spermatogenesis activity

(Torki, 2006).

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Submitted: 23 June 2006

Accepted: 23 September 2007

pp. 136-142

Asiatic Herpetological Research, Vol. 1 1

2008

An Assessment of Solitary and Arribada Nesting of

Olive Ridley Sea Turtles (Lepidochelys olivacea) at the

Rushikulya Rookery of Orissa, India

Basudev TrIPATHY1’2

1 Department of Environmental Sciences, Andhra University, Visakhapatnam - 530 003, India;

2 Wildlife Institute of India, PO Box 18, Chandrabani, Dehradun - 248 001, Uttraranchal, India.

Corresponding author E-mail: tripathyb@yahoo.co.uk

Abstract.- The solitary and arribada population of Olive Ridley Sea Turtles at the Rushikulya rookery of Orissa of

India was monitored for two nesting seasons (2003-04 and 2004-05). Mass nesting population census of turtles was

carried out using standard IUCN/SSC Marine Turtle Specialist Group recommended statistical technique (number of

turtles counted: n = 11024). Curved carapace measurements of egg laying females were recorded (67.16±3.65). There

was a reduction in the size class of nesting females as compare to the data available on turtle morphometry from

Orissa coast in the last decade. The sporadic nesting was documented at the rookery from December to April with a

peak in March and with no major intermediate nesting activities in between. The mass nesting census differs greatly

as compare to the nesting figures projected by the state wildlife authority. While the state wildlife authority projects

a higher figure of nesting turtles, the actual number of turtles that nests during arribada is quite low. Continuous mon-

itoring of the beach for assessment of solitary nesting activities along with accurate methods of mass nesting census

is required for proper assessment of the Olive Ridley Sea Turtle population at the Rushikulya rookery of Orissa.

Keywords.- Lepidochelys olivacea, solitary, arribada, estimation, technique, India.

Introduction

Nesting of Olive Ridley Sea Turtles (Lepidochelys oli-

vacea) takes place either in solitary or in great simulta-

neous aggregations (mass nesting) where upto 100,000

females come onto the beach to lay their eggs also pop-

ularly known as arribada; a Spanish term meaning mass

arrival (Pritchard, 1997). Orissa, a state in India along

the easten coast, harbours three major arribada sites viz.

Gahirmatha, Devi and the Rushikulya rookery (Pandav

et al., 1994; see Fig. 1). Besides solitary nesting all

along the coast of Orissa, more than a hundred thousand

turtles are believed to nest annually at Gahirmatha (Dash

and Kar, 1 990) and tens of thousands nest at other two

locations, i.e. Devi and the Rushikulya rookery (Kar,

1982; Pandav et al., 1994). In spite of its biological

importance, solitary nesting has never been evaluated

adequately in many important nesting rookeries (Castro,

1986). Although some information is available on soli-

tary nesting of L. olivacea at Gahirmatha and Devi rook-

ery (Pandav, 2000), there is little or no information on

solitary nesting activities at the Rushikulya rookery.

Similarly, the mass nesting events at the Rushikulya

rookery have not yet been monitored properly; current

data are from anecdotal accounts (Pandav et al., 1994)

and the imprecise census by the Orissa State Forest

Department due to improper statistical techniques

(Pandav, 2000; Shanker et al., 2003;

http://www.wildlifeorissa.org). The Orissa State Forest

Department have reported mass nesting at this rookery

every year since 2001, but accurate estimates of the

number of nesting turtles in arribadas are not available

in the absence of a standard technique for mass nesting

census (Patnaik et al., 2001; Shanker et al., 2003). The

IUCN/SSC Marine Turtle Specialist Group (MTSG) has

recommended for use of strip transect method for esti-

mating the arribada on mass nesting beaches, on the

basis of successful experiment by Valverde and Gates

(1999).

The Olive Ridley Sea Turtle is an endangered

species according to the protection status of IUCN and

as per CITES prohibited for trade of any kind and also is

included in the schedule I of Indian Wildlife (Protection)

Act (1972) and is legally protected. Flowever, over the

past decade, more than 100,000 dead turtles have been

reported along the Orissa coast due incidental and acci-

dental fishing related casualties in the sea. Whether this

mortality has an impact on the population size of L. oli-

vacea is yet to be known (Pandav and Choudhury,

2000). In this paper, in light of its importance, the spo-

radic nesting and mass nesting census of L. olivacea at

the Rushikulya rookery was evaluated for two season

from November to April (2003-04 and 2004-05) using

standard techniques recommended by the MTSG to

ascertain the actual arribada nesting population of turtles

at this rookery and compare this figure with estimates

from the Orissa State Forest Department.

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Asiatic Herpetological Research, Vol. 1 1

137

INDIA

Map hd t to scale

Figure 1. Map of Orissa coast in India with three arribada sites.

Bay of Bengal

Scale 125.000

Palur canal

Purunabandha*

Figure 2. Map of Rushikulya sea turtle rookery, Orissa.

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 1 . Sporadic and intermediate nesting of L. olivacea

at the Rushikulya rookery of the Orissa coast.

2003-04 2004-05

Being a migratory species L. olivacea are known to

arrive in the Orissa coast during late October and remain

in the coastal waters until May and thereafter migrate

back to the southern Bay of Bengal and Indian Ocean

area. There are no turtle activities after April/May until

October in Orissa. Therefore, the field work was con-

centrated at Rushikulya between November and May.

The mass nesting beach at the Rushikulya rookery is

located on the sand spit along the northern end of the

Rushikulya River mouth. Rushikulya is situated 320 km

south of Gahirmatha mass nesting beach (Lat. 19° 22' N

and Lon. 85° 02' E). Turtle nesting at Rushikulya takes

place along a stretch of ~5 km immediately north of the

Rushikulya River mouth from the village Purunabandha

(1 km north of the Rushikulya River mouth) to

Kantiagada village (Fig. 2).

For systematic coverage, the entire stretch of nest-

ing beach was divided into 100 m segments and was

marked with wooden poles. To monitor nesting activi-

ties, patrolling was done by foot every night between

1700 and 0700 hr from November to April of 2004 and

2005 (1st November to 30th April for both years). Sea tur-

tles are known to nest along the Rushikulya rookery

towards the end of December (Basudev Tripathy, per-

sonal observation) and therefore the chance of missing

out of some crawls during the nesting season was mini-

mal. Turtle crawls onto the beach were classified into

nesting and non-nesting types based on crawl mark pat-

tern and sign of nest (Schroeder and Murphy, 1999).

There is no standard classification of solitary nesting or

arribada nesting based on the number of nests per night.

However, keeping the beach length of the study area (~5

km) in mind, the author classified night with less than 20

nesters (~4 nests/km) as solitary nesting, nesting densi-

ties of 20 to 99 turtles (<20 nests/km) were considered

intermediate nesting, while those with >100 (> 100/km)

or above turtles as arribada nesting. A modified strip

Table 2. Sea turtle ( L . olivacea) mass nesting census at

the Rushikulya rookery.

transect method was used to estimate the mass nesting at

the Rushikulya rookery (Valverde and Gates, 1999).

This method was effective in arriving at an estimate of

the number of nesting females, with a mean, variance

and confidence intervals that provide rigorous statistical

support for the results. A 20 m strip transect was laid at

every 100 m segment of the nesting beach. Only egg-

laying females (turtles in oviposition) within the strip

were counted on hourly intervals starting with the first

individual ascending the beach in the evening until

morning when there were no nesting activities.

The formula below was used for computation of

the mass nesting data (see Valvarde and Gates, 1999):

Estimate of nesting = — ft * H x N —

Wxtx Lx h

Where:

A

H

N

W

t

L

h

Total available nesting area (in m2)

Duration of arribada (in minutes)

Total of number of egg laying turtles

Width of the transect (in m)

Number of sampling period (in days)

Total of length of all transects (in m)

Average time spent by turtles for egg

laying (in minutes)

Size of female L. olivacea was determined by the

measurement of curved carapace length (CCL) at the

time of egg lying. Each turtle was measured down the

midline from the nuchal notch to the posterior carapace

2008

Asiatic Herpetological Research, Vol. 1 1

139

450

December January February March April

Months

Figure 3. Monthly turtle nesting at Rushikulya for L. oli-

vacea (2003-04 and 2004-05).

tip using a flexible measuring tape. Values were rounded

to the nearest 0.5 cm.

Of the 568 nests observed during the 2003-04 nest-

ing season between December 2003 and April 2004,

45.2% of the nesters were intermediate nesters, with rest

being sporadic nesters (Table 1). Similarly, during the

2004-05 nesting season, intermediate nesting was calcu-

lated to be 33.3% (Table 1). There was a distinct pattern

of solitary nesting observed at Rushikulya rookery, with

a peak in activity during March for both the season (Fig.

3). .

A total of 1 5 and 20 transects with 20 m width and

100 m length were established for counting turtles in

arribadas during 2004 and 2005 respectively (Table 2).

During 2005, the topography of the beach changed dras-

tically and mass nesting was extended from the estuarine

mouth and 2 km northward, and therefore, five more

transects were laid. Although arribada took place twice

during 2004 (February 9-10 and March 10-13), the

February arribada could not monitored due to logistic

constraint and hence the census was done only for the

March 2004 arribada. During four days of peak nesting

in March, a total of 23,461 turtles were estimated to

have nested in arribada. However, the 2005 arribada was

larger and was estimated to be 86,688 nesting individu-

als in two nights when censuses were carried out (Table

3).

Nesting females in arribadas at the Rushikulya

rookery had an average CCL of 67. 163.65 cm (n = 5 1 5;

min: 60.8, max: 73.61), a value slightly greater than that

of the solitary nesters at 66.024.34 cm (n = 335; Mann-

Whitney = 505, p = 0.0004). No significant differences

in CCL existed for arribada of February and March 2004

(Kruskal-Wallis %2= 6.9, p = 0.2412) and also between

2004 and 2005 season (Kruskal-Wallis, 5.80, p =

0.1225).

Solitary nesting emergence of L. olivacea is known

to occur almost every month along rest the Orissa coast

(Dash and Kar, 1990). However, solitary nesting is

found in greater numbers during January to May, indi-

cating that this is the main nesting season for this species

(Pandav and Choudhury, 2000). Although year round

sporadic nesting is not known from the Rushikulya

rookery, this study confirms sporadic nesting of olive

ridley turtles at the rookery between December and

April, with a peak in March and is identical to other sea

turtle rookeries along the Orissa coast. Temperature,

weather condition, physiography of nesting beaches and

the adjacent sea, conditions of tide, temperature and sur-

face current circulation all play an important role in

determining female nest selection (Pandav and

Choudhury, 2000). However, this study could not incor-

porate the above variables at the Rushikulya rookery due

to logistic constraints. Unlike Gahirmatha and Devi

(Basudev Tripathy, personal observation) where spo-

radic nesting is almost continuous for the entire season

(> 10 turtles/night), at Rushikulya rookery, sporadic nest-

ing is irregular, with nesting intensity increasing before

the commencement of the arribada. During the other

nights, there is either no nesting or low sporadic nesting

(<5 turtles/night). However, it is likely that the females

emerging on nights with intermediate levels of nesting

are responding to arribada cues (cue such as southerly

strong wind, cloudy weather and strong wave action in

the sea) and are truly arribada nesters. What actually

comprises solitary or arribada nesting must also be eval-

uated in light of the total population for a given beach

(Dash and Kar, 1990). During the present study, there

was no major intermediate nesting events observed at

the Rushikulya rookery except for nine nights in 2004

February and six nights during 2005 March, when nest-

ing per night was over 100. However, it is likely that

these turtles were early arribada nesters, since the arrib-

ada commenced in the rookery few days later.

At the Rushikulya rookery, although arribadas were

reported for many years, precise mass-nesting censuses

have not been carried out. The Orissa Forest Department

report estimates the number of turtles during the arriba-

da every year, but the methods used are unpublished and

unavailable (estimated by Orissa State Forest

Department; Table 4). Furthermore, it is not clear that

methods are standardized, unbiased and therefore com-

parable. The State Forest Department staff counts all

female turtles that remain on the beach during arribada.

However, during arribada emergence, many turtles do

not deposit their eggs (-30-40%) and hence are not part

of true nesting population. While estimating nesting

arribada population, this factor greatly affects the popu-

lation size estimation and leads to bias. In the past 25

years, a variety of approaches and methods have been

used to estimate female populations at arribada beaches

140

Asiatic Herpetological Research, Vol. 1 1

2008

Table 3. Estimates of arribada (nesting number) for the

2004 and 2005 nesting season.

of Orissa (reviewed by Shanker et al., 2003). The pres-

ent study estimated very low nesting populations during

arribadas (using the standard technique as suggested by

the IUCN/MTSG (Valvarde and Gates, 1999) compared

to the figures projected by the Orissa state forest depart-

ment (Tables 3 and 4). While projection of a large nest-

ing figure attracts attention, particularly to the national

and international media and conservation communities

at large, it may results in the downgrading of this species

in the Indian Wildlife (Protection) Act and IUCN’s Red

List.

In recent years (at least between 1996 and 2000), a

small but significant decrease in curved carapace length

(CCL) of female Olive Ridley Sea Turtles has been doc-

umented (Pandav et al., 1994; Kalb, 1999). Similarly,

the average CCL of females at Gahirmatha from 1978 to

1985 were larger than those measured during

1996-2000 (Dash and Kar, 1990; Pandav and

Choudhury, 2000). The present study found that arribada

nesters are significantly larger than the solitary nesters,

with a mean CCL being 1 . 1 4 cm greater, but was within

the range. The decrease in size class (as compare to

1996-2000) was not detected during the current study,

but this could be due to a small sample size, the lack of

sufficient data, and a less accurate measuring technique

(measuring tape for CCL Vs metallic calipers for SCL).

In conclusion, it is apparent that the solitary and

arribada nesters are not different, but from the same pop-

ulation stock. The genetic study on Olive Ridley Sea

Turtles from Orissa also supports this view (Shanker et

al., 2004). Also, sporadic nests contribute equally to the

population recruitment as that of arribada nesters being

hatching success is higher for the later (Castro, 1986,

also see Tripathy et al., 2003). Olive Ridleys along the

Orissa coast are known to exhibit fidelity to their breed-

ing as well as nesting ground (Dash and Kar 1990,

Pandav et al., 2000). Nesting females are known to

exhibit movement between rookeries in Orissa both

within and between seasons (Tripathy and Pandav, in

press; Pandav and Choudhury 2000). Migration and

inter-rookery movement by females during the breeding

and nesting season along Orissa coast has also been doc-

umented (Pandav and Choudhury, 2006). Hence, knowl-

Table 4. Estimates of arribada at the Rushikulya rookery by the Orissa Forest Department and other researchers

2008

Asiatic Herpetological Research, Vol. 1 1

141

edge ot the location and temporal use of nesting grounds

ot Olive Ridley Sea Turtles in Orissa is important in

view of the habitat loss and large-scale mortality of tur-

tles in the offshore waters. Therefore, along with protec-

tion of arribada nesters, it is necessary to monitor the

beach and safeguard the sporadic nesters and their habi-

tat as well.

Prior to this study, there were no proper estimates of

the number of turtles that nest during arribada at the

Rushikulya rookery. Our estimates show that the num-

ber of turtles could be much less than what is projected

by various governmental agencies. Thus, declaration of

a mass nesting population in broad terms (e.g. hundreds

of thousands) without a proper assessment may result in

the reduction of protection required for L. olivacea in

their breeding ground, which is already meager. Hence,

standard and accurate techniques for mass nesting cen-

sus are urgently required for additional years for moni-

toring the status and nesting trends of L. olivacea at the

in Rushikulya rookery of Orissa. As evidence from the

last decade of sea turtle mortality data from Orissa

(Pandav and Choudhury, 2000), reduction of the size

class of individuals participating in arribadas (Shanker

et al., 2004; Tripathy, 2005), and elimination of the older

females from the breeding stock over a period of time.

However, to confirm this, extensive and accurate meas-

urements of the nesting females and clutch sizes need to

be performed to determine if there is a difference/reduc-

tion in size of L. olivacea over the years at rookeries in

Orissa, and thereby a declining of the Olive Ridley Sea

Turtle population of the Indian Ocean area and or the

rest of the world.

Acknowledgments

This study was part of the author’s doctoral work and he

is thankful to Dr. P. S. Raja Sekhar and the Head of the

Department of Environmental Sciences, Andhra

University, Visakhapatnam for approving the work and

support as well as the Orissa Forest Department for per-

mission to conduct field work. The author is thankful to

field assistants and volunteers of Purunabandha village,

Ganjam for their help in data collection during arribadas.

Literature Cited

Castro, J. C. 1986. Contribucion de las tortugas loras

solitarias, Lepidochelys olivacea , en el manteni-

miento de esta especie. Tesis de Licenciatura.

Universidad de Costa Rica.

Dash, M. C. and C. S. Kar. 1990. The Turtle Paradise:

Gahirmatha. Interprint, New Delhi. 295 pp.

Kalb, H. J. 1999. Behavior and physiology of solitary

and arribada olive ridley sea turtle ( Lepidochelys

olivacea) during the intemesting period. Ph.D.

Dissertation, Texas A & M University, USA. 200

pp.

Kar, C. S. 1982. Discovery of second mass nesting

ground for Pacific ridley sea turtles in Orissa, India.

Marine Turtle Newsletter 23:3.

Orissa State Forest Department, Government of Orissa.

http://www.wildlifeorissa.org.in.

Pandav, B. and B. C. Choudhury.2000. Conservation

and management of olive ridley sea turtle

(Lepidochelys olivacea) in Orissa. Project Final

Report, Wildlife Institute of India. 77 pp.

Pandav, B. 2000. Conservation and management of

olive ridley sea turtle (Lepidochelys olivacea) along

the Orissa coast. Unpublished PhD thesis, Utkal

University, Orissa, India. 148 pp.

Pandav, B. and B.C. Choudhury. 2006. Migration and

movement of Olive Ridley Turtles along the east

coast of India. Pp. 365-379. In: Marine Turtles of

the Indian Subcontinent. B.C. Choudhury and K.

Shanker (eds.). Universities Press, Hyderabad, AP,

India, 415 pp.

Pandav, B., K. Banugopan, D.Sutaria, and

B.C. Choudhury. 2000. Fidelity of male Olive

Ridley Turtles to a breeding ground. Marine Turtle

Newsletter 87: 9-10.

Pandav, B., B. C. Choudhury and C. S. Kar. 1994. A sta-

tus survey of olive ridley sea turtle (Lepidochelys

olivacea) and their nesting beaches along the Orissa

coast, India. Wildlife Institute of India, Dehradun,

India. 48 pp.

Pattanaik, S. K., C. S. Kar and S. K. Kar. 2001. A quarter

century of sea turtle conservation in Orissa.

Publication of wildlife wing of forest department,

Government of Orissa publication, Bhubaneswar

34 pp.

Pritchard, P. C. H. 1997. Evolution, phylogeny and cur-

rent status. Pp. 1-28. In\ P.F. Futz and J. A. Musick

(Eds.). The Biology of Sea Turtles. Boca Raton,

Florida, CRC Press, 432 pp.

Schroeder, B. A. and S. Murphy 1999. Population sur-

veys (ground and aerial) on nesting beaches. In:

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Research and management techniques for the con-

servation of sea turtles. K. L. Eckert, K. A.

Bjomdal, F. A. Abreu-Grobois and M. Donnelly

(eds). IUCN Marine Turtle Specialist Group

Publication # 4, CGC, Blanchard, Pennsylvania,

USA. 235 pp.

Shanker, K., B. Pandav and B. C. Choudhury. 2003. An

assessment of the olive ridley turtle ( Lepidochelys

olivacea ) nesting population in Orissa, India.

Biological Conservation 115, 149-160.

Shanker, K., R. K. Aggarwal, J. Rama Devi, B. C.

Choudhury, and L. Singh. 2004. Phylogeography of

olive ridley turtles ( Lepidochelys olivacea) on the

east coast of India: implications for conservation

theory. Molecular Ecology 13:1899-1909.

Tripathy, B. 2002. Is Gahirmatha the world’s largest sea

turtle rookery?. Current Science 83 (11): 1299.

Tripathy, B. 2005. A study on the ecology and

conservation of Olive Ridley Sea Turtle

(. Lepidochelys olivacea) at the Rushikulya rookery

of Orissa coast, India. Unpublished PhD Thesis.

Andhra University, Visakhapatnam, India. 168 pp.

Tripathy, B. and B. Pandav. In Press. Beach fidelity and

inter-nesting movement of olive ridleys

( Lepidochelys olivacea) at Rushikulya, India.

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Hatching success and orientation of Lepidochelys

olivacea (Eschscholtz, 1829) at Rushikulya rook-

ery, Orissa, India. Hamadryad 27(2): 185-1 92.

Valvarde, R. A. and C. E. Gates. 1999. Population sur-

veys on mass nesting beaches. Pp. 56-60. In: K.L.

Eckert, K. A. Bjomdal, F. A. Abrell-Grobois and M.

Donnelly (Editors), Research and management

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Marine Turtle Specialist Group Publication # 4,

CGC, Blanchard, Pennsylvania, USA. 235 pp.

Submitted: 19 October 2006

Accepted: 23 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 143-146

Effects of Starvation on Urinary Nitrogen Composition of Juvenile

Chinese Three-keeled Pond Turtles (Chinemys reevesii)

Jie Wang and Cuijuan Niu*

Ministry of Education Key Laboratory of Biodiversity Science and Ecological Engineering, College of Life

Sciences, Beijing Normal University, Beijing, 100875, People’s Republic of China.

* Corresponding author E-mail: cjniu@bnu.edu.cn

Abstract.- We investigated the effect of starvation on urinary nitrogen composition in the juvenile Chinese three-

keeled pond turtle ( Chinemys reevesii). Under normal conditions, ammonia, urea, and uric acid constitute 2.3, 95.8

and 1.9% ot total urinary nitrogen, respectively. During starvation periods of one to four weeks, the concentration of

urea changed little, while that of ammonia rose sharply and that of uric acid fell significantly. After feeding was

resumed tor four weeks, the levels of ammonia and uric acid returned to control levels. Changes in urinary nitrogen

composition during starvation may be related to the anti-oxidative function of uric acid during periods of stress.

Keywords.- Pond turtle, Chinemys reevesii , bladder urine content, stress, uric acid.

Introduction

The nature of an animal’s nitrogenous waste, including

ammonia, urea and uric acid, is dependent on the envi-

ronment in which it lives (Delaunay, 1931).

Furthermore, the spatial distribution of nitrogenous end

products in the body provides important data on how

that animal responds physiologically to that environ-

ment. The highly diverse excretory function of the

Reptilia has been well-documented (Campbell, 1995),

and although aquatic and semiaquatic turtles are prima-

rily ureotelic, a number of exceptions are known where

the predominant form of nitrogenous waste is ammonia

(Lee et ah, 2007).

The first objective of this study was to determine

the composition of excretory nitrogen in the bladder of

the Chinese Three-keeled pond turtle Chinemys reevesii.

This research will add to the body of literature on nitro-

gen excretion in freshwater turtles, which is currently

very limited (Lee et al., 2007; Singer, 2003).

Nitrogenous end-products have diverse physiologi-

cal functions in different animal groups, including acid-

base regulation, osmoregulation, etc. (Wright, 1995). In

turtles, two of the major factors affecting nitrogen excre-

tion are availability of water and amount of dietary

nitrogen ingested (Singer, 2003), which are of course

highly influenced by starvation and dehydration, which

turtles have developed a magnificent physiological

capability to resist. The aquatic Sonoran mud turtle

(Kinosternon sonoriense ), for example, can aestivate for

1 1 weeks without food or water; during the initial period

of deprivation, urine in the bladder is apparently used

for osmoregulation (Peterson and Stone, 2000). While

ammonia is quite toxic, more inert forms of excretory

nitrogen (e.g. urea and uric acid) are more costly to syn-

thesize (in ATP equivalents; Baze, 1970), making

ammonia the most frequently produced form of waste in

aquatic reptiles because they can lose this waste rapidly

to the environment (Cragg et ah, 1961). Uric acid is con-

sidered to be an endogenous antioxidant, and as such,

might play a role in the clearance of free radicals that are

produced during starvation (Zhu et ah, 2005). The sec-

ond objective of the present work is to investigate how

Chinemys reevesii changes its nitrogen metabolism dur-

ing starvation.

Materials and methods

Experimental animals and diet.- Juvenile turtles were

obtined from a turtle farm in Guangzhou, China and

acclimated to a fixed photoperiod of 12L:12D at a tem-

perature of 29±°C for three weeks in December 2004.

Each of the three turtles were placed in a glass tank with

the dimension of 19 * 23 * 27 cm. Turtles were fed to

apparent satiation twice a day on a commercial formu-

lated feed powder (composition as percentage of dry

matter: crude protein 41.66%, crude lipid 5.84%, ash

18.24%, and water 8.62% of total weight), which was

added to water and extruded to strip pellets before usage.

Each turtle was marked by sawing one to three notches

at the edge of its carapace.

Experimental procedure.- Before the experiment, 108

healthy turtles with a mean body mass of 13.58±1.84 g

(mean ±SD) were chosen, randomly divided into five

groups (C [control], SI, S2, S3 and S4), and placed into

a total of 36 tanks. This experiment lasted eight weeks,

with a period of starvation interrupting periods when the

Table 1 . Percentage of urine in body weight, nitrogen concentration (mmohL1) and partition (percent) formed as ammonia, urea and uric acid in the bladder of fresh-

water turtles ( Chinemys reevesii) at the end of fasting and after feeding for four weeks. Data expressed as Mean ± SE. Different letters denote significant differences

within the same column, p <0.05.

Gioup C was the control group. Group SI was starved during the fourth week; group S2 was starved during the third and fourth weeks; group S3 was starved from

the second to the fourth week; group S4 was starved for the first four weeks.

144

Asiatic Herpetological Research, Vol. 1 1

2008

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2008

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145

turtles were fully fed. The C group included eight tanks

where the turtles were always fed to satiation. The SI,

S2, S3 and S4 groups included seven tanks each. The S4

group was starved in the first four weeks of the experi-

ment. The S3 group was fed in the first week and then

starved tor three weeks. The S2 group was fed for two

weeks and then starved for two weeks. The SI group

was fed for three weeks and then starved for one week.

All groups were fed to satiation from the fifth to eighth

weeks.

Analytical methods.- One turtle was sampled randomly

from every tank at the end of the fourth and eighth

weeks, starved for 24 hours to empty the gut, toweled

off, weighed to within 0.01 g with an electronic balance,

placed in a plastic bag and euthanized at a temperature

of -80°C.

The turtles were dissected and the urinary bladders,

which contained frozen urine, were extracted and

weighed. Urine samples were centrifuged and diluted

twenty times with 0.9% NaCl. Urea and uric acid con-

centrations were determined using a Roche Diagnostics

Cobas INTEGRA 400. The concentration of ammonia

was measured using Roche Modular-P and Integra sys-

tems.

Data processing.- All statistical analyses were per-

formed using the SPSS 13.0 software package. The

Kolmogorov-Smimov test revealed that the data (includ-

ing percentages) followed a normal distribution. A one-

way ANOVA was employed to assess the effects of star-

vation. The Tukey HSD or Games-Howell test was used

for making multiple comparisons between the means of

different groups; p < 0.05 was taken as the level of sig-

nificance.

Results

Table 1 shows the changes in percentage of urine, total

excretory nitrogen concentration, and the proportion of

ammonia, urea, and uric acid among the different groups

at the end of the fasting and refeeding periods.

After fasting, significant differences between the

control and deprived groups were found in the concen-

trations of ammonia (F419= 3.103, p = 0.040)and uric

acid (F4J8= 25.559, p = 0.000), and in the composition of

total excretory nitrogen (F4.l8 = 5.015, p = 0.007;

p4 = 4.789, p = 0.008). Ammonia concentration and its

relative concentration showed a positive relationship

with increading periods of starvation, while those of uric

acid showed the reverse trend. Excretory urea did not

appear to be significantly affected by starvation

(p >0.05).

After feeding for four weeks, urea concentration

and total excretory nitrogen of SI were found to be sig-

nificantly higher in groups S3 and S4, while there were

no clear differences between the other groups for these

two parameters. Furthermore, all groups did not differ

clearly in other parameters measured ip > 0.05). Urinary

nitrogen composition was approximately 2.3% ammo-

nia, 95.8% urea, and 1 .9% uric acid in the control group.

Discussion

In our study, Chinemys reevesii was found to be prima-

rily ureogenic like other freshwater turtles, but it exhib-

ited a relatively higher proportion of urea (about 95.8%)

in excretory nitrogen compared to that of other freshwa-

ter turtles such as Trachemys scripta (about 70%, urine

in ureter; Dantzler and Schmidt-Nielson, 1966) and

Pelodiscus sinensis (54%, water samples; Lee et al.,

2007). Schmidt-Nielsen and Skadhauge (1967) reported

that ureotelic fresh water turtles excreted 45-95% of

their waste nitrogen in the form of urea, comparable to

the results found here.

During food deprivation, the concentration of uric

acid in the turtle’s bladder fell significantly ip = 0.000)

while that of ammonia clearly rose ip = 0.040), even

though they constitute only a small proportion of total

nitrogenous end products. Rapatz and Musacchia (1957)

found that the fasted fresh water turtle Chiysemys picta

(fasted for 6-8 weeks at 22°C) showed characteristic

biochemical properties in decreased liver total fatty

acids, decreased blood glucose and increased urine uric

acid levels. Meanwhile, specimens in cold torpor (4—8

weeks at 4°C) had increased liver total fatty acids, a sig-

nificant increase in liver glycogenolysis and increased

urine uric acid levels. Zhu et al. (2005) found that

Chinemys reevesii retained higher uric acid in its bladder

during cold torpor, but these contents rapidly decreased

when exposed to air because of oxidation. They pre-

sumed that retention of uric acid in the bladder during

cold toipor (they induced hibernation for about one

year) might have a benificial function during long peri-

ods of food deprivation, as uric acid or urate is known to

have antioxidative properties similar to those of vitamin

C and E. This may be the cause of the observed decrease

of uric acid in the bladder during starvation in the pres-

ent study. Conflicting results observed between this and

previous studies may be due to differences in rearing

conditions, the use of a different species iChinemys

reevesii vs. Chiysemys picta ) or ontogenetic differences

(juvenile vs. adult). The increase of ammonia in the

bladder of the turtles may be the result of increased

activity in innate protein catabolism during starvation.

Further research on nitrogen metabolism in stressful

146

Asiatic Herpetological Research, Vol. 1 1

2008

environments should be conducted.

The concentration of total urinary nitrogen and urea

in our experiment did not significantly change with star-

vation, conflicting with prior experiments (Zhu et al.,

2005). This conflict may be due to differences in nitro-

gen metabolism when hibernation is not induced.

Acknowledgments

We thank our colleague Miss Chen-Xi Huang and Dr.

Zhi-Gang Xie for their valuable comments. We are also

grateful to Professor Quan-zhong Shan and Miss Ou Liu

for their help in nitrogen analysis. This research was

supported by the National Natural Science Foundation

of China (No. 30271014, No. 30671598).

Literature Cited

Baze, W. B. and F. R. Home. 1970. Ureogenesis in

chelonian. Comparative Biochemistry and

Physiology 34A: 91-100.

Campbell, J. W. 1995. Excretory nitrogen metabolism in

reptiles and birds. Pp 147-178. In: Walsh P. J. and

P. A. Wright (eds.), Nitrogen metabolism and

excretion. CRC Press, Boca Raton. Cragg, M. M, J.

B. Balinsky and E. Baldwin. 1961. A comparative

study of nitrogen excretion in some amphibia and

reptiles. Comparative Biochemistry and Physiology

3A: 227-235.

Dantzler W. H. and B. Schmidt-Nielsen 1966. Excretion

in fresh-water turtle ( Pseudemys scripta) and desert

tortoise ( Gopherus agassizii ). American Journal of

Physiology 210: 198-210.

Delaunay H. 1931. L’ excretion azotee des invertebres.

Biological Review 6: 265-301.

Lee, S. M. L., W. P. Wong, A. M. Loong, K. C. Hiong,

S. F. Chew and Y. K. Ip. 2007. Postprandial

increases in nitrogenous excretion and urea

synthesis in the Chinese soft-shelled turtle,

Pelodiscus sinensis. Journal of Comparative

Physiology Part B 177: 19-29.

Peterson C. C. and P. A. Stone. 2000. Physiological

capacity for estivation of the Sonoran Mud Turtle,

Kinosternon sonoriense. Copeia 3: 684-700.

Singer M. A. 2003. Do mammals, birds, reptiles and fish

have similar nitrogen conserving systems?

Comparative Biochemistry and Physiology Part B

134: 543-558.

Rapatz G. L. and X. J. Musacchia 1957. Metabolism of

Chrysemys picta during fasting and during cold

torpor. Animal Journal of Physiology 188:

456-J60.

Schmidt-Nielsen B. and E. Skadhauge 1967. Function

of the excretory system of the crocodile

{Crocodylus acutus). Animal Journal of Physiology

212(5): 973-980.

Wright P. A. 1995. Nitrogen excretion: three end

products, many physiological roles. The Journal of

Experimental Biology 198: 273-281.

Zhu Z. R., D. Xiang, Y. Y. Liao, Y. Deng, X. H. Wang

and D. Z. Yin. 2005. Biological characters and bio-

chemical analysis of urine-associated liquid from a

hibernating turtle, Chinemys reevesii. Journal of

Natural Science of Hunan Normal University 28(3):

62-67 . (in Chinese)

Submitted: 01 November 2006

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 147-1 52 1

Survival and Metabolic Responses to Freezing Temperature in the

Northeast Forest Frog Rana dybowskii

Xianghong Xiao*, Dong Zheng, Cuijun Yang and Longhui Chai

Division of Physiology, Northeast Forestry University, Harbin 15004, China.

* Corresponding author E-mail: xiaoxh@vip.045 1 .com

Abstract.- Dynamic changes in water content, crude oil, general proteins, blood sugar and hepatic glycogen during

freezing temperatures in the Northeast forest frog ( Rana dybowskii Gunther, 1 876) were investigated by establishing

frog freeze-tolerant models. Chemical and biochemical analyses showed that a temperature drop from 4°C to -3°C

resulted in (1) increase in integrative water content and decrease in in vivo moisture and dissociative water contents;

(2) decrease in hepatic glycogen and crude oil and significant increase in blood sugar; (3) significant increase

{p > 0.05) in general protein content; (4) mortality below temperatures of -1°C; (5) and increase in blood sugar and

glucose levels in skeletal muscle following injection of glucose at 4°C and -2°C (hepatic glycogen levels showed sim-

ilar increases in test groups injected with 650 mmol/L and 1500 mmol/L glucose-PBS, but not in groups injected with

2000 mmol/L glucose-PBS). These physiological and metabolic responses suggest that the Northeast forest frog

adopts a positive freeze-tolerant strategy in which glucose serves as the primary mechanism by which damage due to

freezing is prevented.

Keywords.- Freezing tolerance, moisture contents, blood sugar, crude oil, general proteins.

Introduction

The Northeast forest frog (Ranidae: Raninae: Rana

dybowskii Gunther, 1876), formerly classified as a

northeastern population of Rana chensinensis David,

1875 (Xie et al., 1999), is found throughout eastern

Asia, with records from Heilongjiang Province, Jilin

Province, Liaoning Province, the northeast of the Inner

Mongolia Autonomous Region, as well as the Russian

Far East, eastern Mongolia, the Republic of Korea, and

Tsushima island (Japan) (Fei et al., 2005). The climate

in the northern province of Heilongjiang, the region

from which research material was collected, is typified

by intermediate and frigid-temperature zones with a

continental monsoon climate. From November to

March, the average temperature is usually less than 0°C,

while in January, temperatures reach -15°C to -30°C.

Due to climatic factors such as west-wind circumflu-

ence, Siberian air mass, Mongolia high pressure and

Baikal cyclone, winters in Heilongjiang Province are

often dry and without snow.

As is typical of northern amphibians facing freez-

ing temperatures, the Northeast forest frog hibernates to

subtly adjust its physiological functions and metabolism

to survive the winter. Although little physiological and

biochemical investigations have been made on this

species, various freeze-tolerant strategies have been

examined in species with similar biochemical metabo-

lisms, including Rana sylvatica LeConte, 1825, Hyla

versicolor LeConte, 1825, Hyla chrysoscelis and Rana

ridibunda Pallas, 1771 (Storey and Storey, 1986;

Voituron et al., 2000). These have included studies on

ecological behavior, genetic characteristics (Jiang and

Zhou, 2001; Yang et al., 2001; Xia et al., 2006), classifi-

cation (Jiang and Zhou, 2001; Xie et al., 1999; Yang et

al., 2001), artificial breeding and reproduction (Wei et

al., 2005) and biochemical composition (Xiao et al.,

2005). To further explore freeze-tolerant mechanisms

and cryobiology in the Amphibia, we here examine the

moisture, crude oil, proteins, blood sugar and glycogen

contents of the Northeast forest frog when subjected to

freezing temperatures.

Materials and Methods

Materials and freeze-tolerant models - Adult male

frogs weighing 20 to 22 g were collected from the

Yichun area of Heilongjiang Province in September.

Following one week of acclimation to room temperature

(25°C), ten randomly-selected frogs were placed into

separate glass boxes (40 * 40 * 40 cm) and transferred

to digitally-controlled refrigerators. Temperature

dropped at a rate of 1°C every 12 h and was held at 4°C

for up to 60 d. One third of the water in each box was

exchanged with pre-cooled fresh water (4°C) every two

days.

Freeze temperature impacting survival ratios.- To

investigate survival ratios at different temperatures, six

test groups (held at 2°C, 1°C, 0°C, -1°C, -2°C, and -3°C,

148

Asiatic Herpetological Research, Vol. 1 1

2008

respectively) and one control group (25°C) were estab-

lished with ten frogs per group. For each test group,

water temperature was lowered from 4°C to the target

temperatures specified above at a rate of 2°C per day.

Frogs in those test groups with target temperatures less

than -1°C were placed on water-soaked sponges.

Survivorship was checked once daily and PT100 ther-

mo-sensors were used to monitor temperatures.

Following 10 d of freezing stress, each test group was

returned to room temperature (25°C) for 24 hrs and sur-

vivorship examined, followed by euthanasia and bio-

chemical analysis. Each test was repeated three times.

Measurement of moisture content, crude oil, general

proteins, blood sugar and glycogen.- The drying

method outlined by Han et al. (2005) was used to meas-

ure dissociative and integrative water content: three 10 g

samples from each frog (recorded as fVw) were incubat-

ed at 70°C for 6 or 7 hrs until the semi-dry samples

reached a constant weight (recorded as W70). ; the semi-

dried samples were incubated at 105°C for 5 or 6hrs

until the samples again reached a constant weight

(recorded as Wl05). Calculated dissociative and integra-

tive water percentages were calculated as follows:

W

Dissociative water (%) =100 — x 100

W

VT w

w - w

Integrative water (%) = — 100

W

VT w

The Soxhlet extraction method was used for meas-

uring crude oil (Wei et al., 2004): for each group, 10 dry,

1 g samples (recorded as W(.d) were placed into the

Soxhlet extraction flask and degreased with low-melt-

point aether/petroleum; samples were packaged and

incubated at 105°C for lhr until the weight again

became constant (recorded as Wfat). Crude oil percent-

age was calculated using the following equation:

Wr - W'r

Crude oil (%) = — x 100

Wfat

General protein content was determined by the

Kjeldahl nitrogen determination method: for each group,

semi-dried 0.5 g samples were transferred into a diges-

tion tube; 2.5 g Na2S04, 0.13 g CuS04, and 10 ml H2S04

were added and digested at 400°C for 3 h until the solu-

tion color changed to pea green; the tubes were then

placed on the Kjeldahl nitrogen determination apparatus

for distillation; 15 ml 1% H3B03 were added to the

Erlenmeyer flask and connected to the condensator exit;

20 ml saturated NaOH was added to the reaction and

methyl red/bromocresol green indicator was added until

the solution turned grey; the solution was finally titrated

with HC1 standard buffer until the solution color

changed from grey to blue. General proteins percentages

were calculated with following equation:

Vs is the volume of HC1 standard buffer added to

adjust the distilled sample solution, V0 is the volume of

General proteins (%) = — - — ' °^-x 0.01 x 0.014 x 6.25 x 100

HC1 standard buffer needed to adjust the control solu-

tion, and W is the dry weight of sample.

The ortho-toluidine o-toloidine colorimetry method

and the anthrone colorimetry method were used for

measuring blood sugar and glycogen, respectively (He

et al., 2004): after 24 h of freezing-temperature stress,

blood sugar was measured from heart tissue (immediate-

ly treated with heparin from phlebotomized specimens

within each group); 1 g of liver tissue was used to meas-

ure glycogen; tissue was homogenated and centrifuged,

the supernatant was collected and added to an equal vol-

ume of ethanol; the solution was centrifuged, and 100

ml of distilled water was added to the precipitated

glycogen, which was then measured.

Exogenous glucose intervention assay.- The method

described by Costanzo et al. (1991) was used to detect

the effects of exogenous glucose on glucose metabolism

in frog liver and muscle tissue, and to demonstrate

whether glucose was involved in any freeze-tolerance

mechanisms. For each target temperature (4°C or -2°C),

ten frogs in three test groups and one control group

(25°C) were examined. For the control group, approxi-

mately 2. 3-2. 5 ml 115 mmol/L PBS was injected into

the dorsal lymph bursa, comprising approximately 6.7 %

of total volume in the bursa. (For the three test groups,

different concentrations (650, 1500, 2000 mmol/L,

respectively) of glucose-PBS (pH 7.4) were injected to

again comprise approximately 6.7% of total bursa vol-

ume. All frogs were euthanized following 72 hrs of

freezing temperatures. One gram of blood, liver and

muscle were immediately collected to analyze glucose

contents by the same methods described above.

Statistical analysis.- SPSS (ver.15.0) software was used

for statistic analysis. Confidence intervals were set to

0.05. Data were presented as means±standard error (SE)

and analyzed using a one-way AN OVA; these results

were subsequently analyzed using the Tukey test.

Results

Freezing temperatures and their impact on survival

ratios - During the temperature decrease from 4°C to

-1°C, ice crystals became visible on the skin of the frogs.

After ten days, mortality was observed only below -1°C

(see Fig. 1).

2008

Asiatic Herpetological Research, Vol. 1 1

149

o

\

-* — -4oc.0qc.-1qc

■X--- -2oq

-C-- -3°q

X>._

V - --

1 day 2 days 3 days 4 days 5-10 days

Figure 1. Impact of freeze temperatures and their dura-

tion on survival ratios of Rana dybowskii. Y-axis is sur-

vival ratios in percentage, X-axis is duration. The contin-

uous line with empty triangles represents data from the

test groups at 4°C, 0°C and -1°C. The dotted line with

crosses is for the test groups at -2°C. The dashed line

with empty diamonds is for the test groups at -3°C.

Changes of moisture contents in vivo, crude oils , gen-

eral proteins and glycogen.- Following the temperature

decrease from 4°C to -3°C, moisture contents in vivo

and dissociative water contents decreased gradually,

whereas integrative water was found to increase; fur-

thermore, blood sugar was found to increase while

hepatic glycogen and crude oil decreased significantly.

During freezing-temperature stress, general proteins

were found to decrease slightly with decreases in tem-

perature (p > 0.05) (see Table 1).

Changes in blood sugar and glycogen levels with addi-

tion of exogenous glucose under freezing-tempera-

tures.- At 4°C and -2°C, blood sugar and glucose con-

centrations in skeletal muscle were always found to

increase with the addition of exogenous glucose.

Hepatic glycogen also increased with increasing concen-

trations of exogenous glucose in the test groups injected

with 650 mmol/L and 1 ,500 mmol/L glucose-PBS, how-

ever, it was found to decrease in the test groups injected

with 2,000 mmol/L, particularly at -2°C (p < 0.01) (see

Table 2-3).

Discussion

Since the 1980s, investigators have studied the freezing-

tolerance mechanisms of amphibians, and have found

that species such as Rana sylvatica can precisely regu-

late their metabolic levels in order to tolerate extracellu-

lar ice crystallization (Storey and Storey, 1988), which

plays as key role in survival and evolution.

Investigations on Rana sylvatica , Pseudacris triseriata

and Rana ridibunda (Churchill and Storey, 1995;

Costanzo et al., 1991; Edwards et al., 2004; Layne and

Jones, 2001; Storey and Storey, 1985) illustrate that in at

least some amphibians, endogenous glucose is used as a

protectant during hibernation.

In the present study, it was found that in Rana

dybowskii, freezing temperatures are associated with

dehydration, an increase in blood sugar and a decrease in

hepatic glycogen; temperatures below -1°C are also

associated with increased mortality. Some investigations

have proposed that endogenous water redistribution aids

in the tolerance of freezing temperatures by changing

dissociative water into integrative water, condensing

extra-cellular solutes and promoting intracellular water

trafficking out of cells. This prevents intracellular icing,

lowers the freezing point of the body, induces antifreeze

Table 1. Effects of lowering temperature on percentage of water, crude oil, general proteins and sugar in Rana

dybowskii.

'Different letters in the same column represent significant differences (p < 0.05).

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 2. Effect of exogenous glucose on blood sugar and

liver glycogen in Rana dybowskii at 4°C.

‘Different letters in the same column represent significant differences

(p < 0.05).

synthesis, and prevents damage to critical organs from

intracellular ice crystallization (Churchill and Storey,

1995; Hermes-Lima and Storey, 1996; Horton, 1996).

Below a certain temperature, however, small ice crystals

enlarge to a point that causes damage to cell membranes

and cellular substructure, thereby causing death.

Stability of the cell membrane would also be compro-

mised, resulting in plasma-membrane fusion, phase

transformation from liquid crystal to gels, membrane

lipids deficiencies, phospholipid separation, etc., and

would destabilize membrane structure (Tong and Nie,

1996). In amphibians glucose is known to function as

antifreeze, lowering the freezing-point of the body,

maintaining cell membranes and stablizing internal

environments (Costanzo and Lee, 1994; Katz, 1989;

King et ai, 1995). In situations involving dehydration,

such as those observed here, hydrogen bond formation

between glucose hydroxyls and the heads of membrane

phospholipids have been found to stabilize lipid bilayers

at the liquid crystalline state, preventing membrane

fusion, phase change, side phase separation, cell leakage

and membrane protein displacement (Tong and Nie,

1996). Simultaneously, freezing temperatures stimulate

the catabolism of hepatic glycogen and crude oils,

increasing blood sugar concentrations, initiating the glu-

cose antifreeze system, stabilize cell membranes, and

enhancing physiological cold-tolerance. These factors

combined provide a physiological and biochemical strat-

egy that the Northeast forest frog uses to survive freez-

ing annual temperatures.

In the present study, it has been illustrated that the

injection of exogenous glucose into the lymph bursa

results in increased levels of blood sugar and skeletal

muscle glucose levels, increasing as higher concentra-

tions of glucose solution are injected, particularly in the

Table 3. Effect of exogenous glucose on blood sugar and

liver glycogen in Rana dybowskii at -2°C.

Skeletal-

Blood sugar Hepatic muScle

(mg %) glycogen (%) glucose (%)

Control 131 .18±0.17a 4.19±0.59a 1.63±0.03a

groups

Test groups 651.69±0.28b 18.43±1.13b 2.54±0.07b

injected with

650 mmol/L

Test groups 7 15.67±2.16c 33.92±1.66c 5.66±0.02c

injected with

1 ,500 mmol/L

Test groups j ,01 2.58±3.90d 20.99±1 ,67b 7.33±0.24d

injected with

2,000 mmol/L

‘Different letters in the same column represent significant differences

(p < 0.05).

test groups kept at -2°C. In comparison, hepatic glyco-

gen levels also showed an increase with higher concen-

tration of the injected glucose solution, although the test

groups injected with 2000 mmol/L glucose-PBS showed

a significant decrease. A possible explanation for this

unexpected result is that extremely high concentrations

of glucose induce diabetes-like symptoms, causing gly-

col-metabolism disorders and decreased glycogen syn-

thesis (Wang and Zhuang, 2001).

Acknowledgments

We thank Liu W. S., Zou Q. and Zeng K.W. for assist-

antship in samples collection. This study was granted by

the Nature Science Fund (C9726) and the Key Projects

of Heilongjiang Province (ZJN0604-02).

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Submitted: 22 November 2006

Accepted: 22 September 2007

2008

Asiatic Herpetological Research, Vol. 11

pp. 153-160

Clone and Sequence Analysis of Sox Genes in Rana tientaiensis

Yan Zhang', Liu Wang Nie1’*, Liang Yan1, Ping Ping Zheng1 and Wen Cheng1

{Life Science College, Anhui Normal University; The Key Laboratory of Biotic Environment

and Ecological Safety in Anhui Province, Wuhu 241000, Anhui, China.

* Corresponding author E-mail: lwnie@mail.ahnu.edu.cn

Abstract.- The Sox genes of Rana tientaiensis were amplified and cloned using highly degenerate primers designed

from the conservative motif (HMG-box) of the human SRY gene. The SSCP technique was used to detect different

clones. Seven distinct Sox gene fragments were obtained from both male and female R. tientaiensis ; no sexual differ-

ences were observed. Seven of these fragments (named RtSox3a, RtSox3b, RtSox3c, RtSox4, RtSoxll, RtSoxl2, and

RtSoxl4) exhibited 95%, 95%, 95%, 97%, 98%, 97%, and 97% similarity (respectively) to the corresponding homol-

ogous human SOX genes. The eighth fragment showed 79% and 77% similarity to the human SOX21 and SOX14

genes, as well as varying levels of similarity to other group B Sox genes. The eighth gene, provisionally named

RtSbx514, may be a new member of the Sox gene family or a derivative of an existing Sox gene. Phylogenetic analy-

sis suggests that the RtSox genes are highly conserved members of the SoxB, SoxC and SoxD gene groups. Sequence

analysis further illustrates that the gene Sox3 found in R. tientaiensis are duplicates of those seen in the mammalian

Sox gene family. Amino acid positions 15-19 are characteristic of each group in the Sox family.

Keywords.- Sox genes, SSCP, Rana tientaiensis, subgroup diagnosis.

Introduction

The Y chromosome-linked gene SRY is a dominant

inducer of testis development in mammals (Sinclair et

al., 1990) and a founding member of a gene family with

sequence homology to the High Mobility Group (HMG)

domain (Fawcett and Klymkowsky, 2004). Since dis-

covery of the SRY gene, many members of the SOX/Sox

(SRY- related HMG box) gene family have been found

throughout the vertebrates, showing at least 60% protein

similarity to the SRY HMG domain. Genes in each sub-

group show over 80% similarity. These SOX/Sox genes

have also been found to be involved in physiological

processes such as sex determination and the develop-

ment of the CNS, neural crest and endoderm (Bowles et

al., 2000). Soxl, Sox2, Sox3 and Soxll, for instance, are

expressed mainly in the developing nervous system

(Collignon et al., 1996; Pevny et al., 1998), and Sox 4 is

essential for heart and lymphocyte development

(Schilham et al., 1996). The SOX/Sox genes have been

divided between ten subgroups, named A to J (Table 1 ),

not all of which occur in the same taxa (Bowles et al.,

2000); groups I and J (containing sox31, sox32 and

sox33 ), for instance, are only found in Zebrafish (Girard

et al., 2001; Lunde et al., 2004).

Some amphibians have ZZ/ZW or XX/XY modes

of sex determination, but most species do not have het-

eromorphic chromosomes, making the study of evolu-

tion and the mechanism of sex determination in these

organisms interesting. Rana tientaiensis (2n = 26) is one

of these species without identifiable sex chromosomes

(Guo et al., 1991). In this paper, we describe the cloning

and sequencing of the eight Sox genes in Rana tientaien-

sis with the aim of researching the diversity and evolu-

tion of this gene family. Sequence analysis indicates that

some of these genes in R. tientaiensis are duplicated

Materials and Methods

Two male and two female Rana tientaiensis were cap-

tured from Taolin and Tingxi Anhui Provinces, China.

Genomic DNA was isolated from muscle tissues using

routine protocols. A pair of degenerate primers (snl:

ATGAAYGCNTTYATGGTNTGG; sn2: GGNCGR-

TAYTTRTARTCNGG) were designed using multiple

alignments of the HGM-box sequence of SRY, corre-

sponding to the MNAFMVW and PDYKYRP motifs

found in the HMG boxes of a wide range of Sox pro-

teins.

PCR reactions were 30 pi in volume, including

18.75 pi ddH20 and approximately 100 ng of genomic

DNA, 1.5mM Mg:7, 120 pM dNTP, 0.3 pM/primer and

1.25 pi Taq polymerase. PCR cycling conditions were 1

cycle for 5 minutes at 97°C, followed by 35 cycles with

40 sec. at 94°C, 40 sec. at 53°C, 1 min. at 72°C, and

finally 72°C for 10 minutes to complete the final reac-

tion.

Clones were genetically sequenced to detect the

positive clones with Sox DNA insertions. PCR products

were detected by 1 .5% agarose gels and cloned by pMD

18-T Vector (purchased from TAKARA). Positive

clones were screened by SSCP (single-strand conforma-

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Asiatic Herpetological Research, Vol. 1 1

2008

Table 1. Classification of the Sox gene family.

Figure 1. Amplified Sox gene fragments from 1: male

Rana tientaiensis, 2: female R. tientaiensis, 3 : Human; 4:

negative control; M: DL2000 marker (TaKaRa).

tion polymorphism) analysis and sequenced with the

universal sequencing primers on an ABI377 auto-

sequencer. DNA sequences were analyzed using the

BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and

CLUSTALX programs. Molecular Evolutionary Genetic

Analysis (MEGA) software was used to construct the

phylogenetic tree.

Results and Discussion

A 203 bp fragment of Rana tientaiensis genomic DNA

was obtained using the degenerate PCR primers listed

above. The fragment was identical to that found in the

human genome (Fig. 1), indicating that these fragments

belong to homologous genes.

Of the 113 white clones (i.e., those with insertions),

64 were positive for the Sox insertion, as confirmed

through nucleotide analysis following genomic amplifi-

cation. Eight distinct positive clones were found in both

male and female Rana tientaiensis ; there were no sexual

differences.

Seven of the eight distinct genes were named as fol-

lows: RtSox3a, RtSox3h, RtSox3c, RtSox4, RtSoxll,

RtSoxl2, and RtSoxl4. The amino acid sequences from

these genes had 95%, 95%, 95%, 97%, 98%, 97%, 97%

and 97% similarity (respectively) to homologous SOX

genes in Human. These seven genes belonged to the

SoxB , SoxC and SoxD subgroups, all of which lack

introns (Bowles et al., 2000). The 9th Sox gene was pro-

visionally named RtSoxB14; the amino acid sequences

from this gene had 79% similarity to the Human SOX21

gene, 77% similarity to the Human Soxl4 gene, as well

as varying levels of similarity to other group B Sox

genes. Nucleotide and putative amino acid sequences for

the eight Sox genes are listed (Fig. 2)

The amino acid sequences from the eight clones

were compared to 44 published Sox gene sequences in

GenBank, including sequences from Human ( HomoSRY,

SOX1, 2, 3, 4, 7, 9, 11, 12, 14, 15, 21, 30), Mouse

(. MusSoxl , 2, 3, 4, 7, 9, 11, 12, 14, 15, 21, 30), Gallus

gallus (GallSox 2, 3, 9, 11 , 14, 21), Danio rerio

( DaniSoxl , 2, 4, 11), Xenopus laevis ( XenopusSox2 , 4,

11), Takifugu rubripes {TakifuguSox 1 , 14b) and Eremias

breuchleyi ( EbSox2 , 4, 11, 12, 14 ,21) (Table 2). All

sequences were analyzed using neighbor-joining (NJ)

methods by MEGA 2.0 (Fig. 3).

Amino acid sequences between the RtSox genes

were highly conserved. Representatives of the Sox gene

in other species were also highly conserved with much

similarity between sequences. Gene duplication has

likely caused most of the diversity seen in the HMG box

superfamily, for which the Sox genes show the highest

mutation rate (Laudet et al., 1993). The high similarity

seen between the Rana and human genes in this study

are certainly indicative of gene duplication.

In the case of Soxl2, amino acid sequences were

nearly identical, although the 10th amino acid was N

instead of H in Human and Mouse (Fig. 2). In Sox 4, the

48th amino acid was R instead of Q in Mouse, and in

Soxll, the 48th amino acid was D instead of N in Mouse,

Human and several other species. The high degree of

similarity amongst these genes suggests that they belong

to the same gene family, and may perform similar roles

amongst taxa. For example, the three highly conserved

genes in group C display overlapping expression pat-

terns, and Sox4 and Soxll display overlapping expres-

sion patterns in the mouse embryonic pancreas

(Lioubinski et al., 2003).

According to Laudet et al. (1993), Sox4 is consid-

ered to be an early offshoot of the SRY gene in the Sox

tamily phylogeny. The conservative nature of Sox4

homologues in non-mammalian amniotes is interesting

because in mammals, the SRY gene exhibits rapid evolu-

2008

Asiatic Herpetological Research, Vol. 1 1

155

Table 2. Sox genes sequence in different species

tion, possibly caused by Y-linked inheritance (Tucker

and Lundrigan, 1993). The limited diversity of this gene

in non-mammalian taxa may be due to the retention of

an ancient conserved function.

It is likely that Sox3 is the closest homologue to the

Srv gene based on nucleotide sequence data.

Furthermore, Sox3 is located on the mammalian X chro-

mosome, and is highly similar to SRY (Sinclair et al.,

1990), suggesting that they arose through duplication of

their common ancestral during differentiation of the sex

chromosomes (Collignon et al., 1996; Foster and

Graves, 1994; Stevanovic et al., 1993). This is signifi-

cant because the X and the Y chromosomes are thought

to have arisen from a common “autosome” ancestor in

the lineage that gave rise to mammals (Wright et al.,

1993). Further examination of Sox genes in lower verte-

brates, Prototheria (monotremes) and Metatheria (mar-

supials) will be necessary to establish the evolutionary

origins of Snc The three conservative Sox3 genes (some-

times found within the same species or individual) can

be identified by the following variations in amino acid

sequence: RtSox3a has an F at position 46 and an H at

position 58; RtSox3h has an F at position 46 and an M at

position 58; RtSox3c has an I on position 46 and an M

on position 58.

The RtSoxB14 is unique among the Sox genes in

having the amino acid sequence VITEH at positions 15-

1 9, a K at position 44 and an S at position 50. In the phy-

logenetic tree (Fig. 3), although RtSoxB14 was more

closely related to subgroup B than other groups, the ori-

gin and classification of this gene is ambiguous.

Previously, genes encoding proteins with more than

60% similarity to the SRY FIMG domain have been

named Sox {SRY box) genes, and Sox genes with at least

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Asiatic Herpetological Research, Vol. 11

2008

Figure 2. (A) Alignment of nucleotide sequences (above), (B) Alignment of amino acid sequences (Opposite page, top),

(C) Percentage amino acid similarity between Rana tientaiensis Sox clones as determined by the sequence identity

matrix function in Bioedit (Opposite page, bottom).

2008

Asiatic Herpetological Research, Vol. 11

157

RtSox3a

RtSox3c

RtSox3b

RtSoxl4

RtSoxBl4

RtSoxl 1

RtSox4

RtSoxl2

MN AF MVWS RG QR RKMA QE NP KIvIHN S E I S KR LG ADWK LLS D AE KR PF I D EA KR LRAVHT KE YP D Y KY R

********************************************** ***********JyJ** *******

^^^^^^^^^^^^^ifc**;***********;!!#:***!***********!^*********

************ *****D***************g*****gY****Y*********C*M**H******

*********V****VI T* H* ****** ***K***Q****G*S**K*****S *****Q*M"7*H******

********KIE***IMEQ$*D**DA*******KR**M*K*S**I***R**E***LK*MAD*******

****** **yj E*** IMEQS*D*Y; *A*******KR****K*;jD*I ***g**E***Lh #141’*******

********QNE***IMDQW*D***A*******RR*Q**Q*S**I**W**E***LK*MAD*******

SoxB

Sox C

SoxD

Figure 2 (continued).

80% similarity have been placed in the same subgroup.

However, as more and more Sox genes are identified, the

ability to accurately classify these genes decreases. For

example, the genes sox30 and Ce-soxj have only 46%

and 48% similarity to Human S7?FHMG. Bowles (2000)

attempted to alternatively diagnose the gene family by

possession of the amino acid sequence “RPMNAF”,

which is highly conserved across genes, however, this

sequence was also found in the taxonomically ubiqui-

tous gene cic, so the sequence “RPMNAFMVW” was

provided as a replacement (Bowles et al., 2000).

Now that a sequence identifying the Sox gene fam-

ily has been identified, can sequences be found to char-

acterize the Sox subgroups? Following analysis of the

available sequences (Figure 4), it appears that positions

15-19 may have be useful for this purpose. The

sequence “MAQE(D)N” may work for group B (except

for HomoSOXS and MusSox3), “IMEQS” for group C,

“IMEQW” (for Soxl2 ) or “ILQAF” (for Sox5, Sox6 and

Sox 13) for group D, “LADQY” for group E, “LAVQN”

(for SoxT) or “LAQQN” (for Soxl7 and SoxlS ) for

group F, “MAQQN” for group G and “LAKAN” for

group H. RtSoxB 14 can be separated from the remaining

Sox genes by the unique sequence VITEH, although in

mammals ( HomoSOX3 and MusSox3) it is changed to

“MALEN”, like the sequence for SRY. This further sup-

ports a close relationship between the SOX3 and SRY

genes.

Several Sox genes appear to have been duplicated

in Rana tientaiensis : RTSox3a, RTSox3b and RTSox3c.

Similar duplications in amphibians are uncommon, but

they are more frequently encountered in teleosts: Soxl,

4, 9 and 14 has been duplicated in the sea bass (Malyka

et al., 2003) and Sox21 has been duplicated in the

Zebrafish (Argenton et al., 2004). The “duplication-

degeneration-complementation” model developed by

Force et al. (1999) suggests that the partition of ancestral

subfunctions is an important mechanism leading to the

preservation of multiple gene copies; this model predicts

that the probability of gene conservation will be higher

in more complex genes with a larger number of subfunc-

tions (Force et al. 1999). Most duplicate genes in Rana

tientaiensis have silent mutations (except RtSox3a ,

which has an encoded amino acid mutation), but it

would appear that the sequences are under selective

pressure and may indeed perform separate subfunctions.

Future studies investigating Sox genes in Rana tientaien-

sis will likely provide much insight into duplicate genes.

Acknowledgments

We are grateful to the reviewers for numerous valuable

suggestions on the manuscript. This research was sup-

ported by National Natural Science Foundation of China

(No. 30640048, No. 30770296), the Natural Science

Foundation of Anhui Education Department

(KJ2007A022).

158

Asiatic Herpetological Research, Vol. 1 1

2008

G5

32

G7

79

7G

61

30

72

77

80

94

51

86

84

85

97

54

99

63

84

97

99

50

82

73

59

53

95

G5

G9

77

99

- GallusSox2

- XenopusSox2

- EbSox2

- DariioSox2

- HomoSox2

- MusSox2

- HomoSoxI

■ MusSoxI

■ DanioSoxI

- TakifuguSoxI

• GallusSox21

■ EbSox21

• MusSox21

■ HomoSox21

■ RTSoxU

• EbSox14

■ HomoSox14

■ MusSoxI 4

■ GallusSox14

■ TakifuguSox14B

■ XenopusSox3

RTSox3a

■ MusSox3

■ HomoSox3

GallusSox3

RTSox3c

RTSox3b

RTSoxB14

B

incertae

sedis

- HornoSox15

- MusSoxI 5

- HomoSry

- EbSox12

- RTSox12

- HomoSox12

- MusSox12

- MusSox4

- RTSox4

- HomoSox4

- DanioSox4

- EbSox4

- DanioSox1 1

- HomoSox1 1

- MusSox1 1

■ GallusSox1 1

■ XenopusSox1 1

■ RTSoxll

■ EbSoxI 1

• HomoSox7

MusSox7

HomoSox9

MusSox9

GallusSox9

mussox30

homosox30

HomoTcf-1

I G

I A

D

C

F

E

H

Outgroup

Figure 3. Phylogenetic analysis of Sox/SOX gene family

2008

Asiatic Herpetological Research, Vol. 1 1

159

imissox30

hom os ox30

MusSox4

RTS ox4

Horn oSox4

DanioSox4

EbS ox4

HomoSoxl 1

MusSoxl 1

GallusSoxll

XenopusS oxll

RTS oxll

EbS oxll

DanioSoxll

HomoSoxl 2

MusSoxl 2

Homosox22

RTS oxl2

EbS oxl2

MusSoxT

Hom oSox?

Hom osoxT

Homosoxl7

Hom osoxl 8

Hom oSox9

MusSox9

GallusSox9

Hom osoxl 0

MusSox3

Hom oSox3

GallusSox3

RTS ox3 c

RTS ox3 a

RTS ox3b

XenopusS ox3

Hom oSoxl

MusSoxl

DanioSoxl

Taki fuguSoxl

GallusSox2

XenopusS ox2

EbS ox2

Hom oSox£

MusSox2

Dani oSox2

MusSox21

Hom oSox21

EbS ox21

GallusSox21

Hom oSoxl 4

MusSoxl 4

GallusSoxl4

EbS oxl4

RTS oxl4

T ak i fuguSox 1 4B

RTS oxB 1 4

Hom oSoxl 5

MusSoxl 5

Hom oSry

Hom 050x5

Hom osoxl 3

Hom osox6

HomoTcf-!

M HAFMVWARIHRP A LAKANP AANHAEIS VQLGLEWNKLS EEQKKFY YDEAQ KIKEEHREE F PGWVYQ F

M NAFMV'WARIHRP A LAKAN 3 AAHNAEIS VQLGLEWHKLS EEQKKPY YDEAQ KLKEKHREE F PGYYVYQ F

MHAFMVWSQIERRK IMEQS ’DMHHAEISKRLGKRWKLLKDSDHPFIQEAE RLRLKHM AD YPDYKYRP

MNAFMVWSQIERRF IMEQS ’DMYNAEISICRLGKRWKLLKDSDKIPFIQEAERLRLKHMAD YFDYKYRF

MHAFMVWSQIERRF IMEQS ’DMHNAEISKRLGKRWKLLKDSDKIPFIREAE RLRLKHM AD YPDYKYRP

M NAFMVWSQIERRF IMEQS 5 DMHNAEIS KRLGKRWKLLKDSDH FF IREAE RLRLKHM AD Y FDYKYRF

MHAFIVWSRIERRF IMEQS I1 DMHMAEIS KRLGKRWKLLKDSDKIPFIQEAERLRLKHMADIFHYKYRP

M HAFMVWS KIERRH IMEQS 3 DMHNAEIS KRDGKRWKMLK DSEKIPF IREAE RLRLKHM AD Y FDYKYRF

MNAFMVWSKIERRF IMEQS ’DMHNAEISFCRLGKRWKMLKDSEKIFFIREAE RLRLKHM AD YPDYKYRP

M HAFMVWS HERRK IMEQS 1 DMHNAEIS KRLGKRWKMLKDSEKIPF IREAE RLRLKHM AD Y PDYKYRP

MNAFMVWSKIERRF IMEQS 3 DMHNAEIS KRLGKRWKMLKDSEKIPF IREAE RLRLKHM AD YPDYKYRP

M HAFMVWS HERRI IMEQS 3 DMHDAEIS KRLGKRWKMLKDSEKIPF IREAE RLRLKHM AD Y FDYKYRF

MHAFIVWSKIERKf IMEQS ? DMHNAEIS KRLGKRWKMLKDSEHFF IREAE RLRLKHM AD Y PDYKYRP

M HAFMVWS HERRF IMEQS ? DMHMAEIS KRLGKRWKMLKDSEKIFF IREAE RLRLQHM AD Y FDYKYRF

M HAFMVWSQHERR* IMDQW 5 DMHMAEIS KRLGRRWQLLQ DSEKIPF VREAE RLRLKHM AD Y FDYKYRF

M HAFMVWS QHERRF IMDQW 5 DMHNAEIS KRLGRRWQLLQ DSEKIPF VREAE RLRLKHM AD Y PDYKYRP

M HAFMVWS QHERRB IMDQW ? DMHMAEIS KRLGRRWQLLQ DSEKIPF VREAE RLRLKHM AD Y PDYKYRP

M HAFMVWSQHERR! IMDQW ’DMHMAEIS KRLGRRWQLLQ DSEKIPF VKEAE RLRLKHM AD Y PDYKYRP

MNAFIVWSQHERRK IMDQW ? DMHMAEIS KRLGRRWQLLQ DSEKIPF VKEAE RLRLKHM AD Y PDYKYRP

M NAFMVWAKDERKF LAVQH ? DLHHAELS KMLGKSWKALT LSQKRFY VDEAE RLRLQHM QD Y FNYKYRP

MNAFMVWAKDERKF LAVQN ? DLHMAELS KMLGKSWKALT LSQKRPY VDEAE RLRLQHM QD Y FNYKYRP

M HAFMVWAKDERKf LAVQH ? DLHHAELS KMLGKSWKALT LSQKRPY VDEAE RLRLQHM QD YFNYKYRP

MHAFMVWAKDERKI LAQQM ? DLHHAELS KMLGKSWKALT LAEKRPF VEEAE RLRVQHM QD HPMYKYRP

MNAFMVWAKDERKFLAQQN’DLHNAVLSKMLGKAWKELHAAEKRFFVEEAERIRVQHLRI HFHYJCYRP

M HAFMVWAQ AARRK LADQY ? HLHNAELS KTD3KLWRLUIESEKRPF VEEAE RLRVQHKKD HFDYKYQP

M HAFMVWAQAARRK LADQY 3 HLHNAELS KTLGKLWRLLHESEKRPF VEEAE RLRVQHKKD HFDYKYQP

MHAFMVWAQAARRi LADQY 3 HLHNAELS KTLGKLWRLLHESEKRPF VEEAE RLRVQHKKD HFDYKYQP

M HAFMVWAQ AARRi LADQY ? HLHHAELS KTLGKLWRLLNESDKRPFIEEAE RLRMQHKKD HFDYKYQP

M HAFMVWS RGQRRK 9ALEN 1 KMHHSEIS KRLGADWKLLT DAE KRPF IDEAKRLRAVHM KE Y PDYKYRP

M HAFMVWS RGQRRF I1ALEN 3 KMHHSEIS KRIGADWKLLTD AEKRPF IDEAKRLRA'/HMKE Y PDYKYRP

M HAFMVWS RGQRRF 9 AQEN 3 KMHHSEIS KRLGADWKLLS DAEKRPF IDEAKRLRAVHM KE Y PDYKYRP

M HAFMVWS RGQRRF 9 AQEN ? KMHHSEIS KRLGADWKLLS DAEKRPF IDEAKRLRAVHM KE Y PDYKYRP

M HAFMVWS RGQRRF 9AQEN? KMHHSEIS KRLGADWKLLS DAEKRPF IDEAKRLRAVHTFI YPDYKYRP

M HAFMVWS RGQRRK W AQEH 3 KMHHSEIS KRLGADWKLLS DAEKRFI IDEAKRLRAVHM KE Y PDYKYRP

M HAFMVWS RGQRRK 9AQEN ? KMHHSEIS KRLGADWKLLS DSDKRPFIDEAKRLRAVHMKD YPDYKYRP

M HAFMVWS RGQRRK 9 AQEN 5 KMHHSEIS KRLGAEWKVMS E AEKRPF IDEAKRLRALHM KE HPDYKYRP

M HAFMVWS RGQRRF 9AQEN 3 KMHNSEIS KRLGAEWKVMS EAEKRPF IDEAKRLRALHM KEHPDYKYRF

M HAFMVWS RGQRRF 9 AQEN 3 KMHHSEIS KRLGAEWKVMS EAEKRPF IDEAKRLRAMHM KEHPDYKYRF

M HAFMVWS RGQRRF 9 AQEN ? KMHHSEISKRLGAEWKVMT EAE KRPF IDEAKRLRAMHM KE HPDYKYRF

M HAFMV'WS RGQRRF 9 AQEN ’KMHHSEIS KRDGAEWKLLS EAEKRPF IDEAKRLRALHM KEHPDYKYRF

M HAFMVWS RGQRRK 9AQEN 3 KMHNSEIS KRLGAEWKLLS EAEKRPF IDEAKRLRALHM KEHPDYKYRF

M HAFIVWS RGQRRK WAQEH 3 KMHHSEIS KRLGAEWKLLS EAEKRPF IDEAKRLRALHM KE HPNYKYRF

M HAFMVWS RGQRRF WAQEN 3 KMHHSEIS KRLGAEWKLLS ETEKRPF IDEAKRLPALHM KE HPDYKYRF

M HAFMV'WS RGQRRK W AQEH 3KMHHSEISKRLGAEWKLLSETEKRFFIDEAKRLRALHM KEHPDYKYRF

M HAFMVWS RGQRRK WAQEH 3 KMHHSEIS KRLGAEWKLLS ESEKRFFIDEAKRLRALHM KEHPDYKYRF

M HAFMVWS KAQRRK WAQEH 3 KMHNSEIS FCRLGAEWKLLT ESEKRPF IDEAKRLRAMHIW KE HPDYKYRF

M HAFMVWS RAQRRK WAQEH 3 KMHHSEIS KRLGAEWKLLT ESEKRPF IDEAKRLRAMHM KE HPDYKYRF

M HAFIVWS RAQRRK 9AQEH 3 KMHHSEIS KRLGAEWKLLT ESEKRPF IDEAKRLRAMHM FCEHPHYKYRP

M HAFMV'WS RAQRRK 9AQEN PKMHHSEIS KRLGAEWKLLS EAEKRPF IDEAKRLRAMHM KE HPDYKYRP

M HAFMVWS RGQRRK 9 AQEH 3 KMHHSEIS KRLGAEWKLLS EAEKRPYIDEAKRLRAQHM ICE HPDYKYRP

M HAFMV'WS RGQRRF 9AQEH 3 KMHHSEIS KRLGAEWKLLS EAEKRPYIDEAKRLRAQHMKE HPDYKYRP

M HAFMVWS RGQRRK 9 AQEH ’KMHHSEIS KRLGAEWKLLS EAEKRPY IDEAKRLRAQHMKE HPDYKYRP

M HAFMV'WS RGQRRK 9 AQEH 3 KMHHSEIS KRLGAEWKLLS EAEKRPY IDEAKRLRAQHM KE KPHYKYRP

M HAFMVWS RGQRRK 9AQDN 3 KMHHSEIS KRLGAEWKLLS EVEKRPY IDEAKFLRAQHM KE HPDYKYRP

M NAFMVWS RGQRRF 9 AQEH 3 KMHNSEIS KRLGAEWKLLS DSEKRPY IDEAKRLRAQHM KE HPDYKYRP

M HAFMV'WS RVQRRF VITEH 3 KMHHSEIS KKUGAQWKILGDSEKKPF IDESKRLRAQHM VI HPDYKYRP

M HAFMVWSSAQRRC 9AQQH 3 KMHHSEIS KRLGAQWKLLD EDEKRPF VEEAKRLRARHLRD Y PDYKYRP

MHAFMVWSSVqRRC9AQQN3KMHHSEISKRDGAQWKLLGDEEKRPFVEEAKRLRAKHLRD YPDYKYRP

M HAFIVWS RDQRRE WALEN 3 RMRHSEIS KQLGY QWEMLT EAEKWPF FQEAQ KLQAMHREK Y FHYKYRF

M HAFMVWA KDERRK IUQAF 3 DMHHSHIS KIIGS RWKAMT HLEFBPY YEEQARLSKQHLEK Y PDYKYKP

MHAFMVWAKDERRF ILQAF ’DMHNSSISKILGSRWKSMTMQEKQPY YEEQARLSRQHLEKYPDYKYKP

MHAFMVWAKDERRF ILQAF 3 DMHHSHIS KIIjGS RWKSMS HQEKQPY YEEQAFLSKIHLEK YPHYKYKP

M YKETVYS AFH — LLHHYPF PSGAGQHPQFQPF LHKANQ PPHGVPQ LSLYE HFNSPHP TP APADIS QKQV

Figure 4. Characteristic Sox amino acid sequences.

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Submitted: 25 October 2006

Accepted: 23 September 2007

2008

Asiatic Herpetological Research, Vol. 1 1

pp. 16 1-205

Cummulative Index to Vols. 1-10 of

Asiatic Herpetological Research

Compiled by

Genevieve V. A. Gee

Flat A09-4 Satria Court, BDC

Kuching, Sarawak, Malaysia

E-mail: jutisha@yahoo.com

Author Index

Aa

Adler, K.

Studies on hynobiid salamanders, with descrip-

tion of a new genus. Asiatic

Herpetological Research 3:37-45. (K.

Adler and E. Zhao)

First records of the pipe snake ( Cylindrophis )

in China. Asiatic Herpetological Research

4:37^1. (K. Adler, E. Zhao and I. S.

Darevsky)

Ahsan, M. F.

The first record of Ptyas korros (Colubridae)

from Bangladesh. Asiatic Herpetological

Research 9:23-24. (M. F. Ahsan and S.

Parvin)

A record of Boiga ochracea walli (Stoliczka,

1870) from Bangladesh. Asiatic

Herpetological Research 10:235. (M. F.

Ahsan and S. Parvin)

Some aspects of breeding biology of the

Bengal Lizard ( Varanus bengalensis) in

Bangladesh. Asiatic Herpetological

Research 10:236—240. (M. F. Ahsan and

M. A. Saeed)

Al-Johany, A. M.

The activity and thermal biology of the fossor-

ial reptile, Diplometopon zarudnyi

(Amphisbaenia: Trogonophiidae) in

Central Saudi Arabia. Asiatic

Herpetological Research 8:1-6.

Ananjeva, N. B.

Historical biogeography of the

Phrynocephalus species of the USSR.

Asiatic Herpetological Research 4:76-98.

(N. B. Ananjeva and B. S. Tuniyev)

Stellio sacra (Smith 1935) - a distinct species

of Asiatic rock agamid from Tibet. Asiatic

Herpetological Research 3:104-115. (N.

B. Ananjeva, G. Peters, J. R. Macey and T.

J. Papenfuss)

See also J. R. Macey (and N. B. Ananjeva)

Anderson, S. C.

Book review: A guide to the fauna of Iran.

Asiatic Herpetological Research 9:149-

150.

Book review: Four recent handbooks for

Turkey. Asiatic Herpetological Research

9:151-152.

Book review: Wild about reptiles. Field guide

to the reptiles and amphibians of the UAE.

Asiatic Herpetological Research 9:153.

Andren, C.

See G. Nilson et al. (and C. Andren)

Arnekleiv, J. V.

See D. Dolmen et al. (and J. V. Arnekleiv)

Assadian, S.

See M. Sharifi (and S. Assadian)

Ataev, C.

See S. Schammakov et al. (and C. Ataev)

Atayev, C. A.

See B. S. Tuniyev et al. (and C. A. Atayev)

Auffcnberg, K.

Studies on Pakistan lizards: Cyrtopodion

stoliczkai (Steindachner, 1867)

(Gekkonidae: Gekkoninae). Asiatic

Herpetological Research 10:151-160. (K.

Auffenberg, K. L. Krysko and W.

Auffenberg)

162

Asiatic Herpetological Research, Vol. 1 1

2008

Auffenberg, W.

Studies on Pakistan reptiles. Pt. 3 Calotes ver-

sicolor. Asiatic Herpetological Research

5:14-30. (W. Auffenberg and H. Rehman)

Calotes versicolor nigrigularis Auffenberg and

Rehman 1993 a junior primary homonym.

Asiatic Herpetological Research 6:27.

Autumn, K.

Preliminary observations on the ecology of

Phrynocephalus axillaris and Eremias

velox in the Turpan Depression, Xinjiang

Uygur Autonomous Region, China. Asiatic

Herpetological Research 2:6-13. (K.

Autumn and Y.-Z. Wang)

Mimicry of scorpions by juvenile lizards,

Teratoscincus roborowskii (Gekkonidae).

Asiatic Herpetological Research 2:60-64.

(K. Autumn and B. Han)

Ayaz, D.

See M. Tosunoglu et al. (and D. Ayaz)

Bb

Balletto, E.

See T. Dujsebayeva et al. (and E. Balletto)

Barth, D.

See M. Schilde et al. (and D. Barth)

Batra, R.

Simplified field technique for obtaining blood

from freshwater turtles. Asiatic

Herpetological Research 6:28-29. (R.

Batra and S. Prakash)

Bauer, A. M.

A preliminary report on the reptile fauna of the

Kingdom of Bhutan with the description of

a new species of scincid lizard (Reptilia:

Scincidae). Asiatic Herpetological

Research 4:23-36. (A. M. Bauer and R.

Gunther)

The systematic relationships of Dravidogecko

anamallensis (Gunther 1875). Asiatic

Herpetological Research 6:30-35. (A. M.

Bauer and A. P. Russell)

Bell, C. J.

See W. G. Joyce (and C. J. Bell)

Beregovaya, S. Y.

See B. S. Tuniyev (and S. Y. Beregovaya)

Biswas, S.

See S. S. Pawar (and S. Biswas)

Biun, A.

See R. F. Inger et al. (and A. Biun)

Blamires, S. J.

Influence of temperature on burrow use by the

monitor lizard Varanus panoptes of the

coastal dunes at Fog Bay, Northern

Australia. Asiatic Herpetological Research

9:25-29.

Bon, C.

See Y. Zhang et al. (and C. Bon)

Borkin, L. J.

See S. N. Fitvinchuk et al. (and F. J. Borkin)

D. V. Semenov (and F. J. Borkin)

Bour, R.

See J. Perala (and R. Bour)

Brown, R. M.

Description of a new species of Pseudorabdion

(Serpentes: Colubridae) from Panay

Island, Philippines with a revised key to

the genus. Asiatic Herpetological Research

8:7-12. (R. M. Brown, A. E. Feviton and

R. V. Sison)

A new snake of the genus Hologerrhum

Gunther (Reptilia; Squamata; Colubridae)

from Panay Island, Philippines. Asiatic

Herpetological Research 9:9-22. (R. M.

Brown, A. E. Feviton, J. W. Femer and R.

V. Sison)

See also, A. C. Diesmos et al. (and R. M.

Brown)

J. W. Femer et al. (and R. M. Brown)

Bursey, C. R.

See S. R. Goldberg et al. (and C. R. Bursey)

Bushkirk, J. R.

New locality records for Chinese non-marine

chelonians. Asiatic Herpetological

Research 2:65-68.

2008

Asiatic Herpetological Research, Vol. 1 1

163

Cc Notes on the diet, survival rate, and burrow

specifics of Uromastyx aegyptius

Cao, Y. microlepis from the United Arab Emirates.

See M. Huang et al. (and Y. Cao) Asiatic Herpetological Research 9:30-33.

Castellano, S.

See T. Dujsebayeva et al. (and S. Castellano)

Cedhagen, T.

Anurans collected in West Malaysia. Asiatic

Herpetological Research 7:1-5.

Chanda, S. K.

See I. Das (and S. K. Chanda)

Chen, B.

The past and present situation of the Chinese

alligator. Asiatic Herpetological Research

3:129-136.

Preliminary research on the function of the

eggshell in the Chinese alligator {Alligator

sinensis). Asiatic Herpetological Research

4:132-136. (B. Chen and B. Liang)

Chen, Y.

Evaluation of snake venoms among

Agkistrodon species in China. Asiatic

Herpetological Research 4:58-61. (Y.

Chen, D. Zhang, K. Jiang and Z. Wang)

Chen, Y.-Y.

See P. Guo et al. (and Y.-Y. Chen)

Chikin, Y. A.

A catalogue of non-metrical variations in skull

bones of Vipera lebetina (Reptilia,

Viperidae). Asiatic Herpetological

Research 7:6-18.

Chou, W.-H.

On the status of Rhacophorus prasinatus Mou,

Risch and Lue (Anura: Rhacophoridae).

Asiatic Herpetological Research 5:11-13.

Ciochon, R. L.

See J. H. Hutchinson et al. (and R. L. Ciochon)

Conant, R.

The type locality of Agkistrodon halys cara-

ganus. Asiatic Herpetological Research

4:57.

Dd

Daniels, R. J. R.

The Ceylonese tree frog Polypedates cruciger

Blyth, a new record for India. Asiatic

Herpetological Research 6:36-37. (R. J. R.

Daniels and M. S. Ravichandran)

Darevsky, I. S.

Two new species of the worm-like lizard

Dibamus (Sauria, Dibamidae), with

remarks on the distribution and ecology of

Dibamus in Vietnam. Asiatic

Herpetological Research 4:1-12.

A new subspecies of the dwarf snake

Calamaria lowi ingermarxi ssp. nov.

(Serpentes, Colubridae) from Southern

Vietnam. Asiatic Herpetological Research

4:13-17. (I. S. Darevsky and N. L. Orlov)

A new gecko of the genus Gonydactylus

(Sauria: Gekkonidae) with a key to the

species of Vietnam. Asiatic Herpetological

Research 7:19-22. (I. S. Darevsky and N.

N. Szczerbak)

See also, K. Adler et al. (and I. S. Darevsky)

Das, I.

Intergradation between Melanochelys trijuga

trijuga and M. t. coronata (Testudines:

Emydidae: Bataguruinae). Asiatic

Herpetological Research 3:52-53. (I. Das

and P C. H. Pritchard)

Cyrtodactylus madarensis Sharma (1980), a

junior synonym of Eublepharis macular-

ius Blyth (1854). Asiatic Herpetological

Research 4:55-56.

Size-gradation in syntopic frogs in South India.

Asiatic Herpetological Research 6:38^14.

Rediscovery of Lipinia macrotympanum

(Stoliczka, 1873) from the Nicobar

Islands, India. Asiatic Herpetological

Research 7:23-26.

Anguis melanostictus Schneider, 1801, a valid

species of Barkudia (Sauria: Scincidae)

from Southwestern India. Asiatic

Herpetological Research 8:13-17.

The dates of publication of amphibian and rep-

tile names by Blanford and Stoliczka in the

Cunningham, P. L.

164

Asiatic Herpetological Research, Vol. 1 1

2008

Journal and Proceedings of the Asiatic

Society of Bengal. Asiatic Herpetological

Research 8:18-24.

A new locality for the rare Bornean skink,

Lamprolepis vyneri (Shelford, 1905)

(Sauria: Scincidae). Asiatic Herpetological

Research 10:241-244.

Leptobrachium smithi Matsui, Nabitabhata,

and Panha, 1999 (Anura: Megophryidae),

an addition to the fauna of Myanmar

(Burma). Asiatic Herpetological Research

10:245-246. (I. Das and S. K. Chanda)

See also, J. L. Grismer et al. (and I. Das)

Diaz, R. E.

A new species of Dibamus (Squamata:

Dibamidae) from West Malaysia. Asiatic

Herpetological Research 10:1-7. (R. E.

Diaz, T. M. Leong, L. L. Grismer and N. S.

Yaakob)

Diesmos, A. C.

Rediscovery of the Philippine forest turtle,

Heosemys leytensis (Chelonia,

Bataguridae), from Palawan Island,

Philippines. Asiatic Herpetological

Research 10:22-27. (A. C. Diesmos, G. V.

A. Gee, M. L. Diesmos, R. M. Brown, P. J.

Widman and J. C. Dimalibot)

Diesmos, M. L.

See A. C. Diesmos et al. (and M. L. Diesmos)

Dimalibot, J. C.

See A. C. Diemos et al. (and J. C. Dimalibot)

Diong, C. H.

Size and shape description of oviductal eggs of

Draco obscurus formosus (Squamata:

Agamidae). Asiatic Herpetological

Research 8:25-28. (C. H. Diong and S. Y.

T. Soon)

Dolmen, D.

Diel activity of Ranadon sibiricus (Amphibia:

Hynobiidae) in relationship to environ-

ment and threats. Asiatic Herpetological

Research 8:29-37. (D. Dolmen, R. A.

Kubykin and J. V. Amekleiv)

Dujsebayeva, T.

On the distribution of diploid and tetraploid

green toads of the Bufo viridis complex

(Anura; Bufonidae) in southern

Kazakhstan. Asiatic Herpetological

Research 7:27-31. (T. Dujsebayeva, S.

Castellano, C. Giacoma, E. Balletto and G.

Odiema)