HARVARD UNIVERSITY

Ernst Mayr Library

of the Museum of

Comparative Zoology

ASIATIC

HERPETOLOGICAL

RESEARCH

VOLUME 3

1990

ISSN 1051-3825

CONTENTS

TUNIYEV, BORIS S., AND SAHAT M. SHAMMAKOV. Coluber atayevi Sp. Nov. (Ophidia,

Colubridae) from the Kopet-Dag Mountains of Turkmenistan 1

CHOU, WEN-HAO. On the Status of Rhacophorus prasinatus Mou, Risch, and Lue

(Anura: Rhacophoridae) 11

AUFFENBERG, WALTER, AND HAFIZUR REHMAN. Studies on Pakistan Reptiles. Pt. 3.

Calotes versicolor 14

ROCEK, ZBYNEK. Holocene anurans from Caucasus 31

Wei, Gang, ning xu, dejun Li, guanfu wu and xiquan Song. Karyotype, C-

Band and Ag-Nors Study of Three Stink Frogs 45

GOLUBEV, M. L. The Variegated Toad Agama in Djungar Gate (Eastern Kazakstan) with

Notes on Certain Systematic Problems of Phrynocephalus versicolor Str. (Reptilia:

Agamidae) 51

MEZHZHERIN, SERGEI AND MICHAEL L. GOLUBEV. Allozyme Variation and Genetic

Relationships within the Phrynocephalus guttotus Species Group (Sauria: Agamidae)

in the Former USSR ." 59

WANG, YUEZHAO AND HUIZHAO WANG. Geographic Variation and Diversity in Three

Species of Phrynocephalus in the Tengger Desert, Western China 65

TUNIYEV, BORIS. S. AND SVETLANA. YU. BEREGOVAYA. Sympatric Amphibians of the

Yew-box Grove, Caucasian State Biosphere Reserve, Sochi, Russia 74

SMITH, BRIAN E. Notes on a Collection of Squamate Reptiles from Eastern Mindanao,

Philippine Islands Part 1 : Lacertilia 85

SMITH, BRIAN E. Notes on a Collection of Squamate Reptiles from Eastern Mindanao,

Philippine Islands Part 2: Serpentes 96

ZHONG, CHANGFU. First Records for Ophisaurus harti and Python molurus bivittatus

from Jiangxi Province, China 1 03

MANILO, VALANTINA V. AND MICHAEL L. GOLUBEV. Karyotype Information on some

Toad Agamas of the Phrynocephalus guttatus Species Group (Sauria, Agamidae) of

the former USSR ' 105

MANILO, VALENTINA V. A Karyosystematic Study of the Plate Tailed Geckos of the

Genus Teratoscincus (Sauria, Gekkonidae) 109

WANG, PEI-CHAO AND JlANG-HUA ZHANG. Resting Metabolic Rate in Three Age-groups

of Alligator sinensis 112

ZHANG, YUN, YULIANG XlONG AND CASSIAN BON. Effects of Chinese Snake Venoms

on Blood Coagulation, Purified Coagulation Factors and Synthetic Chromogenic

Substrates H7

SCHAMMAKOV, SAHAT, CHARI. ATAEV, AND ELDAR. A. RUSTAMOV.

Herpetogeographical Map of Turkmenistan 1 27

LRJ, WAN-ZHAO, AND DA-TONG YANG. A Karyosystematic Study of the Genus

Bombina from China (Amphibia: Discoglossidae) 137

SONG, JIANXING, YlTLIANG XlONG, WANYU WANG, AND XlAOCHUN PU. A Study on

the Purification and Pharmacological Properties of Two Neurotoxins from the

Venom of the King Cobra (Ophiophagus hannah) 143

TARKHNISHVILI, David N., and Irina. A. Serbinova. The Ecology of the Caucasian

Salamander (Mertensiella caucasica Waga) in a Local Population 1 47

Guidelines for Manuscript Preparation and Submission 166

ASIATIC

HERPETOLOGICAL

RESEARCH

VOLUME 6

1995

Asiatic Herpetological Research

Editor

Ermi Zhao

Chengdu Institute of Biology, Academia Sinica, Chengdu, Sichuan, China

Associate Editors

J. Robert Macey

Department of Biology, Washington University, St.

Louis, Missouri, USA

Theodore J. Papenfuss

Museum of Vertebrate Zoology, University of

California, Berkeley, California, USA

Managing Editor

Valeurie E. Friedman

University of California Press, Berkeley, California, USA

Editorial Board

KRAIG ADLER

Cornell University, Ithaca, New Yoik, USA

Natalia B. ananjeva

Zoological Institute, St. Petersburg, Russia

STEVEN C. ANDERSON

University of the Pacific, Stockton, California, USA

KELLAR AUTUMN

Museum of Vertebrate Zoology, University of California,

Berkeley, California, USA

AARON BAUER

Villanova University, Villanova, Pennsylvania, USA

LEOBORHN

Zoological Institute, St. Petersburg, Russia

Bmui CHEN

Anhui Normal University, Wuhu, Anhui, 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

HAJIMEFUKADA

Sennyuji Sannaicho, Higashiyamaku, Kyoto, Japan

CARLGANS

University of Michigan, Ann Arbor, Michigan, USA

HUI-QING GU

Hangzhou Teacher's College, Hangzhou, Zhejiang, China

ROBERT F. INGER

Field Museum, Chicago, Illinois, USA

Mahmoud Latifi

Insutut d'Etat des serums et vaccins Razi, Teheran, Iran

KUANGYANG LUE

National Taiwan Normal University, Taipei, Taiwan, China

RONALD MARLOW

University of Nevada, Las Vegas, Nevada, USA

ROBERT W. MURPHY

Royal Ontario Museum, Toronto, Ontario, Canada

GOREN NTLSON

University of Goteborg, Goteborg, Sweden

HIDETOSHI OTA

Department of Biology, University of the Ryukyus, Nishihara,

Okinawa, Japan

JIONG-HUA Pan

South China Normal University, Guangzhou, Guangdong,

China

YUN-XU TONG

Lanzhou University, Lanzhou, Gansu, China

KE-MING XU

Liaoning Normal University, Dalian, Liaoning, China

yu-hua Yang

Sichuan University, Chengdu, Sichuan, China

KEN-TANG ZHAO

Suzhou Railway Teacher's College, Suzhou, Jiangsu, China

Asiatic Herpetological Research is published by the Asiatic Herpetological Research Society (AHRS) and the

Chinese Society for the Study of Amphibians and Reptiles (CSSAR) 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 outside of China and requests for subscription should be sent to AHR, Museum of

Vertebrate Zoology, University of California, Berkeley, California, USA 94720, or by email to

asiaherp@uclink.berkeley.edu. All correspondence within China should be sent to Ermi Zhao, Editor, Chengdu

Institute of Biology, P.O. Box 416, Chengdu, Sichuan Province, China. Authors should consult Guidelines for

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please notify us, and we will make other arrangements.

Asiatic Herpetological Research Volume 6 succeeds Volume 5 published in 1993, Volume 4 published in 1992,

Volume 3 published in 1990 and Chinese Herpetological Research Volume 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 succeeded 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.

Cover: Batrachuperus pinchonii Elev. 2270 m, 53.2 km north of Hanyuan (29° 21' N 102° 43' E), on the Ya'an to

Hanyuan Rd., Liba Shan (mountain), Ya'an Prefecture, Sichuan Province, China. Photo by J. Robert Macey.

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 1-26

Systematics of the Vipers of the Caucasus: Polymorphism or Sibling

Species?

GORAN NILSON1, BORIS S. TUNIYEV2, NIKOLAI ORLOV^, MATS HOGGREN4 AND CLAES

ANDREN1

'Department of Zoology, Goteborg University, Sweden

2Caucasian State Biosphere Reserve, Sochi, Russia

^Zoological Institute, Russian Academy of Sciences, St. Petersburg, Russia

^Department of Genetics, University of Uppsala, Russia

Abstract. -Inter- and intramorphological variation were examined in sympatric and allopatric polymorphic

and monomorphic populations of the Vipera ursinii and Vipera kaznakovi complexes. The alpine Vipera

dinniki populations in upper Great Caucasus show a pronounced, and to a certain extent geographical,

polymorphism. Color patterns include among others 'kaznakovi', 'tigrina', 'berus', 'bronze', and 'ursinii'

types. Several of these patterns can be represented within the same litter in certain populations. Vipera

dinniki is sympatric with the Caucasian representative of the Vipera ursinii complex in some areas. This last

taxon shows a similar degree of polymorphism, which is unique for this complex, and due to morphological

and molecular distinction, we consider it to be a Caucasian evolutionary species within the ursinii complex -

Vipera lotievi sp.n.

Key Words: Reptilia, Squamata, Viperidae, Vipera dinniki, V. kaznakovi, V. ursinii, V. renardi, V. lotievi

sp.n., Caucasus, Russia, Georgia, taxonomy, morphology, polymorphism.

Introduction

The taxonomy of the vipers of Caucasus

has for a long time been confusing and

contradictory. According to the traditional

view a single species, Vipera kaznakovi, is

distributed in the moist and warm lowlands

of the western Caucasus as well as in the

mountain valleys towards the east. In the

east the habitat is drier and along the range

the vipers gradually change toward Vipera

ursinii in appearance. In the east Caucasus

only this last viper was supposed to occur.

Thus there seemed to be a somewhat clinal

transformation from "pure" V. kaznakovi in

the west to "pure" V. ursinii in the east.

Vipers from the intermediate region could be

difficult to determine. Within the same

locality some specimens look like V.

kaznakovi, other ones are more like V.

ursinii, while still some can be intermediate.

Nikolsky (1913) separated the alpine

populations into the taxon Vipera berus

dinniki, which was based on alpine

specimens of the conventional V. kaznakovi

from high altitudes in the western Caucasus

(Malaya Laba River- terra typica restricta

and Svanetia) as well as from other places.

The name dinniki was long considered as a

synonym of V. kaznakovi until Vedmederja

et al (1986) recognized it as a separate

species inhabiting alpine and subalpine

meadows in the Caucasus, and thus

restricting V. kaznakovi to lower altitudes in

western Caucasus and adjacent moist

lowland habitats along the eastern Black Sea

coast. Thereby the problem of the gradual

transformation from V. kaznakovi in the

west of the Caucasus to V. ursinii in the east

is restricted to the high Caucasus

populations, now including V. dinniki and

V. ursinii. Vipera kaznakovi is well defined

and restricted in distribution, and

geographically separated from all the other

viper species in the region.

The complex history of nomenclature

and taxonomy has been clarified to a great

part in some recent publications (see Orlov

& Tuniyev, 1986; 1990), together with

hypotheses about the phylogeny of this

group. Concurrently the need of genetic

studies was stressed, and this has led to the

present work where we in a series of papers

intend to clarify the taxonomy and evolution

of the vipers of this region. The work is

planned to have a broad perspective

including phenetic and phylogenetic

analyses, habitat choice, niche-breadth, and

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 2

Asiatic Herpetological Research

June 1995

reproduction. Different methods take

different time and in this first paper the

morphology is reexamined, based on

available material in Museum collections and

freshly collected material from a number of

new places, as well as on inheritance of

color pattern. Genetic structures based on

phenetic analyses of allozyme data are also

presented.

The morphological distinction between

Vipera dinniki and V. kaznakovi has been

presented elsewhere (Orlov and Tuniyev,

1986; 1990) and will not be repeated in this

study. In the present paper we are focusing

on patterns of morphological and molecular

variation within and between the different

populations in the Caucasus, and the

taxonomy of these populations.

Material and Methods

The work has mainly been a study of

variation in external morphology, allozymes

and reproduction in order to reveal patterns

of sympatry and sibling species. Additional

genetic studies will follow when material

becomes available in suitable samples

(presently delayed due to political reasons).

Thus live and preserved museum material

has been examined concordantly with

studies of reproduction in the laboratory.

Additional preserved material used in

this study originates from the Natural

History Museum in Goteborg (GNM);

Dipl.-Biol. F. J. Obst, Staatliches Museum

fur Naturkunde, Dresden (MTKD D); Aram

Agasian, Zoological Institute, Academy of

Sciences, Eriwan, Armenia. Abbreviations

for museums as used in the text are: CNR-

Caucasian State Biosphere Reserve,

Collection of Boris Tuniyev at Yew-box

Groove, Sochi; GNM- Goteborg Natural

History Museum, Goteborg; MTKD-

Staatliches Museum for Naturkunde,

Dresden; ZIEr- Zoological Institute,

Academy of Sciences, Eriwan; ZIG-

Department of Zoology, University of

Goteborg; Goteborg- (authors' collection,

which later will be incorporated in GNM);

ZIN- Zoological Institute, Academy of

Sciences, St. Petersburg.

Altogether about 300 preserved or live

specimens of vipers from the Caucasus and

adjacent regions have been seen during the

study. Joint field trips were made in

different parts of the Caucasus in 1990 and

1992, but two of us (Tuniyev and Orlov)

have performed extensive research in the

region prior to that. For morphometric

studies 183 preserved snakes within the

ursinii and kaznakovi complexes have been

examined more carefully, and for most of

these specimens 30 different items of data

have been collected. This information was

used, down to population level, in

morphological descriptions, taxonomical

analyses and conclusions about

zoogeography and range overlap.

Inheritance of color pattern was studied

based on 23 pregnant females and their

offspring.

Data collected were: total length and tail

length; number of preventrals, ventrals,

subcaudals, anterior and mid-body dorsal

scale rows, apical plates, supralabials,

sublabials, circumocular scales, loreals,

second chinshields, mentals, crown scales

(=intercanthals + intersupraoculars), and

zig-zag windings in dorsal band. Further

rostral index (height/breadth) and head index

(breadth/length) were calculated. Division

of parietals, frontal, and nasalia was noted,

as was the color of dorsal and ventral sides,

and iris (in live specimens). Further, upper

preocular size; and head, labial and lateral

body patterns, as well as distinctiveness of

canthus rostralis were examined. Details

about these methods are found in Nilson and

Andren (1986).

Morphologylphenetics

Standard errors accompanying mean

character ratios were used as relative

measurement of dispersion. For the

analysis of intra- and interpopulational

morphological variation (phenetic analysis)

the samples were divided into subsamples

depending on questions raised. Thus

besides an analysis of morphological

variation also a pattern confirming or

rejecting the present taxonomic pattern could

be achieved. This pattern could also be

June 1995

Asiatic Herpetological Research

Vol. 6, p. 3

TABLE 1 . Number of specimens used in the genetic analyses and localities (Russia if nothing else is stated)

for the examined taxa.

kaznakovi:

1. Dagomys, north of Sochi. Six specimens.

2. Rudorova, inland locality, 900 m alt. Four specimens.

dinniki:

3 . Fisht/Oshten, the westernmost locality of the main Caucasus range. Seven specimens.

4. Lake Impsi, 1,980 m. alt., at a tributary to the Little Laba River on the northern slope

of the main range. Seven specimens.

5 . Aishkha-II on the southern slope of the main range. Three specimens.

6. Lake Kardyvach at upper Mzymta River on the southern slope of the main range

Seventeen specimens.

lotievii:

7. Armkhi, Checheno-Ingushetia. 2,000 m altitude. Seven specimens.

berus:

8. Uppsala (terra typica), Sweden. Eleven specimens.

eriwanensis:

9. pooled sample from Asbua and Cildir, Kars, east Turkey; and Sevan, Armenia. Six

specimens.

supported or rejected by the parallel

biochemical studies. Thereby it is possible

to state or reject the occurrence of

convergent or parallel evolution, i.e. sibling

species.

Estimation of the different color pattern

frequencies in local populations was based

on observations during the field work.

Small museum samples collected by others

were not included in this analysis due to

uncertainty of randomness in sampling

(unusual morphs might have been collected

and preserved at a higher degree).

Biochemical data

Enzyme electrophoresis. — Sixty-eight

specimens representing different taxa of

Caucasus vipers, and Vipera berus from

Sweden were examined. The samples were

treated as nine independent operational

taxonomic units (OTUs) in the genetic

analysis, in order to avoid a priori

assumptions of taxonomic relationships

among the eight Caucasus populations

studied (see Table 1, for locality data and

sample sizes). A potential risk of sampling

error due to syntopic occurrence of two taxa

may be avoided by testing observed

genotype distribution within a locality

against Hardy-Weinberg expectations (see

results). Fresh or frozen tissues (-75°C)

from liver and skeletal muscle were

homogenized in distilled water. The extracts

were centrifuged for 10 min at 10,000 rpm

and 4°C and the supernatants were then

stored at -75°C until used. Standard

horizontal starch gel electrophoresis was

carried out, as described by Harris and

Hopkinson (1976) and Murphy et al.

(1990). Gels (11% w/v) were prepared

from Sigma starch (Sigma Chemical Co.,

St. Louis, Mo). Two buffer systems were

used: (A) Gel: 0.03 M tris-0.005 M citric

acid; Electrode: 0.06 M lithium hydroxide-

0.03 M boric acid, pH 8.0 (Ridgway et al.,

1970). (B) Gel: 0.002 M citric acid, pH

6.1; Electrode: 0.04 M citric acid, pH

adjusted with N-(3-amino-propyl)morpholin

Vol. 6, p. 4

Asiatic Herpetological Research

June 1995

TABLE 2. Enzymes and electrophoretic conditions of the polymorphic loci scored in this study.

Nomenclature and commission numbers following the International Union of Biochemistry, Nomenclature

Committee (1984). Abbreviations for tissue sources are: L=liver and M= skeletal muscle.

1) Harris and Hopkinson (1976)

2) Johnson et al. (1970)

3) Shaw and Prasad (1970)

4) Murphy etal. (1990)

5) De Lorenzo and Ruddle (1969)

A) Tris-citrate/lithium hydroxide, boric acid, pH 8.0, lOV/cm, 4h (Ridgway et al., 1970)

B) N-(3-amino-propyl)morpholine/citrate, pH 6.1, lOV/cm, 6h (Clayton and Tretiak, 1972)

(Clayton and Tretiak, 1972). Enzymes

assayed, tissues, modified electrophoretic

conditions and staining references (Table 2)

follow Nilson etal. (1994).

Reproduction

Inheritance of color morphs within

broods was studied by keeping pregnant

females in the laboratory until giving birth.

By this, various hypotheses of

polymorphism and its inheritance patterns

could be evaluated.

Fieldwork

Substantial information has been

gathered during several years in the field in

Russia, Georgia, Armenia and Turkey. A

more intensive field survey was performed

in the western Caucasus in July, 1990, in

order to obtain information of color morphs

in natural populations. Together with

preserved material, this information was the

base for the study of morphological

intrapopulational variation in V. dinniki.

Material was also used for reproductive

studies (see above).

The V. dinniki localities, situated in the

Caucasian State Biosphere Reserve and in

the Sochi Nature State National Park and

listed below were included in this study:

Fisht/Oshten — The westernmost high

mountain area of the main Caucasus range,

and of the Reserve, characterized by the

peaks Mt. Fisht (2868 m. alt.) and Mt.

Oshten (2804 m. alt.). This region is

separated and isolated from the main range

(with Mt. Chugush 3237 m. alt.) by the

forested, moist and warm "Colchis Gate" at

low altitude (not more than 1500 m).

Loyub — Mt. Loyub (2990 m. alt) at the

uppermost part of the Mzymta River in the

eastern part of the Reserve, situated on the

southern slope of the main range.

Aishkha-II (2858 m. alt) — in the same

mountain massif, a little bit further to the

west, and situated on the southern slope of

the main range.

Lake Kardyvach (1850 m. alt) — the

eastern border of the Park, close to Mt.

Loyub but a little further down the Mzymta

June 1995

Asiatic Herpetological Research

Vol. 6, p. 5

FIG.. 1. The typical "dinniki" pattern type of Vipera dinniki, with unicolored lateral sides typical for

Fisht/Oshten (ZIG).

FIG. 2. The "dinniki" pattern type of Vipera dinniki, with a tendency towards the "tigrina" morph. From

Kardyvach (ZIG).

River. Also situated on the southern slope

of the main range.

Lake Impsi — at 1980 m. alt., situated at

the Tsahvoa River, a tributary of the Little

(Malaya) Laba River. The locality is mainly

on the slopes of the Damhorts Range at the

northern part of the Reserve, and partly on

Akaragvarta Mountain situated on the

northern slope of the main range.

In addition much information on Vipera

kaznakovi was gathered at the lowland

Black Sea coast localities of Dagomys, north

of Sochi (Russia) and Hopa, Artvin

Province (Turkey).

Results

The results of the analyses of

morphometries and enzyme electrophoresis

are presented separately.

Intra- and interpopulational variation in

morphology

A great number of different color

morphs are expressed in the Caucasian

vipers. Although several stages of

overlapping and intermediate forms could be

seen, we define the following major pattern

types:

Nilson et al.

Asiatic Herpetological Research

Plate 1

a. The "ursinii" pattern type of Vipera dinniki, from Kardyvach (ZIG).

b. The "tigrina" morph of Vipera dinniki, from Impsi with partly divided transverse bars

(ZIG).

Plate 1

Nilson et al.

Asiatic Herpetological Research

c. The "bronze" morph of Vipera dinniki, from Impsi (ZIG).

d. The "bronze" morph of Vipera lotievi from Itum Kali, Checheno-Ingushetia.

Vol. 6, p. 6

Asiatic Herpetological Research

June 1995

FIG. 3. The "kaznakovi" morph of Vipera dinniki, from Fisht/Oshten (Mt.Oshten - Armenian pass).

A. "dinniki": a more or less continuous

zig-zag band, and pronounced lateral

blotches. Sometimes nebulous in pattern.

Sometimes rather Vipera berus like (Figs.

1,2).

B. "kaznakovi": pronounced black

lateral and dorsal longitudinal bands, and

yellow or orange ground color. The dorsal

band is waving or expressed as a straight

band resulting in a contrasting more or less

bilineated pattern. Besides these we could

define a "bilineated" morph which is similar

to the "kaznakovi" type of pattern but much

lighter. In the analysis below it is included

in the "kaznakovi" type (Fig. 3, compare

Fig. 4).

C. "ursinii": a black-edged continuous

dark brown zig-zag band on a paler ground

color and lighter sides of body (Fig. 5, Plate

la).

D. "tigrina": dorsal pattern fragmented

into broad or narrow transverse bands.

Also spotted pattern could be seen in some

populations. This pattern type is most close

to "tigrina", but differ by being divided

along the vertebral line thus resulting in two

rows of dark spots along the dorsal side of

the body (Fig. 6, Plate lb).

E. "bronze": a uniform greyish to

brownish or blackish ground color covering

all parts of body except the head.

Sometimes with a darker narrow or broad

vertebral stripe (Figs. 7, 8, Plate lc).

F. "melanism": black, with a high

production of melanin covering all other

color patterns.

The different morphs were represented

in different frequencies at the different

localities examined, and some morphs

seemed to be restricted to one or a few

June 1995

Asiatic Herpetological Research

Vol. 6, p. 7

FIG. 4. A typical Vlpera kaznakovi from the inland locality cordon Babuk-Aul at the foothills of Mt. Fisht

(ZIG).

FIG. 5. The "ursinii" pattern type of Viper a

dinniki, with more unicolored lateral sides from Mt.

Fisht (ZIG).

localities (Table 3). Also variation in other

color characteristics was obvious when

comparing populations. The number of

windings in the dorsal zig-zag band varied

markedly with low number in the western

isolated Fisht population and high in the

more eastern populations (Fig. 9). This was

especially pronounced when comparing

Fisht with Kardyvach, characterized by a

high frequency of "tigrina" morphs (Table

3). Almost no overlap was detected as in

Kardyvach the vipers have 68 or more

windings while in the Fisht population the

corresponding figures are 69 or less (Table

4). In general the west Caucasian samples

(except Fisht) have higher number of dorsal

windings or transverse bars than central and

east Caucasian dinniki vipers. Also V.

kaznakovi has fewer windings.

Vol. 6, p. 8

Asiatic Herpetological Research

June 1995

FIG. 7. The "bronze" morph of Vipera dinniki, from Mt. Loyub (ZIG).

TABLE 3. Distribution of color morphs in samples of V. dinniki and V. kaznakovi (Dagomys) in absolute

numbers and in percentage (in some cases) in 1991. Sample size in parenthesis.

Inheritance of color morphs

The different color morphs can be seen

in broods from different types of females

(Table 5), verifying that at these localities a

single polymorphic species is involved. The

sample is not big enough for allowing

definite conclusions about inheritance, but

some indications can be obtained. The three

"bronze" females only gave birth to

"bronze" juveniles (one brood) or mixture of

"bronze" and narrow banded "tigrina"

juveniles (two broods). No other female

than these three (in the total sample of 23

pregnant females) gave birth to "bronze"

juveniles. Further, the "tigrina" pattern

seems dominant as it shows up in several

broods, and is always expressed when the

female has a pattern towards "tigrina". In

the three pure "tigrina" females that gave

birth all juveniles were of "tigrina" type

(N=8). Further (although not seen from the

table) eleven females with other pattern

types than "tigrina" also produced "tigrina"

juveniles together with other morphs. Thus

"tigrina" and "bronze" were the most

frequent juvenile morphs in the dinniki

material.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 9

FIG. 6. The narrow-banded "ligrina" morph of

Vipera dinniki, from Kardyvach (ZIG).

Of the juveniles taken together half

(50%) were "tigrina". When considering

the Kardyvach material alone (10 broods

with 29 juveniles) 52% were "tigrina" while

24% were "bronze". Of 47 adults observed

in the field in July 1991 at this locality 24%

were "bronze". The number of adult

"tigrina" observed (33%) was lower than the

frequency of juvenile "tigrina" produced

while the number of adult "ursinii" was

rather high (27%). This slight reduction of

the "tigrina" pattern between juveniles and

adults can have an ontogenetic explanation

as pattern often fades with age.

In other populations examined the

combinations of morphs were different,

with other patterns dominating. The number

of windings is much lower in Fisht

compared to the Kardyvach sample (with

"tigrina" predominating). "Melanism" was

only observed at Fisht, but it is known also

from Aishkha-II and the Bezymyanka River.

"Bronze" was only observed at Kardyvach

and Impsi. When comparing field

observations and all available specimens in

collections, this morph was not documented

from any other locality along the entire area.

FIG. 8. The "bronze" morph of Vipera dinniki,

from Mt. Loyub (ZIG).

Dorsal bars

FIG. 9. Distribution of number of bars and/or

windings in the dorsal zig-zag band in the different

Vipera dinniki populations, running from west

towards east in high Caucasus (1: Fisht, 2: Loyub,

3: Kardyvach, 4: Impsi, 5: "central Caucasus"

(=Elbrus and surroundings), 6: "east Caucasus"

(=mountains above Lagodechi, Georgia), and V.

kaznakovi populations (7: Sochi-Adler, 8: Hopa,

Artwin).

At Impsi the typical "dinniki" pattern was

dominating (56%).

Analysis ofscalation characters in the

different dinniki populations

Geographic variation in color morph

frequencies is also reflected in scalation

characters (Table 4). Going from the west

Caucasus towards east, certain changes

could be observed. The central and east

Caucasus samples of dinniki have higher

mean number of preventral plates, lower

Vol. 6, p.

10

Asiatic Herpetological Research

June 1995

TABLE 4. Variation in scalation and color pattern characters between different isolated populations of Vipera

dinniki The localities include a series of isolates at close distance in the Caucasus State Biosphere Reserve (1:

Mt Fisht/Oshten; 2: Mt. Loyub; 3: Lake Kardyvach; 4: Lake Impsi), and 5: central Caucasian population

(Mt Elbrus region) and 6: east Caucasian population (the mountain region of Lagodekhi) sample. Given as

Mean value, S.E. and range (except for preventrals and apicals). Number of specimens in parentheses.

Rostral

index

Apicals

Circ urn-

oculars*

1.1+0.1

1.0-1.4

1.1±0.0

1.0-1.3

1.1±0.0

0.9-1.3

1.1±0.1

0.9-1.1

1.0±0.0

0.9-1.3

18.3 ±0.5

15-21

19.1 ±1.0

14-23

18.4 ±0.5

14-22

17.8 ±0.6

13-22

17.3 ±0.9

12-19

1.1±0.1

1.0-1.3

1.46 ±0.14 1.33 ±0.17 1.47 ±0.13 1.50 ±0.13 1.00 ±0.00 1.17 ±0.17

19.2 ±0.8

18-23

* Counted as sum of both sides.

** Unicolored "bronze" specimens as well as completely bilineate and melanistic specimens excluded.

TABLE 5. Phenotypic expression of inheritance of color patterns in 23 clutches of Vipera dinniki. Given as

taxa, female morph, number of clutches, numeric distribution of morphs in group of juveniles of each female

morph.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 1 1

o o

- o o

o o o

o

o o o o o

o o o o o

- o o o o o o -

o o o o o o

o o o o o o

o o o o o o

o o o o o o

o o o o -

0 0 o

o

o o

o o

o

Crownscales 25

FIG. 10. Distribution of ventral numbers in the

different Vipera dinniki populations, running from

west towards east in high Caucasus (1: Fisht, 2:

Loyub, 3: Kardyvach, 4: Impsi, 5: "central

Caucasus" (=Elbrus and surroundings), 6: "east

Caucasus" (=mountains above Lagodechi, Georgia),

and V. kaznakovi populations (7: Sochi-Adler, 8:

Hopa, Artwin).

head index iQO

rostral index

FIG. 12. Head index (breadth/length) and rostral

index (height/ breadth) in sympatric sibling species

of east Caucasus. White circles= V. dinniki ("east-

dinniki"); black squares= V. lotievi.

mean number of apical scales, and lower

number of loreal scales. Also the number of

ventrals showed a slight decrease towards

the east, a pattern also observed between

northwestern and southern populations of

Vipera kaznakovi (Fig. 10).

Analysis of scalation characters in the

different dinniki morphs

In subalpine and alpine mountain belts of the

west Caucasus (eastward to the basin of the

Big Laba River) the "ursinii" morph belongs

to the same species as the rest of the

120 125 130 135 140 145 150 155 160

FIG. 11. Numbers of crown scales and ventral

plates in "ursinii/ bronze" and "dinniki" morphs and

sympatric sibling species of west and east Caucasus.

The dotted line indicate the main separation between

V. lotievi and the V. dinniki from different regions.

Big white circles= the "dinniki" morph of western

Caucasus (V. dinniki); big black squares= the

"ursinii/bronze" morphs of western Caucasus (V.

dinniki); small white circles= the "dinniki" morph of

eastern Caucasus (V. d/nw'/fc/="east-dinniki"); small

black squares= the "ursinii/bronze" morphs of

eastern Caucasus (V. lotievi)

mountain vipers: Vipera dinniki. But in the

extreme eastern part of the west Caucasus,

and the central and east parts of the high

Caucasus, the "ursinii" and "bronze"

morphs belong to a different species. An

examination of the morphology of the

different morphs clearly indicate that in the

eastern half of high Caucasus there are two

sympatric species (Figs. 11 and 12).

Vipera kaznakovi (Fig. 4).

This species also shows some regional

variation, although not so pronounced as in

V. dinniki. When comparing the southern

(Turkish) populations with the northern

(Russian) ones, differences in color pattern

as well as in scalation could be detected.

Specimens from the southernmost

population in Hopa (Turkey) are more

yellowish in ground color compared to the

snakes in the northern parts (Dagomys). To

the contrary, specimens in the north often

have more black areas on the body and

often the orange or reddish ground color is

expressed only as two dorsolateral rows of

spots. Melanism is frequent in this northern

Vol. 6, p. 12

Asiatic Herpetological Research

June 1995

TABLE 6. Variation in scalation and color pattern characters between the northern and southern Vipera

kaznakovi populations. The samples are from northeastern Black Sea regions in Russia (Sochi-Adler); and

from northeastern Turkish Anatolia (Hopa, Artvin province). Given as Mean value, S.E. and range (except

for preventrals and apicals). Number of specimens in parentheses.

Sochi-Adler (17)

Hopa (13)

Preventrals

Ventrals

Rostral index

Apicals

Circumoculars*

Loreals*

Crown scales

Zig-zag windings**

1.4L+0.19

133.8+0.6, 130-138

1.1±0.02, 1.0-1.27

1.50±0.13

20.0±0.48, 16-23

11.06±0.76, 7-16

14.9410.92, 10-23

56.33±0.67, 55-57

1.54±0.18

130.4±0.9, 124-136

1.1±0.06, 0.87-1.5

1.64±0.14

19.31±0.43, 15-21

8.69±0.60, 5-12

17.33+1.08, 11-22

50.75+1.55, 48-55

* Counted as sum of both sides.

** Four completely bilineate and melanistic specimens are excluded.

TABLE 7. Allele frequencies of polymorphic loci (see Table 2 for locus abbreviations). For taxon

abbreviations, sample sizes and localities, see Table 1.

population, a phenomenon never observed

in the southern one. It can be added that

melanistic specimens have been found in

more or less all Russian populations.

During a stay 1992 at an inland locality

along the Psou River we observed ten adult

specimens of which all but three were

melanistic.

In scalation characters there are some

minor differences: e.g. the Hopa population

has a lower mean value in number of

ventrals and crown scales (Table 6).

June 1995

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Vol. 6, p. 13

TABLE 8. Genetic distances (above diagonal) and genetic identities (below diagonal) of eight OTUs of

Caucasus vipers and Vipera berus. 1- kaznakovi, Dagomys; 2- kaznakovi, Rudorova; 3- dinniki, Fisht; 4-

dinniki, Impsi; 5- dinniki, Aishka; 6- dinniki, Kardyvach; 7- lotievii; 8- berus; 9- eriwanensis.

OTU

1

FIG. 13. UPGMA phenogram clustering modified

Nei genetic distances (Hillis 1984) among eight

OTUs of Caucasus vipers and Vipera berus.

Phenetic analysis of electrophoretic data

Of twenty-seven presumptive gene loci

scored, from 14 enzyme systems and one

general protein, only eight were

polymorphic. Allelic products (allozymes)

for each locus were designated numerically

in order of increasing anodal mobility to

identify genotypes (Table 7), with the

relative mobility of the most common allele

in each locus as a standard of reference

("100"). Despite the fact that average

sample sizes were rather small for obtaining

significant results of frequency analysis we

elected not to pool different localities (except

for eriwanensis), for reasons of objectivity

discussed above, although we recognize the

obvious risks of missing rare alleles through

this procedure.

species found in west and east high Caucasus.

Modified Nei genetic distances (Nei,

1978; Hillis, 1984) between OTUs were

computed from allele frequencies (Table 8).

A UPGMA phenogram (Sneath and Sokal,

1973) was constructed from the distance

data (Fig. 13). This phenogram illustrates

only the relative degree of genetic

differentiation among OTUs and should not

be considered as a phylogenetic tree.

Observed genotype distribution of the most

polymorphic locus (Sod-1) in the largest

individual sample (Kardyvach; N=17) was

tested against Hardy-Weinberg expectations

to investigate homogeneity and possible

biased sampling due to potential within-

locality sympatry of taxa (i.e. Wahlund

effect). A chi- square test performed to

obtain the goodness-of-fit between observed

and expected distributions (X2=0.060, 1 df,

P=0.90) strongly support unbiased

sampling. The genetic analysis confirms

that the four populations from the western

Main Caucasus (Fisht, Kardyvach, Aishka-

Vol. 6, p. 14

Asiatic Herpetological Research

June 1995

II and Impsi) belong to one polymorphic

taxonomic unit (V. dinniki).

Discussion

The original question, whether

polymorphism or sibling species are the

prevailing phenomenon in the Caucasian

populations of vipers, must be answered

with yes in both cases. In the western

Caucasus a large number of morphs can be

recognized. We separate six different ones

in that region while the number is restricted

to three in the eastern and central parts of the

mountain range (Fig. 14). As seen in the

reproductive studies all six morphs

("melanism", "bilineat-kaznakovi", typical

"dinniki" (including "nebulosa"), "tigrina",

"bronze", and "ursinii") in the mountain

habitats in the western Caucasus belong to a

single species. This is also verified by

cladistic analyses of biochemical and

morphological data (Nilson et al., 1994).

The different morphs can be seen sympatric

and syntopic in suitable rocky, vegetation-

rich, and moist habitats. There seems to be

a certain inheritance pattern, but in principle

most morphs can occur in the same brood

(Table 5). Certain areas seem to have a

certain range of frequency of the various

morphs, which might be unique for that

particular area. It must be kept in mind that

only a random number of populations have

been investigated, and it is likely that

additional morphs will be described from

other isolated localities. The isolation of the

different mountains in the Caucasus is much

comparable with e.g. the Andes of Ecuador,

or the Galapagos islands, and a high degree

of isolation has obviously taken place

between the different mountain peaks.

How could this particular polymorphic

pattern in the subalpine Vipera dinniki

populations have evolved? The present

warm and wet subtropical Colchis area of

western Transcaucasia at the Black Sea coast

has, as indicated by botanical evidence,

served as a refuge for animals and plants

during the whole of Pliocene and

Pleistocene (Tuniyev, 1990). The total

region has varied much in size during the

Pleistocene glacial and interglacial periods

but remained a permanent relict key area.

The warm and wet adapted Vipera

kaznakovi is distributed in this area today,

and it has been considered that this species

has had an occurrence in this region for a

long time (Orlov and Tuniyev, 1990;

Tuniyev, 1990) due to the climatic stability.

A scenario for the arise of various

polymorphic V. dinniki populations could

have taken place in two steps and been like

this:

1. First, the evolution of Vipera dinniki.

If the Pliocene Colchis region decreased in

range during a Pleistocene glacial period

there are two alternatives for the species in

that part of the old range that have turned

cold: the viper could disappear or it could

adapt. If it disappears it can be done in two

ways: the snake becomes extinct or it is

forced down to the remaining warm zone.

In both these two cases the net result will be

a reduced range for the single species

(kaznakovi) in the remaining warm and wet

Colchis refuge. In the second case, if the

viper is adapted to the new climate, "the cold

zone", it would be a new, physiologically

different race.

In an interglacial period when the climate

becomes warmer again the warm and wet

Colchis zone expands and "the cold zone" is

forced upwards to higher altitudes in the

mountains. Now, if there is a cold-adapted

physiological race, again two alternatives are

open: first, it could adapt, or second, it

could disappear. If it is adapted back to the

new warm environment, it can either go

back into and unite with the old species

(kaznakovi) which under the new climatic

conditions can expand its range, or it could

form a sympatric but physiological distinct

taxon. In the case of sympatry there is a

good possibility that it would disappear due

to competition (if not ecological distinct). In

both these last situations there is a great

probability that the original species

(kaznakovi) would return and again cover its

original range.

If the new physiological race disappears

from the region it could again be done in

two ways: it simply becomes extinct due to

the new "severe" climatic conditions, or it

June 1995

Asiatic Herpetological Research

Vol.6, p. 15

migrates upwards following the pushed up

"cold zone". In the first case again only the

original species (kaznakovi) will remain in

its new expanded (=original) warm and wet

range. In the second case there will be a

number of cold-adapted populations at

higher altitudes (the present different

dinniki, "east-dinniki", darevskii

populations). This would mean a number of

isolated populations as the mountains of the

Caucasus are rather steep and

subalpine/alpine habitats are in many cases

climatologically isolated. Further the

geology is very complex reflecting a high

degree of local endemism among plants and

animals. One can postulate that the different

populations, or groups of populations, must

have become adapted to local conditions.

2. This may have been the prerequisite

for an evolution of a polymorphic Vipera

dinniki. During Pleistocene there were

several glacial and interglacial periods, and

the scenario postulated above would have

been repeated several times, and during each

interglacial the "cold zone" and its cold

adapted viper were forced downwards with

the result of a secondary contact with

neighboring populations. Unique morphs

could by this be spread to adjacent

populations etc.

Today we can see a polymorphic color-

pattern in the cold adapted Vipera dinniki

that to a high degree is unique for one or a

small number of populations in close

connection, e.g. the "bronze" and "tigrina"

morphs, but not seen in all populations.

Vipera dinniki could be a polymorphic

species, constituent of a number of

populations that during periods are isolated

from each other, but irregularly have

secondary contact. In some cases the

isolation could also have been more

permanent resulting in a number of sister

species along the range, a possible

phylogenetic pattern we currently are trying

to solve with genetic studies. But overall,

the relative genetic differentiation between

examined taxa and degree of genetic

polymorphism were low, indicating a rather

recent divergence.

The present geographical distribution

and morphological pattern of Vipera dinniki,

as well as the fact that in all subalpine

mountain regions where V. dinniki is

located today, there also are fragmented

more or less subtropical Colchian refugia at

lower altitudes, inhabited by V. kaznakovi

(Tuniyev, 1990). This supports the

evolutionary pattern postulated above.

Further east in the mountains, the

habitats get drier with moist areas restricted

to stream surroundings and lake shores. In

central Caucasus (Mt. Elbrus) the number of

morphs decreases to two ("ursinii" and

"dinniki") or three in the eastern Caucasus

(where again a form of "bronze" morph

appears) (Fig. 14).

Now, in the region of the central and

eastern Caucasus two different sympatric

species are involved. As shown in the

results section above, at several localities the

two morphs "dinniki" and "ursinii" actually

represents two sympatric species from the

kaznakovi line and the ursinii line

respectively. It is obvious that these two

species are sympatric (but not necessarily

syntopic) in a large number of places in

these eastern and central parts of the main

range of the Caucasus. We have in our

material such records of sympatry from Mt.

Elbrus in the central Caucasus (Figs. 15-

18); mountains north of Lagodechi in the

eastern Caucasus; at Itum- Kali, Checheno-

Ingushetia (Fig. 19); and various records

from Dagestan, besides several isolated

records of both species from the entire

eastern and central Caucasus range.

Vipers of the kaznakovi group are

known from subalpine meadows, and

snakes of the ursinii group have been found

in the semiarid hollows between the main

range and the Skalisty (Rocky) range. At

several places with connection of subalpine

meadows and semiarid hollows the two

species have a sympatric occurrence (and

syntopic along the ecotones of both types of

landscapes). The ursinii line has probably

never been widely represented in the

perpetually humid western Caucasus, as this

taxon is adapted to dry environments, but as

stated earlier, in the extreme eastern part of

Vol. 6, p. 16

Asiatic Herpetological Research

June 1995

FIG. 15. Male of the east Viper a dinniki ("east-

dinniki") at the sympatric locality, Mt Elbrus,

central Caucasus. This specimen was found together

with the Vipera lotievi on figures 17 and 18 (ZIG).

FIG. 17. Female of the Vipera lotievi at the

sympatric locality, Mt Elbrus, central Caucasus.

This specimen was sympatric with the Vipera

dinniki ("east-dinniki") on photo 15 and 16 (ZIG).

the west Caucasus there are some isolated

populations of the ursinii complex (from the

Abishiz-Akhuba Range to Mt. Elburs).

The eastern form of dinniki is not

polymorphic in the same way as the western

populations. Rather the main color pattern

is the typical "nebulosa-dinniki" kind of

pattern. In sympatric areas the ursinii taxon

is more or less typical "mountain ursinii" in

color pattern although with a certain

similarity to the type of bilineate pattern seen

in the southwest European V. seoanei (Fig.

20). In some populations of this taxon in

Checheno-Ingushetia, a certain fraction of

the snakes are also "bronze" colored (Plate

Id). This pattern type has not been

observed in the sympatric populations. The

FIG. 16. Ventral side of the male of the east

Vipera dinniki ("east-dinniki") at the sympatric

locality, Mt Elbrus, central Caucasus from figure 15

(ZIG).

FIG. 18. Ventral side of the female of the Vipera

lotievi at the sympatric locality, Mt Elbrus, central

Caucasus from photo 17 (ZIG).

"nebulosa-dinniki" pattern of eastern dinniki

and the "seoanei-ursinii" pattern of

Caucasian ursinii taxon shows great

similarities, and can in some specimens be

difficult to separate. This is certainly the

reason for much of the confusion in earlier

studies of these vipers.

However, at a closer examination, the

two taxa are possible to identify (Table 9).

In ursinii the belly is lighter and the snout

more concave with raised canthus. The

preocular is large and in contact with the

nasal, and the apical is always single. Also

the crownscales are less fragmented. There

June 1995

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Vol.

6, p. 17

TABLE 9. Main morphological characteristics separating the sympatric Vipera lotievi and "east-dinniki".

White belly

Preocular in contact with nasal

Snout concave

Mean no. of ventrals

Always a single apical

Mean no. of crown scales

Parietal ocellated spot

Iris gold-edged in life

FIG. 19. Vipera dinniki ("east-dinniki")(upper) and Vipera lotievi (lower) from Itum Kali, Checheno-

Ingushctia. These specimens were found together at the same time (ZIEr).

Vol. 6, p. 18

Asiatic Herpetological Research

June 1995

FIG. 20. Female of Vipera lotievi sp.n. from the type locality, the surroundings of Armkhi Village,

Checheno-Ingushetia, Nazranovskiy District.

always seems to be an ocellated spot present

on the parietal plate, and the ventral number

is high. In the "east dinniki" taxon the belly

is blackish and the snout more flat, the

preocular is always separated from the

nasal, and there is a higher fragmentation of

the crown scales. The iris always seems to

be gold-edged in live specimens (as is the

case for the entire V. kaznakovi complex),

and this is specially distinct in younger

specimens. The ventral number is lower.

Although in many ways similar in

pholidosis the kaznakovi lineage and the

ursinii lineage are genetically well separated

and paraphyletic. Immunological

comparisons of blood serum albumins

indicate that Vipera kaznakovi and related

taxa belongs to the berus-aspis branch while

ursinii constitute a distinct evolutionary

lineage (Herrmann et al, 1987; 1992). The

genetic comparisons of the west Caucasian

dinniki and the ursinii taxon from

Checheno-Ingushetia point in the same

direction (this study; Nilson et al., 1994)

except that the closer relation between

kaznakovi and aspis was not supported.

Thus this morphological similarity between

the two lineages in Caucasus might be a case

of convergent adaptation towards a similar

habitat, although Muellerian mimicry might

be involved.

A number of nominal taxa related to

these populations are recognized from this

geographical region (Russian Republic,

Georgia, Azarbaidjan and Armenia): renardi,

June 1995

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Vol. 6, p. 19

FIG. 21. Distribution of the vipers of the Caucasus and adjacent areas discussed in this paper. Light

stippled= Vipera renardi; dark stippled= Vipera kaznakovi; cross-hatching= Vipera (u.) eriwanensis; horizontal

hatching= Vipera lotievi; vertical hatching= Vipera dinniki. Due to environmental reasons the distribution of

all taxa are only fragmented within the depictured ranges, a situation especially pronounced in renardi. Also

occurring in the region and related to the vipers discussed are the north Iranian populations of the ursinii

complex ('ebneri') that penetrates into southeastern Azarbaidjan in the Talysh mountains, Vipera darevskii (of

the kaznakovi complex) which has its known distribution restricted to northwestern Armenia (Mt. Legli), and

V. pontica from the Artwin province in Turkey. Other species of vipers not discussed here occur

sympatrically in the region.

kaznakovi, darevskii, eriwanensis, dinniki

(Fig. 21).

What names are then available for these

two different sympatric central and east

Caucasian taxa? Vipera dinniki was

originally described from Malaya Laba and

Svanetia, localities situated on the western

side of the upper parts of Little Laba River

and the high-mountain basin of the Inguri

River, respectively (Orlov and Tuniyev,

1986). The type locality has been restricted

to Malaya Laba (by selection of 'The

Museum of Natural History of Kharkov

State University specimen no. 26044' as

lectotype; Vedmederja et al., 1986). The

type locality is situated in the western

Caucasus and the polymorphic western

Vol. 6, p. 20

Asiatic Herpetological Research

June 1995

TABLE 10. Variation, given as Mean ±S.E. and range (for apicals in % of specimens with two plates) of

selected morphological characters in Vipera eriwanensis (N=44), Vipera lotievi (N=14, if not otherwise stated),

andVipera renardi (N=42).

* Counted as sum of both sides. ** No specimen with two apicals, but one with three. Also two specimens

with no apical; thus 6.8% total with more than one or without apical. *** N=16. Four unicolored "bronze"

specimens not included. **** reduction from 21 to 19 dorsal scale rows (at ventral number)

dinniki populations must be referred to this

name. The complex picture of separation

and similarities between all the different

isolated mountain populations in the western

Caucasus demands parallel genetic studies.

The name Vipera kaznakowi orientalis

(Vedmederja, 1984; non Vipera orientalis

Seba, 1734-1735; Daudin, 1801-1803;

Shaw, 1802) was given by Vedmederja

(1984) for eastern vipers at Lagodechi. As

stated above at this locality the two species

are sympatric, but the name orientalis is not

available (nomen nudum - Orlov and

Tuniyev, 1986). Besides the color pattern,

the eastern dinniki is as well somewhat

morphologically distinguished compared to

western dinniki (Table 4). It is

geographically separated from the western

dinniki and it might be justified to treat it as

a taxon of its own. The genetic distance has

June 1995

Asiatic Herpetological Research

Vol. 6, p. 21

TABLE 1 1 . Frequency of certain characteristics in the populations (in percentage of investigated specimens)

Divided parietals

Divided frontal

Preocular(s) in contact with nasal

Without upper nasal split

Supralabial dark sutures absent

Lateral body blotches absent

Snout concave on dorsal side

Belly whitish (not dark)

however not yet been calculated as

fundamental material still is lacking.

Thereby we prefer not to draw any

taxonomic conclusion about the "east-

dinniki" populations. We preserve the terra

typica restricta for Vipera dinniki to Malaya

Laba.

The other question is to what taxon does

the viper of the ursinii complex belong. In

principle north of the Caucasus on the dry

steppes renardi occurs while in the

Armenian highlands south of the Caucasus

eriwanensis is found (the species status of

renardi is analyzed and discussed in Joger et

al., 1992). We consider also eriwanensis as

an evolutionary species (Hoggren et al.,

1993; Nilson et al., 1994). The Caucasian

form is geographically separated from these

two taxa, and morphologically distinguished

(Tables 10 and 11, Figs. 17, 19-20, Plate

Id), also supported by genetic distinction

(Tables 7, 8; Nilson et al., 1994).

Taxonomically it does not fit in with these

two allopatric ursinii s.l. taxa, although

morphological similarity with eriwanensis

can be noted. As there is no reason to

believe reduced reproductive cohesion- all

populations traditionally referred to ursinii

would belong to a single species (albeit

divided in several subspecies) according to

the biological species concept (BSC).

However, all here (and elsewhere- Nilson &

Andren, 1994) recognized taxa in this

complex are allopatric and apomorphic in

characters (morphological and/or genetic),

and as we are interested in a taxonomy that

reflects the phylogeny, we find the

evolutionary and phylogenetic species

concepts preferable (Frost and Hillis, 1990;

Frost et al., 1992; see Nilson, 1993, for

application on vipers). We therefore

recognize it as a separate taxon in this group

of vipers.

Taxonomic Account

Vipera lotievi sp.n. (Fig. 22)

Holotvpe and Terra typica: ZIN

20309, Fig. 22, female, Armkhi, Checheno-

Ingushetia, Russia, below Mt. Stolovaya,

2000 m. altitude, 1986-07-20-23. leg. K.

Lotiev.

Paratypes: ZIN 20305, Itum-Kali,

Checheno-Ingushetia, 1990-08, leg. K.

Lotiev; ZIN 20310, Armkhi, Checheno-

Ingushetia, below Mt. Stolovaya, 2000

altitude, 1986-07-20-23, leg. K. Lotiev;

ZIN 20304, vicinity of village Armkhi,

Checheno-Ingushetia, 1988-07, leg.

Gizatulin; ZIG 298-306, river Chanty-

Argun w. Itum-Kali, Checheno-Ingushetia,

Vol. 6, p. 22

Asiatic Herpetological Research

June 1995

FIG. 22. The female holotype of Vipera lotievi (ZIN 20309), Armkhi, Checheno-Ingushetia, below Mt.

Stolovaya, 2000 m altitude.

1986-05-28, leg. B.Tuniyev; ZIN 20307,

Itum-Kali, Checheno-Ingushetia, 1987-08,

leg. Lotiev; ZIN 20312, Armkhi,

Checheno-Ingushetia, 1987-09, leg. Lotiev;

ZIN 20313, Armkhi and Mt. Stolovaya,

Checheno-Ingushetia, 1986-07-20, leg.

Gizatulin; ZIG 297, Mt. Elbrus, 1986, leg

Filippov, coll. Tuniyev; ZIN 18203,

Teberda, State Reserve, Mt. Bolshaya

Hatipara, 1969, leg. Zalslavsky; ZIN

18226, Kabardino-Balkaria, vicinity of

village Terskol, 1970-08-19, leg. Kireev;

ZIN 11996, Caucasus, Gunib, Dagestan,

1909-05-29, leg. Berg; ZIN 20303,

Lagodechi, 1988-07, leg. Bakradze.

Diagnosis and definition: A species

of the Vipera ursinii complex characterized

by polymorphism in color-pattern, including

"bilineate pattern" of the same kind as in V.

seoanei, and "bronze" unimorphs. External

morphology evolved as typical for mountain

taxa of the ursinii complex but not similar to

any of the other in color pattern.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 23

From the sympatric "east-dinniki" it

differs in several scalation characters and in

color of the belly (Table 9, Fig. 11). In

lotievi the belly is generally white, preocular

in contact with nasal, snout concave, 138 or

more ventrals, always a single apical, less

fragmentized crown scales (7-16), parietal

ocellated spot present, iris not gold-edged in

life. In "east-dinniki" the belly is black,

preocular separated from nasal, snout not

concave, 136 or less ventrals, apical single

or divided, more fragmentized crown scales

(10-21), no parietal ocellated spot, iris gold-

edged in life.

From the allopatric renardi it differs

besides color pattern in morphology by

having light supralabials (sutures heavily

colored in black in renardi), a higher rostral

index, smaller size, white belly (dark in

renardi), and a different niche by being

alpine {renardi is a lowland steppe

inhabitant). No future reproductive

cohesion can be postulated.

It is separated from the likewise

allopatric eriwanensis in the Armenian

highlands by the semidesert lowland of the

Kura River Valley, that separates the Big

Caucasus from the Small Caucasus. No

connection can be postulated in an

evolutionary time frame. Besides color

pattern there is a differentiation in

morphology by eriwanensis having a higher

number of crown scales and a somewhat

lower ventral count, and preocular separated

from nasal to a higher degree (Tables 10 and

11).

Description of holotype (Fig.

22): An adult female, total length 422 mm,

tail 41 mm, latter equal to 10.8 % of total

length. Length of head, from posterior

border last supralabial to tip of snout 16.8

mm, from posterior border of parietals to tip

of snout 12.2 mm, breadth of head at

broadest part of head 9.5 mm, at level of the

eyes 8.0 mm, size of eye horizontally 2.5

mm and vertically 2.0 mm, distance between

eye and lip 2.6 mm. Head covered with

rather large scales or plates. Two large

supraoculars and 1 large frontal plate on top

of head, parietals large, frontal separated

from supraoculars by 3 and 2 smaller scales

on right and left side respectively, 1 canthal

and 1 supranasal scale on each canthus

rostralis, but the two supranasals are partly

united with the apical; 3 intercanthals and 6

intersupraoculars. Height/depth of rostral

3.4/2.7 mm (=1.26), it is bordered by 2

supralabials, 2 internasals and the broad

"apical"; eye surrounded by 8 circumoculars

on each side, 5 loreals on each side, upper

preocular in contact with nasal on both

sides, nasal partly divided at upper edge, 8

supralabials, with forth below eye, and 9

sublabials on each side, anterior supralabials

not much enlarged compared to posterior

ones, 6 second chinshields bordering the

anterior ones and 4 scales in the gular row.

Dorsal side of snout concave resulting in a

pronounced and raised canthus rostralis.

Two preventrals and 141 ventrals, 24+1

subcaudals, 21 dorsal scale rows at

midbody and on neck one head-length

behind the head, 17 dorsal scalerows one

head-length anterior to anal. Reduction

from 21 to 19 dorsal scale rows at level of

ventral number 89. Dorsal pattern

consisting of a weakly winding zig-zag band

with 48 windings, lateral body pattern dark

weakly contrasting towards the lighter

dorsal groundcolor. Head pattern consists

of 2 dark oblique bands which do not unite,

and a posterior band from eye to corner of

mouth and somewhat further back along the

lateral sides of neck, no dark pattern on chin

or in labial region although a very weak

dotted pattern at the supralabial sutures can

be imagined, ground color light brown with

dorsal pattern dark brown and black edged,

ventral side light, throat light. Ocellated

spot on frontalia.

Variation: See Tables 5 and 6.

Besides the variation in scalation a

pronounced variation in color and pattern is

expressed. Most striking, and unique for

the entire ursinii complex, is the bronze

morph, which is found in 25 % of the

investigated specimens (N=40) (Plate Id).

Distribution: Vipera lotievi is

distributed in the semiarid 'hollows'

between the northern slope of the main

Caucasian range and Skalisty range from the

upper part of the Kyafar River (range

Abishir-Akhuba) eastward to the interior of

Vol. 6, p. 24

Asiatic Herpetological Research

June 1995

Daghestan (see map). Altitudinal span in

this region goes from 1200 m up to 1600 m

(1800m). Further it is recorded from Mt.

Elbrus in the central Caucasus, and

mountains north of Lagodechi in the eastern

Caucasus, besides several isolated records

from the eastern and central Caucasus range.

Habitats: Typical habitats are

oreoxerophytes landscapes with semiarid

light-forests (like Shibliak), phrygana (with

'tragakant' astragalus) which are very

similar to east-Mediterranean types of

vegetation. On the upper elevation of the

distribution V. lotievi reaches the subalpine

mountain belt.

"Refuge History": The development

of the xerophilous vegetation has taken place

since Pliocene in the eastern part of the

Caucasian Isthmus. Four main refuges are

known: two humid (Colchis and Talysh-

Hyrkanian) and two arid-xerophilous

(Armenian and Dagestanian). The north

Caucasian refuge of oreoxerophits,

including shibliak and phryganas are

situated along the shale-depression between

the main Caucasian Range and the Skalistiy

(Rocky) Range. There are several semiarid

hollows from central Dagestan (Gunibskoe

Plateau) and westward to the beginning of

the Kuban River (at the Mt. Elbrus region).

The vegetation is composed of Juniperus

oblonga, Paliurus spina-christi, Cerasus

incana, Colutea orientalis, Berberis vulgaris,

Astragalus denudatus, Celtis glabrata,

Ephedra procera and others. This vegetation

superficially is very close to the vegetation

of the Armenian highland and the mountains

of the Near East, but the regions share

relatively few species (3-5%). The major

part of the plants of these hollows has an

east Caucasian origin. For example, 25% of

the flora of the Itum-Kali hollow

(Checheno-Ingushetia) has east Caucasian

origin. Altogether, more than 200 species

of plants are endemic to these hollows

(Galushko, 1974).

There have been different interpretations

about the age of the vegetation in these

hollows. Most botanists have been of the

opinion that this vegetation has a Pliocene

origin (Grossgeim, 1948; Krasnov, 1894;

Kuznetsov, 1890), while Galushko (1974)

has the opinion that the semiarid hollows of

Checheno-Ingushetia are younger than the

hollows of Kardino-Balkaria and Osetia in

the west and Dagestan in the east, and

perhaps not older than Holocene. But the

remains of xerophilous flora on the crests

between the semiarid hollows are the

witness of the existence of a united

enormous xerophilous (Mediterranean)

refuge, running from Dagestan to the region

of Mt. Elbrus. Later, in Pleistocene, this

refuge disintegrated to several micro-refuges

which have persisted to different extent until

present. However, it must be pointed out

that although the xerophilous vegetation

(including mountain-steppe) had a wide

distribution along the shale-depression, it

also had the possibility to disperse up to the

mountains along the river valleys. Both

ways could be used by representatives of the

"ursinii-sptcics group" of vipers. Besides

the "ursinii-group" also thermophilous

species like Lacerta strigata, Coluber

najadum, and Elaphe hohenackeri are

present as isolates in these hollows. One

should also pay attention to the occurrence

of relicts of the xerothermal epoch in the

western Caucasus near the mountains

Jatyrgvarta and Magisko (Altukhov, 1966),

but the xerophilous vegetation did never

have any wide development in that area.

Literature Cited

ALTUKHOV, U. D. 1966. [About the high

mountain flora of the limestone of Tryu-Yakirgvart].

Problems of botanik, vol. 8, Leningrad. (In

Russian).

CLAYTON, J. W. AND D. N. TRETIAK. 1972.

Amine-citrate buffers for pH control in starch gel

electrophoresis. Journal of the Fisheries Research

Board of Canada 29:1 169-1 172.

DE LORENZO, R. J. AND F. H. RUDDLE. 1969.

Genetic control of two electrophoretic variants of

glucosephosphate isomerase in the mouse.

Biochemical Genetics 3:151-162.

FROST, D. R. AND D. M. HILLIS. 1990. Species

in concept and practice: Herpetological applications.

Herpetologica 46(1):87-104.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 25

FROST, D. R., A. G. KLUGE AND D. M. HILLIS.

1992. Species in contemporary herpetology:

Comments on phylogenetic inference and taxonomy.

Herpetological Review 23(2):46-54.

GALUSHKO, A. I. 1974. [On the flora to arid

slopes of vicinity of Itumkale (Checheno-

Ingushetia)]. Pp. 5-22. In Flora and vegetation of

the Eastern Caucasus. Ordjonikidze. (In Russian).

GROSSGEIM, A. A. 1948. [Vegetation of the

Caucasus]. Publishing House of the Moscow

Society of Researcher of Nature. (In Russian).

HARRIS, H. AND D. A. HOPKINSON. 1976.

Handbook of Enzyme Electrophoresis in Human

Genetics. North-Holland, Amsterdam.

HERRMANN, H.-W., U. JOGER, G. NILSON AND C.

G. SIBLEY. 1987. First steps towards a

biochemically based reconstruction of the phylogeny

of the genus Vipera.. Pp. 195-200. In J.J. van

Gelder, H. Strijbosch and P.J.M. Bergers (eds.),

Proceedings of the Fourth Ordinary General Meeting

of the Societas Europaea Herpetologica, Nijmegen

1987.

HERRMANN, H.-W., U. JOGER AND G. NILSON.

1992. Molecular phylogeny and systematics of

viperine snakes I. General phylogeny of European

vipers (Vipera sensu strictu). Pp. 219-224. In Z.

Korsos and I. Kiss (eds.), Proceedings of the Fifth

Ordinary General Meeting of the Societas Europaea

Herpetologica, Budapest 1991.

HILLIS, D. M. 1984. Misuse and modification of

Nei's genetic distance. Systematic Zoology 33:238-

240.

HOGGREN, M., G. NILSON, C. ANDREN, N. L.

ORLOV AND B. S. TUNIYEV. 1993: Vipers of the

Caucasus: Natural History and Systematic Review.

Herpetological Natural History 1(2):11-19.

JOGER, U., H.-W. HERRMANN AND G. NILSON.

1992. Molecular phylogeny and systematics of

viperine snakes II. A revision of the Vipera ursinii

complex. Pp. 239-244. In Z. Korsos and I. Kiss

(eds.), Proceedings of the Fifth Ordinary General

Meeting of the Societas Europaea Herpetologica,

Budapest 1991.

JOHNSON, A. G., F. M. UTTER AND H. O.

HODGINS. 1970. Interspecific variation of

tetrazolium oxidase in Sebastodes (rockfish).

Comparative Biochemistry and Physiology 37:281-

285.

KRASNOV, A. N. 1894. [Caucasian chains of

mountains, paralleled to Main Ridge, and its role in

formation of forest and steppe flora of the western

Caucasus]. Transactions of Naturalist Society of the

Kharkov University. (In Russian).

KUZNETSOV, N. I. 1890. [Geobotanical

investigation of the northern slope of the Caucasus].

Reports of the Russian Geographical Society. Vol.

26. (In Russian)

MURPHY, R. W., J. W. SITES, D. G. BUTH AND C.

H. HAUFLER. 1990. Proteins I: Isoenzyme

electrophoresis. Pp. 45-126. In D.M. Hillis and

C. Moritz (eds.), Molecular Systematics. Sinauer,

Sunderland, Massachusetts.

NEI, M. 1978. Estimation of average

heterozygosity and genetic distance from small

number of individuals. Genetics 89:583-590.

NIKOLSKY, A. M. 1913. [Reptiles and

Amphibians of the Caucasus]. The Caucasus

Museum Press, Tiflis, Georgia. 272 pp. (In

Russian).

NIKOLSKY, A. M. 1916. Fauna of Russia and

adjacent countries, Reptiles, Vol. 2. (1964: Israel

Program for Scientific Translations, Jerusalem,

vi+247 pp.).

NILSON, G. 1995. Venomous snakes in west Asia:

-Applicability of current species concepts.

Proceedings of the Asiatic Herpetological Congress.

In Press.

NILSON, G. AND C. ANDREN. 1986. The

mountain Vipers of the Middle East- The Vipera

xanthina complex (Reptilia, Viperidae). Bonner

Zoologische Monografien 20:1-90.

NILSON, G. AND C. ANDREN. 1994. The meadow

and steppe vipers of Europe and Asia, the Vipera

ursinii complex. Submitted.

NILSON, G., M. HOGGREN, B. S. TUNIYEV, N. L.

ORLOV AND C. ANDREN. 1994. Phylogeny of the

vipers of the Caucasus (Reptilia, Viperidae).

Zoologica Scripta. (In press).

ORLOV, N. L. AND B. S. TUNIYEV. 1986. [The

recent areas, their possible genesis and the

phylogeny of three viper species of Eurosiberian

group of the Vipera kaznakowi complex in the

Caucasus]. Pp. 107-135. In: N. Ananjeva and L.

Borkin (eds.), Systematics and Ecology of

Amphibians and Reptiles. Proceedings of the

Zoological Institute, Leningrad, 157. (In Russian).

Vol. 6, p. 26

Asiatic Herpetological Research

June 1995

ORLOV, N. L. AND B. S. TUN1YEV. 1990. Three

species in the Vipera kaznakowi complex

(Eurosibirian group) in the Caucasus: their present

distribution, possible genesis, and phylogeny.

Asiatic Herpetological Research 3:1-36.

RIDGWAY, G. J., S. W. SHERBURNE AND R. D.

LEWIS. 1970. Polymorphism in esterase of

Atlantic herring. Transactions of the American

Fisheries Society 99:147-151.

SELANDER, R. K., M. H. SMITH, S. Y. YANG, W.

E. JOHNSON, AND J. R. GENTRY. 1971.

Biochemical polymorphism and systematics in the

genus Peromyscus. I. Variation in the old-field

mouse (Peromyscus polionotus). Studies in

Genetics VI. University of Texas Publications

7103:49-90.

SNEATH, P. H. A. AND R. R. SOKAL. 1973.

Numerical Taxonomy. W.H.Freeman and Co., San

Francisco. 573 pp.

TUNIYEV, B. S. 1990. On the independence of the

Colchis Center of Amphibian and Reptile

speciation. Asiatic Herpetological Research 3:67-

84.

VEDMEDERJA, V. L., N. L. ORLOV AND B. S.

TUNIYEV. 1986. [On the taxonomy of the three

viper species of the Vipera kaznakowi complex].

Pp. 55-61 In: N. Ananjeva and L. Borkin (eds.),

Systematics and Ecology of Amphibians and

Reptiles. Proceedings of the Zoological Institute,

Leningrad, 157. (In Russian).

SHAW, C. R. AND R. PRASAD. 1970. Starch gel

electrophoresis of enzymes - a compilation of

recipes. Biochemical Genetics 4:297-330.

I June 1995 Asiatic Herpetological Research Vol. 6, pp. 27

Calotes versicolor nigrigularis Auffenberg and Rehman 1993

a Junior Primary Homonym

'Walter Auffenberg and 2Hafizur rehman

'7008 NW 67th Ave, Gainesville, FL. 32653 USA

Zoological Survey Dept. , Karachi 1 , Pakistan

Key Words: Reptilia, Sauna, Lacertilia, Agamidae, Calotes.

We recently described a new subspecies of Calotes versicolor, which we named C. v.

nigrigularis (Auffenberg and Rehman, 1993, Studies on Pakistan Reptiles. Pt. 3. Calotes

versicolor. Asiatic Herpetological Research 5:14-30). Immediately after publication, Dr.

Hidetoshi Ota brought to our attention that the same name has recently been used for a new

species of Calotes from Borneo (Ota and Hikida, 1991, Taxonomic review of the lizards of

the genus Calotes Cuvier 1817 (Agamidae: Squamata) from Sabah, Malaysia. Tropical

Ecology 4:179-192). Thus Calotes versicolor nigrigularis Auffenberg and Rehman is a

junior primary homonym of Calotes nigrigularis Ota and Hikida 1991.

The name we propose for Calotes versicolor nigrigularis Auffenberg and Rehman is

CALOTES VERSICOLOR FAROOQI

It is named in honor of Farooq Ahmed, Director, Zoological Survey Department, Pakistan.

Our extensive herpetological exploration in Pakistan would have been impossible without his

generous logistic support.

© 1995 by Asiatic Herpetological Research

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 28-29 I

Simplified Field Technique for Obtaining Blood from Freshwater Turtles

RESHMA batra and Sant prakash

Department of Zoology, D.EJ., Dayalbagh Agra, 282 005, India

Brief Communication

Studies on the biochemical and

molecular aspects have now been recognized

as essential components of the conservation

program of species. The choice of the tissue

in such studies have invariably been the

blood and this has necessitated researchers

to look for the best procedure for field

sampling without harming or sacrificing the

animal.

Several methods have been proposed to

obtain uncontaminated blood, each having

its merit restricted to the species under study

or to the specific experiment. The most

common method of collection of samples of

blood in reptiles has been by cardiac

puncture (Gandal, 1958; Stephens and

Creekmore, 1983) but has been less popular

in turtles because of their thick plastron.

Cutting off the end of the tail (Duguy,

1970), toenail clipping (Frye, 1991) or

collecting blood from the major veins and

arteries (Maxwell, 1979) have been some of

the other proposals. Each of them has at

least one disadvantage; for example, intricate

dissection of veins/arteries is required

(Avery and Vitt, 1984). The procedure for

obtaining blood samples from the ventral

caudal vein, as suggested by Galbraith

(pers. comm.) and described in alligator

snapping turtles (Powell and Knesel, 1992),

has been initially utilized in our procedure

but we had to discard it as the amount of

blood obtained was not enough for multiple

analyses. Falling back on the oldest method

of heart puncture by inserting a long needle

laterally through the soft tissue between the

plastron and the carapace, we found that the

front leg provides the safest and the shortest

way to reach the heart, thus avoiding the

drilling of the plastron. In addition, our

technique does not require elaborate

equipment and can be used easily in the

field.

syringe

FIG. 1. Method for obtaining blood, h: heart, rfl:

fight fore-leg, s: syringe.

We have applied this technique in the

turtles of the genus Kachuga, K. tentoria

and K. dhongoka. These are primarily

medium sized turtles with males ranging

between 4-8 inches and females between 9-

18 inches in length. Presumably this

technique can be applied to many other

turtles of similar size.

Handling of the turtles, to keep them

docile, is the skill of the field worker and no

standard procedure can be described for it.

However, the turtle has to be suspended in a

manner that the head hangs freely

downward and the foreleg remains

unrestrained. The weight of the body forces

the foreleg to stretch, but this may need

some time. In this position, the left foreleg

can be stretched at an angle of 35° from the

head. The skin joining the leg with the

© 1995 by Asiatic Herpetological Research

April 1995

Asiatic Herpetological Research

Vol. 6, p. 29

carapace is dabbed with 95% alcohol in

order to sterilize the area (Fig. 1).

A 2-inch long 32-gauge hypodermic

needle attached to a 5 ml syringe is inserted

(as shown in the figure) parallel to the

stretched foreleg. The needle is gently

inserted until it reaches the ventricle. The

depth of the needle penetration is often

between 1-1.5 inches. Gentle suction is

applied until the blood spurts into the

syringe and withdrawal pressure is then

slowly increased, until the syringe is at its

full suction capacity. The needle is then

slowly pulled out, with full syringe suction

still being applied. About 2-3 ml of blood is

drawn per sample. No pressure is applied

for the control of bleeding as no visible

bleeding occurs in this procedure.

However, germicidal powder is immediately

sprinkled at the point of the insertion of the

needle, before marking and releasing the

turtles.

Blood samples have successfully been

collected from over 50 freshwater turtles and

several of them have been utilized for

repetitive blood lettings and maintained in

captivity for over 4 months with no apparent

ill-effects. For field sampling, this

procedure provides a safe, practical and

simple technique for obtaining blood in

turtles.

Literature Cited

AVERY, H. W. AND L. J. VITT. 1984. How to

get blood from a turtle. Copeia 1984:209-210.

DUGUY, R. 1970. Numbers of blood cells and

their variation. Biology of reptiles. Pp. 93-109. In

Gans and Parson (Eds). Academic Press, New York.

FRYE, F. L. 1991. Biomedical and surgical aspects

of captive reptiles husbandry, Vol. 1. Kreiger

Publishing Co., Inc., Florida.

GANDAL, C. P. 1958. Cardiac puncture in

anaestized turtles. Zoologica 43:93-94.

MAXWELL, J. H. 1979. Anaethesia and surgery.

Pp. 127-152. In Harless and Morlock (Eds) Turtles:

perspective and research. John Wiley and Sons,

New York.

POWELL, S. C. AND J. A. KNESEL. 1992. Blood

collection from Macroclemys temmincki (Troost).

Herpetological Review 23(1): 19.

STEPHENS, G. A. AND J. S. CREEKMORE. 1983.

Blood collection by cardiac puncture in conscious

turtles. Copeia 1983:522-523.

Acknowledgments

We thank Mr. Dayal Prashad Gupta and

Mr. Akash Mathur for their help in turtle

collection.

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 30-35

The Systematic Relationships of Dravidogecko anamallensis

(Gunther 1875)

AARON M. BAUER1 AND ANTHONY P. RUSSELL2

1 Biology Department, Villanova University, Villanova, Pennsylvania 19085, USA

2Vertebrate Morphology Research Group, Department of Biological Sciences, University of Calgary, 2500

University Drive N.W., Calgary, Alberta, Canada T2N 1N4

Abstract. -The relationships of the monotypic gekkonine genus Dravidogecko are assessed by comparative

evaluation of its external and internal morphology. A suite of shared-derived features is possessed by

Hemidactylus and a variety of satellite genera, including Dravidogecko. These similarities are advocated as

being so compelling, and the ostensible defining features of Dravidogecko to be so weak that the latter is

subsumed as a junior synonym of Hemidactylus. The biogeographic consequences of this taxonomic shift are

considered.

Key words: Dravidogecko, Hemidactylus, Teratolepis, digits, scansors, phalanges, paraphalangeal elements,

muscles, biogeography, India.

Introduction

Dravidogecko is a monotypic genus of

gekkonid lizards endemic to south India.

The single species, D. anamallensis, was

originally described as a member of the

genus Hoplodactylus (Gunther, 1875;

Strauch, 1887), but following the work of

Smith (1933), it was assigned to a new

genus, primarily on the basis of differences

in the distal scansors and in preanal pore

arrangement. Subsequently it has been

demonstrated that Dravidogecko is a

gekkonine gecko, whereas Hoplodactylus

sensu stricto is a diplodactyline

(Underwood, 1954; Kluge, 1967). The

relationships of Dravidogecko have

remained obscure, and the systematic status

of the species has never been investigated

adequately. It is known from only a few

specimens from the Anaimalais, Palnis and

Tirunelveli Hills (Satyamurti, 1962; Murthy,

1985) but is reportedly widely distributed

throughout forested areas of southern

peninsular India (Daniel, 1983).

Russell (1972) considered Dravidogecko

to belong, on morpho-functional grounds,

in the Hemidactylus group, along with

Hemidactylus, Briba, Teratolepis and

Cosymbotus. Kluge (1983) placed it, along

with the other gekkonine genera previously

mentioned, in the tribe Gekkonini on the

basis of the absence of the second

ceratobranchial arch. Russell (1976: 238;

Fig. 14) suggested that Dravidogecko had a

digital structure that was most closely

approached by that of Hemidactylus and its

close allies. While external form of the

digits is particularly sensitive to functional

demands and thus prone to exhibiting

convergence and parallelism (Russell,

1979), details of the internal anatomy are

much more helpful at indicating true

homology and, therefore, affinity (Russell,

1976, 1979; Russell and Bauer, 1990). We

herein present the results of a comparative

survey of both the external and internal

anatomy of the feet and digits in

Hemidactylus (and its close relatives) and

use these to demonstrate both the wide range

of variation present and the shared derived

features that circumscribe this cluster and

help clarify the relationships of the enigmatic

Dravidogecko. We further relegate the

generic name Dravidogecko into the

synonymy of Hemidactylus as there are no

derived features of Dravidogecko that are

not also shared by at least some

Hemidactylus. It is probable that H .

anamallensis is a primitive hemidactyl.

Materials and Methods

Specimens of Dravidogecko were

examined or borrowed from the collections

of The Natural History Museum, London

(BMNH) and the Institute Royal des

Sciences Naturelles de Belgique, Brussels

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol.6, p. 31

(IRSNB). Comparative material of other

gekkonines, especially Hemidactylus, were

borrowed from the BMNH and the

California Academy of Sciences, San

Francisco (CAS). Observations on toe

structure were made using a Nikon SMZ-10

microscope. The specimens examined are

listed below. All numbers refer to BMNH

specimens unless otherwise identified.

Dravidogecko anamallensis 82.5.22.79-84;

IRSNB 1194.

Briba brasiliana 1971.1045.

Cosymbotus craspedotus 1926.12.7.7,

1930.10.9.2

C. platyurus xxi.36a, 97.6.21.4,

97.12.28.10, CAS 18565, CAS 18567

Hemidactylus albopunctatus 1946.8.22.75;

H. ansorgii 1901.1.28.22; 1966.337; H.

barodanus 1905.11.7.1-6; 1937.12.5.215-

216; 1958.1.6.29; 1970.1437-38; H.

bouvieri 66.4.12.3; 75.4.26.10; H .

bowringii 1929.12.1.7-10; 1940.4.26.2-3;

1956.1.11.15-16; H. brookii 1918.1 1.12.2-

10; 1930.10.6.6; 1931.12.10.6-7;

1970.2196-98; 1971.242; H. citernii

1931.7.20.114-119 and 128-130;

1937.12.5.202-204; H. curlei

1946.8.25.41; H. depressus 52.2.19.21;

61.2.21.5; 1948.1.7.35; H. echinus

89.7.6.1; 1903.7.28.1-2; H. fasciatus

1919.8.16.48; 1956.1.11.37-40; 1971.253;

H. flaviviridis 1931.7.20.153-155;

1971.1378-1382; H.forbesii 1946.8.25.43-

Al;H.frenatus 1938.10.2.1; 1952.1.4.30-

31; 1970.1879-1895; H. garnotii

95.11.7.1; 1903.2.21.1-2; 1940.6.3.24-29;

H. giganteus 1908.12.28.27; 1969.828-

829; H. gracilis 74.4.29.1388;

80.11.10.47; H. granti 1957.1.9.52-66; H.

greeffii 93.12.7.1; 98.3.30.21-22; H.

homeolepis 99.12.5.38; 1953.1.7.84-85;

1967.485-489; H. isolepis 1952.1.7.79-80;

H. jubensis 1946.8.23.66; H. karenorum

68.4.3.88-89; 91.11.26.13-14; H.laevis

1946.8.25.42; H. leschenaulti 70.5.18.70-

71; 74.4.29.233-236 (six specimens); H.

longicephalus 1936.8.1.287-305; H.

mabouia 1923.11.9.46-50; 1964.1429-35;

1970.2209-15; H. macropholis

1931.7.20.109; 1937.12.5.250-258; H.

maculatus 69.8.25.15; 1956.1.11.44; H.

megalops 1946.8.25.67; H. mercatorius

1930.7.1.84-90; 1938.8.3.11-15; H.

muriceus 1926.9.24.13; 1966.283; H.

modestus 1946.8.25.37; H. ophiolepis

1937.12.5.324-325; H. oxyrhinus

99.12.5.170-175; 1967.491-494; H .

persicus 1970.250; 1972.716; H. prashadi

1946.8.14.66-69; H. pumilio 1946.8.20.1;

1946.8.25.58-61; H. reticulatus,

1901.3.8.1-3; H. richardsoni 1916.5.29.1;

1919.8.16.49; H. ruspolii 1937.12.5.228-

229; 1937.12.5.239-246; H. sinaitus

97.10.28.83-86; 1937.2.5.293;

1953.1.6.97-98; H. smithi 1931.7.20.85-

89; 1972.745; H. somalicus 1946.8.25.77-

78; H. squamulatus 9 8.1.8.2-3;

1902.5.26.2; 1923.10.9.2; 1923.10.9.14-

15; H. subtriedrus 74.11.11.1; H. taylori

1946.8.23.48; H. triedrus xxi.l9a-b; H.

tropidolepis 1937.12.5.322-323; H .

turcicus 1934.11.8.10- 14; 1971.1143-45;

H. yerburii 99.12.13.43-44; 1903.6.26.3-4;

1945.12.18.12.

Teratolepis fasciata 69.8.28.32;

1933.7.8.37; 1963.1019; 1964.930-931; T.

albofasciatus 1963.613-621

Results

A considerable range of variation in

digital form and subdigital scansor design

exists among members of the genus

Hemidactylus (Fig. 1). This variation is

evident in such aspects as the number of

divided scansors (lamellae), the extent of

their division, the extent of the undivided

lamellar series at the base of the digits, and

the length, form and degree of separation of

the free, distal, claw-bearing segment of the

digits. Figure 2 illustrates the general form

of the ventral aspect of the right pes of

Dravidogecko and provides comparison

with the ventral aspects of the fourth pedal

digit of Hemidactylus reticulatus and

Teratolepis fasciata. While some species of

Hemidactylus, such as H. garnotii and H.

smithii (Fig. 1), have digits with a large

number of completely divided scansors, and

an elongate, free distal, claw-bearing

portion, this is not so for other species, such

as Teratolepis albofasciatus (see Grandison

and Soman, 1963), Hemidactylus somalicus

and H. bouvieri (Fig. 1). In the latter three

cases the number of scansors is small, only

the distal most ones are notched, and the

distal, free, claw-bearing portion of the digit

Vol. 6, p. 32

Asiatic Herpetological Research

June 1995

FIG. 1. The array of digital form in the genera

Hemidactylus and Teralolepis. All illustrations are

of the fourth digit of the pes; a-e, j are of the right

pes, f-h, k-p are of the left pes. The 2 mm scale bar

refers to all specimens except n, to which the 5 mm

scale bar applies. All catalogue numbers refer to the

Natural History Museum, London (BMNH). a.

Teralolepis albofasciatus 1963.617; b. Hemidactylus

bowringii 1929.12.1.6; c. //. garnotii 95.11.7.1; d.

H. barodanus 1970.1438; e. H. turcicus 1971.1144;

f. H. somalicus 1946.8.25.77; g. H. ophiolepis

1937.12.5.324; h. H. mabouia 1964.1431; j. H.

forbesii 1946.8.25.47; k. H. smithii 1931.7.20.85;

1. H. fascial us 1919.8.16.48; m. H. ansorgii

1901.1.28.22; n. //. richardsonii 1916.5.29.1; p. H.

bouvieri 66.4.12.3.

is relatively short. This situation is also

seen in Hemidactylus reticulatus and

Teratolepis fasciata (Fig. 2, b, c). The

almost continuous range of variation in

external digital characters, especially among

the west Asian and Somali species of the

Hemidactylus group of geckos has long

been recognized, and has resulted in the

establishment of several different, largely

arbitrary, generic arrangements (see Parker,

1942 for a discussion). Thus, while

division of the scansors is generally

characteristic of the genus Hemidactylus,

there are many species that express this trait

only marginally.

Russell (1976: Fig. 14) indicated this

potential continuity in scansor form, from

undivided to completely divided, by

FIG. 2. a. Ventral aspect of the right pes of

Dravidogecko anamallensis, BMNH 82.5.22.79. b.

Ventral aspect of the fourth digit, right pes of

Hemidactylus reticulatus, BMNH 1901.3.8.1. c.

Ventral aspect of the fourth digit, left pes of

Teratolepis fasciata, BMNH 1933.7.8.37. Scale bar

in millimeters.

comparing Dravidogecko with

Cyrtodactylus brevipalmatus, Hemidactylus

reticulatus and H. barodanus. While this

was simply a depiction of change of form

assembled as a morphotypic series, it was

also implied that there may be deeper

underlying anatomical clues that are

indicative of the closeness of relationship of

Dravidogecko to Hemidactylus. The

superficial comparisons of the digits(Figs.

1, 2; see above) provide some idea of the

potential range of variation, but should be

treated with caution when being implicated

in arguments about relationship because of

the extreme plasticity of external digital

form. [Such aspects are well exemplified by

the taxonomic history of the taxon that is the

subject of this contribution.] Detailed

examination of the internal anatomy of the

digits provides more convincing evidence

about the affinities of Dravidogecko.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 33

Russell (1976) presented a mechanistic

diagram of the main features of digital

design in Hemidactylus. The chief aspects

of note here are the unusual form and

relationships of the antepenultimate phalanx

of digits III-V of the pes (Russell, 1977),

the distal extent of the dorsal interossei

muscles along the digit, and the means of

tendinous insertion of these muscles onto

the scansors. The pattern of digital

characteristics of Hemidactylus is essentially

repeated in Dravidogecko and is restricted to

only a few other genera (Briba, Cosymbotus

and Teratolepis). This suite of shared-

derived digital features of these taxa (the

short, erect nature of the antepenultimate

phalanx of pedal digits III-V, the distal

extension of the dorsal interossei muscles as

far as the distal end of the antepenultimate

phalanx, and the tendinous insertion of the

dorsal interossei muscles onto the distal

margin of each scansor in turn) unites them

as a distinctive evolutionary unit. Apart

from Hemidactylus, all of the other genera

in this cluster are either monotypic (Briba

and Dravidogecko) or include only two

species (Cosymbotus and Teratolepis).

Dissection of the digits of Dravidogecko

reveals that the dorsal interossei muscles are

well-developed and robust and extend as

fleshy bellies as far distally as the digital

inflection (the point of emplacement of the

reduced, erect antepenultimate phalanx

onmanual digits III and IV and pedal digits

III-V). The dorsal interossei muscles send

individual tendons to the distal borders of

the scansors as they do in Hemidactylus (see

Russell, 1976) and Cosymbotus. This

situation also pertains in Teratolepis and

Briba (Russell, 1972). Dravidogecko also

shares with Hemidactylus, Briba and

Cosymbotus the particular morphology and

placement of paraphalangeal elements

(Russell and Bauer, 1988).

The above comparisons indicate that

Dravidogecko shares with other members of

the Hemidactylus radiation (Hemidactylus,

Briba, Cosymbotus, Teratolepis) all of the

derived digital features that distinguish these

taxa from all other geckos. However,

apomorphic features characteristic of many

Hemidactylus species, such as those

associated with the complete division of the

scansors, are lacking in Dravidogecko. It is

therefore likely that D. anamallensis is a

relatively plesiomorphic member of this

radiation. As such, it is probable that the

recognition of Dravidogecko renders

Hemidactylus as presently construed

paraphyletic. In order to maintain

monophyletic generic units we hereby place

Dravidogecko into the synonymy of

Hemidactylus Gray, 1825. The correct

designation for the single known species

formerly referred to this genus thus becomes

Hemidactylus anamallensis (Giinther 1875),

new combination.

Discussion

Many lizard families include monotypic

genera. Although in some cases these

represent independently evolving lineages,

in most they are relatively primitive or

highly derived members of other lineages,

and their recognition renders the latter

groups paraphyletic. Hemidactylus is the

most specious genus in the Gekkonidae,

with 75 species currently recognized

(Kluge, 1991). Relationships within the

genus are very poorly understood (Parker,

1942; Loveridge, 1947; Kluge, 1969;

Bastinck, 1981) and a general uniformity

among most forms (Russell, 1976) has

rendered casual attempts at investigating its

phylogeny unsuccessful. The placement of

Dravidogecko anamallensis into this morass,

of course, does nothing to aid this

confusion. It does, however, ensure that

Hemidactylus anamallensis is taken into

account if and when a generic revision of all

Hemidactylus is accomplished.

It is not only in the interest of

maintaining monophyletic groups that the

revaluation of monotypic genera is

undertaken. Current nomenclatural usage

has implications for non-systematists. As

an endemic Indian subcontinent form,

Dravidogecko might be used to support

arguments about the uniqueness and

antiquity of the Indian biota. The use by

biogeographers of classification schemes

that do not adequately reflect phylogenetic

patterns has been shown to lead to the

erection of demonstrably false hypotheses

Vol. 6, p. 34

Asiatic Herpetological Research

June 1995

(Bauer, 1989). Clearly, biogeographic

interpretations must be based upon the

phylogenetic relationships of the organisms

considered. Some other Hemidactylus

group geckos sharing with H. anamallensis

at least partially undivided scansors are also

Indian forms (e.g., Teratolepis albofasciatus

from the Ratnagiri District, Maharashtra,

Hemidactylus gracilis from the Madhya

Pradesh, Maharashtra and Andhra Pradesh

(Smith, 1935;Murthy, 1985), and H.

reticulatus from Tamil Nadu, Andhra

Pradesh and Karnataka (Smith, 1935;

Murthy, 1985)). Teratolepi fasciata is also

from the Indian subcontinent (Anderson,

1964; Minton, 1966) and it appears likely

that the hemidactyls, as a group, have

undergone a long period of evolution and

diversification within the region.

Although the geographic ranges of some

forms of Hemidactylus are indicative of

relatively recent expansions (Kluge, 1967,

1969), most Indian species are moderately

to highly circumscribed in their distribution

and hold the promise of contributing

substantially to biogeographic hypotheses of

area relationships within peninsular India.

However, both biogeographic analyses and

meaningful studies of the evolution of the

pedal characteristics that have made

Hemidactylus sensu lato so successful in

India (and elsewhere) must await the

ultimate resolution of phylogenetic

relationships within the genus. In

subsuming Dravidogecko within

Hemidactylus we concur with the sentiments

expressed by Loveridge (1947: 97) in

discussing the African members of this

radiation, "Any arrangement that would

break up so unwieldy a genus as

Hemidactylus is worthy of careful attention .

. ." Such an arrangement must be

phylogenetically based, and at present

insufficient data are at hand to attempt this.

However, we regard the identification of all

members belonging to the Hemidactylus

clade as a necessary first step in the process.

Acknowledgments

We thank E. N. Arnold, Mathias Lang

and Jens Vindum for access to specimens.

E. Prickley and S. F. Milone provided

general assistance in the laboratory.

Literature Cited

ANDERSON, J. A. 1964. A report on the gecko

Teratolepis fasciata (Blyth 1853). Journal of the

Bombay Natural History Society 61(1):161-171.

BAST1NCK, J. 1981. Phyletische analyse van

dertien kenmerken verspreid over degeslachten der

Gekkoninae. Unpublished thesis, University of

Amsterdam, The Netherlands.

BAUER, A. M. 1989. Reptiles and the

biogeographic interpretation of New Caledonia.

Tuatara 30:39-50.

DANIEL, J. C. 1983. The Book of Indian Reptiles.

Bombay: Bombay Natural History Society, x +

141 pp.

GRANDISON, A. G. C. AND P. W. SOMAN. 1963.

Description of a new geckonid lizard from

Maharashtra, India. Journal of the Bombay Natural

History Society 60(2):322-325.

GUNTHER, A. 1875. Second report on collections

of Indian reptiles obtained by the British Museum.

Proceedings of the Zoological Society of London

1875:224-234 + 5 pis.

KLUGE, A. G. 1967. Higher taxonomic categories

of gekkonid lizards and their evolution. Bulletin of

the American Museum of Natural History 135:1-59.

KLUGE, A. G. 1969. The evolution and

geographical origin of the New World Hemidactylus

mabouia-brooki complex (Gekkonidae: Sauria).

Miscellaneous Publications of the Museum of

Zoology, University of Michigan 138:1-78.

KLUGE, A. G. 1983. Cladistic relationships

among gekkonid lizards. Copeia 1983:465-475.

KLUGE, A. G. 1991. Checklist of gekkonid

lizards. Smithsonian Herpetological Information

Service 85:1-35.

LOVERIDGE, A. 1947. Revision of the Africn

lizards of the family Gekkonidae. Bulletin of the

Museum of Comparative Zoology, Harvard

University 98(1): 1-469.

MINTON, S. A. 1966. A contribution to the

herpetology of West Pakistan. Bulletin of the

American Museum of Natural History 134:27-184.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 35

MURTHY, T .S. N. 1985. A field guide to the

lizards of Western Ghats. Records of the Zoological

Survey of India Miscellaneous Publications,

Occasional Paper 72: 1-51.

PARKER, H. W. 1942. The lizards of British

Somaliland, with an appendix on topography and

climate by Capt. R. H. R. Taylor, O. B. E.

Bulletin of the Museum of Comparative Zoology,

Harvard University 91:1-101.

RUSSELL, A .P. 1972. The foot of gekkonid

lizards: a study in comparative and functional

anatomy. Unpubl. Ph. D. thesis, University of

London, England.

RUSSELL, A. P. 1976. Some comments

concerning interrelationships amongst gekkonine

geckos. Pp. 217-244. In A. d'A. Bellairs and C.

B. Cox (Eds.), Morphology and Biology of

Reptiles. London, Academic Press.

RUSSELL, A. P. 1977. The phalangeal formula of

Hemidactylus Oken, 1817 (Reptilia:Gekkonidae): a

correction and a functional explanation. Zentralblatt

fur Veterinar Medizin, Reihe C. Anatomia,

Histologia Embryologia 6:332-338.

RUSSELL, A. P. 1979. Parallelism and integrated

design in the foot structure of gekkonine and

diplodactyline geckos. Copeia 1979(1):1-21.

RUSSELL, A. P. AND A. M. BAUER. 1988.

Paraphalangeal elements of gekkonid lizards: a

comparative survey. Journal of Morphology

197:221-240.

RUSSELL, A. P. AND A. M. BAUER. 1990. Digit I

in pad-bearing gekkonine geckos: alternate designs

and the potential constraints of phalangeal number.

Memoirs of the Queensland Museum 29(2):453-472.

SATYAMURTI, S. T. 1962. Guide to the lizards,

crocodiles, turtles and tortoises exhibited in the

reptile gallery, Madras Government Museum, pp. 1-

45. Government of Madras.

SMITH, M. A. 1933. Remarks on some Old World

geckos. Records of the Indian Museum 35:9-19.

SMITH, M.A. 1935. The Fauna of British India,

including Ceylon and Burma. Reptilia andAmphibia,

vol. II, Sauria. London, Taylor & Francis.

STRAUCH, A. 1887. Bemerkungen iiber die

Geckoniden-Sammlung im zoologischen Museum

der Kaiserlichen Akademie der Wissenschaften zu St.

Petersburg. Memoires de l'Academie Imperial des

Sciences, St-Petersbourg. (7)35(2): 1-38.

UNDERWOOD, G. 1954. On the classification and

evolution of geckos. Proceedings of the Zoological

Society of London 124:469-492.

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 36-37 j'

The Ceylonese Tree Frog Polypedates cruciger Blyth, a New Record for

India

R. J. Ranjit Daniels1 and m. S. Ravichandran2

'M. S. Swaminathan Research Foundation, 3rd Cross Gtreet, Taramani Institutional Area, Madras, India

600113

Zoological Survey of India, 100 Santhome High Road, Madras, India 600028

Abstract. -The Ceylonese tree frog Polypedates cruciger was considered endemic to the island of Sri Lanka.

However, recent surveys in the Western Ghats, India has revealed that the species is widespread outside its

originally reported range. P. cruciger differs from both the Indian species P. maculatus and P. leucomystax in

morphology and ecology. P. cruciger is an addition to the amphibian fauna of India.

Key words: Amphibia, Anura, Polypedates cruciger, India, Western Ghats, distribution.

FIG. 1. Polypedates cruciger from the Western

Ghats of India.

Introduction

During a recent survey of amphibians in

the Western Ghats and southwestern India,

three specimens of tree frogs were obtained

in June 1990 from a private estate in the hills

of Kanyakumari district (c. 8° 15' N; 77°25'

E). Later in the year, two more specimens

of the same species were observed in parts

of Karnataka State around 14" N; 75° E.

The specimens have been deposited in the

ZSI (Madras) and BNHS (Bombay)

museums.

Discussion

Based on the extent of webbing between

the toes and fingers, the specimens were

assigned to the genus Polypedates (Liem,

1970; Daniel and Sekar, 1989). In India,

only two specimens of Polypedates have

been hitherto reported. These are P .

leucomystax (Gravenhorst) and P.

maculatus (Gray) (Inger and Dutta, 1986).

The examples from the Western Ghats

differed from both leucomystax and

maculatus in the skin of the head adhering to

the nasal and frontoparietal bones, the

tympanum being two-thirds the diameter of

the orbit, the three-fourth webbing on toes

and in the hour-glass shaped marking on the

dorsum. These characters agree well with

Rhacophorus (-Polypedates) cruciger of

Boulenger (1890) described from Sri Lanka.

Further, the hour-glass marking on the

dorsal side commencing from the middle

level of eyes to mid-body terminating in the

form of a trident is unmistakably similar to

that in Rhacophorus cruciger illustrated by

Kirtisinghe (1957). Considering the above

characteristics, we confirm the identity of

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol. 6, p. 37

our specimens as Polypedates crucigerfFig.

1).

These specimens mark the first record of

this species in India outside its range (Sri

Lanka). The first three animals were located

within a clove plantation at an elevation of

400 m above MSL. These hills receive an

average annual rainfall of about 2000 mm.

Several males were observed calling on a

rainy night in June, perched on low

branches (c. 1 m above ground). The calls

consisted of a harsh tre... chuck repeated by

rival males followed by a series of chucks of

low intensity. In captivity, the males

continued to call during overcast and rainy

days. They fed well on a variety of live

insects including grasshoppers and

cockroaches.

The specimens observed in

southwestern Karnataka were in evergreen

forests in regions of 3000-6000 mm rainfall.

One individual was observed on October

1990 crossing a forest road and moving

towards a stream at midday. A second

individual was observed on November 1990

in a patch of dense forest, resting on a tree

trunk at c. 2 m above ground in the

company of a few smaller Philautus spp.

Specimens of Polypedates cruciger observed

in Karnataka were found at lower elevations

between 50 and 250 m above MSL.

The habitat preference, altitudinal and

geographical range, of this species in

southwest India suggest that this species

may be widespread in the Western Ghats

and has been overlooked by surveys until

recently. This species seems to be most

active during the rains and much of the

Western Ghats are inaccessible at this time.

This partly explains why the species has not

been reported from nearly 500 km of

intervening hills in its known range in India.

Acknowledgments

The authors wish to thank the Director,

Zoological Survey of India, Calcutta, Dr. K.

V. Lakshminarayana, Officer-in-charge,

Southern Regional Station, ZSI, Madras and

the Centre for Ecological Sciences, Indian

Institute of Science, Bangalore for the

facilities provided. Thanks are due to Dr.

R. S. Pillai, Scientist Emeritus (ZSI,

Madras), Mr. Romulus Whitaker, and Mr.

Harry Andrews of the Madras Crocodile

Bank for critically reviewing the manuscript.

Literature Cited

BOULENGER, G. A. 1890. Fauna of British India:

RepUlia and Batrachia, Taylor and Francis. London,

541 pp.

DANIEL, J. C. AND A G. SEKAR. 1989. Field

Guide to the Amphibians of Western India: Part

Four. Journal of the Bombay Natural History

Society 86:194-202.

INGER, R. F. AND S. K. DUTTA. 1986. An

overview of the amphibian fauna of India. Journal

of the Bombay Natural History Society 83

(supplement): 135- 146.

KIRTISINGHE, P. 1957. The Amphibia of Ceylon.

Colombo. 112 pp.

LIEM, S. S. 1970. The morphology, systematics

and evolution of the old world tree frogs

(Rhacophoridae and Hyperoliidae). Fieldiana

Zoology 57:1-145.

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 38-44 :

Size-gradation in Syntopic Frogs in South India

INDRANEIL DAS

Animal Ecology Research Group, Department of Zoology, University of Oxford, South Parks Road, Oxford

0X1 3PS, England

Present address: Centre for Herpetology, Madras Crocodile Bank, Post Bag No. 4, Mamallapuram-603 104,

Tamil nadu, S. India

Abstract. -An assemblage of eight metamorphosed anuran amphibians were studied at a seasonal locality in

South India to examine patterns of body size-gradation. In general, body shape was found to be closely

correlated to ecological characteristics of the species. The mean ratio of linear dimensions of the body in

adjacent species, when arranged in a size -series was 1.3 (range 0.83-1.88), as predicted by Hutchinson (1959)

for closely-related sympatric species. It was concluded that competition may be at work in producing these

ratios.

Key words: Amphibia, Anura, India, body size, competition.

Introduction

The body forms and sizes of organisms

have been considered, since the time of

Darwin (1859), to be determined by the

powerful forces of natural selection, and

may mirror a wide range of ecological

interactions often too complex to

comprehend in their entirety. The concept

of a form-function relation appears to have

stood the test of time, and is central to the

problem of organismic evolution (Gans,

1988). Animal size, for example, may be an

evolutionary response to demands of the

immediate environment, related to key life

history traits, such as fecundity, foraging,

locomotion, and reduction of predation,

desiccation, heating and cooling. Some

workers (e.g. Hutchinson, 1959; Schoener,

1986a) have argued that smaller species are

generally more specialized, as reflected in

their restricted diets and geographical

ranges. Gould (1966), however,

considered both large and small species to

be viable strategists, optimal size, according

to Phillipson (1981) being dependent on

competitive abilities and survival

probabilities of the various size and age

classes. However, large species tend to

appear later in a group's evolutionary

history, an exception being the Amphibia

(Peters, 1983).

Within ecological communities, if

environmental resources are partitioned

according to dimension, as documented by

Schoener (1965) and subsequent workers

(reviewed by Schoener, 1974; 1986b),

differences in the comparative sizes of the

organisms are to be expected. In fact, such

apparent differences were reported much

earlier by Hutchinson (1959), who found

constant differences in the ratios of linear

dimensions of the trophic (feeding)

apparatus among closely-related sympatric

species in his now controversial "Homage to

Santa Rosalia, or why are there so many

kinds of animals?" paper. Hutchinson

argued that similar species could coexist if

the ratios of linear dimensions and weights

of presumed competing species are around

1.3 and 2.0 respectively. While possible

causal factors remain unclear, such ratios

have been found in a wide range of both

invertebrate and vertebrate communities,

including beetles, spiders, frogs, lizards and

birds (reviewed by Schoener, 1986a).

In this paper, I report patterns of body

size and shape, and of size-gradation

observed in metamorphosed anurans from a

locality in South India. Specifically, I

searched for patterns of size gradation

within the eight syntopic frog species.

Materials and Methods

Eight species of anuran amphibians

occur in sympatry in the coastal scrublands

of Chengai Anna District, Tamil Nadu State,

South India. These, along with their mean

snout-vent lengths have been listed below:

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol. 6, p. 39

Tomopttma rolandat

(2 82 pat

Microhyla rubra

(1.566 gm)

Uperodon tystorna

(13 0 gm)

Microhyla ornaia

(0 362 gm)

Rana htiadacryla

(99 64 gm)

FIG. 1. Shapes and masses of the species studied.

Figures in brackets are mean weights.

Microhyla ornaia (4.1 mm), M. rubra (7.2

mm), Tomopterna rolandae (10.9 mm),

Uperodon systoma (12.4 mm), Polypedates

maculatus (20.9 mm), /?a/za cyanophlyctis

(16.1 mm), fl. crassa (22.9 mm) and R.

hexadactyla (33.4 mm).

Abbreviations used include SVL (snout-

vent length), HW (head width at the angle of

the jaws, perhaps better defined as the

gape), TBL (tibia length, the distance from

the convex surface of knee to the convex

surface of heel, with both tibia and tarsus

flexed) and WT (wet body weight). Linear

measurements were taken to the nearest 0.1

mm with a Mitutoyo Dial Vernier Caliper,

weights were taken to the nearest 0.1 gm

with an Acculab Electronic Balance (Model

333).

To interpret shape changes, logarithmic

transformations of the dimensions of the

organ of each species were used in the

function log y = b log x + log a (where x

and y are the morphological variates), which

has been considered to approximate shape

change in most organisms (see Gould,

1966, for justification).

Observations

The assemblage of eight anuran species

studied display a range of body forms

(Figure 1) and sizes (Table 1). Except for

the diminutive species of the genus

Microhyla (maximum SVL 17.4 and 26.5

mm) and the large ranid, Rana hexadactyla

(maximum SVL 132.2 mm), all species

were small to medium, with maximum SVL

between 43.2 to 93.1 mm (mean 68.2).

A general impression is that some

species are comparatively short and squat,

while others are long and thin. To quantify

differences, WT was divided by the cube of

the SVL for individuals of each species.

Three morphological groups along a

continuum were recognized based on the

patterns of body form (length-weight data in

Table 2), which comprise species with

similar ecological preferences, including

heavy-bodied, terrestrial forms; light bodied

aquatic forms; and an arboreal form of

intermediate body mass.

The relationships of untransformed

values of WT/SVL3 (Figure 2.1), as well as

the arcsine-transformed data (Figure 2.2)

can be described as linear, the slope b being

0.38 and 1.89 respectively, not differing

significantly (r-test, P>0.05) from isometry,

indicating that large frogs are not likely to be

comparatively lighter.

The ratios of differences in linear

dimensions (SVL, HW, ED and TBL) and

weight (WT) of frogs from this study,

arranged in a size series (mean dimensions

divided by corresponding figures for

Microhyla ornata, the smallest member of

the assemblage) are shown in Figure 3. The

range of ratios of SVL (1.57-6.17) appear

greater than shown by Neotropical Anolis

lizards, 1.01-1.46 (Duellman, 1978), or

Vol. 6, p. 40

Asiatic Herpetological Research

June 1995

TABLE 1 . Morphometric data on the eight species of anurams studied. References to species: MO,

Microhyla ornata; MR, Microhyla rubra; TR, Tomopterna rolandae; US, Uperodon systoma; RC, Rana

cyanophlyctis; PM, Polypedates maculatus; RCR, Rana crassa; RH, Rana hexadactyla. References to body

parts: SVL, snout-vent length; HW, head width; TBL, tibia length; WT, weight. Linear dimensions in cm;

wights in gm.

Philippines scincid lizards, 1.41-4.33

(Auffenberg and Auffenberg, 1987). Ratios

of HW (here considered the measure of the

trophic apparatus) which may be more

meaningful for understanding differences in

morphology that may be the result of

differences in feeding patterns, in the

present study were somewhat greater than

SVL ratios, the mean 4.3 (range 1.61-8.15).

Mean values of the SVL, HW and WT ratios

appear to be arranged in pairs that do not

reflect phylogenetic affinities, and the

implications, if any, are unclear. The

incremental increase in SVL from the

smallest to the largest species varied

between 2.93-60.99, and no regular pattern

of increment between adjacent species seems

evident, although Auffenberg and

Auffenberg (op. cit.) found that the greatest

size differences exist among the smallest

species in the skink community they

studied.

June 1995

Asiatic Herpetological Research

Vol.6, p. 41

TABLE 2. Weight to cube of snout-vent length in the eight species of anurans studied.

Ratios of linear dimension of the body in

adjacent species, when arranged in a size-

series (Table 2) appear close to the

Hutchinsonian ratio, mean values for all

species ranging between 0.83-1.88 (mean

1.32± SE 0.09). Weight ratios were,

however, 0.83-4.60, the mean 2.63 (±SE

0.56) substantially larger than 2.0 as

predicted by Hutchinson.

Discussion

Morphologically similar species tend to

display similar ecological need, and if they

occur in sympatry, it is assumed that they

compete.

Duellman (1978), in his study of the

Ecuadorian herpetofaunal communities

discovered no size groupings among frog

and lizard species, using SVL ratios of

differences between sympatric species,

showing that there is, instead, a steady

increase in SVL. Auffenberg and

Auffenberg (1987) found that Philippines

skinks show greater ratios of differences

between species, and presumed predation on

a significantly greater size range of prey by

the scincids.

With Microhyla ornata, the smallest

species in the area, was used as a standard

against which all others were compared, the

range of ratios of SVL (1.57-6.17) was

found to be considerably greater than figures

reported from other studies on tropical

herpetological assemblages, including those

of Duellman (1978) and Auffenberg and

Auffenberg (1987), suggesting that

members of the assemblage of South Indian

amphibians under investigation feed on a

greater size range of prey.

For coexisting frog species, the ratio of

mean head width among species pairs

appear close to the ratio for closely-related,

sympatric and presumably competing

species described by Hutchinson (1959) in

studies from Canada (McAlpine and

Dilworht, 1989) and Peru (Toft, 1980),

indicating the potential for competition. The

mean values (±SE) of the ratios of SVL,

HW and WT for adjacent species in the

South Indian amphibians were 1.31

(±0.09), 1.37 (±0.09) and 2.63 (±0.56),

respectively.

The 1.3 ratio concept has recently come

under close scrutiny, leading some workers

(e.g. Strong et al., 1979; Simberloff and

Boeklen, 1981) to doubt its constancy. In

fact, Simberloff and Boecklen, op cit.)

attempted to show that the methods utilized

by workers reporting the constancy of the

Vol. 6, p. 42

Asiatic Herpetological Research

June 1995

SVL

0 2

0 4 0 6

log SVL

0 8

FIG. 2. Relationship between snout- vent length

(SVL) and the ratio of weight to cube of snout-vent

length (Wt/cube SVL) in the species studied. 2.1.

Untransformed data; 2.2. Log-transformed data.

Species abbreviations as in Table 1 .

ratios have been erroneous, although

subsequent workers have demonstrated that

the evidence is, in many cases, quite strong

on examination of fresh data (e.g. Schoener,

1984) or even on the basis of the tests

conducted by Simberloff and Boecklen (see

Losos et al., 1989). Maiorana (1978)

suggested that if ecological segregation of

two species (or age classes) requires a

minimum level of overlap in their degree of

morphological variability, the linear

displacement in mean size will also be

relatively constant, and argued that the

presence of similar ratios in many man-made

objects, as discovered by Horn and May

SVL

300

200

<

100 -

SVL

FIG. 3. Size-gradation in the species studied (mean

dimensions divided by corresponding figures for the

smallest species in die assemblage, Microhyla

ornata. 3.1. Snout-vent length (SVL); 3.2. Head

width (HW): 3.3 Weight (Wt).

(1977) may be associated with human

perceptional abilities derived from the

natural world.

The constancy of Hutchinsonian ratios

has been shown for a large number of

June 1995

Asiatic Herpetological Research

Vol. 6, p. 43

invertebrate and vertebrate groups (reviewed

by Schoener, 1984; 1986a), which is

suspected to be indicative of interspecific

competition. To complicate matters further,

ratios have been shown to be size-

dependent, a function of allometry, and may

thus change with growth (Roth, 1981).

These are also thought to vary

geographically, assemblages in the tropics

show smaller ratios than temperate ones

(Klopfer and Mac Arthur, 1961), suggestive

of greater niche overlap in tropical

assemblages. In the present study, HW,

considered a measure of the trophic

apparatus of frogs, showed negative

ontogenetic allometry, the slopes b of the

regressions of SVL and HW (on log paper)

scaling allometrically in all eight anuran

species that comprise the assemblage under

investigation, indicating differences in the

relative size and shape of the trophic

apparatus among the different size-classes

and sexes within a species may be

influenced by food size.

Acknowledgments

The staff of Madras Crocodile Bank

Trust helped with field work, and provided

laboratory space. In particular, I would like

to thank Romulus Whitaker, Harry Andrew,

Romaine Andrews, Manjula Tiwari and

Aurofilio Schiavina. My research on the

ecology of frogs in South India was

supported by Inlaks Foundation, Madras

Crocodile Bank Trust, Trinity College,

Oxford and an Overseas Research Student

(ORS) Award. I thank R. Avery, M. Coe,

and C. Perrins for comments.

Literature Cited

AUFFENBERG, W. AND T. AUFFENBERG. 1987.

Resource partitioning in a community of Philippine

skinks. Florida State Museum Bulletin 32:151-

219.

DARWIN, C. 1859. On the origins of species by

means of natural selection. Murray, London.

DUELLMAN, W. E. 1978. The biology of an

equatorial herpetofauna in Amazonian Ecuador.

Miscellaneous Publications of the Museum of

Natural History, University of Kansas 65: 1-352.

GANS, C. 1988. Adaptation and the form-function

relation. American Zoologist 28:681-697.

HORN, H. S. AND R. M. MAY. 1977. Limits to

similarity among coexisting species. Nature

270:660-661.

HUTCHINSON, G. E. 1959. Homage to Santa

Rosalia, or why are there so many kinds of animals?

American Naturalist 93:145-159.

KLOPFER, P. H. AND R. H. MACARTHUR. 1961.

On the causes of tropical species diversity: Niche

overlap. American Naturalist 95:223-226.

LOSOS, J. B., S. NAEEM AND R. K. COLWELL.

1989. Hutchinsonian ratios and statistical power.

Evolution 43:1820-1826.

MCALPINE, D. F. AND T. G. DILWORTH. 1989.

Microhabitat and prey size among three species of

Rana (Anura: Ranidae) sympatric in eastern Canada.

Canadian Journal of Zoology 67:2244-2252.

MAIORANA, V. C. 1978. An explanation of

ecological and developmental constants. Nature

273:375-377.

PETERS, R. H. 1983. The ecological implications

of body size. Cambridge University Press,

Cambridge.

PHILLIPSON, J. 1981. Bioenergenetic options and

phylogeny. Pp. 20-45. In C.R. Townsend & P.

Calow (Eds). Physiological ecology: An

evolutionary approach to resource use. Blackwell

Scientific Publishers, Oxford.

ROTH, V. L. 1981. Constancy in the size ratios of

sympatric species. American Zoologist 118:394-

404.

SCHOENER, T. W. 1965. The evolution of bill

size differences among sympatric congeneric species

of birds. Evolution 19:189-213.

SCHOENER, T. W. 1974. Resource partitioning in

ecological communities. Science 185:27-39.

SCHOENER, T. W. 1986a. Kinds of ecological

communities- Ecology becomes pluralisitic. Pp.

467-479. In J. Diamond & T.J. Case (Eds).

Community ecology. Harper & Row, Publishers,

New York.

SCHOENER, T. W. 1986b. Resource partitioning.

Pp. 91-126. In J. Kikkawa & D.J. Anderson (Eds).

Vol. 6, p. 44 Asiatic Herpetological Research June 1995

Community ecology: Patterns and processes.

Blackwell Scientific Publications, Melbourne.

SIMBERLOFF, D. AND W. BOECKLEN. 1981.

Santa Rosalia reconsidered. Evolution 35:1206-

1228.

STRONG, D. R., L. A. SZYSKA, AND D. S.

SIMBERLOFF. 1979. Tests of community-wide

character displacement against null hypothesis.

Evolution 33:897-913.

TOFT, C.A. 1980. Feeding ecology of thirteen

syntopic species of anurans. Oecologiia 45:131-

141.

I June 1995

Asiatic Herpetolo^ical Research

Vol. 6, pp. WW

Observations on Arboreality in a Philippine Blind Snake

MAREN Gaulke

Muhliusstrasse 84, 24103 Kiel 1, Germany

Abstract. -Five blind snakes were observed in June 1990 in the rain forests of Sibutu Island in the Sulu

Archipelago, Philippines. Contrary to the usually fossorial habits of typhlopids, Ramphotyphlops suluensis

(Taylor, 1918) shows arboreal habits. It climbed through trees at night using the prehensile tail and

hindbody. When caught they extruded a strong smelling liquid from their cloaca. Relatively long tails are

found in some other rain forest dwelling typhlopids, which may also have arboreal habits.

Key words: Reptilia, Ophidia, Typhlopidae, Ramphotyphlops suluensis, Philippines, ecology.

and the relative humidity between 70 and

Introduction 95%.

Little is known of the behavior of blind

snakes (Typhlopidae). Information is

normally generalized and consists of little

more than that typhlopids are small,

burrowing snakes, which live in decaying

logs, humus and leaf litter, and feed mainly

on ants and termites, especially their grubs,

pupae and eggs (e. g. Taylor, 1922;

Loveridge, 1946; Gruber, 1980).

This gap in observations is certainly due

to a number of different factors. Typhlopids

are very inconspicuous and rather dull

looking, and as such, arouse the interest of

few people, even among herpetologists.

About 168 species are known (Hahn,

1980). Many are found infrequently, and

often are known from one or a few

specimens only. Due to their size,

coloration, and secretive habits, they are

hard to observe. However, observations

reported here on a rain forest dwelling blind

snake in the Philippines, indicate that at least

not all of them are as secretive as generally

assumed.

Methods

In June 1990 a three week field survey

was conducted on Sibutu, a small island in

the Sulu-Archipelago, a few miles off the

northeast coast of Sabah, Borneo (04°

46.4'N, 119° 28.8'E). Observations and

collections of amphibians and reptiles were

made within a forested area (primary and

secondary lowland forest of the molave type

sensu Dickerson, 1928). Short, but heavy,

rains fell every two to three days, the

temperature ranged between 25 and 32°C,

The nomenclatural history and taxonomy

of the typhlopids observed and caught on

Sibutu is discussed in Gaulke (in press),

where the species, previously synonymized

with Ramphotyphlops olivaceus (Gray,

1845), is revalidated. Ramphotyphlops

suluensis reaches a length of approximately

40 cm, the eyes are distinctive, and the tail is

more than twice as long as broad. The

dorsal side is gray, the ventral side is cream,

with bright white scales along the median

row.

Observations

Although a considerable amount of time

was spent turning and splitting decaying

logs, and digging in humus and leaf litter in

search of blind snakes, all efforts were

unsuccessful. However, a few days before

I had to leave Sibutu Island, the luck turned.

While looking for geckos with a flashlight

between 2200 and 0200 hours, the first

blind snake was observed, not on the

ground as expected, but on a tree on an

almost leafless twig approximately 3 m

above the ground. While trying to reach it,

the disturbed animal let itself drop to the

ground, and vanished into the leaf litter.

During the following three nights, four more

specimens were observed, all on branches

and twigs above my head. Being more

careful now, it was quite easy to catch them.

All four reacted to the capture with the

excretion of a pungent musk from their

cloaca, the stench of which adhered to the

skin for some time.

Before capture, the mode of locomotion

of the climbing blind snakes was observed

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 46

Asiatic Herpetological Research

June 1995

FIG. 1. Ramphotyphlops suluensis climbing in an avocado tree.

FIG. 2. Ramphotyphlops suluensis making searching movements with its stiffened forepart while climbing.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 47

for some time. While the tail and hindbody

is tightly coiled around a twig, they crawl

forward with the free part of their body.

Depending on the thickness of the twig, they

may use protrusions as resistance and hold,

or make serpentine movements, with parts

of their body hanging loosely down on both

sides of the twig. After the forepart is

secured, the tail/body grip is released and

then dragged forward, and anchored further

along. Compared to typical arboreal snakes,

like whip snakes or flying snakes, they

move very slowly, but are nevertheless

skilled and effective climbers. During their

movements, they stop relatively often and

demonstrate a conspicuous behavior. While

the hind part is coiled in the tree, the

stiffened forepart is stretched into the air,

making slow circular movements. When

they discover another twig or branch within

their reach with this searching movements,

they might climb over to it. All the while

they are tongue flicking. (Fig. 1, 2).

Two of the snakes captured were

preserved and transferred to the

Forschungsinstitut und Naturmuseum

Senckenberg (SMF 74247/8). The stomach

of the larger snake (total length 357 mm,

weight 11.5 g) contained part of an

unidentified earthworm, with a surprisingly

large girth in relation to the tiny mouth of the

snake. The two other specimens were kept

alive for further observation. They are

strictly nocturnal. Larvae of the moth,

Galleria mellonella, were offered as food,

but they were never observed feeding.

Nonetheless, their good condition after

several months in captivity indicated some

food uptake. For video records they were

placed on a small avocado tree during

daytime. Here the same movements could

be observed and recorded, as described

above. When released in the middle of the

avocado stem, they more often climbed up

than down, searching for a resting place

within the branches. However, sometimes

they climbed down and started to burrow

into the soil of the flower pot, proving that

they are as effective in digging as in

climbing.

Discussion

Characteristic features of typhlopid

snakes are: a cylindrical body, smooth

small scales throughout the entire body, a

small narrow head with a solid cranium, a

short broad tail ending in a sharp spine, and

reduced eyes covered by much larger scales.

These adaptations for fossorial life are in

almost complete contrast to the

characteristics of typical arboreal snakes,

such as a laterally compressed or triangular

body, a thin prehensile tail, and medium

sized to large eyes. However, as shown by

R. suluensis, it can be erroneous to interpret

the mode of living from the habitus alone.

Only the relatively longer tail compared to

other typhlopids (in most typhlopids the tail

is about as long as wide) might be

interpreted as an adaptation towards

arboreality.

The question remains, why are they

climbing in trees, as the disadvantage is

obvious. They are more exposed to

predation from nocturnal animals, such as

owls or cat snakes, than their relatives

which only seldomly leave their burrows.

Few blind snakes were observed climbing

on trees before. A Ramphotyphlops

nigrescens was found 5 m above the ground

in a tree (Shine and Webb, 1990),

arboreality is discussed for R. braminus

(Swanson, 1981), and it is reported for

some Leptotyphlopidae (Vanzolini, 1970).

Shine and Webb (1990) discuss arboreality

in scolecophidians as a feeding strategy.

They assume that there may be little

difference for them to follow ant-trails

underground or on trees. However, the

observations on R. suluensis indicate that

this species is not incidentally climbing up

trees, but might be more or less specialized

on an arboreal life. All specimens on Sibutu

were found climbing, and none on the

ground. It can be assumed that R. suluensis

is not the only blind snake specialized on

arboreality. Taylor (1922) collected several

Philippine typhlopids in the root balls of

aerial ferns, on felled trees. He concluded

that they are living and hunting within these

root balls. I assume it is much more likely

that they are using epiphytes, etc., only as

daytime retreats, actively searching for food

Vol. 6, p. 48

Asiatic Herpetological Research

June 1995

in the twigs and branches of the tree during

night time. Those blind snakes found in

epiphytes have unusually long tails for

typhlopids, being four to seven times as

long as broad. In view of the skilled way

R. suluensis uses its much shorter tail for

climbing, they should be even better

equipped for an arboreal live.

The function of the cloacal sac

substance, which R. suluensis used as a

defense mechanism against capture, was

investigated in the leptotyphlopid

Leptotyphylops dulcis. Gehlbach et al.

(1968) found that it repelled attacking army

ants, upon which these snakes feed.

Furthermore the substance was found to

repel sympatric ophiophagous and

insectivorous snakes, a much more serious

danger to the blind snakes. On the other

hand, L. dulcis are attracted to their own

colacal sac substance (Watkins et al., 1969),

so it has different functions, as interspecific

repellent, and as intraspecific attraction. It

can be assumed that it has similar complex

functions in R. suluensis.

Acknowledgments

The field study was supported by the

Senckenbergische Naturforschende

Gesellschaft, Frankfurt a. Main. Video

records were done in cooperation with Dr.

A. Altenbach, Kiel University. I thank Dr.

W. R. Branch, Port Elizabeth, for reviewing

the manuscript.

Literature Cited

GEHLBACH, F. R., J. F. WATKINS, AND H. W.

RENO. 1968. Blind snake defensive behavior

elicited by ant attacks. Bioscience 18:784-785.

G RUBER, U. 1980. Blindschlangen,

Wiihlschlangen und Warzenschlangen. Pp. 362-366

In B. Grzimek (Ed), Grzimeks Tierleben. Band 6.

Kriechtiere. Deutscher Taschenbuch Verlag,

Miinchen.

HAHN, D. E. 1980. Liste der rezenten Amphibien

und Reptilien. Anomalepidae, Leptotyphlopidae,

Typhlopidae. Das Tierreich 101:1-93. Walter de

Gruyter, Berlin.

LOVERIDGE, A. 1946. Reptiles of the Pacific

world. Macmillan Co., New York. 259 pp.

SHINE, R., AND J. K. WEBB. 1990. Natural

history of Australian typhlopid snakes. Journal of

Herpetology 24(4):357-363.

SWANSON, S. 1981. Typhlina bramina, an

arboreal blind snake? Northern Territory Naturalist,

Darwin, N.T. 4:13.

TAYLOR, E. H. 1922. The snakes of the

Philippine Islands. Philippine Bureau of Science,

Manila. 312 pp.

VANZOLINI, P. E. 1970. Climbing habits of

Leptotyphlopidae (Serpentes) and Wall's theory of

the evolution of the ophidian eye. Pap. Avul. Zool.

23:13-16.

WATKINS, J. F., F. R. GEHLBACH, AND J. C.

KROLL. 1969. Attractant-repellent secretions of

blind snakes (Leptotyphlops dulcis) and their army

ant pray (Neivamyrmex niggrescens). Ecology

50(6): 1098-1 102.

DICKERSON, R. S. 1928. Distribution of life in

the Philippines. Philippine Bureau of Science,

Manila, Monograph 21:1-322 pp.

GAULKE, M. (in press). Die Herpetofauna von

Sibutu Island (Philippinen), unter Beriicksichtigung

zoogeographischer und okologischer Aspekte.

Senckenbergiana biologica

|Junc 1995

Asiatic Herpetological Research

Vol. 6, pp. 49-52

On the Distribution of Emydid Turtles and the Anuran Genus Microhyla in

the Philippines

Maren Gaulke

Muhliusstr. 84, 24103 Kiel, Germany

Abstract. -Cuora amboinensis kamaroma Rummler and Fritz and Microhyla annectens Boulenger are

reported from the Sulu Archipelago in the southwestern Philippines. The Philippines range of Cyclemys

dentata (Gray) is extended to the Sulu Archipelago. The distribution of emydids and the genus Microhyla in

the Philippines is discussed.

Key Words: Reptilia, Testudines, Emydidae, Cuora amboinensis amboinensis, Cuora amboinensis

kamaroma, Cyclemys dentata, Amphibia, Anura, Microhylidae, Microhyla annectens, Philippines,

distribution.

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Sanga^PV

uBongao

f" Sibutuft ^

' Sitanki D)) ^

<L

^

*

FIG. 1 Generalized map of the Sulu Archipelago.

Introduction

The Philippines are separated into

different faunistic regions (Brown and

Alcala, 1970, 1980; Dickerson, 1928; Inger,

1954; Leviton, 1963). The Sulu

Archipelago, an island chain situated

between Basilan and Mindanao to the east,

and Sabah, Borneo, to the west, is one of

these regions. Faunistically it is more

closely related to Borneo than to the

Philippines. The only comprehensive work

on the herpetofauna of the Sulu Archipelago

is from Taylor (1918). Additions to it's

herpetofaunal record include for example

Gaulke (1993, 1994), Leviton (1963) and

Taylor (1922a, 1922b). Almost nothing is

known of the emydid turtles of the region,

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 50

Asiatic Herpetological Research

June 1995

and knowledge on the anurans is also

scarce. This work contributes to both of

these subjects, and discusses the distribution

of taxa in the groups in other parts of the

Philippines.

Material and Methods

The author has conducted field surveys

in different regions of the Philippines since

1984. In the years 1990, 1991 and 1992 the

following islands of the Sulu Archipelago

were visited: Bongao, Sanga Sanga, Siasi,

Sibutu, Tawitawi and Jolo (Fig. 1). Most

of the turtles found were photographed and

released after examination.

Three turtles (one Cuora amboinensis

kamaroma, two Cyclemys dentata) are kept

alive by the author, the microhylid frog

(SMF 74908) and one C. dentata shell

(SMF 74909) are deposited in the

herpetological collection of the

Forschungsinstitut und Naturmusem

Senckenberg, Frankfurt am Main,

Germany. The C. amboinensis subspecies

were identified after Rummler and Fritze

(1991), and the frog after Inger (1966).

Results and Discussion

Emydidae

Cyclemys dentata.- On June 9, 1990, an

adult specimen of C. dentata was caught in

an undisturbed swampy lowland forest at

Languyan on Tawitawi. It's faeces showed

that it had fed on ripe figs before capture.

In 1991 and 1992 several, mainly

juvenile, specimens of C. dentata were

found in swamps and small creeks in

different areas of Tawitawi (Batu Batu,

Magsaggaw), proving that it is not a rare

turtle on the island. It has not yet been

found on any other island of the Sulu

Archipelago.

Previously the known range of C .

dentata in the Philippines included only the

Palawan Province (Balabac, Palawan,

Calamian Islands) and Leyte (Alcala 1986;

Taylor 1921). While it is common

throughout the Palawan Province, records

from Leyte are rare. Palawan and Leyte

belong to different faunistic regions, and

were never connected by a land bridge.

Therefore it is surprising that C. dentata is

known from both regions, but from

nowhere in between. Three explanations

can be offered for this disjunct distribution:

1. C. dentata exists on other Philippine

Islands, but has not been recorded (or

reported) until now. 2. C. dentata

previously occurred on other Philippine

islands, but became extinct subsequently on

most of them, with a relict population

surviving on Leyte. 3. C. dentata never

reached the more eastern Islands of the

Philippines, and the Leyte records rely on

specimens introduced by man. This is not

unlikely, since people often keep turtles as

pets and for medicinal purposes (as rheuma

remedy) leading to their translocation. C.

dentata kept in captivity on Negros and

Cebu were obtained on Palawan (pers.

obs.).

The disjunct Philippine distribution of

C. dentata is remarkably echoed by the third

emydid turtle from the Philippines, the

endemic Heosemys leytensis (Taylor) which

is also reported from Leyte and Palawan,

but no in between island. Since both

distribution records of this rare turtle rely on

single individuals (Timmermann and Auth

1988; Taylor 1921), no assumptions on the

reasons for the disjunct distributions can be

made.

The Sulu Archipelago and Palawan lie in

both Philippine entryways from Borneo,

suggesting that the C. dentata populations

have their origin on Borneo, where this

turtle is widespread.

Cuora amboinensis.- In 1990 the author

found Cuora amboinensis on the islands of

Sanga Sanga and Tawitawi, and in 1992 on

Jolo. During this period C. amboinensis

was reported from Jolo (Rummler and Fritz

1991), who identified two specimens from

Jolo (MNHN 6152:1-2, Musium Nationale

d'Histoire Naturelle Paris) as C. a.

amboinensis, as they did all Philippine

June 1995

Asiatic Herpetological Research

Vol. 6, p. 51

FIG. 2. Plastral view of three Cuora amboinensis

from Jolo, showing that almost no dark markings

are present on the plastron, as is typical for the

subspecies C. a. kamaroma Rummler and Fritz.

material they studied. However, the

specimens found during my trips on Sanga

Sanga, Tawitawi and Jolo do not belong to

the nominate form, but to the recently

described C. a. kamaroma Rummler and

Fritz. They have none, or very few, dark

patches on their plastron (Fig. 2) and a high

carapace, as is typical for this subspecies.

U. Fritz, who kindly examined photos of

some of the turtles captured on Jolo,

mentioned that they are slightly flatter than

other C. a. kamaroma he had seen, and the

marginal scutes were slightly more visible

from above. Nevertheless he confirms the

determination as C. a. kamaroma, assuming

that some interbreeding with Philippine C.

a. amboinensis may have occurred.

C. a. kamaroma, the most widespread of

the three subspecies of C. amboinensis, is

known from Borneo, the Southeast Asian

mainland, and now the Sulu Archipelago.

C. a. amboinensis has a wide distribution

over the Philippines, being reported from

Bacoo, Cebu, Dinagat, Leyte, Luzon,

Mindanao, Mindoro, Negros, Polillo and

Samar (Boettger 1886; Rummler and Fritz

1991; Taylor 1921), but occurs on other

Philippine Islands as well. The author, for

example, has collected a few specimens on

Masbate (currently being kept alive).

Outside the Philippines, C. a. amboinensis

is known from Sulawesi and the Moluccas.

The Palawan Province is presently the only

known Philippine Province not inhabited by

any subspecies of C. amboinensis.

Rummler and Fritz (1991) consider the

high domed C. a. kamaroma and the flat C.

a. amboinensis as the evolutionary extremes

of C. amboinensis, whilst the third

subspecies C. a. couro (Schweigger), which

occurs on Sumatra and Java, shows

intermediate characteristics. They believe it

possible that in the future high domed and

flat forms of C. amboinensis may be found

sympatric on Borneo, proving that they

belong to different species. The record of

C. a. kamaroma from the Sulu Archipelago

shows that the Bornean form could reach the

outskirts of the Philippines, but does not

resolve the question of their phylogenetic

affinities. It is surprising that within the

Philippines two significantly different forms

are found. C. a. amboinensis must either

have developed from C. a. kamaroma, or

reached the Philippines, via Mindanao, from

the Moluccas or Sulawesi.

Microhylidae

On March 18, 1991, the author collected

one specimen of Microhyla annectens

Boulenger (SMF 74908) in leaf litter beside

a small pond in a lowland forest at

Magsaggaw, Tawitawi. The species has a

wide distribution on Borneo, Malaysia and

Thailand, but was previously unknown

from the Philippines.

Only one other member of the genus

Microhyla has been recorded from the

Philippines. Fischer (1885) reported

Microhyla achatina (Boie) from Southern

Mindanao, based on a single specimen.

Boettger (1886) listed this frog, but

mentioned that the specimen, according to a

comment by G. A. Boulenger, most

probably is not M. achatina but a Kaloula

species. Taylor (1921) listed M. achatina

without questioning the determination.

However, he doubted the accuracy of the

locality record, assuming that it might have

come from another country. He also

assumed that it might be from one of the

high, little explored mountains on Southern

Mindanao and therefore had not

subsequently been found. Inger (1954) and

Alcala (1986) finally do not include M .

Vol. 6, p. 52

Asiatic Herpetological Research

June 1995

achatina in the Philippine amphibian fauna

anymore.

Whether the record of M. achatina from

the Philippines is true or not (the specimen

is no longer available) must remain open,

however, the new record shows that the

genus Microhyla has reached the

Philippines.

Acknowledgments

I am grateful to Mr. R. Miller and Mr.

Th. Borromeo, who were helpful in finding

turtles on the Sulu Archipelago, and to the

Provincial Department of Environment and

Natural Resources in Bongao for allowing

me to conduct surveys in their area, and to

collect a few specimens. Dr. W. Branch

kindly revised the manuscript, giving many

helpful comments.

Literature Cited

ALCALA, A. C. 1986. Guide to Philippine flora

and fauna. Vol. X. Amphibians and reptiles. JMC

Press, Quezon City. 195 pp.

BOETTGER, O. 1886. Aufzahlung der von den

Philippincn bekannten Reptilien und Batrachier.

Senckenbergische Naturforschende Gesellschaft

1886:91-134.

BROWN, W. C, AND A. C. ALCALA. 1970. The

zoogeography of the herpetofauna of the Philippine

Islands, a fringing archipelago. Proceedings of the

Californian Academy of Science, fourth series

38(6): 105-130.

BROWN, W. C, AND A. C. ALCALA. 1980.

Philippine lizards of the family Scincidae. Silliman

University Press, Dumaguete City. 264 pp.

DICKERSON, R. S. 1928. Distribution of life in

the Philippines. Philippine Bureau of Science

Monograph 21:1-322.

FISCHER, J. G. 1885. A list of reptiles and

batrachians of Mindanao. Jahrbuch der

WissenschafUichen Anstalt Hamburg 1885(2):80-81.

GAULKE, M. 1993. First record of the polydont

snake Sibynophis geminatus geminatus (Boie,

1826) from the Philippines, with a discussion of

Sibynophis bivittatus (Boulenger, 1894).

Herpetological Journal (3): 151-152.

GAULKE, M. 1994. Contribution to the snake

fauna of the Sulu Archipelago, with the description

of a new subspecies of Dendrelaphis caudolineatus

(Gray, 1834). Herpetological Journal.

INGER, R. F. 1954. Systematic and zoogeography

of Philippine amphibia. Fieldiana: Zoology

33(4):183-531.

INGER, R. F. 1966. The systematics and

zoogeography of the amphibia of Borneo. Fieldiana:

Zoology 52(1966): 1-402.

LEVITON, A. E. 1963. Remarks on the

zoogeography of Philippine terrestrial snakes.

Proceedings of the Californian Academy of Science,

fourth series 31(15):369-416.

RUMMLER, H.-J., AND U. FRITZ. 1991.

Geographische Variabilitat der Amboina-

Scharnierschildkrotc Cuora amboinensis (Daudin,

1802), mit Beschreibung einer neuen Unterart, C. a.

kamaroma subsp. nov. Salamandra 27(1/2): 17-45.

TAYLOR, E. H. 1918. Reptiles of Sulu

Archipelago. Philippine Journal of Science

13(5):233-269.

TAYLOR, E. H. 1921. Amphibians and turtles of

the Philippine Islands. Philippine Bureau of

Science Monograph 15-1-193.

TAYLOR, E. H. 1922a. Additions to the

herpetological fauna of the Philippine Islands, 2.

Philippine Journal of Science 21(2): 161-206.

TAYLOR, E H. 1922b. Additions to the

herpetological fauna of the Philippine Islands, 3.

Philippine Journal of Science 22(5):5 15-557.

TIMMERMANN, W. W. AND D. L. AUTH. 1988.

Geographic distribution. Heosemys leytensis.

Herpetological Review 19(1):21.

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 53-61

Ecology and Conservation of Onychodactylus fischeri (Caudata, Hynobiidae)

in the Russian Far East

Paul C. Griffin1 And Vasili A. Solkin2

1 Department of Forestry and Natural Resource Management, University of California at Berkeley, Berkeley,

California, 94720, USA

^Pacific Institute of Geography, Far East Branch of Russian Academy of Sciences, Vladivostok, 690032,

Russia

Abstract. -Onychodactylus fischeri (Boulenger, 1886) is a lungless salamander with a larval development

time of over four years and lifespan that may extend over 18 years. O. fischeri develops and spawns only in

cold torrential brooks. In the Russian Primorski Krai of the Russian Far East O. fischeri lives in undisturbed

Ussuriland montane taiga forest. Adults migrate annually to breed. In homing experiments adults which were

ready for breeding migrated over 800m of land to breeding sites. Homing experiments showed that sexually

mature adults may demonstrate stream fidelity. Males were more frequently encountered at surface waters than

females. There are seasonal peaks to breeding-adult activity. High-impact forest harvesting is now typical in

headwater forests and river valleys across the range of 0. fischeri, causing disturbance and siltation in

spawning brooks and surrounding forest habitat. Pinus koreansis trees comprised a dominant component of

the forest canopy in spawning drainages.

Key Words: Amphibia, Caudata, Hynobiidae, Onychodactylus fischeri, Russian Far East, Ussuri taiga,

conservation, ecology.

Introduction

This study set out to discover aspects of

Onychodactylus fischeri biology by

collecting standardized morphological and

color pattern data during the course of adult

and juvenile mark-recapture surveys, by

documenting microhabitat location, by

determining ages of adults and larvae, by

moving marked sets of salamanders and

recording their return to home brooks, and

by continuing early attempts at inducing

captive breeding, and to assess movement of

individuals between spawning streams.

Ussuri taiga has been heavily harvested

from plains and river valleys in this century

to the point that headwaters comprise some

of the least disturbed examples of this forest

type. High-impact, wasteful forest

harvesting is now occurring in the

elevational range of O. fischeri range in an

inefficient manner, causing catastrophic

disturbance and siltation to its spawning

brooks and surrounding forest habitat. O.

fischeri is an endangered species in the Red

Data Book of the USSR but is afforded no

protection under current Russian

environmental law.

FIG. 1. Map of Asia and the Russian Far East

showing study location in boxed area.

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 54

Asiatic Herpetological Research

June 1995

FIG. 2. Dorsal Color Patterns of 0. fisc fieri.. Percent frequencies out of 351 salamanders in "Chinese"

brook were: A, 1%; B and B2, 36.4%; C, 46.7%;D, 5%; F, 4.8%; L, 0.3%

Methods and Materials

Our studies were within the range of

Onychodactylus fischeri in the Sixote-Alin

Mountain Range of the Primorskii Krai,

Russia. These mountains are forested by

Usuriland taiga. We conducted our surveys

and experiments at elevations between 500

m and 1000 m., at a site 10 km west of the

Pacific Ocean in headwater brooks of the

Mysovka River (Lat. 135°, Long. 41°).

This river flows into the Tinfura River, a

tributary of the Ol'ga Bay (Fig. 1). Climate

is typical of the maritime Russian Far East,

June 1995

Asiatic Herpetological Research

Vol. 6, p. 55

with humid summer days averaging 25° C in

July and cold winters down to -30° C. The

summer monsoon and winter snows provide

approximately 120 cm

precipitation (Fullard, 1972).

of annual

We surveyed the brook tributaries at the

headwaters of the upper Mysovka watershed

and gave them the following arbitrary

names: "Chinese," "Zapovednik," "Mutnii,"

"Himalayan," "Burned," and "Tetkin." We

characterized the forest canopy cover in the

entire study site in terms of dominant tree

species and also inventoried subdominant

trees of the forest canopy in "Chinese"

drainage. In 1993 we noted changes in soil

and forest structure that resulted from forest

harvest activity taking place in the Mysovka

watershed.

To estimate the sex ratio and age structure in

a breeding population we marked and

recorded data from all individuals

encountered in a one kilometer stream

transect of "Chinese" brook, divided into 20

sections, during measurements from 1990 to

1993. Between one and three people with

headlamps walked the transect once every

night for a two to three hour observation

period in June 1990, May and June 1991,

September 7-14, 1991, and June 3-13 1993.

We began each transect at a downstream

gauging station where we measured relative

humidity, brook depth and temperature.

Each individual yielded data for the

following categories: sex, maturity class,

biotope, transect section, fat condition, skin

pattern, tail wounds, and morphometric

lengths for snout-vent, tail, head length and

head width. We distinguished adult sex and

reproductive stage by the secondary sexual

characters discussed in Serbinova and

Solkin (1992). We assigned salamanders

unique identification numbers by toe-

clipping, and reclipped regenerating toes

only when we were sure of the original

mark. We designated the color pattern for

individuals according to our categorization

of light-colored spot placement on the neck,

back and tail (Fig. 2). To determine exact

ages for some individual salamanders we

sent a random subset of 118 clipped adult

and larval toe bones to Moscow for

FIG. 3. Timber harvest operations in the Mysovka

floodplain downstream from the study site.

skeletochronological analysis (Smirina,

1972).

To examine stream fidelity we released

20 breeding adults from "Zapovednik"

brook into "Chinese" brook in 1991. We

released 100 salamanders with a range of

secondary sexual characters from

"Zapovednik" brook into "Burned" brook.

In 1992 we released 31 breeding males, 43

breeding females, and 52 non-breeding

adults from "Chinese" brook into

"Zapovednik" brook. We searched for

marked individuals in "Zapovednik" and

"Burned" brooks on nights when we were

not surveying the "Chinese" brook transect.

In 1993 we collected three males and

females that were ready for breeding from

"Chinese" brook. We injected synthetic

leutenizing and releasing hormone into them

to induce courtship, egg-laying and

fertilization. Their aquaria were plastic,

with rocks, moss, and 3 to 5 cm of standing

water. Air and water temperature in the

aquaria were dependent on ambient stream

temperature.

Results

Onychodactylus fischeri spawn in "Mutnii",

"Zapovednik", "Himalayan", and "Chinese"

brooks. "Chinese" brook flows over a

substrate of 1.5 meter deep rock cobble

which is covered by moss and shallow

rooted plant growth. "Zapovednik" brook

has much less rock cobble, with smooth

Vol. 6, p. 56

Asiatic Herpetological Research

June 1995

45

40

35

30 -|

| 25

d 20

15

10

5

Number of individuals caught

>>

■%

E

3

X

u

OS

0

100

95

90

85 -

80 -

75

70

65

60

55

Oi

I T ? ' T

a

40

50

60

70

80

Daily Relative

0

> ■ i

10

-r'r-i — t—

20

12

1 1 - Daily Water Temperature

10 -

a. 9-

E 8

t 7-

S 6-

£ 5-

4 -

3

30 40 50

count day

60

i — i — i — i — i — i — |

70 80

0 10

May

20

30

i— I — i— i— r— i— I—

40 50

60

<— r-

70

80

June

September

FIG. 4. Daily number of salamanders captured, relative humidity and stream temperature in "Chinese" brook

during May, June and September 1991.

bedrock as the substrate for some sections.

In the study area predaceous fish only

occurred in "Burned" and "Tetkin"

drainages, which were both affected by a

forest canopy-removing wildfire 40 years

ago and by ensuing landslides.

Pinus koreansis accounts for a

significant portion of the dominant canopy

cover in the brook drainages where O.

fischeri does occur. Dominant forest trees

in the drainage of "Chinese" brook reflects a

strong slope effect; Manchurian oak

(Quercus mongolica ) and P. koreansis

dominate the southeast-facing slope while P.

koreansis, fir {Abies sp.), birches (Betula

spp.), maples (Acer spp.), elms (Ulmus

spp.), and linden (J ilia spp.) comprised

much of the canopy of the north-facing

slope.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 57

"i Individuals Caught in Water

SO ?

S 60

3 40

20

0

50 60 70 80 90 100

Rel Humiditv

"*u "1 Individuals caught ai water edge

35 -

30

25

§20

5 15

10

5

0

40 50 60 70 80 90

Rel Humidity

0 -i Individuals caught in waterfall splash zone

6 -

2 -

50 60 70 80 90 100

Rel Humiditv

60 . Individuals caught on land

50 -

S40

o 30 -

°20-

40 50 60 70 80 90 100

Rel Humidity

FIG. 5. Histograms divided according to ten-

percent atmospheric relative humidity classes

showing number of salamander captures in water,

water edge, splash zone and land biotopes. Note that

few salamanders were active on land when relative

humidity was below 90%.

A six man work team from the local

forest production ministry harvested timber

downstream in 1993 with one tank- treaded

bulldozer and one rubber-tired loader

servicing a logging truck (Fig. 3). Their

road building often crossed the small river in

the floodplain. The bulldozer skidded logs

down slopes of greater than 35 degrees.

There was severe disturbance to soil

structure caused by these activities. Soils

were brown podbels with an illuvial B

horizons ending less than 50 cm below the

litter surface (Ivanov, 1976). Pinus

koreansis was being harvested from the

river plain and hills despite current

regulations outlawing harvest of that

species. Other harvested species included

Abies sp., Tilia sp. and Populus tremula.

The river channel was widened in 1992,

apparently as a subsequent outcome of

harvest activities.

We first encountered breeding adults at

spawning brooks during snowmelt in late

April and early May. Adult males captured

in surface waters outnumbered adult females

in all seasons. Breeding adult activity was

highest in May and June, when stream

temperatures were low (Serbinova and

Solkin, 1992). In September 1991 the

breeding population rose during a small

peak composed of adults aged 6 to 9 years;

at that time 1 1 out of 20 males and 5 out of 9

females exhibited secondary sexual

characters and juveniles represented 45.3 %

of all captures.

Around spawning brooks we found O.

fischeri in pools and riffles, on dry and wet

rocks in the watercourse, on rocks in the

misty zone near waterfalls, and on the

shore. Salamanders were most active at the

surface during evenings of high relative

humidity (Fig. 4). 535 captures provided

data for biotope location. We found

salamanders most often in the water (54.0 %

of total) and on the stream bank (16.4 % of

total). On nights of low relative humidity

salamanders were often located on rocks in

the misty splash zone near waterfalls (6.0 %

of total). On land salamanders were most

often within 50 cm of the water (11.3 % of

total). Salamanders were rarely active on

land if relative humidity was lower than 90

percent (Fig. 5). Juveniles were active on

land markedly less than adults. In the brook

channel we uncovered 1 gravid female when

we dismantled a 0.5 cubic meter rock matrix

where brook water seeped through moss-

covered rocks. We occasionally observed

O. fischeri on land between streams and on

ridges during times of rainfall and high

relative humidity.

We found abundant larvae in surface

waters during nocturnal transect surveys.

Larvae were also active diurnally. During

two one-hour periods in June 1993 we

observed Onychodactylus fischeri larvae

Vol. 6, p. 58

Asiatic Herpetological Research

June 1995

95% Confidence Bands for Tail to Snout-vent Allometry

Males

Females

50 55 60 65 70 75 80 85 90 95 100

Lsv

FIG. 6. Allometric relationship showing relatively

longer tail size (L cd) in adult males at given snout-

vent length (L sv). The difference in the slopes is

significant (ANCOVA; F = 18.21; df = 1; p <

0.0001).

hunting on flat rocks under 3 cm of water in

the late afternoon. By means of their limbs

the larvae locomoted to approach aquatic

Gammarid casings and lunged when the

invertebrates exposed their legs and head. It

appeared that larvae used their forelimbs in

conjunction with a gape-and-suck feeding

technique to apprehend the prey.

The number of transect captures was an

index of active adult population size,

generally decreasing over the course of the

summer except for a small increase in early

fall. Analysis of our mark-recapture data by

the Jolly-Seber stochastic method (Donnely

and Guyer, 1994) yielded standard errors

too large for individual population estimates

of males, females, and juveniles.

Males had significantly longer tails than

did females once the effect of snout to vent

length was accounted for (ANCOVA; F =

8.99; df = 1; P - 0.003). Also, tail length

increased more rapidly with body size in

males than in females (ANCOVA; F =

18.21; df = 1; p < 0.0001) (Fig. 6). We

observed tail wounds on 23 out of 350

males and 19 out of 166 females. It

appeared that females in "Chinese" brook

had a thinner fat layer than females in

"Mutnii" brook, where the brook substrate

includes more bedrock. According to our

10

! 6-

u

4 -

Distribution of Captured Male Age. 1993

jfh.

!5 5 7.5 It)

15 17 5 20

6 -

i -

Distribution of Captured Female Age. 1991

xp.

, m, , n,

0 2.5 5 7 5 10 12 5 15 17 5 20 22.5

Age

FIG. 7. Histogram showing relative abundance of

ages in a subset of captured males and females.

designation of color pattern 351 individuals

in "Chinese" brook, the dominant patterns

of bright flecking on grey to brown

backgrounds were C (46.7 % of total) and B

(36.4% of total).

Our data on age and development times

support the hypothesis that Onychodactylus

fischeri is a slow developing, long-lived

salamander. Cross sections of larval bones

indicate that larvae may develop up to four

years before metamorphosis to terrestrial

juveniles. The mean age of breeding males

in 1991 was 8.65 years (Std Dev. 2.87);

mean breeding female age was 8.75 years

(Std. Dev. 2.34) . Maximum ages were at

least 16 years for males, and 18 years for

females (Fig. 7). Ages approximated by

counting winter rings may underestimate

true age because of endosteal resorption

(Leclair 1990).

In 1991 at "Zapovednik" brook we

recaptured one of the 20 salamanders that

we had taken from there for release into

"Chinese" brook; we recaptured none of

these in "Chinese" brook. In 1991 we

recaptured none of the 100 salamanders

which we had released into "Burned" brook

from "Zapovednik" brook. We also

June 1995

Asiatic Herpetological Research

Vol. 6, p. 59

FIG. 8. Onychodactylus fischeri female lays eggs

in two gelatinous sacs.

recaptured none of these in their original

stream. In 1992 at "Chinese" brook we

recaptured 8 of 31 breeding males, 15 of 43

breeding females and none of the 52 non-

breeding adults out of the total 126

salamanders which we moved in 1991 from

"Chinese" brook to "Zapovednik" brook.

The breeding adults which we caught had

returned within a month to "Chinese" brook

over 1 km of land or by a longer route along

the Mysovka river.

Hormone injection in captives

successfully induced courtship, egg-laying

(Fig. 8) and fertilization (Fig. 9). One

female produced a clutch of two egg sacs in

1993. Eggs were 6 to 8 mm in diameter,

with 5-10 eggs in each sac. We observed

deflation of female cloacal labia in the six

days after egg-laying. Egg death probably

resulted from a severe rise in pH of available

water.

Discussion

Onychodactylus fischeri is limited by the

availability of suitable torrential streams for

FIG. 9. Onychodactylus fischeri male grasps and

fertilizes egg sacs with hind limbs.

reproduction and development. The

seasonal activity pattern which we observed

in adults seems limited by water

temperatures over 10° C. The burst of

breeding activity observable in late spring

may signify the most strategic time for egg-

laying in view of the short warm season in

the Russian Far East; it is logical that eggs

laid in the late spring develop faster than

eggs laid in early fall because water

temperatures drop below 5° C in the winter.

The higher proportion of males caught in

our surveys suggests that males may wait on

the surface rocks and waters to seek

females. Egg sacs and larvae seem to be

located most often in intersticial rock habitat,

sheltered from surface predators in areas of

continuous water flow (Regel and Epshtein,

1975). Iwasawa and Kira observed that O.

japonicus eggs developed slowly for 143

days in water of 10° C (1980). Longer

winters may cause the larval stage in O.

fischeri occasionally to extend longer than

the three years observed in O. japonicus

(Hayase and Yamane 1982).

Lunglessness is a synapomorphy of

salamanders in the Onychodactylus clade,

which have a long larval period in torrential

streams as a requisite portion of their

ontology. Bruce et al. (1992) supported the

hypothesis that lunglessness in amphibians

is a larval adaptation to torrential streams by

showing that experimentally lungless

salamander larvae were displaced

Vol. 6, p. 60

Asiatic Herpetological Research

June 1995

downstream less when released in fast-

flowing water than were control larvae with

lungs.

The reproductive potential of an

individual Onychodactylus fischeri seems

low, despite their long lifespan, considering

that a female living 12 years may only

produce 140 eggs. Additionally, many of

the adults we found did not have secondary

sexual traits indicative of reproduction.

Akita (1989) notes that not all adult O.

japonicus breed every year. The low clutch

size and large egg mass suggest that each

egg is a significant reproductive investment

relative to other hynobiids. Kuzmin showed

that the sympatric hynobiid Salamandrella

keyserlingii is more general in its feeding

and breeding habitat preferences (1990).

Our 1992 experiments suggest that

successful homing was only by adults

which were ready for breeding, indicating

that adults may have a preference for

particular spawning brooks. It is also

possible that non-breeding adults may also

have returned to their original brooks and

not been recaptured, or that the breeding

adults that did not return may have spawned

in a different stream. Onychodactylus

fischeri may migrate over land through

forested corridors. We speculate that the

connectedness of suitable habitat between

large watersheds is important for dispersal

after landscape level disturbances.

The pressure to harvest trees in the

fragile taiga is promoted by an international

wood and fiber market that devalues natural

capital by not factoring in negative

externalities such as anthropogenic fire,

landslides and species loss. Repeated

highgrading is the most widespread harvest

method in the Russian Far East and has

lasting effects on forest ecology. Forested

slopes and floodplains are exposed to

catastrophic erosion for years after tractors

are used to skid out widely spaced trees.

Reduction of canopy cover to levels to

below 40% alters the evapotranspiration

regime in a way which allows catastrophic

wildfire. The hard currency that goes to the

Primorskii krai in return for the Ussuri taiga

logs does not compensate for the permanent

loss of seasonal employment in fur,

mushroom, honey, berry, ginseng, and

game meat gathering and preparation

(Schumacher, 1973). The best conservation

strategy for Onychodactylus fischeri appears

to be exclusion of high-impact timber

harvest activity from key spawning

watersheds, given that regional timber

harvesters, Hyundai and other multinational

timber interests generally ignore existing

rules of the Russian Federation's Ministry

of Forests. It would be informative to

delineate metapopulation boundaries in order

to assess the adequacy of existing preserves.

Onychodactylus fischeri is an indicator

species of high-quality old growth forest

habitat in upper slopes of Ussuri taiga.

Because eggs and larvae develop in cold

running water year round under the cover of

rocks, undisturbed stream substrate and a

specific hydrological regime are critical for

quality spawning habitat. Long-term

stability of stream hydrology, which is

needed to support O. fischeri breeding and

development, is dependent on undisturbed

complex forest structure. Shade and large

woody debris affect stream temperature and

riffle-pool ratio. Extensive root systems

retain soil structure and decaying logs on the

forest floor regulate soil moisture. The deep

roots of conifers provide more soil stability

on slopes than shallow-rooted deciduous

trees. Acid soils locally associated with

Pinus koreansis litter may regulate stream

pH and nutrient availability. We observed

that catastrophic disturbance to spawning

brooks occurred through natural processes

of wildfire and landslides and through

anthropogenic road building and logging in

the watercourse, upslope, and downstream.

Tree harvest on slopes above watercourses

causes stream sedimentation of fine

materials and organics. Such increased

sediment loads associated with logging have

negative effects on aquatic amphibians

(Corn and Bury, 1989). As is the case on

"Burned" creek, siltation eliminates the

spaces between rocks which are critical for

egg deposition and larval development.

Disturbed brooks may be suitable to O.

fischeri spawning only after adequate forest

structure regenerates and interstitial spaces

filled with silt and sand are cleared.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 61

Acknowledgments

The authors would like to thank

Vladimir Aramilev for his invaluable

assistance in Vladivostok and in the field;

Dmitri Pikunov for support from the

Laboratory of Wild Animals of the Pacific

Institute of Geography; Irina Olenicheva for

her assistance with the skeletochronological

work; and David Wake and Mario Garcia-

Paris for their constructive criticism of the

manuscript.

Literature Cited

AKITA, Y. 1989. Breeding Cycle of

Onychodactylus japonicus on Mt. Hodatsu, with

Special Reference to Biannual Spawning. P. 305.

In M. Matsui, T. Hikida and R. C. Goris (Eds),

Current Herpetology in East Asia. Herpetological

Society of Japan, Kyoto.

BRUCE, R. C, C. K. BEACHY, P. G. LENZO, S. P.

PRONYCH AND R. J. WASSERZUG. 1994. Effects

of Lung Reduction on Rheotactic Performance in

Amphibian Larvae. The Journal of Experimental

Zoology 268:377-380.

CORN, P. S. AND R. B. BURY. 1989. Logging in

Western Oregon: Responses of Headwater Habitats

and Stream Amphibians. Forest Ecology and

Management 29:39-57.

DONNELLEY, M. A. AND C. GUYER. 1994.

Estimating Population Size: Mark Recapture. In

Heyer, W.R. et al. (Eds.), Measuring and

Monitoring Biological Diversity: Standard Methods

for Amphibians. Smithsonion Institution Press,

Washington and London.

FULLARD, H. (ED.). 1972. Soviet Union In Maps.

George Philip and Son, Ltd., London.

HAYASE, N. AND S. YAMANE. 1982. [Life

History During the Aquatic Life Period of a

Salamander, Onychodactylus japonicus (Houttuyn),

at Mts. Tsukuba, Ibaraki, Japan]. Japanese Journal

of Ecology 32(3):395^03. (In Japanese).

IVANOV, G. I. 1976.

south of the Far East],

pages. (In Russian).

[Soil Formations in the

Moscow. Science. 200

IWASAWA, I. AND Y. KIRA. 1980. [Normal

Stages of Development of he Japanese Lungless

Salamander, Onychodactylus japonicus (Houttuyn)].

Japanese Journal of Herpetology 8(3):73-89. (In

Japanese).

KUZMIN, S. L. 1990. [Feeding of Sympatric

Species of Hynobiidae in the Primorye]. Zoological

Journal 69(5):7 1-75. (In Russian).

LECLAIR, R. 1990. Relationships between relative

mass of the skeleton, endosteal resorption, habitat

and precision of age determination in ranid

Amphibians. Annales des Sciences Naturelles

Zoologie et Biologie Animale 1 1:205-208.

REGEL, E. D., AND S. M. EPSHTEIN. 1975.

[Some Patterns of Biology in Onychodayctulus

fischeri]. Zoological Journal 63(7): 1515-1524. (In

Russian).

SCHUMACHER, E. F. 1975. Small is Beautiful:

Economics as if People Mattered. Harper and Row.

New York. 305 pages.

SERBINOVA, I. A. AND SOLKIN V. A. 1992.

Reproduction of the Ussuri Lungless Triton

(Onychodactylus fischeri)]. Zoological Journal

71(7):68-74. (In Russian).

SMIRINA, E. M. 1972. [Annual Layers in bones

of R ana temporaria}. Zoological Journal 51:1529-

1534. (In Russian).

I June 1995'

Asiatic Herpetological Research

Vol. 6, pp. 62-68

Studies on the Distribution of Trace Elements in

Agkistrodon blomhoffii brevicaudus Stejneger

JIN LIN1, KE-MIN XU> AND DONG-GEN LIU2

1 Department of Biology, Liaoning Normal University, Dalian, Liaoning, 116022 China

^Kunshan Snake Museum, Suzhou, Jiangsu, 215334 China

Abstract: -The distribution of trace elements in various organs and tissues of Agkistrodon blomhoffii

brevicaudus Stejneger was studied by ICP-AES. The results show that there are more than 17 kinds of trace

elements in the snake, namely, Cu, Zn, Fe, Mn, Se, Sr, Al, Si, Al, Ni, Ba, Co, Pb, Ge, Cd, Mo, La, etc.

The amount of each kind of trace element is quite different in the various organs and tissues. The contents of

Zn, Fe, Al and Cr have the highest value; Cu and Mn have the medium value; Se, Sr, Si, Ba, Pb, Ge and Mo

have the lower value; and Ni, Cd, Co and La have the lowest value. The results suggested that the

distributional characteristics of trace elements in Agkistrodon blomhoffii brevicaudus Stejneger correspond

with the snake's physiological and biochemical functions.

Key words: Reptilia, Serpentes, Agkistrodon blomhoffii brevicaudus Stejneger, China, trace elements.

Introduction

According to recent literature, there are

four species which belong to the genus

Agkistrodon in northeast China. They are

A. blomhoffii brevicaudus Stejneger, A.

saxatilis Emelianov, A. shedaoensis Zhao,

and A. ussuriensis Emelianov (Zhao and

Adler, 1993). Xii et al. (1993) reported the

distribution of trace elements in A .

shedaoensis Zhao and A. ussuriensis

Emelianov and the results showed that there

existed interspecific differences. This paper

reports the quality and quantity observations

of trace elements in the various organs and

tissues of A. b. brevicaudus Stejneger. It

will provide experimental data for

comparative physiology and contribute also

to the classification of pit- vipers.

Materials and Methods

Four specimens of Agkistrodon

blomhoffii brevicaudus were provided by

Kunshan Snake Museum at Kunshan

County, Jiangsu Province, China in

December, 1993. The snakes were

decapitated and various organs and tissues

were freshly removed. All samples, except

serum, plasma and bile, were stored in an

incubator at 105°C for 5 hrs., cooled down

and weighted after incubation. Then, the

samples were immersed in mixed acid

(HN03 : HC1=4 : 1) for 24 hrs, then heated

continuously until clear and transparent, and

transferred into a 10 ml graduated tube by

adding double distilled water.

The trace elements of all samples were

measured through ICP-AES (Leeman Co.,

USA). The data was analyzed with a micro-

computer, then they were randomly

arranged as a trace elements graph.

Results

The results show that there are more

than 17 kinds of trace elements, such as Cu,

Zn, Fe, Mn, Se, Sr, Al, Si, Ni, Ge, Cd,

Ba, Mo, Co, Pb, Cr and La in various

organs and tissues of A. b. brevicaudus.

The contents of each kind of trace elements

in the various organs and tissues are as

follows:

Skin, Muscle and Skeleton.

The trace element graphs of the skin,

muscle and skeleton are very similar, but

they still have their own characteristics. The

contents of Zn, Fe and Al are higher in skin,

the order is Al > Fe > Zn, and the Al content

in skin is the highest among all organs and

tissues. The contents of Cr, Mn, Cu, Pb

and Ba have a medium value, and Se, Sr,

Ni, Ge, Cd, Mo and La have a lower value

in the skin. The trace elements showed

different contents in the different segments

of the skin (Figs, la, lb, and lc).

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol. 6, p. 63

The skeletal muscle has more Fe and Zn

and less Al, compared with those of the

skin. Other elements have no more

differences. Ge can not be tested out (Fig.

2).

There are more Zn, Sr and Ba and less

Fe, Al and Cr found in the skeleton than in

the skin and skeletal muscle. The skeleton

has more Sr than in other organs and

tissues. But, Ge and La can not be tested

out in the vertebrae (Figs. 3 and 4).

Cardio-Vessel System, Lung and Trachea.

The similarity of elements distribution in

the cardio-vessel system, lung and trachea is

the presence of more Zn, Fe, Al and Cr,

then Cu and Mn, and less Ni, Ge, Cd, Mo,

Co, and La (Figs. 5-10).

There are more contents of Cu, Zn, Fe

and Al found in the heart, vessel and lung

than in the blood and trachea. But, the

contents of Ge and Se are the opposite. The

Cu content in the heart is ahead of the other

organs, and La and Ge can not be tested out

in the lung and trachea.

It reveals that most trace elements are

found in bile, e.g., Cu, Mn, Se, Sr, Al, Ge,

Ba, Mo, Pb, Cr, especially Cu, Ba, Mo,

Pb, and Cr are much more than those in the

serum. The Cr content is the highest among

all samples. The La and Cd can not be

tested out in the serum (Figs. 6 and 10).

Digestive Tract, Liver, and Pancreas.

The contents of Zn, Fe, Al, Mo and Cr

found in the digestive tract and mesentery

have a medium value, then the Cu, Mn, Se,

Sr, Ba, and the other elements are very

small (Figs. 16-21). The Al content is

obviously less than that of the skin.

The liver, spleen and gall-bladder have

similar contents of trace elements. They

contain considerable amounts of Zn and Fe.

The other elements, like Se, Sr, Si and Ge,

are more than those of the digestive tract,

mesentery and other organs. There are no

difference of Cu, Mn, Se, Ni, Ba, Co, Pb

and La among them (Figs. 11-15).

Besides, the contents of Fe and Mo in

the liver and Zn in the pancreas are the

highest among all the samples. It suggests

that the liver is the important place to store

Fe.

Kidney and Reproductive Organs.

There is a large amount of Mn, Se, Ba

and Cr in these organs, but very little of Si,

Ni, Cd, Mo and Co (Figs. 22-25).

Furthermore, there are more Zn, Fe, Al, Cu

and Pb in the oviduct, Fallopian tube and

uterus than in the kidney.

The Al content of the kidney is similar to

that of the digestive tract, but clearly less

than that of the skin.

Brain, Spinal Cord and Poisonous Gland.

The graph of the brain resembles that of

the spinal cord; both contain abundant

elements in the order of Zn > Fe > Cr > Cu

> Al > Mn > Se > Sr > Ni > Ba. The Fe

content of the brain is just lower than that of

the liver, but the Ge and La in the brain can

not be tested out (Figs. 26 and 27).

The Zn content in the poisonous gland is

higher than that of the brain and spinal cord,

and the La content is the highest among all

samples. Se, Ge and Mo can not be

examined (Fig. 28).

Discussion

There are 17 kinds of trace elements

found in Agkistrodon blomhoffii

brevicaudus through ICP-AES. The

contents of each element are quite different

in the various organs and tissues. The

results showed that the trace elements are the

important parts of life substances in the

snake. And, the distribution characteristics

of trace elements correspond with the

animal's physiological and biochemical

functions.

There are abundant amounts of Zn, Fe,

Al and Cr, then Cu, Mn, Se and Sr found in

the organs and tissues, especially in the

cardio-vessel system, lung, liver, spleen,

brain, oviduct, etc. It suggests that these

Vol. 6, p. 64

Asiatic Herpetological Research

June 1995

ng'g

200-

150-

100

50

0

Fig. la Skin of Neck

Fig. lb Skin of Thorax

Hgg

200-

L50-

100-

50-

0

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La ( u Zn Fc Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr U

Hg g Fig. lc. Skin of Abdomen ug g Fig. 2 Skeletal Muscle

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr lj& ( u Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr I .a

Fig 4 Rib

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Fig. 6. Serum

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr I.a Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr I.a

June 1995

Asiatic Herpetological Research

Vol. 6, p. 65

Hg 8

200-

150

100

50 H

0

Pig 7. Plasma

Fig. 8. Vessel

u n " * \

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr la Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Fig. 9. Lung u.g g Fig. 10 Trachea

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr la Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Fig 11 Spleen ug g Fig. 12 Bile

( u Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo ( "o Pb Cr la

Cu Zn Fe Mn Se Sr Al Si Ni (}c Cd Ba Mo Co Pb Cr La

Fig 14 Gall Bladder

( 'u Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo ( o Pb Cr La Cu Zn Fe Mn Se Sr Al Si Ni Cie Cd Ba Mo Co Pb Cr La

Vol. 6, p. 66

Asiatic Herpetological Research

June 1995

(2058) Fig 15 Liver

Fig. 16. Oesophagus

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Or La Cu Zn Fe Mn Se Sr Al Si N*i Ge Cd Ba Mo Co Pb Cr la

Fig. 17. Stomach

Fig. 18. Duodenum

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Hg g Fig. 19. Ileum ng g Fig. 20. 1-arge Intestine

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr la

Fig. 21 Mesentery

Hgg

Fig 22. Iterus

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

June 1995

Asiatic Herpetological Research

Vol. 6, p. 67

Fig 24 Oviduct

( 'u Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Hg g

200-

150-

100

50 H

0

Fig. 25. Kidney

Fig 26 Spinal Cord

^\

( u Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr \ja Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Fig 28. Poisonous Gland

( u Zn Fc Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr U Cu Zn Fe Mn Se Sr Al Si Ni Ge Cd Ba Mo Co Pb Cr La

Vol. 6, p. 68

Asiatic Herpetological Research

June 1995

elements have a close relationship to the

growth and development, the metabolic

process, the enzyme synthesis and

reproduction. But, how the trace elements

take part in the regulation of physiological

and biochemical functions and which is the

essential or non-essential element needs to

be further studied.

There contains much more Al (just less

than Zn and Fe) in most organs and tissues,

especially in the skin; and the Al contents in

the heart, lung, spleen etc. are higher than

those in the kidney and digestive tract. It

show that the skin can collect Al. The

authors thought that the skin of A. b.

brevicaudus may be the place to store and

excrete Al. It is known that Al is harmful to

humans and mammals, but what is the

reason for the high Al content in the body of

the snake is still a question to be solved.

The characteristics of element graphs in

bile is the balanced content of most

elements, and there contain more Cu, Cr,

Ge, Pb, Sr, Cd and Mo than those in other

the organs and tissues. The ratio of Cu/Zn

(2.00) is the highest among the organs. The

results may be due to the concentrating

mechanism of bile.

Literature Cited

XU, K. M., Y. K. ZHAO, J. LIN, AND J. L. LI.

1993. Studies on the interspecific differences in the

distribution of trace elements in various organs

between Agkistrodon shedaoensis Zhao and

Agkistrodon saxatilis Emelianov. Pp. 68-74. In

Proceedings of the First Asian Herpetological

Meeting (eds. Zhao, E. M., B. H. Chen, and T. J.

Papenfuss). China Forestry Press, Beijing. [In

Chinese].

ZHAO, E. M. AND K. ADLER. 1993. Herpetology

of China. SSAR in cooperation with CSSAR,

Contributions to Herpetology 10. 522 pp.

In addition to morphological differences,

there are also changes in metabolic activity

between species due to the divergence of the

genetic constitution, ecological surroundings

and living habits. So, the authors suggest

that trace element detecting is a way to help

classify the pit-vipers.

I June 1995

Asiatic Hcrpctological Research

Vol. 6, pp. 69-72

A Study on Morphological Similarity between the Genera Nanorana and

Altirana (Amphibia, Anura, Ranidae)

Shun-qing Lu and da-tong Yang

Kunming Institute of Zoology, Accidentia Sinica, Kunming, China

Abstract. -Through Wilk's stepwise discriminant analysis, 16 of 18 indices of Nanorana ventripunctata, N.

pleskei, and Altirana parkeri were selected and used in a numerical taxonomy study with their weights given as

the following formula: W=Cxl/U. The result of clustering analysis of the Euclidean Distances between the

three species reveals that N. ventripunctata is more similar to A. parkeri than to N. pleskei in morphology.

Key words: Amphibia, Anura, Ranidae, Nanorana, Altirana, China, Transhimalaya Mountains, stepwise

discriminant analysis, numerical taxonomy.

TABLE 1 . Number, locality, and altitude of species used.

Species Groups Number

N. ventripunctata 1 10 M, 10 F

N. pleskei 2 10 M, 10 F

A. parkeri 3 10 M, 10 F

Zhongdian, Yunnan

Kangding, Sichuan

Bashu, Xizang

Altitude

3350 m

3260 m

4100m

Introduction

Nanorana ventripunctata, N. pleskei,

and Altirana parkeri are three species of

frogs in two genera that are distributed in the

Transhimalaya Mountains of China. Except

for morphological identification and

chromosome research, we know of no other

studies on these frogs that has been

published.

Nanorana and Altirana have a close

relationship (Su et al, 1985: Hu et al,

1986), and some distinguishing characters

between them are vague since the discovery

of N. ventripunctata (Fei and Huang, 1985).

It is necessary to reexamine the two genera.

In this paper, based on 18 external

morphological indices, the authors use

stepwise discriminant analysis and

numerical taxonomy to compare the three

species.

Materials and Methods

The number, locality, and altitude of the

specimens used are shown in Table 1.

Nineteen external morphological

characters were measured from each

specimen, and changed to eighteen ratios,

i.e. 18 indices: HEL (head length)/SVL

(snout vent length), HEW (head

width)/SVL, SNL (snout length)/HEW,

BND (distance between noses)/HEL, BED

(distances between eyes)/HEL, ELW (eyelid

width)/HEL, EYD (eye diameter)/HEL,

FED (distance between front angles of

eyes)/HEL, AHL (hand and front arm

length)/SVL, ARW (front arm width )/HEL,

HAL (hand length)/SVL, SVL/LFL (leg

length, TIL (tibia length)/HEL, TIW (tibia

width)/HEL, TFL (tarsalia and foot

length)ATSVL, FOL (foot length)/SVL, NSL

(length from nose to the top of snout)/HEL,

and SPN (snout process length)/HEL.

Results

After stepwise discriminant analysis,

sixteen of the 1 8 indices were selected, their

Wilk's statistic measure U (from the first

step of the stepwise discriminant analysis)

are shown in Table 2.

The weights of the 16 indices are given

by the following formula:

W=Cxl

U

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 70

Asiatic Herpetological Research

June 1995

TABLE 2. Selected indices and their U after discriminant analysis.

Indices

FED

HAL

HEL

HEW

SVL

0.7937

EYD

HEL

0.9677

TABLE 3. The weights of the 16 indices.

HAL

SVL

0.2381

TFL

SVL

0.1724

HEL

SVL

0.1332

BED

HEL

0.2986

HEW

SVL

0.1260

EYD

HEL

0.1033

SPN

HEL

0.1807

AHL

SVL

0.1987

TABLE 4. The matrix of weighted measures of the 16 indices.

TABLE 5. The Euclidean Distances among the three species. Unit: 10"^

1

2

3

Natwrana ventripunctata

0

0.8868

0.1707

Nanorana pleskei

0.8868

0

0.8787

Altirana parkeri

0.1707

0.8787

0

Here C is a coefficient used to regulate

the size of weight, which is given according

to the condition, and U is the Wilk's statistic

measure. Its calculating formula is: U =

IWI/TI = IWI/(IWI + IBI), where W is the

variance in group, B is the variance between

groups, T is the total variance. This formula

reveals that the smaller the U, the more

important the index. So the weighting

formula used in this paper is agreeable with

the weighting principles. In addition, it has

some merits when compared to other

weighting formulas used in the literature: 1)

No negative weights appear; 2) As a

measure of the importance of characters, U

has been accepted commonly, and as a

measure of weights, it may be accepted

easily; 3) Convenient for calculation.

The calculated weights of the 16 indices

are shown in Table 3.

Multiplying the measures of the 16

indices with their weights, a numerical

matrix is given as shown in Table 4.

June 1995

Asiatic Herpetological Research

Vol.6, p. 71

1.00 0.8 0.6 0.4 0.2 0.0

FIG. 1. The UPG MA phonogram of the three species based on Table 5.

. A. parked

N. ventripunctata

_ N. pleskei

Table 6. Some identification characters between the three species.

Nanorana pleskei

tympanum under skin, but visible;

columella exists

nasals separate, not connected with

fTontal-parietal

precoracoid ossified incompletely

clavicle short, not attach epiconicoid

the first low labial teeth of tadpole

shorter than the second obviously

N. ventripunctata and Altirana parked

tympanum and columella absent

nasals connected with each other and connected

with frontal-parietal

precoracoid ossified completely

clavicle long, attach epiconicoid

the first low labial teeth of tadpole slightly shorter

than the second

The Euclidean Distance is selected in this

paper to measure the morphological

differences between the three frogs. The

formula is: Dij= Vx(Xik-Xjk)2. The

calculated Euclidean distances among the

three frogs are shown in Table 5.

Figure 1 detects that the distance

between N. ventripunctata and A . parked is

the shortest. The two frogs meet together at

the distance 0.1703, then they meet with N.

pleskei at the distance of 0.8827. The

morphological similarity of N .

ventripunctata and A parked is closer than

that of N. ventripunctata and N. pleskei.

Discussion

Up to the present, the differences

between the genera Nanorana and Altirana

reported on by Tian and Jiang (1986)

contained the most details. But the genus

Nanorana as they meant, did not contain N.

ventripunctata, so it was just the differences

between N. pleskei and A. parked that they

noted.

The characters of N. ventripunctata

show that this species is more similar to the

genus Altirana than to the genus Nanorana

as shown in Table 6. The numerical

taxonomy research of this paper and the

biochemical systematic study (Lu and Yang,

1994) show the same results. It seems

logical to take ventripunctata out of genus

Nanorana and place it in genus Altirana, but

the biochemical systematic study reveals that

the Nei's (1972) genetic distances between

the three frogs are 0.30, 0.57, 0.57,

respectively, smaller than 1.05 obviously,

but larger than 0.15. We feel that these

differences are at the species level, not the

generic level (Thorpe, 1983). Thinking of

the principle: in order to avoid more

monogenera, the interruption of a genus

with other genera should be anti-relative

Vol. 6, p. 72

Asiatic Herpetological Research

June 1995

with the size of the genus, i.e. the number

of species contained in this genus, the

authors suggest that the genus Altirana be

cancelled and the species parkeri be placed

in the genus Nanorana..

Acknowledgments

We are grateful to Mr. Dingqi Rao for

collecting specimens together with one of us

(Lu). Thanks also due to Mr. Ruliang Pan

and Fahong Yu for offering us the computer

program for stepwise discriminant analysis.

Literature Cited

FEI, L. AND Y. Z. HUANG. 1985. A new species

of the genus Nanorana (Amphibia Ranidae) from

northwestern Yunnan, China. Acta Biologica

Plateau Sinica 1985(4):71-75. (In Chinese).

HU, Q. X., Y. M. JIANG, AND E. M. ZHAO. 1985.

Studies on the influence of the Hcngduan Mountains

on the evolution of the amphibians. Acta

Herpetologica Sinica 1985, 4(3):225-233. (In

Chinese).

LU, S. Q. AND D. T. YANG. 1994. A study of

relationships among ranid frogs of the genera

Nanorana and Altirana in the Transhimalaya

Mountains of China. Asiatic Herpetological

Research. (In Press).

SU, C. Y., D. T. YANG, AND S. Q. LI. 1986.

Studies on vertical distribution of Amphibians in

the middle section of the Hengduan Mountains.

Acta Herpetologica Sinica 1986, 5(2):134-144. (In

Chinese).

THORPE, J. P. 1983. Enzyme variation, genetic

distance and evolutionary divergence in relation to

levels of taxonomic separation. Pp. 131-152. In G.

S. Oxford and D. Rollonson (Eds.), Protein

polymorphism: Adaptive and taxonomic

significance. Academic Press, London.

TIAN, W. S. AND Y. M. JIANG (EDITORS),

ASSISTED BY G. F. WU, Q. X. HU, E. M. ZHAO,

AND Q. Y. HUANG. 1986. Identification manual

for Chinese species of amphibians and reptiles.

Science Press, Beijing. 164 pp. (In Chinese).

I June 1995'

Asiatic Herpetological Research

Vol. 6, pp. 73-77

A Study of Relationships among Ranid Frogs of the Genera Nanorana and

Altirana in the Transhimalaya Mountains of China

Shun-qing Lu and Da-tong Yang

Department of Vertebrate Zoology, Kunming Institute of Zoology, Academia Sinica,

Kunming, Yunnan, China

Abstract. -Nanorana ventripunctata,N. pleskei and Altirana parkeri were examined electrophoretically to

investigate the intraspecific genetic relationships. Twelve isozyme loci were assayed and their allele

frequencies were calculated. The result of UPGMA clustering, when corrected by the Present-Day Ancestor

Method and based on the allele frequencies, detected that the genetic relationship between N. ventripunctata and

A. parkeri is closer than that between N. ventripunctata and N. pleskei. The authors suggest that the genus

Altirana should be canceled and that A. parkeri be placed in the genus Nanorana.

Key words:

Mountains.

Anura, Ranidae, Nanorana, Altirana, genetic relationships, isozyme, China, Transhimalaya

TABLE 1 . The location, altitude, date and number of specimens collected.

Introduction

Materials and Methods

The genera Nanorana and Altirana

include three species, which are distributed

in the Transhimalaya Mountains of China.

They are considered to be closely related,

and some identification characters between

the two genera have been vague since the

description of N. ventripunctata (Fei and

Huang, 1985). Except for some

morphological identification and

chromosome studies, there are no other

studies published on these genera. In order

to re-study the two genera, the authors here

use starch gel electrophoresis on extracts

from liver and muscle to determine genetic

distances in order to better understand the

genetic relationships among the three species

of frogs.

For comparison, the species of Rana

shuchinae and R. chensinensis were selected

as out-groups. Part of the distribution of

these two frogs is the same as Nanorana and

Altirana.

The collecting locality, altitude, date and

number of living specimens of the five

species are shown as in Table 1 .

The specimens were killed in the field,

the liver and thigh muscle of each specimen

were taken and placed in 1.5 ml plastic

micro centrifuge tubes with several drops of

physiological saline, then preserved in liquid

nitrogen, and taken back to the laboratory.

Tissues were washed with distilled

water and physiological saline, the volume

ratio is tissue: physiological saline = 1:1.5,

homogenized and centrifuged. These

samples were run in horizontal starch gels

using gel buffers described by Pasteur et al

(1988) as follows: Tris-Borate-EDTA (pH

8.6), Tris-Citrate (pH 6.7). Isozyme stains

used were also described by Pasteur et al

(1988). The following enzyme systems

were stained: alcohol-dehydrogenase

© 1995 by Asiatic Herpetological Research

ADH-2

GLC-1

MDH-1

a

b

c

d

e

1.0000

0.4333

0.3667

1.0000

0.5000

0.5000

1.0000

0.0333

0.2667

0.3000

0.3333

0.0667

0.0667

0.6000

0.1333

0.1000

0.1000

0.5000

0.5000

0.1667

0.8333

0.3750

0.3333

0.2083

0.0833

0.3125

0.3125

0.3750

MDH-2

a

b

c

d

e

0.0769

0.8077

0.1154

0.2333

0.4333

0.3333

0.3636

0.0808

0.5000

0.0808

0.0808

0.5000

0.5000

0.5000

0.1250

0.3333

0.0417

June 1995

Asiatic Herpetological Research

Vol. 6, p. 75

TABLE 4. The corrected distances with R. shuchinae as the present-day ancestor.

N. ventripunctata

N. pleskei

A. parkeri

N. ventripunctata

N. pleskei

-1.4498

A. parkeri

-2.0496

-1.4589

. N. ventripunctata

A. parkeri

_ N. pleskei

0 -1.00 -2.00

FIG. 1 . The UPGMA phenogram of corrected distances with R. shuchinae as the present-day ancestor.

(ADH, Ec 1.1.1.1). esterase (EST, Ec

3.1.1.1), NAD-glucose-dehydrogenase

(GLC, Ec 1.1.1.1), lactate-dehydrogenase

(LDH, Ec 1.1.1.27), malate-dehydrogenase

(MDH, Ec 1.1.1.37) malic enzyme (MOD,

Ec 1.1.1.40), sorbitol-dehydrogenase

(SDH, Ec 1.1.1.14).

Results

Twelve isozyme loci were resolved and

scored. Their allele frequencies are shown

as in Table 2.

From Table 2, the Nei's (1972) genetic

distances and similarities among the five

species was calculated and is shown in

Table 3.

It is obvious from Table 3 that the

evolutionary rates of the five species are

unequal, so it is necessary to make a

correction before UPGMA clustering. The

Present-Day Ancestor Method (Li, 1987)

was selected in this paper, the correcting

formula is: D'ij=Dij.DjX) here D'ij is the

corrected distance, the Dij is the original

distance, x represents the supposed present-

day ancestor. At first, with R. shuchinae

from the out-group selected as the present-

day ancestor, the corrected distances among

the three frogs in the genera Nanorana and

Altirana are are shown in Table 4.

The UPGMA clustering phenogram of

the corrected distances among the three

species in Table 4 is shown in Figure 1 .

When R. chensinensis is selected as the

present-day ancestor, the corrected distances

are shown in Table 5.

Vol. 6, p. 76

Asiatic Herpetological Research

June 1995

TABLE 5. The corrected distances with R. chensinensis as the present-day ancestor.

N. ventripunctata

N. pleskei

A. parked

N. ventripunctata

N. pleskei

-1.4332

A. parked

-1.6854

-1.1975

_ N. ventripunctata

.A. parked

N. pleskei

0 -1.00 -2.00

FIG. 2. The UPGMA phenogram of corrected distances with R. chensinensis as the present-day ancestor.

FIG. 3. The genealogical phenogram among the three species.

N. ventripunctata

A. parked

N. pleskei

Based on Table 5, we prepared a

UPGMA phenogram which is shown in

Figure 2.

Not minding the difference of distances

among the frogs, we find that they are

similar in both Figure 1 and Figure 2,

though they are based on the different

present-day ancestors, so we synthesized

and simplified them as shown as in Figure

3.

Figure 3 shows that the genetic

relationship between N. ventripunctata and

A. parkeri is closer than that between N .

ventripunctata and N. pleskei.

Discussion

Up to the present, the differences

between Nanorana and Altirana described by

Tian and Jiang (1986) are the most detailed,

but the genus Nanorana of their meaning

does not contain N. ventripunctata. It is just

/V. ventripunctata that confuses the

distinction between the two genera, and the

study of morphological similarities among

the three species of the two genera shows

the same result that N. ventripunctata and A.

parkeri are more similar than N .

ventripunctata and A', pleskei (Lu and Yang,

1994). Both of the results of biochemical

systematics and morphological similarity

studies do not support the presently

recognized generic assignments and we

suggest that N. ventripunctata should be

taken out of the genus Nanorana and placed

in the genus Altirana.

From Table 3, we know Nei's (1972)

genetic distances among the three species are

0.5709, 0.5693 and 0.2979. This is larger

than 0.15, but much smaller than 1.05.

These differences are at the species level,

June 1995

Asiatic Herpetological Research

Vol. 6, p. 77

but not the generic level (Thorpe, 1983.

Also, we know that there is a principle: in

order to avoid more mongenera, the

interruption of a genus with other genera

should be anti-relative with the number of

species in this genus. Thinking of these and

the vague line between Nanorana and

Altirana, according to the principle of

priority of the International Code of

Zoological Nomenclature, the authors

suggest that it is perfect to cancel the genus

Altirana, and that the species parkeri should

be placed in the genus Nanorana.

Acknowledgments

We thank Mr. Dingqien Rao for

collecting specimens with one of us (Lu)

and his help in the laboratory. Thanks also

are given to Professor Jingyan Li for his

suggestions concerning the Present-Day

Ancestor Method.

Literature Cited

FEI, L. AND Y. Z. HUANG. 1985. A new species

of the genus Nanorana (Amphibia Ranidae) from

northwestern Yunnan, China. Acta Biologica

Plateau Sinica 1985(4):71-75. (In Chinese).

HU, Q. X, Y. M. JIANG, AND E. M. ZHAO. 1985.

Studies on the influence of the Hcngduan Mountains

on the evolution of the amphibians. Acta

Herpetologica Sinica 1985, 4(3):225-233. (In

Chinese).

LI, J. Y. 1987. The essence of the Present-Day

Ancestor Method for constructing evolutionary trees

from sequence data and the puzzle encountered in

applying this method. Zoological Research

9(2): 141-150. (In Chinese).

LU, S. Q. AND D. T. YANG. 1994. A study on

morphological similarity between the genera

Nanorana and Atltirana (Amphibia, Anura, Ranidae).

Asiatic Herpetological Research. In Press.

PASTEUR, N. 1988. Manual technique de

genetrique par electrophorese des proteines.

Lavoisier TEC & DOC., Paris. 215pp.

THORPE, J. P. 1983. Enzyme variation, genetic

distance and evolutionary divergence in relation to

levels of tax onomic separation. Pp. 131-152. InG.

S. Oxford and D. Rollonson (eds.), Protein

polymorphism: Adaptive and taxonomic

significance. Academic Press, London.

TIAN, W. S. AND Y. M. JIANG (EDITORS),

ASSISTED BY G. F. WU, Q. X. HU, E. M. ZHAO,

AND Q. Y. HUANG. 1986. Identification manual

for Chinese species of amphibians and reptiles.

Science Press, Beijing. 164 pp. (In Chinese).

I June 1995

Asiatic Herpetological Research

Vol. 6, pp. 78-84

Fibrinogenase from the Venom of Trimeresurus mucrosquamatus

JIAN-PING MAO1, WAN-YU WANG1, YU-LlANG XlONG1 AND LIANG LU2

'Kunming Institute of Zoology, Academia Sinica, Kunming, 650223 China

2 Yunnan Provincial Hospital, Kunming, 650021, China

Abstract. -A new fibrin(ogen)olytic protease (FP) was purified from the venom of Trimeresurus

mucrosquamatus by DEAE-Sephadex A50, Sephadex-G75, CM-Sepharose CL-6B and mono-s (FPLC)

column chromatography. The molecular weight was 22,000 Da and the isoelectric point was 9.2. It was a

glycoprotein composed of 194 amino acid residues. FP could hydrolyze casine, finbrin, fibrinogen and also

showed hemorrhagic subcutaneously, no phospholipase A activity, arginine esterase activity which existed in

the crude venom. The enzyme could be inhibited by ethylenediamine tetra-acetate (EDTA) and cysteine, but

not by phenylmethyl sulfonyl fluoride (PMSF). FP cleaved the BB-chain of fibrinogen first following the

Aa-chain. In vivo, thrombolytic activity was tested on artificial thrombus placed in the cerebral artery of

rabbits. Thrombolysis was then characterized by angiographic techniques over several intervals. The

fibrinolytic activity resulted in thrombolytic recanalization of two dosage groups. Of four rabbits of 0.2

mg/kg, one achieved recanalization in 12 hrs. and three in 24 hrs. Of another four under the dosage of 0.4

mg/kg, three recanalized successfully in 5 hrs. and one in 9 hrs.

Keywords: Venom, Fibrin(ogen)olytic Protease, Thrombolysis.

Introduction

Fibrinolytic and fibrinogenolytic activity

had been described in the venoms of a

number of snake species, including

members of the Crotalinae, Viperinae, and

Elapidae families (Ouyang and Teng, 1976;

Willis et al., 1988; Daoud et al., 1987;

Evans and Barrett, 1988). The

fuibrin(ogen)olytic enzymes in snake

venoms had also been reviewed previously

(Seegers and Ouyang, 1979; Hellmann,

1968; Markland, 1988; Markland, 1991).

Several fibrin(ogen)olytic enzymes were

isolated from the venom of Trimeresurus

mucrosquamatus: two fibrinogenases

(Ouyang and Teng, 1976), two hemorrhagic

principals HT-a and HT-b (Nikai et al.,

1985) and three proteinases (Sugihara and

Mori, 1985). Willis, et al. (1989) evaluated

the thrombolytic potential of anticoagulant

proteases in Crotalus atrox venom using

rats. In the present study, we purified a

new fibrinogenase from the venom of

Chinese habu snake and studied its

characteristics.

Materials And Methods

Lyophilysed Trimeresurus mucro-

squamatus venom was obtained from

Yuanlin Farm (Hunan, China) and stored at

-20°C. Human thrombin was purchased

from Shanghai Hospital. Fibrinogen,

BAEE (N- benzoyl- L- arginine ethyl ester),

PMSF were from the Shanghai Institute of

Biochemistry, Academia Sinica. Urokinase

(UK) came from Nanjing University.

DEAE-Sephadex A50, Sephadex-G75, CM-

Sepharose CL-6B and mono-s HR5/5

(FPLC) were purchased from Phrmacia Fine

Chemicals (made in Uppsala, Sweden).

The other chemicals used were analytical

grade from commercial sources.

Isolation Procedure: Isolation of FP

was achieved by a combination of gel

filtration and ion-exchange chromatography

at 4°C (Fig. 1). One gram of crude venom

was dissolved in 5 ml of 50 mM Tris, pH

8.8. The insoluble material was removed by

centrifugation (2000 g) for 10 min. The

supernatant was fractionated thus: first, on

DEAE-A50 (3 X 100 cm), 50 mM Tris-HCl

pH 8.5; second, Sephadex -G75 (2 X 100

cm), 20 mM Tris-HCl pH 7.5; third, CM-

Sepharose CL-6B (2 X 30 cm), 10 mM

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol. 6, p. 79

Vsc C i

III !0 JO 40

*M FPLC Mono-S

10 !0 30 40 50 lak, Nui

10 40

Tubr Nuaber

FIG. 1. Fractionation of Trimeresurus mucrosquamatus venom. 1st, DEAE-A50 (3X100 cm) anion

exchanging, 50mM Tris-HCL pH 8.5; 2nd, Sephadex G-75 gel filtration (2X100 cm), lOmM ammonium

acetate pH 7.0; 4th, mono-s cation exchanging, lOmM sodium acetate pH 5.8.

ammonium acetate pH 7.0; and fourth, on

mono-s HR 5/5, 10 mM sodium acetate pH

5.8.

Characterization of FP: Assay for

hemorrhagic activity assay for gross

observation was performed as reported

previously (Bjarnason and Fox, 1983).

Proteolytic activity was assayed by a method

using casine of Kunitz (1947). The

inhibition of EDTA, Cysteine, and PMSF

was also tested with this method..

Fibrinogenase activity was measured by the

method of Ouyang and Huang (1979).

Fibrinolytic activity was tested with the

fibrin plate method of Astrup and Mullertz

(1952), and also with fibrin clot from

fibrinogen with thrombin. Arginine ester

hydrolytic activity was assayed using BAEE

as a substrate. BAEE 50 mM (containing 1

mM CaC12) was prepared with 50 mM Tris-

HC1 (pH 8.0) buffer. Trypsin was taken as

the control. The phospholipase activity was

qualitatively assayed with the substrate of

yolk. The pH of the substrate was

modulated to 8.0; after the fraction was

added, the pH decreased by the

phospholipase activity, and was adjusted to

that of the original by 10 mM NaOH. The

values of sodium hydroxide was taken to

present the activity. Amino acid

compositions were carried on a Model 835-

50 Hitachi high speed automatic analyzer by

the method of Simpson et al. (1976).

Twenty-four h, 48 h hydrolysates were

used. Phanolanaline was used as the

minimum residue to calculate the number of

amino acid residues.

Thrombosis assay by angio-

graphy: For FP dosage determination, 0.2

ml rabbit plasma made clot with 3U

thrombin, 50(lg FP was used to test in vitro

activity. In 4.5-5.5 hours, the milky white

Vol. 6, p. 80

Asiatic Herpetological Research

June 1995

TABLE 1 . Summary of purified FP from T. mucrosquamatus venom (n=6).

Recovered protein (mg)

Hemorrhage (mm X mm)

(25±3 gm mice 100 ug sample)

Caseinolytic activity

Units/mg.min

Fibrinolytic activity

(Fibrin heated plate mm2)

Arginie esterase activity

Phospholipase A activity

• *»

W tffl

ft

12 3 4 5 6 7

FIG. 2. SDS-PAGE of reduced fibrinogen after

incubation with FP (lOug) lane 1; fibrinogen; lane

2-7; fibrinogen with FP for 5, 10, 20, 30; 45; and

60 min. respectively.

clot could become clear. Thrombolytic

activity by FP was tested on artificial

thrombus catheterized in the rabbit. Sixteen

rabbits, ranging from 2.2 to 2.7 kg, were

used. 0.5 ml of blood was drawn from the

ear vein of the rabbits and made thrombus as

20 mm (long) X 1.00 mm (diameter). The

rabbits were then anesthetized with an

intravenous injection of a ketamine at a

dosage of 2 mg/kg weight. The left

common carotid artery was isolated, and a

polythylene catheter (1.00 ID X 1.40 OD)

was inserted into the artery. By digital

subtraction angiology (DSA, Angiotran

cmp, Siemens, Germany), normal

angiographic was recorded with angiografin

solution (meglucamine diatrizoate 65% 30

ml X "Schering AG", made in Germany) of

3 ml. The thrombus was catheterized into

the artery, then the thrombosis graph was

recorded. Thirty minute after the thrombus

induction, the 16 rabbits, separated into four

groups, were injected with saline, U.K.

1,000 IU/kg weight, 0.2 mg FP/kg weight,

0.4 mg FP/kg weight transcatheterically

respectively, then the four angiogrames

were taken after 5, 9, 12 and 24 hours.

Results

Fourteen fractions were achieved after

DEAE-Sephadex A50 chromatography, and

three fractions, which contained fibrinolytic

activity, were further fractionated. Fraction

3 was equilibrated with 20 mM Tris-HCl

buffer (pH 7.5) by dialysis and loaded onto

the Sephadex-G75 column. Fraction 2

June 1995

Asiatic Herpetological Research

Vol.6, p. 81

f i

u-nai

FIG. 3. Aniographs of in vivo thrombolysis (from left). Control group! A, Normal graph. B, Thrombosis

performed (X). C, Treated with saline, 24 hours. FP group: D and G, Normal graphs. E and H, Thrombosis

performed (X). F, Treated with FP at 0.2 mg/kg dosage, recanalization occured in 24 hrs. I, Treated with FP

at 0.4 mg/kg dosage, recanalization occured in 5 hrs. U. K group (positive control): J, Normal graph. K,

Thrombosis performed (X). L, Recanalized within 9 hours of U. K administration (1 ,000 IU/kg).

obtained in this step yielded fibrinolytic

activity. This fraction was dialyzed against

10 mM aminium acetate (pH 7.0) and loaded

on the CM-Sepharose CL-6B column and

was separated into three fractions, the

fibrinolytic activity was located in the 2nd,

this fraction was rechromatographied on

FPLC mono-s column and eluted with three

phases of gradient: 0-30 ml (0 M NaCl);

30-80 ml (0-0.1 M NaCl) and 80-100 ml

Vol.

6, p. 82

Asiatic Herpetological Research

June 1995

TABLE 3. Proteases from the venom of T. mucrosquamatus.

TABLE 4. Proteases from the venom of T. mucrosquamatus.

(1 M NaCl). The fibrinolytic activity was

in the 6th fraction and the principal was

qualified as FP.

Properties of FP: The molecular

weight of the purified FP was determined

to be 22,000 Da by SDS-polyacrylamide

slab gel electrophoresis. The isoelectric

point obtained by isoelectric focusing disc

polyacrylamide gel was 9.2. The enzyme

is a glycoprotein as shown by periodic

acid-Schiff's agent staining after low pH

(pH 4.3) polyacrylamide gel

electrophoresis. The amino acid residues

of FP was 194, the combined number of

Glx and Asx were 49. Yet, the isoelectric

point of the proteinase was basic. This

enzyme possessed proteolytic activity

hydrolyzing casine, fibrin and fibrinogen,

but did not have BAEE hydrolase,

June 1995

Asiatic Herpetological Research

Vol. 6, p. 83

phospholipase A activity. The enzyme

also had hemorrhagic activity (Table 1).

Heat and pH stability: The

proteinase, at a concentration of 40 |ig/ml

in 10 mM acetate buffer (pH 5.8)

containing 10 mM NaCl, were incubated

for 30 minutes at various temperatures and

then quickly cooled down to room

temperature. The caseinolytic activity was

then determined. The enzyme was fully

active at 37°C, showed little activity at

50°C, and almost lost complete activity at

55°C. Incubation of FP at pH values

below 5.0 and above 10.0 for 30 min lead

to a sudden decrease in proteolytic

activity.

Biological activity: The effects of

some reagents on the proteolytic activity of

FP were examined. This activity was

inhibited by ethylenediamine tetraacetic

acid (EDTA), cysteine but not by PMSF.

The effects of some divalent and trivalent

ions on the proteolytic activity of FP were

also assayed. The enzyme (40 |ig/ml) and

ions at a concentration of 10 mM in 10

mM acetate buffer were first incubated at

37°C for 30 min. before the proteolytic

activity was assayed. The proteolytic

activity increased in the presence of

bivalent ions in the following order: Ca++

< Cu++ < Zn++ < Co++. The increase

by Fe+++ and A1+++ were lower than that

of Zn++ but higher than that of Cu++,

Ca++ (Table 2). When fibrinogen was

incubated with FP, this enzyme cleaved

the Bb-chain of fibrinogen and followed

the Aa-chain as shown in Figure 2. The

degrading of fibrinogen by FP was

measured as 36.5 mg per mg enzyme in

one minute.

Thrombolysis: None of the four

rabbits of saline administration reached

recanalization. Of the FP 0.2 u.g/kg

group, one achieved recanalization in 12

hrs. and three in 24 hrs.; of the other four

of 0.4 mg/kg, three recanalized

successfully in 5 hrs. and one in 9 hrs. Of

the four rabbits treated with 1,000 IU/kg

of U.K., thrombolytic recanalization

occurred in two in 6 hrs. and two in 9 hrs.

(Fig. 3).

Discussion

The venom of the Crotalinae species

contained much proteinases, which had

proteolytic and esterase activities. Several

enzymes were isolated from Trimeresurus

mucrosquamatus venom (Table 3 and 4).

Our results showed that FP is a new

fibrinogenase existing in Trimeresurus

mucrosquamatus venom. Compared with

the others, this enzyme, is a metallo-

proteinase which attacks the Bb-chain of

fibrinogen preferentially. As we know,

the enzymes which had fibrinogenolytic

activity were classified as a-fibrinogenases

and b-fibrinogenases; most of the a-

fibrinogenases can degrade Aa-chain of

fibrinogen and usually are metallo-

proteinases. Of most of the b-

fibrinogenases degrading the Bb-chain of

fibrinogen, little of them could also

degrade the Aa-chain inhibited by DFP or

PMSF. They are serine proteinases. HT-

a, the first example of which hydrolyzed

Bb-chain of fibrinogen and was inhibited

by EDTA. The followed FP was the

second report of these enzymes. Three

proteinases from the Agkistrodon halys

blomhoffii were activated by Ca++ and

Co++ (Satake et al., 1963). Ca++ and

Zn++ were also needed for the proteolytic

activity of protein G from Bothrops asper

(Ortiz and Gubensak, 1987). FP was

activated by Co++ and Zn++. For these

metallo-proteinases, these bivalent cations

were important for their stability and their

activities. FP had marked activity on the

plasma clot in vitro, also 0.4 mg FP/kg

dosage occurred thrombolysis in 3/4 in

vivo. It was a good trial for FP

thrombolytic potential. Hemorrhage was

caused when injected subcutaneously, but

did not occur within the heart, liver,

kidney and lung after FP injection in

rabbits at the dosage of 1 mg/kg in mice.

Vol. 6, p. 84

Asiatic Herpetological Research

June 1995

Literature Cited

ASTRUP, T. AND S. MULLERTZ. 1952. The

fibrin plate method for estimating fibrinolytic

activity. Archives of Biochemistry and

Biophysics 40:346-349.

BJARNASON, J. B. AND J. W. FOX. 1983.

Proteolytic specificity and cobalt exchange of

hemorrhagic toxin e, a zinc protease isolated from

the venom of the western diamondback ratdesnake

(Crolalus atrox). Biochemistry 22:3770-3778.

DAOUD, E., H. Y. HALIM AND F. M. EL-

ASMAR. 1987. Further characterization of the

anticoagulant proteinases, cerastase F^4, from

Cerastes cerastes (Egyptian sand viper) venom.

Toxicon 25:891-897.

EVANS, J. H., AND A. J. BARRETT. 1988. The

action of protease F 1 from Naja nigricollis

venom on the Aa-chain of human fibrinogen. Pp.

213-222. In Pirkle H. and Markland F. S. (eds.).

Hemostasis and Animal Venoms. Marcel Dekker,

New York.

HELLMANN.K. 1968. Naturally occurring

anticoagulants and fibrinolysis. Pp. 254-265. In

The Scientific Basis of Medicine Annual Reviews.

Oxford University Press, New York.

MARKLAND, F. S. 1988. Fibrin(ogen)olytic

enzymes from snake venoms. Pp. 149-172. In

Pirkle H. and Markland F. S. (eds.). Hemostasis

and Animal Venoms. Marcel Dekker, New York.

MARKLAND, F. S. 1991. Inventory of a and 6-

fibrinogenases from snake venom. In Hemostasis

and Animal Venoms. F. K. Schattauer

Verlagsgesellschaft mbH (Stuttgart), 65(4):438-

443.

NKAI, T., N. MORI AND M. KISHIDA. 1985.

Isolation and characterization of hemorrhagic

factors a and B from the venom of the Chinese

habu snake (Trimeresurus mucrosquamatus).

Biochimica et Biophysica Acta 838:122-131.

ORTIZ, F. A. AND F. GUBENSAK. 1987.

Characterization of a metallo-proteinase from

Bothrops asper (Terciopelo) snake venom.

Toxicon 25:759-763.

OUYANG, C. AND C. M. TENG. 1976.

Fibrinogenolytic enzymes of Trimeresurus

mucrosquamatus venom. Biochimica et

Biophysica Acta 420:298-308.

SEEGERS, W. H. AND C. OUYANG. 1979.

Snake venoms and blood coagulation. Handb.

Exp. Pharm. 52:648-750.

SIMPSON, R. J., M. R. NEUBERGER AND T. Y.

LIU. 1976. Complete amino acid analysis of

proteins from a single hydrolysate. Journal of

Biological Chemistry 25:1936-1940.

SUGIHARA, H. AND N. MORI. 1985.

Comparative study of three proteinases from the

venom of the Chinese habu snake (Trimeresurus

mucrosquamatus). Comparative Biochemistry and

Physiology 82B(l):29-35.

WILLIS, T. D. AND A. T. TU. 1988.

Purification and characterization of Atroxase. A

non-hemorrhagic fibrinolytic protease from

western diamondback rattlesnake venom.

Biochemistry 27:4769-4777.

WILLIS, T. D., A. T. TU AND C. W. MILLER.

1989. Thrombosis with a snake venom protease

in a rat model of venous thrombosis. Thrombosis

Research 53:19-29.

I June 1995

Asiatic Herpetolo^ical Research

Vol. 6, pp. 85-96

Digital Pad Morphology in Torrent-living Ranid Frogs

ANNEMARIE OHLER

Laboraloire des Reptiles el Amphibiens, Museum national d'llistoire nalurelle,

25 rue Cuvier, 75005 Paris, France

Abstract. -Digital pads of 24 species of ranoid frogs (Raninae, Dicroglossinae, Ranixalinae, Rhacophorinae,

Hyperoliinae) were studied by scanning electron microscopy. In many species of Raninae the cells of the

adhesive pad are differentiated (elongated and wearing projections). Functional aspects of cell morphology and

digital pad expansion are discussed in relation with sticking condition in aquatic medium.

Key words: Amphibia, Anura, morphology

Introduction

Digital pads occur in most of advanced

anuran families. This organ seems to be of

multiple origin and of difficult use in

systematics (Noble and Jaeckle, 1928;

McAllister and Channing, 1982). Digital

pads occur in arboreal anurans (Hyla), but

they can also be observed in torrent-living

frogs (Amolops), and in some fossorial

species (Kaloula). In Asian and African

frogs of the family Ranidae, several genera

and species groups belonging to different

subfamilies have fingers and toes bearing

digital pads.

There exists no strong hypothesis of

phylogeny of ranids as a whole.

Phylogenetic analyses were undertaken only

for geographic and taxonomic limited

groups (Liem, 1970; Clarke, 1981; Hillis,

1985; Emerson and Berigan, 1993). The

broadly accepted classification (Frost, 1985)

is based on Boulenger's works dating from

the beginning of this century (Boulenger,

1882; 1920). Recently Dubois (1986,

1992) tried to review the entire group and

proposed a tentative classification which he

sees as a working hypothesis. In this

hypothesis ranids are split into several

families, subfamilies and tribes (Dubois,

1992). Species that are enclosed in the

genus "Rana" in Frost (1985) are in Dubois'

classification distributed in several

subfamilies (Table 1).

Results from study of skeleton showed

several major lines in "Rana" (Deckert,

1938; Clarke, 1981). Study of the

morphology of the digital pads (Ohler and

Dubois, 1989) confirmed that two of these

lines could be distinguished by their digit

morphology. Ranines have digital pads

with a latero-ventral groove, often separated

terminally. Dicroglossines have digital pads

showing a dorso-terminal groove.

The histological structures of the digital

pads were first described by Schuberg

(1895) and Siedlecki (1910). Noble and

Jaeckle (1928) undertook a comparative

histological analysis of 47 species of

anurans. The fine structure of the epidermal

cells in the digital pad has been observed by

transmission electron microscope (Komnick

and Stockem, 1969; Ernst, 1973 a-b).

Scanning electron microscopy has been used

to describe morphology of digital pads,

often in view of taxonomic utilisation or

functional interpretation (Welsch, Storch

and Fuchs, 1974; Green, 1979, 1980, 1981;

Emerson and Diehl, 1980; Mc Allister and

Channing, 1983; Green and Simon, 1986;

Green and Carson, 1988).

The epidermis of anurans has a

superficial layer of hexagonal or pentagonal

squamosal cells, which are disposed in a

regular way (Tyler and Miller, 1985).

Differentiation of the pad leads to prismatic

epithelial cells. Their surface is usually

hexagonal or pentagonal, as is that of

generalized cells, but their height is more

important than in the latter. They are

separated in their distal part forming deep

crypts.

In the dermis of amphibians both

mucous and venomous glands are present.

Their aperture is situated between the

epithelial cells of the epidermis. On the pad

1995 by Asiatic Herpetological Research

Vol. 6, p. 86

Asiatic Herpetological Research

June 1995

TABLE 1. Classification of Dicroglossinae and Raninae as proposed by Dubois (1992) and numbers of

species studied here. D: digital pad or expanded digit tip present in some species at least; M: some species at

least in the genus Micrixalus in the classification given by Frost (1985); R: some species at least in the

genus Rana in the classification given by Frost (1985); the number indicates the number of species here

studied by morphometry, external morphology and/or scanning electron microscopy.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 87

FIG. 1 . Generalized plan of digital pad. Left dorsal view; right ventral view, c: cover; df: dorsal fold; tk:

terminal knuckle; eg: circumferential groove; p: pad; bg: basal groove.

FIG. 2. Digital pad of Raninae with latero-ventral groove (Rana (Hylarana) erythraea, MNHN 1987.3343,

Thailand), a: dorsal view of finger III; b: ventral view of finger III; stippled area corresponds to the pad with

prismatic cells.

only openings of mucous glands can be

observed.

The first authors (Schuberg, 1895;

Siedlecki, 1910; Noble and Jaeckle, 1928)

supposed that the products of the mucous

glands were implicated in sticking function.

To complete sticking the epidermal cells

would allow attachement to natural surfaces

that are covered with irregularities (Welsch,

Storck and Fuchs, 1974), somehow close to

the mechanism of clinging in lizards. But

lizards differ substantially from amphibians

in having a dry or setal adhesive system

(Green and Carson, 1988).

Emerson and Diehl (1980) and Green

(1981) independently showed that surface

tension was mechanically responsible for the

adhesive abilities of treefrog digital pads.

As the surfaces of plants have usually a low

surface tension, the structure of the pad cells

assures humidification responsible for

adhesion. The grooves surrounding the pad

Vol. 6, p. 88

Asiatic Herpetological Research

June 1995

FIG 3. Digital pad of Dicroglossinae with dorso-tcrminal groove (Limnonectes (Bourretia) doriae, MNHN

1987.3130, Thailand), a: dorsal view of toe IV; b: ventral view of toe IV; stippled area corresponds to the pad

with prismatic cells.

serve as a reservoir for the fluid wetting

agent (McAllister and Channing, 1983).

Numerous frog species with enlarged

digital tips have been studied (Hyperoliinae,

Hylidae, Telmatobiinae, Rhacophorinae,

and others), as well as the digital tips of

some species without enlarged digital tips.

Only some species of the family Ranidae

have been studied in this respect, including

only species without digital pad. Here I will

present the structure of digital pads and

digital pad cells of subfamilies of ranoids

according to Dubois' (1992) classification,

Ranixalinae, Dicroglossinae, Raninae,

Rhacophorinae and Hyperoliinae. They

include arboreal ("Hylarana") and torrent-

living frogs (Amolops) that have digital pads

with grooves and modified cells. For the

torrent-living frogs a mechanism of sticking

is proposed and the correlation of cell

morphology, digit tip enlargement and

biology of these frogs is outlined.

Material and methods

Specimens representing 15 of 34 genera

and subgenera, possessing digital pads, as

recognised by Dubois (1992) were chosen

in the collection of MNHN (see Table 1,

Appendix I). They had been generally

formalin fixed and all had been stored in 70

% alcohol. Finger II or IV or toe III were

cut on the terminal articulation. Cleaned

with ultrasonic sounds, they were

dehydrated in alcohol. After critical point

drying, they were gold covered (2-4 A).

Specimens were observed with the Scanning

electron microscope (JSM-840) of the

MNHN SEM facilities. Photographs were

taken on 120 Ilford FP4 film.

Measurements were taken with a slide

caliper (SVL) or a binocular microscope

(FW): SVL - snout-vent length; FW - third

finger width (maximum width of tip of third

finger). To eliminate size factor, FW is

given as a ratio of SVL (per thousand).

Terminology of digital pad

morphology (Fig. 1, 2, 3)

(1) The circumferential groove (Green and

Simon, 1986) (Fig. 1) surrounds the digit

tip latero-terminally and separating a dorsal

part from a ventral part. The groove may be

complete or open (with a distal zone of

contact between the dorsal and ventral part).

This is the generalized groove that is

modified in various manners according to

the group of frogs observed.

June 1995

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Vol. 6, p. 89

- »i

FIG. 4. Squamosal cells with short spinulae,

ventral view, proximal of pad of finger III

(Batrachylodes vertebralis, MNHN 1970.1407,

Salomon Islands).

FIG. 5. Squamosal cells with microridges, ventral

view, outside the circumferential groove of finger HI

(Amolops marmoratus, MNHN 1988.2787, Nepal).

r "V* '.J* tf"SS

FIG. 6. Squamosal cells with spongious

structures, dorsall view, on subunguis close to the

dorso-tenninal fold of finger III (Ingerana tasanae,

MNHN 1987.2002, Thailand).

(a) The latero-ventral grooves (Ohler

and Dubois, 1989) (Fig. 2) close the pad,

that is of triangular shape, laterally. In some

species they join distally and close to a

unique groove arround an oval or rounded

pad.

(b) The dorso-terminal groove

(Ohler and Dubois, 1989) folds on the

dorsal part of the digit. The pad is of oval

or rounded form. In species where the

groove is more pronounced its lateral parts

can be observed ventrally (Fig. 3).

FIG. 7. Sqamosal cells with hallow tubercles,

ventral viw, proximal of pad of finger III {Ingerana

tasanae, MNHN 1987.2002, Thailand).

(2) The basal groove (Fig. 1) is the basal

limit of the digital pad. Fusion of this with

the circumferential groove results in a

circumplantar groove. The latter is not

present in all digital pad types.

(3) The ventral part is the proper

adhesive organ, the pad (Savage, 1987)

(Fig. 1). Its latero-terminal limits are

usually distinct formed by the groove. Its

basal limit is intergrading, and the basal

groove, if present, is not the limit of the

functional part as indicated by presence of

modified cells still beyond this limit distally.

Vol. 6, p. 90

Asiatic Herpetological Research

June 1995

TABLE 2. Distribution of prismatic cell types and relative width of tip of third finger in ranoid frogs. - Cell

differentiation: L: elongated prismatic cells; R: regularly outshaped prismatic cells; H: cells of heterogeneous

shape; P: projections on proximal border of prismatic cellls; S: small projections on proximal border of

prismatic cells; N - no projections on prismatic cells; -: no prismatic cells in the digit tip. - Relative width of

tip of third finger, measured by FW/SVL: x: mean; s: standard deviation; n: number of specimens measured;

EV: extreme values of ratio FW/SVL in group.

(4) The dorsal part, the cover (Savage,

1987) (Fig. 1), does not show histological

specialisation.

(5) Proximally the cover is limited by the

dorsal fold (Fig. 1).

(6) Dorsally on fingers and toes a

terminal knuckle (Lynch, 1979) (Fig. 1) is

present in the area of distal articulations.

(7) The pad is generally entirely masked

by the cover, but in some cases it projects

distally. The part of the pad that is then

visible dorsally is called the subungis

(Lynch, 1979).

Results

The study of digital pads in Ranidae

gave interesting results concerning gross

morphology and its use for phylogeny

already published (Ohler and Dubois, 1989)

as well as new results concerning the type of

prismatic cells observed in the digital pad.

These microstructural results are exposed

below and a functional hypothesis is

proposed.

June 1995

Asiatic Herpetological Research

Vol.6, p. 91

FIG. 8. Regular outshaped prismatic cells with

mucous gland pore on pad of finger III (Hyperolius

viridiflavus karissimbiensis, MNHN 1988.1055,

Rwanda).

FIG. 9. Elongated prismatic cells with disatal

projections on pad of finger III {Amolops sp. 1,

MNHN 1987.2163, Thailand).

FIG. 10. Elongated prismatic cells with distal

projections on pad of finger III {Amolops sp. 3,

MNHN 1987.2140, Thailand).

FIG. 1 1. Elongated prismatic cells with small

distal projections on pad of finger III {Amolops

marmoratus, MNHN 1988.2787, Nepal).

FIG. 12. Orientation and distribution of prismatic

cells on distal part of the digital pad of finger III of

Rana (Odorrana) andersoni (MNHN 1938.57,

Vietnam).

FIG. 1 3. Distribution of prismatic and sqamosal

cells on the extreme distal part of the digital pad of

finger III of Rana (Sylvirana) sp. (MNHN

1987.3471, Thailand).

Vol. 6, p. 92

Asiatic Herpetological Research

June 1995

The epidermal cells

On the tips of the digits one observes

two major cell types (squamosal cells and

prismatic cells) with intermediary cells that

occur in high numbers in the proximal pad

zone.

(1) Squamosal cells. This is the

generalized cell type, covering the body of

amphibians (Tyler and Miller, 1985). The

cells often show short spinulae (Fig. 4) or

structures called microridges (Fig. 5). In

Ingerana tasanae the surface of the

squamosal cells is extremely rough and can

show spongious structures (Fig. 6). On

other parts of the epiderm the surface of the

squamosal cells of Ingerana tasanae shows

hallow tubercles (Fig. 7). The squamosal

cells cover fingers and toes outside the pad.

The groove is generally the border, but

sometimes the squamosal cells are present

on the border of the pad {Rhacophorus

leucomystax) or in the contrary they are

pushed back by the prismatic cells even

outside the groove (Amolops).

(2) Prismatic cells. The prismatic cells

are present on the pad. They are of regular

outline in all the species already studied

(Green, 1979; Green and Simon, 1986;

McAllister and Channing, 1982; Richards et

al., 1977; Welsch, Strock, and Fuchs,

1974). Among the species studied here,

Hyperolius vividiflavus karissimbiensis and

Rhacophorus leucomystax have prismatic

cells of regular outline (Fig. 8) like those

found by previous authors. Also some

other species of ranids (Limnonectes

(Bourretia) doriae, Batrachylodes

vertebralis) have this kind of prismatic cells.

However, in most of the ranid species

investigated (Table 2) in this study, the

prismatic cells are not of regular outline but

elongated. Their long axis is oriented in the

proximo-distal direction on the digital pad.

The ratio of the width to the length of these

cells is smaller than 60%, while in normal

prismatic cells this ratio is over 80%, often

close to 100%. On their narrow distal side,

the elongated cells have more or less

developed projections.

In the species of the genus Amolops,

this kind of cells is present with well

developed projections (Fig. 9, 10). These

were also observed in different "subgenera"

of the genus Rana (Odorrana, Amnirana,

Hylarana, Chalcorana) and in Indirana

gundia (Ranixalinae). The prismatic cells of

these species vary in their elongation, in the

size of the projection, and in the degree of

regularity. They are often rather regularly

hexagonal, rounded proximally, with small

distal projections, as in Rana (Chalcorana)

chalconota and in Ingerana tasanae. In some

species the prismatic cells are elongated,

rounded proximally without projections

{Rana (Hylarana) erythraea). In other

species outlines are very variable among

neigbouring cells; the cells are elongated

forming a somehow triangular outshape

wearing a single or two distal projections

(Fig. 11). In all species of Amolops of this

study, this kind of elongated cells with

heterogeneous outlines was observed.

The prismatic cells are present outside

the latero-ventral grooves in Rana

(Odorrana) andersoni and in Amolops sp. 3.

Observation of direction of the channels

formed by the prismatic cells shows a

generalized alignement in the direction of the

space between the pair of lateral grooves

(Fig. 12). In other species the border of pad

is formed by squamosal cells, but a contact

between the ventral and dorsal part of digital

tip remains (ex. Rana (Sylvirana) sp., Fig.

13).

The development of the toe pad

The measurements of the digital width

(Table 2) show an important variation that

can be divided in several units. The species

Amolops formosus and Amolops

marmoratus show the most enlarged finger

pads (FW/SVL = 68 p.m.). Other species

of Amolops, but also Rana (Chalcorana),

Ingerana tasanae and Rhacophorus

leucomystax have very well developed

digital pads (FW/SVL = 47-57 p.m.). The

frogs of the subgenera Rana (Amnirana) and

Rana (Odorrana) show moderately enlarged

digital pads (FW/SVL = 35-43 p.m.). The

species of Rana (Sylvirana) and Rana

(Hylarana), as the species of the subgenus

June 1995

Asiatic Herpetological Research

Vol. 6, p. 93

FIG. 14. Scheme of liquid fluid on a digital pad with regular outshaped cells (left) and with elongated cells

(right).

Limnonectes (Bourretia) have very little

enlarged finger pads (FW/SVL = 22-28

p.m.). The species studied that show no

digital pad formation have the lowest ratios

(FW/SVL = 17-20 p.m.).

Discussion

The elongated cells here described in

some species of Ranidae have not been

described in other anuran families. In fact

the species that have been studied until now

are "treefrogs", and no torrent-living frogs

have yet been investigated. Considering the

ecology of the studied species, five types

can be distinguished: (1) torrent-living frogs

of the genus Amolops, Rana (Odorrana) (the

possible sister-group of Amolops) and Rana

(Amnirana); Ingerana tasanae should be

placed in this group; (2) aquatic frogs, like

Limnonectes (Limnonectes) kuhlii and

Phrynoglossus laevis; (3) terrestrial frogs of

the genus Limnonectes (Bourretia) and Rana

(Hydrophylax); (4) ground/vegetation living

frogs of the genera Rana (Hylarana), Rana

(Sylvirana), and Rana (Chalcorana); (5)

arboreal frogs (Hyperolius, Rhacophorus).

Actually the Raninae, the Dicroglossinae

and the Ranixalinae do not include strictly

arboreal species. The closest group of

treefrogs are the Rhacophorinae, an other

subfamily of Ranidae. Hyperolius

viridiflavus karissimbiensis is another

ranoid treefrog studied. Rhacophorus

leucomystax, Hyperolius viridiflavus

karissimbiensis and the species studies by

the previous authors (Green, 1979; Green

and Simon, 1986; Richards et al., 1977;

McAllister and Channing, 1982; Welsch,

Strock, and Fuchs, 1974) have prismatic

cells of regular outshape. This kind of

regular cells was here also observed in

Limnonectes (Bourretia) doriae and

Limnonectes (Bourretia) pileata, two

terrestial species. Elongation of digital pad

cells in Amolops, Rana (Odorrana), and

Rana (Amnirana) might be in relationship

with their mode of life. The presence of

elongated cells in Rana (Hylarana) and in

Rana (Sylvirana) might indicate

Vol. 6, p. 94

Asiatic Herpetological Research

June 1995

phylogenetic relationship to Amolops. The

heterogeneous cells in some of these species

might indicate a regression in comparison to

the elongated cells with projections in

Amolops. The functional analysis of cell

morphology underlines this interpretation.

The major sticking force of tree frogs is

surface tension (Emerson and Diehl, 1980;

Green, 1981). It is a kind of wet adhesion,

where two surfaces are hold together by an

interlaying liquid. The prismatic cells, the

channels and the mucous glands are required

in the humidification mechanism necessary

for sticking. For torrent-living frogs the

surfaces to stick to are already humid or in a

liquid medium. In liquid the force is no

more proportional to the surface, but to the

squared surface which reduces the sticking

force to its square root (Emerson and Diehl,

1980). When sticking to glass at an angle of

90 to 180 , a treefrog is inmerged in water,

it will separate almost immediately (Emerson

and Diehl, 1980). The force of attachment

in liquid medium is inversely proportional to

the distance of the two surfaces, separated

by the liquid.

To provide a good sticking in water, the

surface of the pads should be enlarged.

Some of the species of Amolops, as

Amolops formosus or Amolops marmoratus

have in fact very much enlarged digital pads

(Table 2). A correlation between the digital

pad development, as defined by the groups

A, B, C, D and E see Table 2), and the

ecology of the species may be found. The

terrestial species and the aquatic frogs

belong to the group A. The group B

includes ground/vegetation-living frogs.

The torrent-living frogs are distributed in

three groups: C {O dorr ana, Amnirana and

Huia), D (Amolops, Ingerana), E (Amolops

formosus and Amolops marmoratus). The

treefrogs (Rhacophorus, Hyperolius) are all

members of the group D, thus not the

species with the largest digital pads.

Elimination of the distance between the

pad and the surface to stick to will increase

attachment force equally and more distinctly.

In treefrogs the regular cells guide the fluids

in all directions, thus humidifying the whole

pad in a regular manner and optimizing the

use of liquid (Fig. 14). The elongated cells

of Amolops guide the liquid in the disto-

proximal direction. The digital pad is not

closed posteriorly and often also anteriorly

by a groove, and prismatic cells are not

restricted to the pad surface, but are also

present in the groove and outside to it.

Water can flow out of the pad and distance

from pad to sticking surface is minimized,

thus increasing the sticking force inversely.

It would be interesting to compare the

cell morphology of torrent-living frogs of

other anuran families, like Ansonia

(Bufonidae), Petropedetes

(Phrynobatrachidae), some Litoria and Hyla

(Hylidae) and Heleophryne

(Heleophrynidae) to what is here described

in Raninae. A more detailed morphological

analysis of surface of digital pads should be

undertaken to compare sticking surface in

tree and torrent-living frogs.

Acknowledgments

The photos of this work were realized

with the precious help of Jean Menier in the

facilities of the "Service Commun de

Microscopie electronique" of the Museum

national d'Histoire naturelle of Paris. I

express my gratitude to Alain Dubois for

advice and discussion.

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Batrachia Salientia s.Ecaudata in the collection of

the British Museum. London, Taylor and Francis: i-

xvi+ 1-503, pi. I-XXX.

BOULENGER, G. A. 1920. A monograph of the

South Asian, Papuan, Melanesian, and Australian

frogs of the genus Rana. Rec. Indian Mus. 20:1-

126.

CLARKE, B. T. 1981. Comparative osteology and

evolutionary relationship in the African Raninae

(Anura Ranidae). Monit. zool. ital. (n. s. ) 15

suppl. :285-331.

DECKERT, K. 1938. Beitrage zur Osteologie und

Systematic ranider Froschlurche. Sber. Ges. naturf.

FreundeBerl. 1938:127-184.

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DUBOIS, A. 1987. Miscellanea taxinomica

batrachologica (I). Alytes 5:7-95.

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EMERSON, S. B. AND D BERRIGAN. 1993.

Systematics of southeast Asian ranids: multiple

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Limnonectes (Fitzinger). Herpctologica 49:22-3 1 .

EMERSON, S. B. AND D DIEHL. 1980. Toe pad

morphology and mechanisms of sticking in frogs.

Biol. J. Linn. Soc. 13:199-216

ERNST, V. V. 1973 a. The digital pads of the

treefrog Hyla cinerea. I. The epidermis. Tissue Cell

5:83-96.

ERNST, V. V. 1973 b. The digital pads of the

treefrog Hyla cinerea. II. The mucous glands.

Tissue Cell 5:97-104.

FROST, D. R. (ed. ) 1985. Amphibian species of

the world. Lawrence, Allen Press and Assoc. Syst.

Coll.: (i- iv) + i - v + 1 - 732.

GREEN, D. M. 1979. Treefrog toe pads:

comparative surface morphology using scanning

electron microscopy. Canadian J. Zool. 57:2033-

2046.

GREEN, D. M. 1980. Size differences in adhesive

toe-pad cells of treefrogs of the diploid-polyploid

Hyla versicolor complex. J. Herpet. 14:15-19.

GREEN, D. M. 1981. Adhesion and the toe-pads of

treefrogs. Copeia 1981:790 - 796.

GREEN, D. M. AND J. CARSON. 1988. The

adhesion of treefrog toe-pads to glass: cryogenic

examination of a capillary adhesion system. Austr.

J. nat. Hist. 22:131-135.

GREEN, D. M. AND M. P. SIMON. 1986. Digital

microstructure in ecologically diverse sympatric

microhylid frogs, genera Cophixalus and

Sphenophryne (Amphibia: Anura), from Papua New

Guinea. Austr. J. Zoo. 34:135-145.

HILLIS, D. M. 1985. Evolutionary genetics and

systematics of New World frogs of the genus Rana:

an analysis of ribosomal DNA, allozymes, and

morphology. Thesis, The University of Kansas, i-

vi+ 1-304.

KOMNICK, H. AND W. STOCKEM. 1969.

Oberflache und Verankerung des Stratum comeum an

mechanisch stark beanspruchten Korperstelllen beim

Grasfrosch. Cytobiologie 1:1-16.

LIEM, S. S. 1970. The morphology, systematics,

and evolution of the Old World treefrogs

(Rhacophoridae and Hyperoliidae). Fieldiana: Zool.

57: i-vii + 1-145.

LYNCH, J. D. 1979. A new genus for Elosia

duidensis Rivero (Amphibia, Leptodactylidae) from

Southern Venezuela. Amer. Mus. Novit. 2680:1-8.

MCALLISTER, W. AND A. CHANNING. 1983.

Comparison of toe-pads of some southern African

climbing frogs. S. Afr. J. Zool. 18:110-114.

NOBLE, G. K. AND M. E. JAECKLE. 1928. The

digital pads of the tree frogs. A study of the

phylogenesis of an adaptive structure. J. Morphol.

Physiol. 45:259-292.

OHLER, A. AND A. DUBOIS. 1989.

Demonstration de l'origine independante des

ventouses digitales dans deux lignees

phylogenetiques de Ranidae (Amphibiens, Anoures).

C. r. Acad. Sci. Paris 309 (3):4 19-422.

RICHARDS, C. M., B. M. CARLSON, T. G.

NONNELLY, S. L. ROGERS AND E. ASHCROFT.

1977. A scanning electron microscopic study of

differentiation of the digital pad in the Kenyan reed

frog Hyperolius viridiflavus ferniquei. J. Morphol.

153:387-396.

SAVAGE, J. M. 1987. Systematics and distribution

of the Mexican and central American rainfrogs of the

Eleutherodactylus gollmeri group (Amphibia:

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javanischen Flugfrosches (Polypedates reinwardtii).

Bull. Anat. Rec. 185:253-257.

TYLER, M. J. AND C. A. MILLER. 1985. Surface

architecture of the dorsal epidermis in Australian

frogs. Trans. Roy. Soc. South Austr. 109 (2):45-

48.

WELSCH, U., V. F. STORCH, AND W. FUCHS.

1974. The fine structure of the digital pads of

rhacophorid treefrogs. Cell Tissue Res. 148:407-

416.

Vol. 6, p. 96

Asiatic Herpetological Research

June 1995

APPENDIX I

Specimens studied by scanning electronmicroscopy (origin and reference in the

catalogue of the Museum national d'Histoire naturelle of Paris).

Species studied

Origin

Collection Number

Amolops (Amolops) formosus

Amolops (Amolops) marmoratus

Amolops (Amolops) sp. 1

Amolops (Amolops) sp. 2

Amolops (Amolops) sp. 3

Amolops (Huia) kinabaluensis

Amolops (Huia) nasicus

Batrachylodes vertebralis

Rana (Amnirana) albolabris

Rana (Amnirana) lepus

Rana (Chalcorana) chalconota

Rana (Hydrophylax) galamensis

Rana (Hylarana) erythraea

Rana (Odorrana) andersoni

Rana (Sylvirana) sp.

Ingerana tasanae

Limnonectes (Limnonectes) kuhlii

Limnonectes (Bourretia) doriae

Limnonectes (Bourretia) pileatus

Phrynoglossus laevis

Platymantis corrugatus

Rhacophorus leucomystax

Hyperolius viridiflavus karissimbiensis

Indirana gundia

Namdu Khola, Nepal MNHN 1994.5559

Timal, Nepal MNHN 1988.2787

Khao Chong, Thailand MNHN 1987.2163

Doi Inthanon, Thailand MNHN 1987.2082

Phu Kradung, Thailand MNHN 1987.2140

Kina Balu, Borneo MNHN 1889.240

Hanoi region, Vietnam MNHN 1938.70

Bougainville, Solomon MNHN 1970.1407

Islands

Liberia MNHN 1989.3456

Central African Republic MNHN 1968.247

Khao Chong, Thailand MNHN 1987.3490

"Afrique Orientale Francaise" MNHN 1920.145

Chiangmai, Thailand MNHN 1987.3343

Vietnam MNHN 1938.57

Doi Pui, Thailand MNHN 1987.3471

Khao Phra Tiu, Thailand MNHN 1987.2002

Phu Kradung, Thailand MNHN 1987.3332

Khao Chong, Thailand MNHN 1987.3130

Phu Kradung, Thailand MNHN 1987.3140

Khao Chong, Thailand MNHN 1987.2944

New Guinea MNHN 1989.3461

Khao Chong, Thailand MNHN 1987.3544

Gihirwa river, Rwanda MNHN 1988.1055

Gundia, India MNHN 1985.607

1. Formerly Amolops afghanus: see Dubois (1992: 340).

I June 1<W5

Asiatic HerpetoloRical Research

Vol. 6, pp. 97-110

Social Organization and Demography in the Rock Agama, Stellio caucasius

EUGENY N. PANOV AND LARISA Y. ZYKOVA

Severtsov Institute of Animal Evolutionary Morphology and Ecology, Russian Academy of Sciences,

Leninsky Prospect 33, Moscow 117071 , Russia

Abstract. -We studied the social organization and demography of the rock agama Stellio caucasius in a

natural population, located in Gobustan (eastern Azerbaijan, approximately 60 km south of Baku) and in an

introduced population in the small Karadag Range near Krasnovodsk (western Turkmenistan). We found that

these populations are highly stable with low turnover. This appears to be the result of delayed reproduction,

longevity and a sedentary life style. Population growth is relatively slow due to high juvenile mortality and

low immigration rates from adjacent subpopulations. The age structure of all subpopulations studied was

dominated by older age classes. Rock agamas exhibit those natural history and population characteristic of a

K selected species.

Key words: Agamidae, age, Azerbaijan, density dependence, K-strategy, Lacertilia, ontogenetic trajectories,

polygyny, population dynamics, social behavior, spacing, Stellio caucasius, survivorship, mortality,

territoriality, translocation, Turkmenistan.

FIG. 1. Adult (10+ years old) male Stellio caucasius perched on basking site (left); adult female S.

caucasius (right).

Introduction

Long-term mark and recapture studies of

natural populations have become important

in recent decades due to their great power in

demonstrating population parameters.

These investigations permit the testing of

hypotheses concerning the mechanisms

governing population parameters. The

ability to follow particular individuals

through time reveals the scale of behavioral

heterogeneity within the local population. It

also allows the examination of these

population parameters as a function of age

or changing social status.

This approach has already gained firm

position in the population studies of birds

and mammals, but it has only recently been

broadly employed in reptile studies.

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 98

Asiatic Herpetological Research

June 1995

FIG. 2. Regional distribution (cross hatched area)

of Stellio caucasius (upper plate). Specific study

sites in Azerbaijan and the Turkmen Republic: 1)

Gobustan; 2) Krasnovodsk; 3) Bol'shoi Balkhan

Range; 4) Kyurendag Range; 5) lower Sumbar

River; 6) Parkhai Gorge; 7) Kalaligez; 8) Aidere

(lower plate).

Although this method is used in modern

herpetology rather frequently, it is oriented

mainly toward answering the questions of

traditional population ecology (dynamics of

numbers and sex-age composition of local

populations, modes of spacing in the context

of resource utilization by communities and

species, etc.). In behavioral ecology, and

particularly with respect to the fates of

particular individuals and their social

relationships, the reptiles in general and

lizards in particular remain poorly studied

(for review see Semenov, 1989).

The rock agama, Stellio caucasius, is an

ideal subject for the study of behavioral

aspects of population organization in reptiles

that are K strategists. This is a long-lived

lizard attaining an age of ten or more years

(Zykova and Panov, 1991). Many

populations are characterized by high and

very high density. Most individuals

demonstrate strong home area fidelity.

Adults live in small social groups in which

the lizards form long lasting pair bonds

(Panov and Zykova, 1985; 1989).

This paper examines the social

interactions within local settlements of rock

agamas and analyzes the role of social

behavior as a regulator of demographic

processes.

Methods and Materials

Rock agamas are large lizards with an

overall length of up to 30 cm. and weighing

up to 160 gm. Males are on average larger

than females and have a heavier build.

General background color is a mixture of

gray, brown and olive with a darker, dull

spotted pattern on the back and sides.

During the breeding season males differ

from females by having black on the breast,

contrasting with pale or pinkish-gray on the

throat (Fig. 1). In males, there is light gray

epidermal holocrine gland in the center of

the blackish-gray belly. These lizards are

typical inhabitants of stony landscapes,

although some populations have become

adapted to the life on the steep slopes of clay

canyons or even at the margins of the sandy

desert (Ananyeva and Ataev 1984; Panov,

Zykova, Glauzer and Vasil'ev, 1987).

The bulk of data presented here was

obtained during a comparative study of two

populations of rock agama: a natural

population, located in Gobustan (eastern

Azerbaijan, approximately 60 km south of

Baku) and in an introduced population in the

small Karadag Range near Krasnovodsk

(western Turkmenia) where rock agamas

were known to be absent earlier (Fig. 2). In

the second area, on 17 May 1985, we

introduced into an abandoned quarry 13

adult males, 19 adult females, 13 two year

old lizards and 25 juveniles born in the

preceding year. All these animals were

caught in the Blocky Balkan Range situated

some 160 km from the introduction site.

The latter lies in view of Krasnovodsk

Plateau known to be a part of rock agama

June 1995

Asiatic Herpetological Research

Vol. 6, p. 99

1 «"2 c'3 94

FIG. 3. Territory structure in Gobustan (A) and Aidere (B): 1) territory boundaries; 2) breeding males; 3)

subordinate males; 4) females.

breeding range (Ataev, 1985). The

introduction site is similar to the dry

semidesert habitats of rock agamas found in

Gobustan. The predominately stony surface

is covered by very sparse grasses, but trees

and bushes, in contrast with Gobustan, are

wholly absent. The experimental plot was

situated in a broad, dry valley with steep

slopes broken by narrow ravines and rifts.

The numerous cavities and cracks under and

between the stones provided abundant

temporary and permanent shelters for the

lizards. Prior to release all introduced

agamas were marked by toe clipping.

In Gobustan, on the natural plot of 0.65

hectares, capturing, marking and

observations were conducted in April 1987

and 1988; in spring of 1986 and 1989 we

performed censuses and selective capture of

lizards. In Krasnovodsk, on the introduced

population, field work was carried out 24

April - 6 May 1986, 22 - 25 March and 30

April - 18 May 1987, 26 April - 17 May

1988, 5 - 10 April and 28 April - 23 May

1989, 15-25 May and 10-11 September

1990 (total of 103 days). Some

observations were made during short visits

in the summer and fall from 1985 to 1989.

Important additional data were obtained

during the course of field studies conducted

on two marked populations in western

Kopetdag near Kara-Kala settlement (Sjunt-

Khasardag State Reserve). An

observational plot in Parkhai Gorge was

inspected in the spring months of 1986 and

1989 and in September - October 1986 -

1988; a population in the Kalaligez area was

investigated in the fall of 1984 - 1987 (total

of 25 days).

In all of the above study plots we carried

out total censuses of lizards in the areas

under study. Most of the agamas observed

on the plots were captured. They were

measured with a ruler and calipers according

to standard procedures, weighed and

Vol. 6, p. 100

Asiatic Herpetological Research

June 1995

0115

ES3! S2 E33 \^4 S5

• 6 07 38 -9 ^10 [2311

FIG. 4. Krasnovodsk experimental study plot. A. Map of study site. B. Cross-section of the study . C.

Distribution of rock agamas in 1985. D. Distribution of rock agamas in 1986. E. Distribution of rock

agamas in 1988. F. Distribution of rock agamas in 1989. 1. Dry creek bed. 2. Large rocks and boulders.

3. Cross-section illustrated in Fig. 4B. 4. Talus. 5. Communal winter shelter. 6. Adult male. 7. Adult

female. 8. Second-year lizard. 9. Yearling. 10. Territory boundary. 11. Subordinate male home range

boundary. Numbers refer to individual lizards.

photographed. Animals caught for the first

time were marked by toe clipping. Prior to

release all agamas received individual color

marks made with dye. The locations of all

individuals on the plots were mapped.

The ages of agamas were estimated

using standardized size criteria (Zykova and

Panov, 1991). We assumed that the age of

individuals corresponds to the number of

winter hibernations. So, the ages of agamas

captured in spring will be slightly over-

estimated. Our "yearlings" in May are

actually about 10 months old; "two year old"

lizards in May are approximately a year and

10 months old, etc.

Altogether 426 agamas were captured

and marked, 144 of them were observed a

total of 273 times during subsequent years.

June 1995

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Vol. 6, p. 101

Results and Discussion

Spacing Patterns in Rock Agama

Settlements

The baseline of spacing pattern of a

whole social organization in Rock Agama is

a mosaic of mutually exclusive territories

owned by mature adult males. Mature

females have either mutually exclusive or

overlapping home ranges situated within

territories of those males with which the

given female is tied by the family bonds.

The adult female, as a rule, does not leave

the territory of "her" male. Therefore, the

territory of adult breeding male, if there is

female(s) living in it (which is not

necessarily the case) in the same time the

territory of pair or a family group defended

as a whole by the breeding male only.

The home ranges of immature lizards

one and two years of age may lie both

within the territory of the family group or

outside in the neutral zones separating such

territories. The home range of an immature

male during the first years of life within an

adult male territory, shifts to the periphery

of this territory as the immature male

becomes older. In contrast, the home range

of an immature female adjacent to that of an

adult male shifts with time toward the male's

territory.

In saturated habitats all suitable space is

shared among adult males, so that

neighboring territories have common

boundaries. The size of territories in such

saturated habitats depends on the substrate

and local food abundance (Fig. 3). In

barren habitats of Gobustan with low

microrelief (absence of talus mounds, in

particular) the size of adult male territories

was found to vary from 100 to 210 m2

(140.0 15.8 m2 on average). In the middle

altitudes of the Western Kopetdag

Mountains (Canyon Ai-Dere, see Fig. 3B)

where the climate is more humid and the

vegetation is rich and diverse and the

substrate includes jumbles of fallen rocks

and boulders, the size of territories was

found to range from 28 to 136 m2 (94.0 ±

16.3 m2 on average), for details see Panov

and Zykova (1985).

Formation of Territorial Structure

We followed the establishment of

territorial structure in the course of our long-

term observations on the Krasnovodsk

introduced population. Here 13 male

"founders" were released into an area that

would have supported a population of a

density comparable to that in natural

colonies of rock agamas. Males were

released into deep holes and crevices which

seemed to us to be similar to hollows

normally used by rock agamas as their

permanent dens.

However, contrary to our expectations,

the majority of introduced males left the area

where they had been released, and moved

away at distances ranging from 60 to 500

m. Only three males remained in proximity

to the release site by the next spring. At that

time the territory of only one male (N 56)

overlapped the release site. The boundaries

of two other males (NN 36 and 31)

territories were 60 and 100 m respectively

from the release site (Fig. 4 c, d).

The process of territory establishment

adjacent to the release site is shown in Fig. 4

c-f. During 1986, the year following

introduction, adult male territories were

large and had no common boundaries (Fig.

4d). Since male rock agamas do not patrol

the borders of their territories (as, for

example, males of steppe agama, Trapelus

sanguinolentus, do (see Panov and Zykova,

1986), it is difficult to locate precisely the

boundary between territories and, therefore

to estimate exactly the real territory size.

The greatest territory diameter was estimated

in 1986 on the experimental plot as 140 m,

with the width of the neutral zones

separating neighboring territories as some

20-60m. The small, indistinctly demarcated

home ranges of three immature lizards

(males NN 30 and 61, and female N 41, all

less than two years old) were situated in

these neutral zones (Fig. 4e). Other

immatures approximately two years old

established their home ranges within the

territories of mature males, as well as four

Vol. 6, p. 102

Asiatic Herpetological Research

June 1995

lizards born already in place of introduction

in preceding year.

In 1988, the third year after

introduction, most of the area that in 1987

was neutral zone was shared among

relatively young males of about four years

of age (NN 30 and 61) and three years of

age (N 1 15 already born on the introduction

site). Although these males apparently had

attained maturity, they seemed to be

bachelors at that time. Three year old male

N 1 15 and four year old female N 74 were

repeatedly seen in 1988 in close proximity

(10-15 m) to each other, but we did not

witness immediate contacts between them.

In 1989, the fourth year after

introduction, slight changes in territorial

structure were observed (Fig. 4e). There

was some tendency toward clumping of

territories toward each other, possibly

because of the increasing density of lizards.

Male N 61 (about five years old) left his

adolescent bachelor home range and

occupied the territory of deceased male N 31

and established a bond with his former

mate, a female of eight or nine years of age.

The corpse of male N 31 indicated that he

had died between June 1988 and April

1989. Relatively young male N 115

occupied the territory of five to six year old

male N 4a after he disappeared. Female N

74 moved into the territory of 10 to 11 year

old male N 3.

Dynamics of Space Utilization Within a

Home Area

During the hot periods of the year, each

individual had at least one permanent den

and one to several observational posts (used

also as basking sites) where it spent

considerable time, except during periods of

foraging. In individuals about one year old

or younger the den and basking site were the

same or immediately adjacent. Usually the

shelter was situated under a boulder or rock

on which the lizard basked. Foraging

activities of juveniles and yearlings take

place within a radius of several meters of the

individual dens. Many subadults of both

sexes and some adult females behave in a

similar way, although during foraging they

often move greater distances from the den,

up to several tens of meters.

During the day adult males range more

widely. The several posts of a male are

connected by a network of relatively

permanent pathways. The territory is

utilized unevenly with some points located

along the pathways receiving regular use

while others, situated at some distance from

the pathways being visited only occasionally

or not used at all during a given season.

This pattern of territorial use can result

in changes in territorial boundaries. For

example, by comparing the positions "c",

"d", and "e" in Fig. 4, one can see that in

1988 the border of the territory of male N 36

shifted 30-40 m eastward from its position

in 1986. Such shifts are possible in non-

saturated habitats only. In established rock

agama settlements with dense populations

the boundaries remain constant from year to

year.

In relatively sparse Krasnovodsk

population the cases of mutual intrusions by

neighboring males into neutral zones

separated their territories and even into

peripheral parts of these territories

themselves are possible. The above said

does not hold in respect to male-

"pretenders", or "satellites". Their home

ranges may broadly overlap the peripheral

parts of two or more territories of adult

"resident" males (see, for example, home

ranges of male-pretenders NN 30 and 61 in

Fig. 4). In saturated habitats home ranges

of satellites are practically always situated

within the territories of resident males.

By the winter the agamas leave their

summer home areas and migrate to

communal hibernation shelters. Migrations

begin when air temperatures are relatively

high (25° C and above). Communal winter

dens may be situated up to 500 m from an

individual's home area. On 23 March 1987

on the Krasnovodsk experimental plot,

when only a few agamas had returned to

their summer areas from the communal

hibernation shelter (daily temperatures

ranged from 2° C to 16.5° C) we found in

that shelter males NN 21, 27, 36, 61,

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Asiatic Herpetological Research

Vol. 6, p. 103

females NN 22 and 58 (ages from two to

seven years) and subadult male N 105.

Summer residences of these lizards are

shown in Fig. 4, home areas of others were

located 100 - 500 m from the communal

winter den.

Individual Ontogenetic Trajectories

An ontogenetic trajectory is defined as a

sequence of changes in social status and of

social roles of an individual during its life

(Wiley 1981). In rock agamas social

behavior and social status of individuals of

both sexes appear to be similar during the

first two years of their lives. But thereafter

ontogenetic trajectories of males and females

become progressively divergent.

It is not known if lizards remain in the

vicinity of their birth place during their lives.

Among those juveniles (n=61) that were

captured on the two plots in the western

Kopetdag at the end of September and the

beginning of October (i.e. at the age of two

to three months), only five (8.2%) were

observed near the places of their first capture

(within a radius of 25 m) in next year. The

proportion of such recaptures on one of the

two plots was as high as 25%, while on

another plot none of 41 juveniles caught

during preceding fall was observed later on.

Unfortunately of all those juveniles that

were captured during the first months of

their lives, it remains unknown whether they

remained near their birthplaces or if they

dispersed by the time of capturing

(dispersion of juveniles just after hatching

has been described for Anolis aeneus ;

Stamps, 1988).

More definitive results were obtained

from recaptures of those first year lizards

that were initially captured soon after their

first winter hibernation, in April and May.

Of those lizards 64.5% (20 of 31) were

observed regularly within a radius of 10 to

50 m from the place of their first capture, in

some cases over a period of several years.

First year animals initially occupy small

home ranges of about 10 m in diameter both

outside the territories of adult males (5 males

and 5 females in Krasnovodsk population

in 1985) and within such territories (4 males

and 5 females). When the home ranges of

first year lizards were immediately adjacent

their interactions appeared to be agonistic.

In some cases between adjacent home

ranges a well defined boundary was

established and both neighbors displayed

pronounced territorial behavior toward each

other. In other cases the home ranges

overlapped and a stable rank order was

formed so that one lizard appeared to

dominate the other in the overlap zone.

Generally, the mature members of the

settlement behave indifferently toward

yearlings. However, in periods of high

sexual activity adults may chase the

yearlings short distances.

After the second winter the young

agamas returned from communal den to their

original summer home area where they

knew the topography of the place and their

foraging routes became longer and

pioneering of new feeding places and new

temporary shelters began. Apparent

differences between social behaviors of

young males and females began to emerge

only after their third winter, at an age of

more than 30 months.

Male Ontogenetic Trajectories

Males participate in reproduction only

after they have acquired a territory. Males

continue to reproduce until the end of their

lives. For example, on the Krasnovodsk

plot male N 3 in 1989 at an age of

approximately 12 years had a large territory

that included four adult females of different

ages and two immature females. In addition

the same year we observed interactions

between this male and immature female N

161 in the border zone between his territory

and that of male N 6 1 .

If, on the given territory, the only one

female lives, in the case of her

disappearance a holder of territory becomes

a widower. He however, subsequently

does not try to search for females outside his

territory. In Gobustan, such case of

widowhood was observed in about 6 years

old male. A more young male, evidently,

may also turn out to be a widower.

Vol. 6, p. 104

Asiatic Herpetological Research

June 1995

TABLE 1. Composition and age structure of family groups of the experimental introduced

population at Krasnovodsk and the natural population at Gobustan.

Krasnovodsk

Gobustan

Year

1986

1987

1988

1989

1990

1986

1987

1988

minimum estimated age.

Female Ontogenetic Trajectories

Female rock agamas are able to reproduce at

about three years of age. By this age to be a

successful breeder, a female should

establish a stable bond with a male territory

holder and obtain constant shelters within

his territory. For a female that had a

juvenile home area within a male's territory

the process of assimilation into a family

group and establishment of a bond with the

male is very direct. An example of this was

female N103 who was observed for the first

time in 1986 as a first year juvenile living on

the territory of male N 56 where she

continued to stay at least until 1989 (see Fig.

4d-f and Table 1A).

Females that had juvenile home areas on the

periphery of an adult male's territory, in the

home range of a non-territorial satellite male,

or in a neutral zones where sexually active

males were totally absent usually left and

attempted to establish a bond with a territory

holding male. This process of selection of a

male and territory may take several years

and involve short stays in the areas of

several males. For example, female N 101,

originally having frequented the territory of

male N 56,

June 1995

Asiatic Herpetological Research

Vol. 6, p. 105

1985 1986 1987 1988 1989 1990

Cl J* „

B

Jj

1987 1988

D

. I

1986 1989

1987 1988 1989

1985 1986 1987

mmi CZI2 EE33 VHZ34

FIG. 5. Population structure of study populations.

A. Krasnovodsk. B. Gobustan. C,. Parkhai

Gorge (spring). Cr Parkhai Gorge (fall). D.

.Kalaligez. 1. Yearlings. 2. Second-year lizards.

3. Adult males. 4. Adult females.

subsequently over a period of 3 years

(1988-1990) was resident of the neutral area

visited at different time by two adult

territorial males (NN 56 and 36). Female N

74 resided at ages of a little less than 4 years

temporal home range of 3 year old male N

115 in next year moved on to territory of

male N 3 (Fig. 4 e,f) who

became a permanent target for her courtship

displays.

It is useful to describe shortly a peculiar

courtship behavior of females addressed by

them towards males, which we regard as a

very important mechanism contributing to

establishment and maintenance of personal

bonds between mating partners. At sight of

a male young female moves to him and at

once tries to climb onto his back. A male,

as a rule, during first minutes of contact

tolerates these actions of female who is

crawling over him in different directions and

makes insistent attempts to crawl under him.

After that male behaves as if he is inclined to

retire, while a female pursues him and

repeats her actions. Such a behavior is quite

characteristic of females younger age

classes, even of those lesser than 2 years

old. The behavior retains in older puberal

females, although, contrary to expectation, it

almost never occurs prior to actual sexual

interactions, i.e. copulations. Once a female

has selected a male she begins to co-habit

his shelters. Where there are several

females on a territory usually the oldest

female cohabits with the male.

The home ranges of adult females

overlap broadly, especially if the territory is

large. However, some older females exhibit

territorial behavior in the vicinity of their

shelters, basking sites and foraging areas

when approached by other older female.

Females leave the territory of their

family groups only for egg laying and to

migrate to communal winter shelters. We

did not observe emigration or dispersal of

females. Over the five year study of the

Krasnovodsk population the time of

residence of adult females introduced in

1985 ranged from one to five years (mean =

2.5 ± 0.6, n=6).

Family Groups

The mode of sexual relations in rock

agama settlement may be defined as a

territorial facultative polygyny. As many as

four females may establish bonds with a

territorial male. In the natural Gobustan

population there were 1.73 females per male

on territories. In the introduced

Krasnovodsk population the average

number of females per male was increasing

as population structure matured. Over the

duration of our study the average number of

females within the family groups was 1.33

(1988), 1.86 (1989) and 2.43 (1990).

The term "family group" is not quite

precise since each female enters into such a

unit independently from other females. Any

personal or functional bonds between female

members appear to be absent. Only

relations between the territorial male and

each of the females may be regarded as

bonds.

The stability of such breeding units is

determined primarily by an association its

Vol. 6, p. 106

Asiatic Herpetological Research

June 1995

TABLE 3. Survival of rock agamas introduced as adults in the Krasnovodsk experimental plot in

May 1985. The number of lizards alive is presented; the number is parentheses is the per cent

surviving since the previous year.

TABLE 2. Life table of a cohort of rock agama yearlings introduced into the experimental

population at Krasnovodsk.

1 =

_ f

_X1_

xl

dx = 1xi

lxi+1; qx=£x; px=l-qx(Caughley, 1977)

TABLE 5. Sex ratios in different rock agama populations (n).

Location

1985

1986

Year

1987

1988

1989

1990

Gobustan (spring)

1:1.1(31) 1:0.9(33)

Western Kopetdag

Parkhai Gorge

spring

fall

Kalaligez (fall)

1:0.9(21)

1:1(12) 1:0.8(18)

1:0.7(10)

1:1.2(11)

1:0.8(16)

1:1.6(13)

1:0.7(10)

Krasnovodsk (spring)

1:0.8(20) 1.1(30) 1.1(40) 1:0.9(47) 1:1.2(41)

Grand sex ratio for all localities summed = 1:0.98 (393).

member to certain shelters and to its home

range (or territory) as a whole. All this

seemingly explains why females accept so

readily a new male partner after the death

of a previous mate. For example, male N

36 established bonds with a single adult

female and two immature females (all from

the initial cohort of lizards simultaneously

introduced to the study plot) immediately

upon introduction (Table 1).

Subsequently, upon the deaths of these

females replacement females were

recruited from those subsequently born in

the colony.

Population Dynamics

Rock agamas are long lived and

predominately sedentary. Populations are

characterized by stable membership with

recruitment as the primary source of new

members. Emigration and immigration are

of minor importance. Each of these

factors were examined in the Krasnovodsk

population.

June 1995

Asiatic Herpetological Research

Vol. 6, p. 107

The general picture of changes in the

relative proportions of immatures, matures

and sex ratio is shown in Fig. 5. It is

necessary to repeat that in 1985 the

original colonizing cohort was composed

primarily of immatures, this may have

resulted from capture sampling error. In

1986 the proportion of immatures to adults

was similar but in 1987 and 1988 the ratio

of immatures to adults was nearly equal.

Since 1988 adults have predominated.

Comparison with natural populations (Fig.

5) shows that adults predominate though

relative proportions may vary significantly

among areas and years. Sex ratio, males

to females, is generally equal (Fig. 5;

Table 5).

Recruitment

Surveys of juveniles were conducted

during the spring (Fig. 5) since few

lizards were active during the hottest

weather in summer and early fall.

Differences in the numbers of over

wintering juveniles may be attributable to

embryonic mortality or hatchling mortality

in the first months after emergence or over

the first winter.

Post Hatching Mortality and Survivorship

The survivorship of 25 juveniles

introduced into the Krasnovodsk

experimental plot are presented in Table 2.

We assumed that those lizards that

disappeared, that is, those not recaptured,

had died. Of the initial 25 hatchlings, 15

(10 females, five males) survived to

sexual maturity (three years) and of this

cohort four males (80%) and four females

(40%) survived six years, to the end of the

study. The annual survival rate (px)

ranged from 0.72 to 0.83.

We believe the high survivorship of

lizards in the Krasnovodsk experimental

plot during the first three years of life are

comparable to natural populations. We

tested this by comparing survivorship in a

cohort of 20 hatchlings, hatched in 1986,

1987, and 1988, that were first captured in

1987, 1988 and 1989 as yearlings.

Among these lizards 15 (75%) survived to

two years and 13 (65%) to three years of

age. Of those surviving to three years

were three males and 10 females.

Since the duration of this study was

approximately half the life span of a rock

agama, survivorship in animals older than

six years is based on indirect evidence.

We estimated the age, based on

standardized size criteria (Zykova and

Panov 1991) of adult lizards captured for

introduction onto the Krasnovodsk

experimental plot. Such age estimates are

at best imprecise but the adults in this

group ranged from three to 1 1 years with

78% being four to six years old (Table 3).

The maximum rate of disappearance

(which we attribute to mortality, not

emigration) occurs in the first year (Table

3) after introduction. From the second

year after introduction adult male

survivorship is nearly constant and

comparable to survivorship during the first

six years of life (Table 2). The more

variable mortality rates for females may be

due to small sample size.

We combined cohorts of lizards of

four, five and six years of age and

calculated survivorship and estimated

survivorship to the 9-11 year age class

(Table 4).

In general, survivorship in rock

agamas after the first winter following

hatching is relatively constant until the

eighth year. After the eighth year

mortality increases. The maximum

estimated age of for males was 12-14

years (n=4) and for females it was 9-10

years (n=2).

Sex Ratio

Although the rock agama social system

is territorial polygyny, the sex ratio among

adults does not differ significantly from

even one (Table 5). This relationship was

found at several localities throughout the

range and for all localities taken together.

Vol. 6, p.

108

Asiatic Herpetological Research

June 1995

FIG. 6. Expansion of the introduced colony at

Krasnovodsk from 1985 to 1990. 1. Unsuitable

habitat. 2. Boundary of unsuitable habitat. 3.

Principal ridges. 4. Initial introduction site. 5.

Principal study area under regular observation. 6.

Subpopulation established by an introduced adult

male. 7. Subpopulation established by an

introduced immature male. 8. Subpopulation

established by offspring of introduced lizards. 9.

Single sighting of rock agama. 10. Boundary of

populated area in 1990.

Movement and Dispersion

Rock agamas display a high level of home

area fidelity. Juveniles that were marked

after their first winter were found to

remain within 50 m of that location up to

two years or until sexually mature (at two

to three years of age). Some data on the

capture of juveniles before their first

winter suggests that home site fidelity may

extend from the age of three or four

months to the end of life. Four of six

juveniles (66%) marked in October 1988

in the Parkhai Gorge (Western Kopetdag)

were found in the same place the next

spring. The maximum movement of

immature lizards was not greater than 300

m. A juvenile male first captured in

October 1985 before his first winter was

recaptured in 1987, 200 m from the first

capture location. He was recaptured in the

spring of 1988 as a mature male with a

territory 250 m from the previous capture

location and 100 m from the initial capture

point 3 1 months before.

As the population increases, new areas

will be pioneered, mainly by young

dispersing lizards. Given the sedentary

habits of these agamas we would expect

such expansion to be relatively slow.

Such an expansion occurred in the

introduced population at Krasnovodsk

(Fig. 6). From 1985 to 1990 the initial

introduced population occupying an area

of approximately 300 m2 dispersed into

adjacent areas of approximately 25 ha. Of

the seven subpopulations formed during

this period two (Fig. 6, points 1 and 2)

were founded by introduced mature adult

lizards after the initial introduction. Two

other subpopulations (Fig. 6, points 3 and

6) were founded by lizards that were

immature when introduced. Finally, three

subpopulations were established by the

offspring of the original introduced

lizards. In 1988 (the fourth year after

introduction) there were 33 individuals (13

adult males, 14 mature females and 10

immatures) in these seven subpopulations.

Individual lizards were observed to

leave their home areas only for collective

winter shelter. Females may leave their

home areas looking for places appropriate

for egg laying (Danieljan and Grigorjan

1975; Ananyeva and Danieljan 1987).

Conclusions

As it can be seen, the characteristic

features of Rock Agama social

organization and demography are high

stability of breeding individuals'

contingent and low population turnover.

This is evident consequence of sedentary

way of life characteristic for males of all

age classes, longevity of these lizards, and

postponed onset of breeding in early

lifetime of young agamas.

The latter may be especially applied to

males. Although they are capable to

reproduce already at the age of a little

under three years (after their third

wintering), most of them begin to breed,

actually, only at the age of four or five

June 1995

Asiatic Herpetological Research

Vol. 6, p. 109

years. How it may be seen, a male attains

the status of a breeder only after he had

taken possession of territory of his own.

So each maturing male faces with obvious

difficulties since many features of species'

social organization of settlement (high

density together with strong territoriality

of males retaining control over his home

area until his death) lead to deficiency of

vacancies which might be used by young

male-recruits. Temporary exclusion of

part of mature (non-territorial) males from

process of reproduction may, in principle,

decrease the whole reproductive potential

of the local population.

Besides such social regulators of

population growth, rapid increase of

population size is retarded also by rather

slow recruitment of new deme members.

Although breeding productivity of Rock

Agama is relatively high (from seven to

ten eggs per mature female during

breeding season- see Ataev, 1985), only

a small number of new-born agamas die.

Even if these losses (especially the latter

figure) is overestimated, the analysis of

demographic structure of all demes under

study shows the numerical preponderance

of mature individuals over immature ones.

This is in good agreement with the general

conclusion about the low rate of

population growth in Rock Agama.

Another argument in favor of this

conclusion is a quite slow expansion of

growing population into new, early

unoccupied areas.

To conclude, it may be stated that

Rock Agama give us a good example of

lizard species practicing a typical K-

strategy. It was to be expected providing

large size of individuals and the ecological

peculiarity of the species- in particular, its

pronounced omnivorousness with

prevalence in diet (at least in respect to

biomass) of diverse plant objects. It is

noteworthy that in Rock Agama, like in

many species of higher vertebrates (birds

and mammals) practicing K-strategy,

among deme members there are

considerable number of mature male being

excluded from reproduction by density-

dependent social factors.

Acknowledgments

We are indebted to Drs. Mira E.

Gauzer, Vladislav I. Vasiljev

(Krasnovodsk State Reserve) and Nikolaj

Andreev (Sunt Khasardag State Reserve)

for their help in organization of field work

and for participation in catching lizards.

We also wish to express our sincere

thanks to Prof. Ilya S. Darevsky, Drs.

Natalia B. Ananjeva (Zoological Institute,

Russian Academy of Sciences, St.

Petersburg) and Valentina F. Orlova

(Zoological Museum, Moscow

University) for their assistance and help

during our work with museum collections.

The final stage of our research was

supported by the Soros Foundation.

Literature Cited

ANANYEVA, N. B. AND C. ATAEV. 1984.

[Stellio caucasius triannulatus ssp. nov. - a new

subspecies of the Caucasian agama from

southwestern Turkmenia]. Trudy

Zoologicheskogo Instkuta Akademii Nauk SSR

124:4-11. (In Russian).

ANANYEVA, N. B. AND F. D. DANIELJAN.

1987. [Seasonal migrations of rock agama,

Stellio caucasius Eichwald, 1981, in Armenia.].

Trudy Zoologitcheskogo Institute Akademii Nauk

SSR 158:33-38. (In Russian).

ATAEV, C. 1985. [Reptiles of the mountains of

Turkmenistan!. Ylym Publishing House,

Ashkabad. 343 pp. (In Russian).

DANIELJAN, F. D. AND E. S. GRIGORJAN.

1975. [On the population number and migrations

of the rock agama Stellio caucasius Eichwald in

Armenia]. Pp. 62-63 In [Fauna and its protection

in the Transcaucasian republics].. Erevan. (In

Russian).

PANOV, E. N. AND L. Y. ZYKOVA. 1985.

[Comparative biology the steppe and rock agamas

{Agama sanguinolenla, A. caucasica) in the

Sumbar River basin (Western Kopetdag)]. Pp.

185-204 In [Vegetation and animal world of the

western Kopetdag]. Ashkhabad. (In Russian).

PANOV, E. N. AND L. Y. ZYKOVA. 1986.

[Notes on the behavior of the steppe agama,

Agama sanguinolenta. I. General features of the

biology, spatial structure of the population and

Vol.6, p. 110

Asiatic Herpetological Research

June 1995

social behavior]. Zoologichesky Zhurnal,

Moscow 65:99-109. (In Russian).

PANOV, E. N. AND L. Y. ZYKOVA. 1989.

[Dynamics of social relations in a population of

rock agamas, Stellio caucasius Eich.]. Pp. 184-

185 In [Questions of Herpetology. Abstracts of

the Reports at the Seventh All Union Conference

of Herpetology, Naukova Dumka, Kiev]. (In

Russian).

PANOV, E. N., L. Y. ZYKOVA, M. E. GLAUZER

AND V. I. VASIL'ER. 1987. [Zone of

intergradation of different forms of Stellio

caucasius compex in South-western Turkmenia].

Zoologichesky Zhurnal, Moscow 66: 402-411.

(In Russian).

SEMENOV, D. V. 1989. [Spatial organization in

populations of reptiles. Achievements of science

and technology, ser. Vertebrate Zoology]. VINITI

17: 101-134. (In Russian).

STAMPS, J. 1988. Conspecific attraction in

territorial species. American Naturalist 131:329-

347.

WILEY, R. H. 1981. Social structure and

individual ontogenesis: problem of description,

mechanism and evolution. Perspectives in

ethology 4:105-133. Plenum Press, New York.

ZYKOVA, L. Y. AND E. N. PANOV. 1991.

[Long-term study of growth in the rock agama,

Stellio caucasius ]. Zoologichesky Zhurnal,

Moscow 70: 81-90. (In Russian).

I June 1995

Asiatic Herpctological Research

Vol. 6, pp. 111-113 |

A Study on the Comparative Cytology of Some Endocrine Glands in

Rana plancyi between Hibernation and Post-hibernation

JlQlNG

Department of Biology, Xuzhou Teachers College, Xuzhou, Jiangsu, 221009 China

Abstract: -This paper studies the ultrastructure of the pituitary gland cell and the adrenal cortex cell in Rana

plancyi between hibernation and post-hibernation. The results show that the two kinds of cells mentioned above

are much less inactive during hibernation than during post-hibernation. The significance of those results is also

discussed.

Key words: Amphibia, Anura, Rana plancyi, pituitary gland, adrenal cortex, comparative cytology.

Introduction

Hibernation is an adaptive strategy of

frogs for keeping out of the cold during the

winter. Studying the hibernation biology of

frogs is beneficial to protecting the frogs and

making use of frog resource. No paper

related to the comparative cytology of the

endocrine glands in frogs has been reported

in China for many years. This paper reports

the results of studying the comparative

cytology of the pituitary gland cell and the

adrenal cortex cell in Rana plancyi between

hibernation and post-hibernation by using a

transmission electron microscope.

Methods

According to the regular pattern of

hibernation in the locality, many specimens

of Rana plancyi were collected from a little

river in October 1992, in the suburbs of

Xuzhou City, put into a box, and then laid

outdoors during the winter. Some

specimens, which represent the hibernation

group, were fetched from the box and used

for the experiment on January 8, 1993.

Some specimens representing the post-

hibernation group were collected from the

same little river mentioned above and were

used to do the experiment on May 17, 1993.

Four specimens were collected from the

two groups, regardless of their sex. The

specimens were killed and dissected so that

the pituitary gland and the adrenal gland were

obtained. The two glands were fixed with

4% glutraldehyde and embedded in Epon-

812. Ultra-thin sections were doubly stained

with uranyl acetate and lead citrate by the

standard method, and the specimens were

examined with a Hitachi 600-A-II electron

microscope.

Results

Pituitary gland cell

The pituitary gland cell of the hibernation

group has plenty of glycogen particulates (PI.

1:1) but few rough endoplasmic reticulum and

mitochondrium (PI. 1:2), while the pituitary

gland cell of the post-hibernation group has

plenty of rough endoplasmic reticulum and

Golgi bodies but no glycogen particulates

(PI. 1:3). The pituitary gland cell of the post-

hibernation group also has plenty of secretory

granules (PI. 1:4), and some secretory

granules can sometimes be seen moving to

blood capillary (PI. 1:5).

Adrenal cortex cell

According to the result of the examination

of the semi-thin sections, the adrenal cortex

in the adrenal gland was defined. The

adrenal cortex cell has tubular cristate

mitochondrium as its marking. The adrenal

cortex cell has plenty of lipid drops, some

smooth endoplasmic reticulum and few

mitochondrium (PI. 1:6, 7) during

hibernation, but has plenty of mitochondrium

(PI. 1:8), few lipid drops and plenty of

expanded smooth endoplasmic reticulum (PI.

1:9).

Discussion

The pituitary gland is the most important

endocrine gland in the frog and plays an

© 1995 by Asiatic Herpetological Research

Vol. 6, p. 112

Asiatic Herpetological Research

June 1995

Plate I

1. Pituitary gland cell showing glycogen particulates during hibernation (arrows). X 12 000

2. Pituitary gland cell showing mitochondrium (Mt), rough endoplasmic reticulum (RER), Golgi bodies (GB)

and secretory granules (SG) during hibernation. X 15 000

3. Pituitary gland cell showing Mt, RER and GB during post-hibernation. X 20 000

4. Pituitary gland cell showing SG during post-hibernation. X 17 000

5. Pituitary gland cell showing SG near the blood capillary (BC) during post-hibernation. X 20 000

6. Adrenal cortex cell showing lipid drops (LD) and Mt during hibernation. X 20 000

7. Adrenal cortex cell showing smooth endoplasmic reticulum (SER) during hibernation. X 20 000

8. Adrenal cortex cell showing Mt during post-hibernation. X 20 000

9. Adrenal cortex cell showing expanded SER and LD during post-hibernation. X 20 000

O

GB

Mt

-

?6

»*

SG

Mt"

GB

RER

.--■

£*£$&■ •-■..._•■ ■

■>•■.

fe

N

••

•^SG

fc-

BC

*.

1 SG

■ ••

■

5 f~*t

Mt

SER

Mt

%

SER

jgg

June 1995

Asiatic Herpetological Research

Vol. 6, p. 113

important role in its hibernation. It can

secrete, releasing hormone which can

regulate the activity of the other endocrine

glands. The studies on frog cytology have

not concerned the pituitary gland for many

years. Previous work (Daguy, 1963; Saint

Girons, 1975) has showed that the pituitary

gland in some reptiles increase in activity

several weeks before the end of hibernation.

This investigation shows that the pituitary

gland cell is inactive during hibernation, but

active during post-hibernation. It also

suggested that the behavior of the pituitary

gland in frogs is similar to the same gland in

reptiles during hibernation and post-

hibernation.

The adrenal cortex can secrete

glucocorticoid and mineralocorticoid. The

two kinds of hormones can regulate

glucometabolism and mineralometabolism.

These two kinds of hormones are both

synthesized in the smooth endoplasmic

reticulum, so that the number of smooth

endoplasmic reticulum can indicate the

secreting level of the two kinds of hormones.

Previous work (Robertson et al., 1959; Chan

et al., 1971) have shown that the adrenal

cortex is inactive during the winter and active

in the summer. This investigation also

shows that the adrenal cortex cell is active

during post-hibernation and inactive during

hibernation. According to this investigation,

it is considered that the activity of the adrenal

may begin from the end of hibernation.

Acknowledgments

The author is deeply indebted to

Professor Shou-chang Zou for having

supplied the materials and for his helpful

advice throughout the research. The author

also wishes to thank Professor She-chao

Chen for his excellent technical assistance.

Literature Cited

CHAN, S. W. C. AND J. G. PHILIPS. 1971.

Seasonal variations in production in vitro of

corticosteroids by frog (Rana rugulosa) adrenal.

Journal of Endocrinology 5:1-17.

DAGUY, R. 1963. Biologie de la latence hibernale

chez Vipera aspis. Vie Milieu 1963 14:311-443.

ROBERTSON, O. H. AND B. C. WEXLER. 1959.

Histological changes in the organs and tissues of

migration and spawning Pacific salmon (Genus

Oncorhynchus). Endocrinology 66:222-239.

SAINT GIRONS, H. 1975. Le cycle annuel des

glandes endocrines chez les serpents des regions

temperas. Bulletin Zoology of France 100:654-655.

I June 1995'

Asiatic Herpetological Research

Vol. 6, pp. 114-119

Reproductive Behavior in the Long-tailed Salamander (Onychodactylus

fischeri Boulenger).

I. A. Serbinova1 and V. A. Solkin2

'Moscow Zoo, B. Gruzinskaya 1 , Moscow 12342 Russia

2Pacific Ocean Institute of Geography, Far East Branch of Russian Academy of Sciences, Vladivostok,

690032, Russia

Abstract. - We studied reproductive behavior in wild and captive Onychodactylus fischeri. Reproductive

behavior and physiology is triggered by warming water temperatures in the spring. Pair formation begins at

7° C and spawning at 9° C water temperature. Fertilization is external. The development of distinctive

femoral musculature and skin on the posterior surface of the hindlimbs in the male is for grasping the eggs

during fertilization. Pairs of salamander experimentally injected with a synthetic analogue of gonadotropin

releasing hormone exhibited typical mating behavior and produced fertile clutches of eggs.

Key words: Onychodactylus fischeri , salamander, reproduction, external fertilization,

courtship, gonadotropin releasing hormone.

Introduction

The long-tailed salamander,

Onychodactylus fischeri , is one of the most

poorly known amphibians in Russia.

Emeliyanov (1947) noted that spawning

takes place from the time salamanders

appear in the spring until the middle of July.

Regel and Epshtein (1975) concluded that

reproduction in this species is not restricted

to a specific season. The discovery of a

clutch with some hatched larvae indicated

that oviposition probably occurred in the

spring (Kozik, 1991). It is difficult to

establish the precise time of oviposition

since the length of embryonic development

for this species is unknown. The length of

embryonic development for the related

species O. japonicus is 120 days (Hayase

and Oseki, 1983) and it breeds in the winter

(Akita, 1989). The absence of precise

information on the timing and method of

reproduction of the long-tailed salamander

prompted us to undertake this study.

Methods and Materials

This study was conducted from May

through July, 1991 on the Primorsky krai a

tributary of the Mineral'naya River.

Daylight observations of the location and

reproductive state of salamanders in nature

were made along a 1000m transect along

Kitaisky Spring. The search effort was

directed to the time of maximum diurnal

activity. All captured salamanders were

individually numbered by toe-clipping using

standard techniques. A total of 601

salamanders were captured of which 102

were recaptured. All salamanders were

measured and weighed on a triple beam

balance. We noted the degree of

development of such male secondary sexual

characters as the distinctive femoral

musculature and skin development on the

posterior surface of the hind limbs. We

recorded such female characteristics as the

relative size of oocytes observed through the

abdomen, their passage into the oviducts

and the condition of the ventral opening to

the cloaca.

The long-tailed salamander has secretive

habits and we determined that observation of

natural oviposition was unlikely. Therefore,

we stimulated reproductive behavior

hormonally in captive individuals.

Salamanders were housed outdoors in the

shade of an awning in 8 liter aquaria with 3-

5 cm of running water and shelters.

Aquarium water was changed daily. Each

of seven males and seven females were

injected with 1-5 |ig/individual/day of a

synthetic analogue of gonadotropin releasing

hormone for 7-12 days. All individuals

were determined to be ready for breeding

before injection. Males and females were

kept separate until ovulation began; then

they were paired.

© 1995 by Asiatic Herpetological Research

June 1995

Asiatic Herpetological Research

Vol.6, p. 115

number of animals

temperature

1 4 7 10 13 16 19 22 25 28 31 3 6 9

may June

— — water temperature — B~ non-breeding animals

-*- breeding animals

FIG. 1 . Pattern of Onychodactylus fischeri activity and water temperature during the spring..

number of males

- *- non-breeding — s— breeding

FIG. 2. Frequency occurrence of breeding and non-breeding male Onychodactylus fischeri.

Vol.6, p. 116

Asiatic Herpetological Research

June 1995

number of females

21

may

with opened cloaca

oocytes in oviducts

5 8

June

body cavity oocytes

spawned

14

FIG. 3. Frequency occurrence and reproductive state of female Onychodactylus fischeri.

Results and Discussion

The first observations of active

salamanders were made in late April to early

May at a water temperature of approximately

3° C and while sections of the stream were

still covered with ice. The number of

animals active each day increased as

temperatures warmed during the spring

(Fig. 1). The first salamanders observed

did not exhibit readiness for breeding.

Oocytes were not visible through the

abdomen wall of females and males did not

exhibit hindlimb muscle and skin

development. All animals were in a poor

nutritional state. The peak number of non-

reproductively ready salamanders occurred

on 12 May when water temperature reached

6° C. Not all salamanders leave their winter

shelters simultaneously due to differential

warming rates along the slopes and this is

reflected in Fig. 1. After 12 May the

number of non-reproductive salamanders

decreased to a constant level by 20 May.

The peak number of reproductively

ready salamanders was encountered on 17

May when the last stretches of the stream

were ice free. These animals appeared to be

well nourished. From 15 May to 15 June a

majority of the males encountered were in

breeding condition (Fig. 2).

By 20 May post-hibernation

aggregations of salamanders began to

disintegrate and the individuals dispersed

along the stream. This marked the

beginning of the breeding season.

By 10 May we began encountering

females with opened cloacas (Fig. 3). By

the end of May females with ovulated

oocytes in the body cavity were observed

and by 10 June we found salamanders with

oocytes in the oviducts. We first observed a

female that had spawned or oviposited her

eggs on 9 June. In early June we observed

3 male-female pairs of salamanders with at

least one member in reproductive readiness.

These observations agree with previous

accounts (Regel and Epshtein, 1975).

In the hormonally treated females the

caudad displacement of oocytes usually took

April

1995

Asiatic Herpetological Research

Vol. 6, p. 117

FIG. 4. Courtship behavior of Onychodactylus

fischeri with the male (light salamander)

approaching the female's (dark salamander) vent,

rubbing against the female's abdomen and straddling

the female.

two to four days after injection. In another

day oocytes appeared in the body cavity and

three days later they filled the oviducts.

From hormone injection to readiness for

spawning usually took from six to 10 days.

We observed 6 cases of spawning, two of

which were followed by spermatophore

deposition.

In the hormonally treated animals

approximately one to two days before

spawning the female salamander ceased

most activity and assumed a typical pre-

spawning posture on a stone with the rear

third of the body in the water. At this time

the male moves about the aquarium,

periodically approaches the female touching

his snout to her vent, rubbing his body on

hers and resting beside her (Fig. 4). The

FIG. 5. Comparison of the male Onychodactylus

fischeri hindlimbs and tail in normal posture and in

the pre-fertilization posture with the legs extended.

Note the distinctive heavy femoral musculature and

skin development on the posterior of the hindlimbs.

male then extends his rear legs and holds

this position (Fig. 5).

When spawning begins the female

attaches a mucous cord to the stone with a

pair of egg sacs each containing one to eight

eggs. The eggs are five to six mm in

diameter. The mucous capsule is thick and

strong unlike that of Salamandrella

keyserlingii and Ranodon sibiricus. During

egg deposition (30-40 min.) the male

remains sitting beside and in contact with the

female. When the egg sacs appear the male

enters the water and approaches the female's

vent with his snout. In this position the

male's body begins to undulate. When the

male nudges the egg sac with his snout this

causes a burst of excited thrashing from side

to side by his snout. This dislodges the egg

sac from the female. The male moves

immediately over the egg sac with his legs

extended and grasps the egg sac with his

forelimbs, positioning it between his

hindlimbs. The male then grasps the egg

sac with the hindlimbs and pulls the base of

the sac against his vent and deposits the

spermatophore (Fig. 6).

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Asiatic Herpetological Research

June 1995

F IG. 6. Male Onychodactylus fischeri grasping

the egg sac with the hindlimbs in apposition with

the vent during spermatophore deposition. Note the

distinctive heavy femoral musculature and skin

development on the posterior of the hindlimbs.

Those individuals that are ready for

breeding emerge from hibernation later than

those that are not reproductively ready. We

believe that the triggering mechanism for

breeding is the elevation of the stream

temperature as can be seen in the increased

activity of reproductively ready salamanders

when the water temperature reaches 6 to 7°

C (Fig. 1). At this time females with open

cloacas begin to appear. When water

temperatures have reached 8° C most

females have opened cloacas and many have

oocytes in the body cavity. At 9° C water

temperature some females have oocytes in

the oviducts. By the time water temperature

reaches 10° C most females have oocytes in

the oviducts and some are beginning to

spawn (Fig. 3). We believe increasing

temperature is the primary triggering