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Amphibian & Reptiie Conservation 8(1) [Special Sec]: 1-7.

Rediscovery of Andinophryne o/a//a/ Hoogmoed, 1985 (Anura,

Bufonidae), an enigmatic and endangered Andean toad

^Ryan L. Lynch, ^Sebastian Kohn, ^Fernando Ayala- Varela, ^Paul S. Hamilton, and ^Santiago R. Ron

^The Biodiversity Group, Tucson, Arizona, USA ^Rw Manduriacu Cooperative, Quito, ECUADOR ^Museo de Zoologia, Escuela de Biologia,

Pontificia Universidad Catolica del Ecuador, Quito, ECUADOR

Abstract . — ^We report the rediscovery of Andinophryne olaiiai, an endangered species only known

from a single specimen, collected in 1970. At the type locality, Tandayapa, Pichincha Province,

numerous follow-up surveys after 1970 failed to record the species suggesting that the population

is extinct. The rediscovery of A. oiailai took place in 2012 at Ri'o Manduriacu, Imbabura Province,

Ecuador. Two surveys suggest that a healthy population of A. olaiiai survives at the site, with

observations of froglets, juveniles, and adults across numerous stream systems. However, the

extent of known occupancy of the population is small (<1 km^). Further data are presented to update

knowledge of the distribution, ontogeny, morphology, and conservation status of the species. The

population at Ri'o Manduriacu is surrounded by logging, mining, and hydroelectric developments

that could compromise its future survival. There is an urgent need to establish a monitoring program

and to protect its remaining population and habitat in the region.

Key words. Andinophryne olaiiai, rediscovery, Tandayapa Andean toad, Andinosapo de Olalla, Bufonidae, Endan-

gered species, Ecuador

Citation: Lynch RL, Kohn S, Ayala-Varela F, Hamilton PS, Ron SR. 2014. Rediscovery of Andinophryne olaiiai Hoogmoed, 1985 (Anura, Bufonidae), an

enigmatic and endangered Andean toad. Amphibian & Reptile Conservation 8(1) [Special Section]: 1-7 (e75).

Introduction

The small and understudied toad genus Andinophryne

(Bufonidae) is restricted to the western slopes of the

Andes in Colombia and Ecuador. Three species of An-

dinophryne have been described: Andinophryne atelo-

poides (Lynch and Ruiz-Carranza 1981), Andinophryne

colomai (Hoogmoed 1985), and Andinophryne olaiiai

(Hoogmoed 1985). Until recently, all three species were

only known from five or fewer adult individuals at the

type localities: A. atelopoides (Cauca Department, Co-

lombia, 1980), A. colomai (Carchi Province, Ecuador,

1984), and A. olaiiai (Pichincha Province, Ecuador,

1970).

The paucity of information available on Andinophryne

has led to many questions about the taxonomic and con-

servation status of all three species. Andinophryne at-

elopoides, the only species endemic to Colombia and

only known from two specimens, was originally placed

in the genus Bufo by Lynch and Ruiz-Carranza (1981).

Four years later, following the discovery of two similar

bufonid species (A. colomai and A. olaiiai) in northern

Ecuador, and the reexamination of information presented

on B. atelopoides by Lynch and Ruiz-Carranza (1981),

Hoogmoed (1985) created the genus Andinophryne (Bu-

fonidae), and placed all three species within the new ge-

nus.

Despite numerous attempts by trained scientists and

over 150 search hours, subsequent visits to the type lo-

calities of A. colomai and A. olaiiai in Ecuador have

failed to record either species (Coloma et al. 2004; Ron

and Frenkel 2013). Then, in 2005, Murillo et al. (2005)

reported a 160 km range extension for A. olaiiai in Rio

Nambi, Department of Narino, Colombia. This observa-

tion marked the first record of any Andinophryne species

in more than two decades. However, as part of our recent

work with Andinophryne, a member of our team recently

examined a specimen from Rio Nambi and determined

that it was not Andinophryne olaiiai but a different spe-

cies (Santiago Ron, unpubl. data). This identification has

been confirmed by additional fieldwork and specimens

collected at Rio Nambi by Paul David Gutierrez-Carde-

nas (pers. comm.). Therefore, A. olaiiai is the rarest of all

Andinophryne species, with the only known record being

the original type specimen from Tandayapa, Pichincha

Province, Ecuador in 1970.

Forty-three years after the original description of

Andinophryne olaiiai, we report the rediscovery of a

population of A. olaiiai from Rio Manduriacu (herein

Manduriacu), Imbabura Province, Ecuador. We also pro-

Correspondence. ^ryan@ biodiversitygroup.org (corresponding author); ^sebastiankohn® hotmail.com; ^fpayala2000@ gmail.

com; Hamilton @ biodiversity group, org; ^Santiago, r. ron@ gmail. com

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (1) February 2014 | Volume 8 | Number 1 | e75

Lynch et al.

-a 1,0 'M.O -?9,0 -7?,0

-B1.0 -BO.a -70.0 -78.0 -77.0

Fig. 1. Known records of Andinophryne olallai in northwest

Ecuador; Tandayapa: Type Locality, Manduriacu: New Local-

ity.

vide the first information on the species’ natural history,

geographic range, ontogeny, and conservation status, and

present the first published color photos of live individu-

als across different age classes.

Materials and Methods

Our surveys took place in the premontane tropical for-

est and cloud forests of Manduriacu in NW Ecuador

(1,100-1,400 m), 40 km N of the type locality of A. olal-

lai and near the south border of the Cotacahi-Cayapas

Ecological Reserve (Eig. 1). Surveys were conducted

on 18 November 2012 (original rediscovery) and 13-15

May 2013 using Visual Encounter Surveys (VES) along

stream transects between 19:00 and 01:00 h.

The objectives of the surveys were: (1) determine the

population status of A. olallai', (2) determine the extent

of its occupancy in Manduriacu; and (3) obtain informa-

tion about the behavior and natural history of the species.

Surveys were carried out along small rocky streams with

overhanging herbaceous vegetation (Eig. 2). A total of

three nights were spent surveying four stream systems

neighboring the site of initial discovery (approximately

100 m between streams; < 1 km^ area total).

Information collected in the field included: air temper-

ature (°C), relative humidity (%), time of encounter (24

hr), perch height (cm), snout- vent length (SVL, mm), sex

(when possible), and age class (froglet, juvenile, adult).

Froglets (i.e., recently metamorphosed individuals) were

defined as individuals with heavily patterned dorsum,

lack of pronounced parotoid glands, and SVL between

10-20 mm. Juveniles were defined as individuals with

faint dorsal patterning, more pronounced parotoid glands,

and SVL between 20-30 mm. Adults were defined as

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (2)

individuals with no dorsal patterning, very pronounced

parotoid glands, presence of large cream- tan colored tu-

bercles on the flanks, and SVL above 30 mm.

Perch height for each individual was measured using

a marked meter stick and SVL measurements were taken

using dial calipers. Climate information was recorded us-

ing a handheld Kestrel 3500 Weather Meter. Individual

toads were only handled when necessary, and always

with use of latex gloves to prevent transferring pathogens

such as amphibian chytrid fungus (Batrachochytrium

dendrobatidis).

Results and Discussion

During the first survey of Manduriacu on 1 8 November

2012, two adult A. olallai were encountered perched on

leaves overhanging a small running stream. Elevation

of the observation site was 1,253 m, and perch heights

of the individuals were 1.5 m and 2.0 m above ground.

Both individuals appeared to be females, based on size,

with SVL of 57 and 58 mm, however sex could not be

determined with complete certainty in the field because

no secondary sexual characteristics are evident in live

Table 1. Reptiles and amphibians associated wiihAndinophyne

olallai at Manduriacu, Imbabura Province, Ecuador and their

current (August 2013) lUCN and EaunaWebEcuador Red List

status (NE - Not Evaluated, DD - Data Deficient, LC - Least

Concern, NT - Near Threatened, V - Vulnerable, EN - En-

dangered). lUCN Red List available at: http://www.iucnredlist.

org/; EaunaWebEcuador Red List available at: http://zoologia.

puce.edu.ee/vertebrados/anfibios/EspeciesEstadoConserva-

cion.aspx.

Species

Fauna Web

Ecuador Red

List

lUCN Red

List

Caecilia guntheri

DD

Centrolene peristictum

NT

V

Epipedobates darwinwallacei

EN

NE

Espadarana prosoblepon

LC

Hyloscirtus alytolylax

NT

Pristimantis achatinus

LC

Pristimantis calcarulatus

LC

V

Pristimantis labiosus

NT

LC

Pristimantis luteolateralis

NT

Pristimantis muricatus

V

Pristimantis scolodiscus

DD

EN

Rulyrana orejuela

DD

A lopoglossus festae

NT

NE

Anolis aequatorialis

NT

NE

Anolis gemmosus

LC

Basiliscus galeritus

NE

Bothriechis schlegelii

NT

NE

Cercosaura vertebralis

DD

NE

Diaphorolepis wagneri

NT

NE

Lepidoblepharis conolepis

EN

NE

February 2014 | Volume 8 | Number 1 | e75

Rediscovery of Andinophryne olallai

animals. This initial observation yielded two signifieant

findings: the first evidence of an A. olallai population in

43 years and the second known locality for the species

extending its known range 40 km N from its type locality.

During the course of the survey in May 2013 a total

of 18 A. olallai were observed across four stream sys-

tems. Average nightly environmental conditions during

the three nights of surveys in May were: air temperature

18.3 °C and relative humidity 92.8%. We recorded the

presence of adults, juveniles, and froglets, indicating on-

going population recruitment (Fig. 3). Eleven of the nine-

teen individuals encountered were adults, and although

their sex could not be determined, eggs were visible in

the abdomen of two gravid females. The sex of one pre-

served adult male (QCAZ-A 55561) was confirmed by

internal gonad examination. The confirmed adult females

had SVL of 57 mm and 60 mm, considerably larger than

the SVL reported by Hoogmoed (1985) for the holotype

($, 39.6 mm). The single confirmed male had a SVL of

36.5 mm. Mean SVL for adults with unknown sex was

47.1 mm (n = 8).

All individuals encountered were perched on branch-

es or leaves overhanging or bordering the streams. Mean

perch height was 1.4 m (n = 18), with adults generally

perching higher than younger individuals. Maximum ob-

served perch height was four meters. Although no official

surveys were conducted during the day, no individuals

were observed along streams during random daytime

walks. Although further behavioral work needs to be con-

ducted, this observation suggests that A. olallai may be

actively foraging during the day in the forests surround-

ing streams. At night, they remain immobile perched on

leaves overhanging the streams. Lack of movement may

protect them from predators.

Ontogeny and Morphology

All information on A. olallai reported by Hoogmoed

(1985) was based on two adult specimens. Our obser-

vations of froglets and juveniles mark the first reported

information on the species’ pre-adult morphology and

ontogeny. Ontogenetic change in color pattern is con-

siderable (Fig. 3), and is one of the few reported cases

of such an extreme change in bufonids in Ecuador (see

Hoffman and Blouin 2000). We observed a total of two

froglets (mean SVL 13.1 mm) and five juveniles (mean

SVL 26.6 mm). Eroglets have a copper, gold, and white

dorsum with a mottling pattern reminiscent of some

species of Atelopus (Eig. 3: A, B). This contrasts with

the patternless brown dorsum of the adults. The venter

of froglets have a series of white undulating lines that

extend the length of the body (Eig. 4). The iris in frog-

lets and juveniles is more vibrantly red than in adults,

which have a yellow copper-colored iris that is darker

medially near the horizontally oval pupil. Froglets also

differ from adults in lacking tubercles and parotoid

Fig. 2. Andinophryne olallai habitat from Rio Manduriacu,

Imbabura Province, Ecuador. All individuals encountered were

found perched on branches or leaves along streams similar to

the stream pictured here.

glands. Juveniles retained some of the mottling pattern

seen in froglets (primarily posteriorly on the hind legs)

and lacked the conspicuous tubercles on the flank (Fig.

3: C, D). However, they begin to show adult traits like

pronounced parotoid glands, tan-brown coloration, and

strongly webbed fingers.

Morphological characteristics of the adults match

those of the holotype of A. olallai (comparisons based

on photographs of the holotype, available at Link/URL:

Amphibiaweb Ecuador, and Hoogmoed 1985). The holo-

type and the observed specimens of the population from

Manduriacu differ from the other species of the genus

in having more developed parotoid glands, larger body

size, strongly webbed fingers, and conspicuous yellow-

ish glands scattered on the flanks and arranged in rows

or in irregular patterns (Fig. 3: E, F) (Hoogmoed 1985).

The dorsal texture varies from smooth to mildly tuber-

culate. One individual had abundant tubercles on the

anterior half of the dorsum and large scattered tubercles

on the posterior half. The description of coloration given

by Hoogmoed (1985) was of an animal in preservative;

however, the color description falls within the variation

observed in life at Manduriacu. The only notable differ-

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (3)

February 2014 | Volume 8 | Number 1 | e75

Lynch et al.

Fig. 3. Ontogenetic transformation of color and pattern in Andinophryne olallai from Rio Manduriacu, Imbabura Province, Ecuador.

(A) Froglet (11 mm SVL; in situ), (B) Froglet (15.1 mm SVL; in situ), (C) Juvenile (26.3 mm SVL; in situ), (D) Juvenile (28.1 mm

SVL; in situ), (E) Adult (44.6 mm SVL; ex situ), (F) Adult (53.3 mm SVL; in situ). Note the progressive ontogenetic change in dor-

sal patterning from heavily mottled to no pattern; lack of parotoid glands and tubercles along the flank to presence of conspicuous

parotoid glands and tubercles along the flank; a darkening of color from copper, tan, and white to dark brown; and iris color change

from vibrant crimson to copper-orange.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (4)

February 2014 | Volume 8 | Number 1 | e75

Rediscovery of Andinophryne olallai

Fig. 4. Ventral pattern of froglets of Andinophryne olallai.

Manduriacu, Imbabura Province, Ecuador.

ence is that dorsal and flank coloration is not uniform in

all individuals; the head and dorsum were darker brown

than the light brown-tan flanks in most live animals ob-

served at Manduriacu.

Sympatric Species

During our herpetofaunal surveys of Manduriacu we re-

corded observations of all amphibian and reptile species

occurring at the site (Table 1). Most of these species are

mid-elevation (1,000-2,500 m) inhabitants of premon-

tane and cloud forests of the eastern Andes. A number of

the species (i.e., Lepidoblepharis conolepis, Pristimantis

scolodiscus) are either nationally or internationally listed

as Endangered, and two of the species are categorized

as Data Deficient or have not yet been assessed (i.e., Di-

aphorolepis wagneri, Epipedobates darwinwallacei) and

very little is known about their biology or conservation

status due to few available records or localities.

Conservation and Threats

Andinophryne olallai is currently classified as Data

Deficient by the lUCN Red List (Coloma et al. 2010).

However, more recent assessments considers A. olallai

as Endangered based on its restricted range, the appar-

ent extirpation of the species from the type locality and

Fig. 5. A recently deforested plot of land that is less than one km from the population of Andinophryne olallai in Manduriacu, Im-

babura Province, Ecuador.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (5)

February 2014 | Volume 8 | Number 1 | e75

Lynch et al.

extensive habitat degradation (Coloma et al. 2011-2012;

Ron and Frenkel 2013). The rarity of known distribution

and a very small population size likely warrants an lUCN

Red List status of Endangered.

Although we found evidence of a seemingly healthy

population of A. olallai at Manduriacu, with the presence

of all size classes across numerous stream systems, the

extent of known occupancy remains extremely small (< 1

km^). At present, pristine habitat still exists at Manduria-

cu, however, the surrounding forest is rapidly disappear-

ing due to a variety of anthropogenic factors (i.e., inten-

sive logging, mining, and hydroelectric development).

These activities are expanding quickly and resulting in

extensive habitat fragmentation and loss (Fig. 5). The

apparent extirpation of A. olallai from the type locality,

a site where forest has been lost and fragmented, sug-

gests that the species is sensitive to anthropogenic habi-

tat change. Urgent conservation measures and population

monitoring are needed in order to ensure the survival of

A. olallai in nature. It is our hope that the rediscovery

of A. olallai will result in immediate support for greater

protection of the forests in and around Manduriacu, and

provide assistance in creating biological corridors be-

tween the neighboring reserves of Los Cedros and Co-

tacachi-Cayapas.

Acknowledgments. — We thank Juan and Monica

Kohn for purchasing and protecting the land at Rio

Manduriacu. Programa Socio Bosque provides support

for conservation of the forests of Manduriacu. Pontifi-

cia Universidad Catolica del Ecuador provided logistical

support for our fieldwork. The Biodiversity Group pro-

vided support for RLL research, and Belisario Cepeda

Quilindo gave access to their 2005 publication on A.

olallai. Paul Gutierrez-Cardenas provided access to

specimens and photographs of A. colomai. This work

was conducted under Ministerio del Ambiente permit #

005-12- IC-EAU-DNB/MA.

Literature Cited

Coloma LA, Guayasamin JM, Menendez-Guerrero P

(editors). 2011-2012. Amphibian Red List of Ecua-

dor, AnfibiosWebEcuador. Otonga Foundation, Quito,

Ecuador.

Coloma LA, Ron SR, Cisneros-Heredia DF, Yanez-Mu-

noz MH, Gutierrez-Cardenas PD, Angulo A. 2004.

Andinophryne olallai. In: lUCN 2011. lUCN Red List

of Threatened Species. Version 2011.2. Available:

http://www.iucnredlist.org. [Accessed: 27 December

2013].

Hoffman EA, Blouin MS. 2000. A review of colour and

pattern polymorphisms in Anurans. Biological Jour-

nal of the Linnean Society 70: 633-665.

Hoogmoed MS. 1985. A new genus of toads (Amphibia:

Anura, Bufonidae) from the Pacific slopes of the An-

des in northern Ecuador and southern Colombia, with

the description of two new species. Zoologische Med-

edelingen 59: 251-274.

Link/URL: Amphibiaweb Ecuador. Available: http://zoo-

logia.puce.edu.ec/vertebrados/anfibios [Accessed: 25

August 2013].

Lynch JD, Ruiz-Carranza PM. 1981. A new species of

toad (Anura: Bufonidae) from the Cordillera Occiden-

tal in southern Colombia. Lozania 33: 1-7.

Murillo Pacheco J, Cepeda Quilindo B, Elorez Pai C.

2005. Andinophryne olallai (Tandayapa Andes toad).

Geographic distribution. Herpetological Review 36:

331.

Ron SR, Frenkel C. 2013. Andinophryne olallai. In: Ron

SR, Guayasamin JM, Yanez-Munoz MH, Merino-

Viteri A (editors). AmphibiaWebEcuador. Version

2013.1. Available: http://zoologia.puce.edu.ee/ver-

tebrados/anfibios/FichaEspecie.aspx?ld=l 140 [Ac-

cessed: 26 July 2013].

Received: 09 December 2013

Accepted: 24 January 2014

Published: 03 February 2014

Ryan L. Lynch is the lead biologist and photographer for Ecuadorian programs for The Biodiversity

Group in Quito, Ecuador. He received his M.S. in wildlife ecology and conservation from the University of

Elorida where he used occupancy modeling to determine the status of anurans across the Elorida everglades

landscape. Ryan’s current research interests focus on the ecology, distribution, and conservation of rare,

threatened, and new species of reptiles and amphibians in Ecuador.

Sebastian Kohn is the administrator for the Antisanilla-Sunfohuaico Reserve run by the Jocotoco Eounda-

tion in Ecuador. He received his B.A. in biology and environmental studies at Whitman College in Wash-

ington State, USA. He currently directs the Rio Manduriaco Cooperative in Imbabura, Ecuador, as well as

the llitio Wildlife Rescue Center and Hacienda llitio in Cotopaxi, Ecuador. Sebastian is a founding member

of the Andean Condor Conservation Group of Ecuador (Grupo Nacional de Trabajo del Condor Andino)

and has been working with, and researching, both wild and captive condors for ten years.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (6)

February 2014 | Volume 8 | Number 1 | e75

Rediscovery of Andinophryne olallai

Fernando Ay ala- Varela is the director of the herpetology collection at the Pontificia Universidad Catolica

del Ecuador in Quito. He received his diploma at the Pontificia Universidad Catolica del Ecuador, Quito in

2004. He has been interested in herpetology since childhood and has dedicated a lot of time studying the

lizards of Ecuador, specifically the taxonomy and ecology of Anolis species. His current research interests

include reproductive biology and ecology of lizards and snakes in Ecuador.

Paul S. Hamilton is the founder and executive director of The Biodiversity Group in Tucson, Arizona,

USA. He holds a master’s degree in biology from the University of California, Riverside, and a Ph.D. in

biology from Arizona State University, and has conducted field studies in evolutionary, behavioral and

conservation ecology both in the tropics and the desert southwest. In addition to his research interests in

ecology and conservation of overlooked species such as amphibians, reptiles, and invertebrates, he is also

, __ a well published scientific and artistic photographer.

t Santiago R. Ron is the curator of amphibians and professor at the Pontificia Universidad Catolica del

Ecuador in Quito. His research focuses on the evolution and diversity of neotropical amphibians with

emphasis on Ecuador. Areas covered include evolution of animal communication, sexual selection, sys-

tematics and taxonomy. In the area of conservation biology Santiago is interested in the study of amphibian

extinctions in the Andes. Santiago also oversees the ex situ amphibian conservation project Balsa de los

Sapos at the Pontificia Universidad Catolica del Ecuador in Quito.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (7)

February 2014 | Volume 8 | Number 1 | e75

the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported

License, which permits unrestricted use for non-commercial and education purposes only

provided the original author and source are credited. The official publication credit source:

Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

[Special Section] 8(1) : 8-24.

A new Andean anole species of the Dactyloa clade

(Squamata: Iguanidae) from western Ecuador

Ternando P. Ayala- Varela, ^Diana Troya-Rodn'guez, ^Xiomara Talero-Rodn'guez

and ''Omar Torres-Carvajal

^■^■^■^Escuela de Ciencias Biologicas, Pontificia Universidad Catolica del ECUADOR, Avenida 12 de Octubre 1076 y Roca, Apartado 17-01-2184,

Quito, ECUADOR

Abstract . — ^We describe a new species of Anolis from the western slopes of the Andes of Ecuador,

province of Bolivar. It is referred to (1) the aequatoriaiis series based on its moderate size and

narrow toe lamellae, and (2) the eu/aemus sub-group based on having a typical Ano/is digit, in which

the distal lamellae of phalanx III distinctly overlap the proximal subdigital scales of phalanx II. The

new species is most similar morphologically to A. otongae and A. gemmosus, both from similar

elevations on the western Andean slopes of Ecuador, but differs from these species in morphology

and color patterns. We present a phylogeny based on DNA sequence data as additional evidence

supporting delimitation of the new species. The new species and A. gemmosus are sister taxa

within the “western Dactyloa clade.”

Key words. Clade Dactyloa, DNA, lizard, phylogeny, South America, systematics

Citation: Ayala-Varela FP, Troya-Rodriguez D, Talero-Rodriguez X, Torres-Carvajal O. 2014. A new Andean anole species of the Dactyloa clade (Squa-

mata: Iguanidae) from western Ecuador. Amphibian & Reptile Conservation 8(1) [Special Section]: 8-24 (e76).

Introduction

With nearly 490 described species, anole lizards {Anolis)

have proliferated impressively in the Americas (Nich-

olson 2002; Poe 2004), possibly prompted by ecologi-

cal opportunity (Losos 2009). Although the diversity of

these lizards has been extensively studied in the West

Indies (Losos 2009), the same is not true for the main-

land radiation, which is probably greater than previously

thought. For example, all but two — Anolis ruibali Navar-

ro & Garrido 2004 and 4. sierramaestrae Holahova et al.

2012 — of the 31 new species of Anolis described during

the last decade (2003-2013) occur in mainland Central

and South America (Uetz and Hosek 2014). Improving

knowledge concerning the diversity of mainland anoles

is crucial to understanding the nature of this radiation.

Anole lizards represent the most species-rich clade

traditionally recognized as a genus in Ecuador, with 37

species reported to date (Torres-Carvajal et al. 2014).

The diversity of anole lizards in Ecuador is remarkably

greater west of the Andes, with more than twice the num-

ber of species that occur east of the Andes (25 and 12

species, respectively). Of these, five species have been

described during the last six years from both sides of the

Andes as a result of both careful examination of exist-

ing collections and recent collecting in poorly explored

areas. Here we contribute to that growing body of taxo-

nomic knowledge with the description of a new species

of Anolis endemic to the western slopes of the Andes in

Ecuador. We present molecular evidence supporting rec-

ognition of the new species by performing phylogenetic

analyses of mitochondrial DNA sequence data.

Materials and Methods

Morphological data

All known specimens of the new species described in

this paper are included in the type series, and were de-

posited in the Museo de Zoologia, Pontificia Universi-

dad Catolica del Ecuador, Quito (QCAZ). Specimens of

other species of Anolis examined in this study are listed

in Appendix 1. We follow previously proposed terminol-

ogy (Williams et al. 1995) for measurements and squa-

mation. Nine morphological measurements were taken

with digital calipers and recorded to the nearest 0.1 ncnn:

head length, head width, head height, forelimb length,

hindlimb length, snout- vent length, jaw length, axilla-

groin length, and snout length. In addition, tail length

measurements were taken with a ruler and recorded to

the nearest millimeter; regenerated or broken tails were

not measured. Sex was determined by noting the pres-

ence of hemipenes, which were everted in all male speci-

mens during preparation.

Statistical analyses

Given that the new species is very similar in morphol-

ogy to Anolis gemmosus and A. otongae we performed

Correspondence. fpayala2000@yahoo.com (Corresponding author); ^dianatrl 7@gmail.com; ^xiomy.talero@gmail.com;

^omartorcar@gmail. com

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Ayala- Varela et al.

Fig. 1. Head of the holotype (QCAZ 3449) of Anolis poei sp. nov. in dorsal (top), ventral (middle), and lateral (bottom) views

[Scale bar =10 mm]. Photographs by F Ayala-Varela.

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A new species ofAnolis lizard from western Ecuador

a Principal Component Analysis (PCA) to determinate

whether separation in morphological space between

those species was statistically significant. Principal com-

ponents (PCs) were extracted from a covariance matrix

of the raw and rescaled data. The new species is most

similar to A. gemmosus, for which we also used Mests to

evaluate quantitative differences between both species.

One of the assumptions of the t-test for two samples is

that the variances of both samples are equal: therefore,

F-tests also were performed for each character to test for

equality of variances. If the variances were not the same

(i.e., P < 0.05), an unequal variance t-statistic was used.

Statistical analyses were performed in SPSS Statistics 17

(SPSS Inc. 2008).

The distribution map was prepared in ArcMap 9.3

(ESRI, Inc.); WGS84 is the datum for all coordinates

presented below.

DNA sequence data

Total genomic DNA was digested and extracted from liv-

er or muscle tissue using a guanidinium isothiocyanate

extraction protocol. Tissue samples were first mixed with

Proteinase K and a lysis buffer and digested overnight

prior to extraction. DNA samples were quantified using

a Nanodrop® ND-1000 (NanoDrop Technologies, Inc),

re-suspended and diluted to 25 ng/ul in ddH20 prior to

amplification.

Using primers and amplification protocols from the

literature (Folmer et al. 1994; Kumazawa and Nishida

1993; Macey et al. 1997; Schulte and Cartwright 2009)

we obtained 2807 nucleotides (nt) representing the nucle-

ar gene recombination-activating gene 1 (RAGl, 8 lint),

as well as the mitochondrial genes Cytochrome c oxi-

dase I (COl, 655nt) and a continuous fragment includ-

ing the NADH dehydrogenase subunit 2 (ND2, 1038 nt),

tRNATrp, tRNAAla, tRNAAsn, tRNACys (282nt), and

the origin of the light-strand replication (Ol, 29nt). The

new sequence data were obtained for three individuals of

the new species described herein, two of A. gemmosus,

and two of otongae. In addition we used sequence data

generated by Castaneda and de Queiroz (2011) for 20 in-

dividuals of the clade Dactyloa, as well as one sequence

of A. occultus, which was used as the outgroup in the

phylogenetic analysis. Gene regions of taxa included in

phylogenetic analyses along with their GenBank acces-

sion numbers are shown in Table 1.

Phylogenetic analyses

Editing, assembly, and alignment of sequences were

performed with Geneious ProTM 5.3 (Biomatters Ltd.

2010). Genes were combined into a single dataset with

eleven partitions, three per protein coding gene corre-

sponding to each codon position, one with all tRNAs, and

one with the Ol. The best partition strategy along with the

corresponding models of evolution were obtained in Par-

titionFinder 1.1.1 (Lanfear et al. 2012) under the Bayes-

ian information criterion.

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Phylogenetic relationships were assessed under a

Bayesian approach in MrBayes 3.2.0 (Ronquist and

Huelsenbeck 2003). Four independent analyses were

performed to reduce the chance of converging on a lo-

cal optimum. Each analysis consisted of 20 million

generations and four Markov chains with default heat-

ing values. Trees were sampled every 1,000 generations

resulting in 20,000 saved trees per analysis. Stationarity

was confirmed by plotting the -In L per generation in the

program Tracer 1.6 (Rambaut et al. 2013). Additionally,

the standard deviation of the partition frequencies and the

potential scale reduction factor (Gelman and Rubin 1992)

were used as convergence diagnostics for the posterior

probabilities of bipartitions and branch lengths, respec-

tively. Adequacy of mixing was assessed by examining

the acceptance rates for the parameters in MrBayes and

the effective sample sizes (ESS) in Tracer. After analyz-

ing convergence and mixing, 2,000 trees were discarded

as “bum-in” from each mn. We then confirmed that the

four analyses reached stationarity at a similar likelihood

score and that the topologies were similar, and used the

resultant 72,000 trees to calculate posterior probabilities

(PP) for each bipartition on a 50% majority mle consen-

sus tree.

Systematics

The taxonomic conclusions of this study are based on the

observation of morphological features and color patterns,

as well as inferred phylogenetic relationships. We con-

sider this information as species delimitation criteria fol-

lowing the general species concept of de Queiroz (1998,

2007).

Anolis poei sp. nov.

urn:lsid:zoobank.org:act:712687F6-CF33-4969-815D-E4600D01FB4C

Proposed standard English name: Telimbela anoles

Proposed standard Spanish name: Anolis de Telimbela

Holotype

QCAZ 3449 (Figs. 1, 2), adult male, Ecuador, Provincia

Bolivar, Telimbela, 01.65789°S, 79.15334°W, WGS84

1,354 m, 10 June 2011, collected by Fernando Ayala- Va-

rela, Jorge H. Valencia, Diana Troya-Rodriguez, Francy

Mora, and Estefama Boada.

Paratypes (1 5)

ECUADOR: Provincia Bolivar: QCAZ 3444-3448,

3451-3455, 4359, same data as holotype, ex-

cept 0.1658440°S, 79.157150°W, 1,310 m; QCAZ

6781-6783 Telimbela, Escuela Elisa Marino de Carva-

jal, 0.1665857°S, 79.172096°W, 27 July 2004, collected

by Edwin Carrillo-Ponce and Morley Read; QCAZ 9219

Guaranda, Salinas, Recinto Tres Cmces, 01.431380°S,

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Ayala- Varela et al.

Fig. 2. Anolis poei sp. nov. Holotype, adult male (SVL = 59.67 mm, QCAZ 3449, A), eye close-up (SVL = 60.31 mm, QCAZ 3448,

B), subadult male (SVL =52.12 mm, QCAZ 3455, C, D), adult male (SVL = 59.02 mm, QCAZ 3451, E, F), adult male (SVL =

60.31 mm, QCAZ 3448, G, H). Photographs by L. Bustamante (A), and O. Torres-Carvajal (B, C, D, E, F, G, H).

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A new species ofAnolis lizard from western Ecuador

Fig. 3. Male dewlap of Anolis poei sp. nov. (holotype, QCAZ 3449, A; paratype, QCAZ 3455, B); A. otongae (QCAZ 4661, C;

QCAZ 11791, D); and^. gemmosus (QCAZ 4385, E; QCAZ 4352, F; QCAZ 9452, G; QCAZ 11850, H). Photographs byL. Busta-

mante (A), O. Torres-Carvajal (B, C, D, E, F, H), and S. R. Ron (G).

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Ayala- Varela et al.

Table 1. Species of Anolis sequenced in this study, voucher

specimen numbers, collecting localities, and GenBank acces-

sion numbers.

Species

Voucher

Locality

GenBank Number

A. gemmosus

QCAZ 4385

Ecuador, Car-

chi, Rio San

Pablo near

Chical

ND2: KJ854205

COI: KJ854219

RAGE KJ854212

QCAZ 4406

Ecuador, Car-

chi, Maldo-

nado, Teldibi

Ecological

Trail

ND2: KJ854206

COI: KJ854220

RAGE KJ854213

A. otongae

QCAZ 11790

Ecuador,

Pichincha,

Biological Re-

serve Otonga

ND2: KJ854207

RAGE KJ854214

COI: KJ854221

QCAZ 11791

Ecuador,

Pichincha,

Biological Re-

serve Otonga

ND2: KJ854208

COI: KJ854222

RAGE KJ854215

A. poei

QCAZ 3444

Ecuador,

BoKvar,

Telimbela

ND2: KJ854209

COI: KJ854223

RAGE KJ854216

QCAZ 3445

Ecuador,

BoKvar,

Telimbela

ND2: KJ854210

COI: KJ854224

QCAZ 3448

Ecuador,

BoKvar,

Telimbela

ND2: KJ854211

COI: KJ854225

RAGE KJ854217

QCAZ 4359

Ecuador,

BoKvar,

Telimbela

RAGE KJ854218

79.097970°W, 2,628 m, 28 May 2009, collected by Eli-

cio E. Tapia, Silvia Aldas- Alarcon, and Eduardo Toral-

Contreras.

Diagnosis

We assign Anolis poei both to the aequatorialis series,

based on moderate to large body size, narrow toe lamel-

lae, small head scales, smooth ventral scales, and uni-

form dorsal scalation; and to the eulaemus-subgroup,

based on a typical Anolis digit, in which the distal la-

mellae of phalanx III distinctly overlap the first proximal

subdigital scale of phalanx II (Williams 1976; Williams

and Duellman 1984; Castaneda and de Queiroz 2013).

At present ten species are recognized within the eulae-

mus-subgroup: Anolis anoriensis Velasco et al. 2010,

A. antioquiae Williams 1985, A. eulaemus Boulenger

1908, A. fitchi Williams & Duellman 1984, A. gemmo-

sus O’Shaughnessy 1875, yf. maculigula Williams, 1984,

A. megalopithecus Rueda-Almonacid 1989, A. otongae

Ayala- Varela & Velasco 2010, A. podocarpus Ayala-

Varela & Torres-Carvajal 2010, and A. ventrimaculatus

Boulenger 1911. Anolis poei differs from them mostly

in dewlap features. The dewlap in males of A. poei has a

yellowish-green (or both yellow and green) gorgetal re-

gion, light blue border, and white sternal and marginal

regions (Eig. 3). It has a blackish gorgetal region, and

creamy white sternal region with light brown scales in A.

anoriensis; brown gorgetal region, and pale brown mar-

ginal region in A. eulaemus; bluish-gray gorgetal region,

orange stripes, pale bluish-rose anterior third, and white

sternal region becoming pale blue toward the belly in A.

maculigula; sepia background, with red narrow and ir-

regular stripes on each side of rows in A. megalophitecus;

white, pale yellow, or greenish-yellow gorgetal region,

with white or pale-yellow marginal and sternal regions

in A. otongae (Eig. 3); dull yellowish-green or light blue

gorgetal region, shading to dull cream, greenish yellow

or orange on the marginal region, with white or bluish

green gorgetal rows with or without brown spots and

with yellowish white, yellow or orange sternal region in

A. gemmosus (Eig. 3). The dewlap in males of A. poei

has wide rows of 3-7 scales separated by naked skin; the

width of these rows is one scale in A. fitchi, 2-5 granular,

minute scales in A. podocarpus, 1-2 scales in A. ventri-

maculatus, 3-6 scales in A. otongae, and 2-3 scales in A.

gemmosus. In addition, females of the new species lack

a dewlap, which is present in females of A. anoriensis,

A. antioquiae, A. eulaemus, A. fitchi, and A. podocarpus.

Anolis poei is most similar morphologically to A.

otongae and A. gemmosus (Eig. 4). Erom the former

species (character states in parenthesis) A. poei differs

in having small dorsal chevrons in females (large dorsal

chevrons extending onto flanks), pale yellowish-brown

iris (iris dark blue), interparietal scale (if present) sur-

rounded by small swollen scales (interparietal scale

surrounded by relatively enlarged flat scales), enlarged

postanal scales separated by 3-5 scales (postanal scales

separated by 1-2 scales), and in lacking a dark stripe on

side of head (dark coppery -brown stripe present). Ad-

ditionally, PCA analyses suggested that specimens of A.

poei have shorter jaws, as well as lower and narrower

heads than A. otongae (Table 2, Fig. 5), with PCI (39%

of total variation) represented mainly by head height,

head width, and jaw length.

The new species can be distinguished from A. gem-

mosus (Table 3) in having fewer scales between sec-

ond canthals (11-14, mean = 12.08 and 12-21, mean

= 15.25, respectively; t = 5.31, P<0.005); fewer scales

between supraorbital semicircles (1-3, mean =1.62 and

1-5, mean = 3.13, respectively; t = 4.46, P<0.005); more

lamellae under phalanges III-IV of fourth toe (18-19,

mean = 18.92 and 14-18, mean = 17.33, respectively;

t = -7.86, P<0.005); a narrower head (head width =

7.84-8.84, mean = 8.29 and 6.97-17.41, mean = 10.82,

respectively; t = -7.03, P<0.005); lower head (head

height = 6.54-7.48, mean = 6.92 and 5.42-15.96, mean =

9.51, respectively; t = -6.96, P<0.005); and shorter snout

(snout length = 6.75-7.30, mean = 6.92 and 5.79-14.95,

mean = 10.58, respectively; t = -11.74, P<0.005).

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A new species ofAnolis lizard from western Ecuador

Fig. 4. Part 1. Five species of Anolis from western Ecuador. A. aequatorialis: male (QCAZ 11861, A) and female (QCAZ 3443,

B); A. binotatus: male (QCAZ 3434, C, D); A.fasciatus: male (QCAZ 3450, E, F); A. otongae: male (QCAZ 11790, G) and female

(QCAZ 11791, H).

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Ayala- Varela et al.

Fig. 4. Part 2. A. gemmosus: male (QCAZ 4352, 1, J), male (QCAZ 4385, K, L), male (QCAZ 11849, M, N), and female (QCAZ

4393, O, P). All photographs by O. Torres-Carvajal, except A, M, N (S. R. Ron).

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A new species ofAnolis lizard from western Ecuador

Description of hoiotype (scores for para-

types in parentheses)

Male (Figs. 1, 2); SVL 59.7 mm (46.5-60.3 mm); tail

length 150.2 mm (146.2-163.4 mm); head length 15.9

mm (14.8-16.5 mm); head width 8.4 mm (7. 8-8. 8 mm);

head height 7.2 mm (6. 5-7. 5 mm); internasal distance

2.0 mm (1. 2-2.1 mm); interorbital distance 2.4 mm

(2.2-2. 5 mm); interparietal absent (present, interparietal

length 0.8-0. 9 mm; second largest scale length near in-

terparietal 0. 3-0.4 mm); ear opening maximum length

1.6 mm (1. 6-2.1 mm); snout length 6.8 mm (6. 8-7.3

mm); jaw length 11.7 mm (11.7-14.4 mm); axila-groin

distance 27.7 mm (27.4-30.6 mm); femur length 14.8

mm (14.4-15.6 mm); 4th toe length 12.5 mm (10.6-12.8

mm); 4th toepad width 1.2 mm (1.0- 1.3 mm); forelimb

length 36.2 mm (21.8-36.2 mm); hindlimb length 42.6

mm (42.6-52.7 mm).

Head scales multicarinate (same, unicarinate, or ru-

gose) on frontal region and unicarinate (same, multicari-

nate or rugose) on supraocular disc; 11 (10-14) scales

between second canthals; 13 (11-15) scales between first

canthals; 6 (5-7) scales bordering the rostral posteriorly;

anterior nasal in contact with rostral (same or inferior

nasal in contact with rostral); supraorbital semicircles

separated by two (0-3) scales; supraocular disk with

scales heterogeneous in size; one elongate superciliary

followed by a series of granules (same or one small scale

instead of granules); 6 (5-8) loreal rows on left side; 49

(25-53) loreal scales; interparietal absent (same or, when

present, the interparietal smaller than ear opening, with

4-7 scales between interparietal and semicircles on each

side, and 8-15 scales between interparietal and nape

scales); suboculars in contact with supralabials; 6 (5-7)

supralabials counted up to a point below center of eye;

6 (5-7) infralabials counted up to a point below center

of eye; 7 (4-7) postmentals; one enlarged sublabial on

each side.

Table 2. PCA loadings conducted on nine morphological vari-

ables of Anolis gemmosus, A. otongae and A. poei.

Raw

Rotated

1

2

3

1 2 3

Head height

-0.96

0.21

-0.02

-0.97

0.16

-0.07

Head length

0.24

0.34

0.05

0.22

0.35

0.05

Head width

-0.96

0.20

-0.03

-0.96

0.15

-0.07

Jaw length

0.98

0.06

-0.06

0.98

0.11

-0.03

Snout length

0.82

0.33

-0.07

0.81

0.37

-0.06

Forelimh length

-0.01

0.80

0.04

-0.05

0.80

0.00

Hindlimh length

-0.01

0.85

0.02

-0.05

0.85

-0.03

Axilla-groin length

-0.01

-0.50

-0.02

0.01

-0.50

0.01

Snout- vent length

0.06

-0.04

0.99

0.02

0.01

1.00

Eigenvalue

3.54

1.93

1.00

3.53

1.93

1.01

% van explained

39.31

21.42

11.16

39.23

21.45

11.21

Species

O A. gemmosus

# A (Aongao

G poei

Fig. 5. Distribution of Anolis gemmosus, A. otongae and A. poei

sp. nov. along the first and second principal components axes.

Dorsal crest or enlarged middorsal row absent; dorsal

scales keeled, 11 (9-11) dorsal scales in 5% the length

of SVL contained in the dorsal midline at the level of

the forelimbs; flank scales more or less separated by

skin; ventrals smaller than dorsals, 13 (8-13) longitudi-

nal rows in 5% the length of SVL; ventrals smooth and

granular, arranged in diagonal rows.

Toepads overlap the first phalanx in all toes; 19

(18-19) lamellae under phalanges III and IV of fourth

toe (character 27 in Williams et al. 1995 and character

9 in Poe 2004); supradigitals multicarinate; tail with a

double row of middorsal scales; postanals present (same

or absent), with a slightly enlarged scale laterally on each

side.

Nuchal fold present (absent in females and juveniles);

dorsal folds absent; dewlap extending posteriorly to a

point halfway between fore and hindlimbs (absent in fe-

males); dewlap with five longitudinal rows of 3-7 swol-

len scales, similar size to ventrals, separated by naked

skin.

Sexual variation of meristic and morphometric char-

acters in A. poei is presented in Table 4.

Color in life

Hoiotype (QCAZ 3449; Figs. 2, 3): background of head,

body, limbs and tail green; head with light bluish green,

dark green, and light grey irregular spots dorsally; dor-

sal surface of body with six light grey, small irregular

blotches; dorsal surface of neck with two light grey,

small irregular blotches; limbs with dark green and yel-

lowish-cream spots; lateral surface of head with a white

stripe extending posteriorly from loreal region, through

subocular region, to a point anterior to the tympanum;

white blotch with yellow center above tympanum; eye-

lids yellowish green with first row of upper and lower

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Ayala- Varela et al.

palpebrals black, second and third rows both yellow and

green; lateral surface of neck with dark green dots; body

flanks green, with rows of yellow-centered white spots

oriented ventro-posteriorly; ventral surface of head yel-

lowish green with light yellow blotches; ventral surface

of body white with bluish-green reticulations; ventral

surface of limbs white with several transparent scales

and dark brown reticulations; ventral surface of tail white

with dark green spots anteriorly, and yellowish-green

transverse bands posteriorly; dewlap skin light blue, dark

yellowish green on gorgetal region, light blue on ster-

nal region; gorgetal scales light yellowish green; mar-

ginals and stemals white; iris dark brown with a white

inner ring. When stressed, the dorsal background color

switched from green to yellowish brown.

Subadult male (QCAZ 3455, Figs. 2,3, differences

from holotype): head with dark green and white irregu-

lar small spots dorsally; dorsal surface of body and neck

with white and dark green small spots, and larger pale

yellow spots; lateral surface of head yellowish green with

a white stripe extending posteriorly from loreal region,

through subocular region, to upper border of tympanum;

lateral surface of body with rows of white and dark green

small spots, and larger pale yellow spots; ventral surface

of head with white blotches and light blue spots; ventral

surface of body with dark green reticulations; ventral sur-

face of limbs with brown or green reticulations; ventral

surface of tail with blackish green reticulations anteri-

orly; dewlap skin white, yellow on gorgetal region, white

on sternal region; throat, edge of mouth, and tongue pink-

ish white (Fig. 6). When stressed, rust-colored blotches

appeared on dorsal surface of head, body, limbs and tail.

Adult female (QCAZ 3454, Fig. 7): dorsal surface

of head, body and tail yellowish green; dorsal surface

of body with six narrow brown chevrons, each one de-

limited posteriorly by a grayish white blotch; limbs yel-

lowish green with dark green spots arranged in bands,

and pale yellowish spots; tail with two brown chevrons

anteriorly; lateral surface of head yellowish green; loreal

region yellow; lateral surface of neck and body yellowish

green with brown dots; ventral surface of head pale yel-

low with yellowish green reticulations, short white lon-

gitudinal stripe on throat; ventral surface of body and tail

white with black reticulations laterally; ventral surface

of limbs white with some transparent scales and brown

reticulations on hindlimbs; ventral surface of tail with

brownish green reticulations anteriorly; iris brown with

a pale white ring.

Subadult female (QCAZ 3446, Fig. 7, differences with

QCAZ 3454): occipital and temporal regions with brown

and white small blotches; dorsal surface of neck with a

distinct brown chevron delimited posteriorly by a grayish

white blotch; lateral surface of body yellowish green dor-

sally and light blue ventrally, with white or cream spots;

dorsal surface of tail with two brown chevrons, each one

delimited posteriorly by a grayish white blotch.

Fig. 6. Tongue of Anolis poei sp. nov., subadult male (QCAZ

3455, top); A. gemmosus, adult male (QCAZ 4347, middle); A.

otongae, adult male (QCAZ 4661, bottom). Photographs by S.

R. Ron (top), O. Torres-Carvajal (middle, bottom).

Color in preservative

Holotype (QCAZ 3449): dorsal background of head,

body, limbs and tail grayish brown; dorsal surface of

head with metallic green, dark green, blue, gray and

white cream irregular spots; dorsal surface of body with

six black small chevrons, each delimited posteriorly by a

white irregular blotch; limbs with dark brown and white

spots; lateral surface of head with a white stripe extend-

ing posteriorly from loreal region, through subocular re-

gion, to a point anterior to the tympanum; upper border

of tympanum with a white spot; eyelids purple with first

row of upper and lower palpebrals black, second and

third rows white and purple; neck flanks with black dots;

body flanks grayish brown, with dark brown diagonal

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A new species ofAnolis lizard from western Ecuador

Table 3. Summary of morphological characters of Anolis poei sp. nov. and A. gemmosus from Ecuador. For each quantitative character, the F-value,

t-value, and corresponding P-values are given. Range and sample size (in parenthesis) followed by mean + standard deviation are given.

Character

A. gemmosus

A. poei sp. nov.

F-value

P

t-value

P

Scales between second canthals

12-21 (24) 15.25 + 1.98

11-14(13) 12.08 + 1.12

2.59

0.12

5.31

<0.005

Postrostrals

5-7 (24) 5.79 + 0.72

5-7 (13)5.92 +0.64

1.37

0.25

-0.55

0.59

Row of loreals

6-10(24)7.25 + 1.15

5-8(13) 6.31 + 1.18

0.03

0.86

2.36

0.02

Scales between supraorbital semicircles

1-5 (24)3.13 + 1.23

1-3 (13) 1.62 + 0.77

5.27

0.03

4.46

<0.005

Scales between interparietal (if present) and

semicircles

3-8 (24) 5.67 + 1.27

4-7(6)5.83 + 1.17

0.18

0.67

-0.29

0.77

Supralabials

5-7 (24) 6.08 + 0.50

5-7(13)6 + 0.41

1.29

0.27

0.51

0.61

Postmentals

4-8 (24) 6.13 + 1.03

4-7(13)5.77 + 0.93

0.18

0.67

1.03

0.31

Lamellae under phalanges III-IV of fourth toe

14-18 (24) 17.33 + 0.92

18-19(13) 18.92 + 0.28

8.71

0.01

-7.86

<0.005

Head length

13.23-18.12 (94) 15.46+ 1.07

14.79-16.5 (7) 15.67 + 0.51

4.67

0.03

0.93

0.37

Head width

6.97-17.41 (94) 10.82 + 3.24

7.84-8.84 (7) 8.29 + 0.36

32.16

<0.005

-7.03

<0.005

Head height

5.42-15.96 (94) 9.51 +3.32

6.54-7.48 (7) 6.92 + 0.38

31.04

<0.005

-6.96

<0.005

Jaw length

7.31-17.43 (94) 12.32 + 3.02

11.73-14.36 (7) 12.44 + 0.91

19.25

<0.005

0.26

0.80

Snout length

5.79-14.95 (94) 10.58 + 2.93

6.75-7.30 (7) 6.92 + 0.19

41.30

<0.005

-11.74

<0.005

Forelimb length

23.41-34.34 (94) 29.43 + 2.28

21.84-36.18 (7) 28.57 + 4.25

0.02

0.89

-0.12

0.90

Hindlimb length

41.51-63.80 (94) 52.82 + 4.13

42.56-52.68 (7) 49.01 + 3.33

1.00

0.32

-2.38

0.02

Axilla-groin length

20.73-33.51 (94) 26.74 + 2.07

27.35-30.61 (7) 28.54+ 1.30

0.95

0.33

2.26

0.03

Snout- vent length

46.71-66.21 (94) 58.34 + 3.65

46.47-60.31 (7) 56.87 + 4.85

0.35

0.56

-1.00

0.32

Tail length

94.94-191 (94) 154.59 + 18.66

146.21-163.37 (7) 154.74 + 6.32

3.82

0.05

0.02

0.98

Table 4. Sexual variation in lepidosis and measurements (mm) of Anolis poei sp. nov. Range followed by mean + standard devia-

tion are given.

Character

Males

Females

n = 4

n = 3

Scales between second canthals

11-13 11.75 + 0.96

12-13 12.67 + 0.58

Postrostrals

5-6 5.75 + 0.5

6-7 6.33 + 0.58

Row of loreals

6-8 7 + 1.15

5-6 5.33 + 0.58

Scales between supraorbital semicircles

1-2 1.75+0.5

1-2 1.67 + 0.577

Scales between interparietal and semicircles

Interparietal absent

6-7 6.50 + 3.78

Supralabials to below center of eye

6

Postmentals

4-7 5.25 + 1.5

6-7 6.33 + 0.58

Lamellae under phalanges II-III of fourth toe

19

Head length

15.8-16.5 15.95 + 0.38

14.8-15.62 15.29 + 0.44

Head width

7.84-8.84 8.31 +0.41

8.05-8.66 8.26 + 0.34

Head height

6.67-7.48 7.02 + 0.39

6.54-7.27 6.8 + 0.41

Jaw length

11.73-12.65 12.25 + 0.38

11.86-14.36 12.70 + 1.43

Snout length

6.75-7.04 6.87 + 0.12

7.82-7.30 7 + 0.26

Forelimb length

27.94-36.18 30.75 + 3.72

21.84-28.19 25.68 + 3.37

Hindlimb length

42.56-52.68 49.35 + 4.59

47.50-49.56 48.57 + 1.03

Axilla-groin length

27.35-28.17 27.76 + 0.33

27.94-30.61 29.57 + 1.43

Snout- vent length

58.80-60.31 59.45 + 0.68

46.47-58.48 53.43 + 6.22

Tail length

150.20-163.37 157.89 + 5.89

146.21-155.38 150.53 +4.60

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Ayala- Varela et al.

A ^

B

'I7\

f *-

9

c

D

E

F

Fig. 7. Anolis poei sp. nov. Adult female (SVL = 46.47 mm, QCAZ 3454, A, B), subadult female (SVL = 47.99 mm, QCAZ 3446,

C, D), juvenile male (SVL = 26.85 mm, QCAZ 3453, E, F). Photographs by O. Torres-Carvajal

bands oriented ventro-posteriorly and intercalated with Adult male (QCAZ 6783): dorsal surface of head and

white spots; ventral surface of head white with light blue

reticulations; ventral surface of body white with faint

grayish purple reticulations; ventral surface of limbs

grayish cream with dark brown reticulations; ventral sur-

face of tail white anteriorly with a metallic green tint and

grayish purple spots, and gray posteriorly; dewlap skin

with a turquoise gorgetal region and white sternal region;

gorgetal scales light brown with a gold tint internally, and

dark brown externally; dewlap marginals and sternals

white; throat, edge of mouth and tongue white.

body dark brown with gray dots; dorsal surface of limbs

dark brown, with gray dots on forelimbs; lateral surface

of head dark brown with white cream dots dorsal and an-

terior to tympanum; body flanks dark brown with faint

white dots arranged on diagonal lines that reach venter;

ventral surface of head with bluish-purple infralabial

and sublabial regions, and light purple gular region with

white irregular spots; ventral surface of body white with

faint purple reticulations; limbs creamish gray with dark

brown reticulations; ventral surface of tail white with

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (19) May 2014 | Volume 8 | Number 1 | e76

A new species ofAnolis lizard from western Ecuador

purple mottling anteriorly, and gray posteriorly; dewlap

skin with a light blue gorgetal region and white ster-

nal region; gorgetal scales purple; sternal and marginal

scales white.

Adult female (QCAZ 3454): dorsal surface of head

brown with metallic blue and green frontal and supraocu-

lar regions; dorsal surface of body brown with six nar-

row black chevrons, each one delimited posteriorly by

a white blotch; forelimbs bluish brown with white spots

arranged in stripes; hindlimbs brown with dark brown

bands and dots; tail with two black chevrons anteriorly;

lateral surface of head brown with purple tint; labial re-

gion light purple; lateral aspect of neck and body purple

with black dots; ventral surface of head white with purple

brown stripes; ventral surface of body white with dark

brown dots laterally; ventral surface of limbs grayish

cream with dark brown reticulations on hindlimbs; ven-

tral surface of tail white with dark brown dots.

Subadult female (QCAZ 3446, differences with

QCAZ 3454): occipital and temporal regions with dark

brown, small blotches; dorsal surface of neck with a dis-

tinct dark brown chevron; dorsal surface of body with six

distinct, dark brown chevrons; dorsal surface of tail with

two dark brown chevrons.

Phylogenetic relationships

The data matrix analyzed in this study contained 1,065

unique site patterns. Of the 2,807 nucleotide characters

included in our analysis 1,703 were constant, 224 par-

simony uninformative, and 880 were parsimony infor-

mative. The 50% majority rule consensus tree resulting

from the Bayesian analysis (Fig. 8) is generally congru-

ent with the phytogeny of the clade Dactyloa presented

by Castaneda and de Queiroz (2011). Both the new spe-

cies described here and A. otongae are members of the

aequatorialis series of Castaneda and de Queiroz (2013),

which corresponds roughly to the “western clade” of

Castaneda and de Queiroz (2011). Our phytogeny sup-

ports strongly (PP = 0.99) a sister taxon relationship be-

tween Anolis poei and A. gemmosus, as well as the ex-

clusivity (de Queiroz and Donoghue 1990; de Queiroz

1998) of both species. They form a clade sister (PP =

0.89) to A. otongae. The clade formed by the three spe-

cies is sister (PP = 1) to a clade formed by A. aequatoria-

lis and^. anoriensis.

Distribution and ecology

Anolis poei inhabits low montane evergreen forest (Sierra

1999) on the western slopes of the Andes in central Ecua-

dor, Provincia Bolivar, between 1,310-1,354 m (Fig. 9).

f c u SN M 1 xy 1

A. agasshi KEN2<l04_2

A. iuciae USNM321960

A- transversaUx QCAZ 5 6

^ r A. eiiskiikiTiiiri MBLUZy34

I A. enxkaterriari MBLUZ925

*

Fig. 8. Phytogeny of the “western Dactyloa clade” sensu

Castaneda and de Queiroz (2011), which is part of the ae-

quatorialis series of Castaneda and de Queiroz (2013), and

representatives of the heterodermus series {A. euskalerri-

ari), punctatus series (A. transversalis), roquet series (A. lu-

ciae), latifrons series {A. agassizi), and a. non-Dactyloa Ano-

lis (A. occultus). The tree is a majority rule (50%) consensus

tree of 72,000 trees obtained from a Bayesian analysis of the

mitochondrial genes COl, ND2, and adjacent tRNAs, and

the nuclear gene RAGl. Asterisks correspond to posterior

probability values > 0.99. Voucher information is presented

in Castaneda and de Queiroz (2011) and Table 1.

C

A. p^mcccte QC AZ6&69

A- pcrvccac QC A Z6R79

A fusuiti QCAZ6930

A.L'hhris MRCI26

A. chhris QCAZftR77

A. cftloiis OCA26920

'A. venirifnucitlufns MRC09I

A. venirimciL'nlcitiix MRC1J2

■ A. anqiiatoiiaiis QCAZ(iS55

— A, (tctfuuifn'iijtix QCAZ6SK3

- A. afitiriatixix MiiUAT5l7

r A. cinurKttxfx MHUAl 156S

T- A. anoriensis MHUAT5I6

r A. (itonpae QCAZ] I79<)

1= A. oion^ae QCAZ] i7y]

A. poei QCAZ3445_4359

A. poei QC A Z3444

A. poei QCAZ344S

rl

n.na

4

A - petnnuisiss QCAZ43S5

A. gemmosus QCAZ4406

A. gemmosus QCAZ6X51

A. gemmosus QC A ZtSHB 4

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Ayala- Varela et al.

The new species occurs in sympatry with A. aequa-

torialis, A. binotatus, and A. fasciatus at its type locality

(Fig. 4). Specimens of the new species were collected

along the border of a road, close to rivers, in second-

ary forest, and on shrubs within pastures. All individu-

als were found between 20h00 and 22h00 sleeping with

their heads up, or in a horizontal position on branches or

vines, 0.5^.5 m above ground or streams. The smallest

individual QCAZ 3453 (SVL = 26.9 m; TL = 67.6 mm)

was collected on 11 June 2011.

Etymology

The specific name is a noun in the genitive case and is

a patronym for Steve Poe, who has published important

contributions to the systematics and evolution of Ano-

lis lizards (Poe 2004, 2011). During his collecting trips

to Ecuador in 2009 and 2010, Poe trained several young

herpetologists in field collecting techniques and inspired

them to explore the diversity of anole lizards. This paper

is one of the products resulting from that inspiration.

Acknowledgments. — We thank Jorge H. Valencia,

Francy Mora, and Estefama Boada for assistance in the

field; Santiago Ron and Lucas Bustamante for the pho-

tographs; Paulina Santiana and Andrea Varela for assem-

bling some of the figures; Melissa Rodriguez for helping

with the map. Special thanks to Kevin de Queiroz and

two anonymous reviewers for commenting on previous

versions of this manuscript. OTC received funds from

Secretaria de Educacion Superior, Ciencia, Tecnologia

e Innovacion (SENESCYT). Specimens were collected

under collection permit OOl-IC-FAU/FLO/DRZCHI/MA

and 008-09 IC-EAU-DNB/MA issued by Ministerio de

Ambiente del Ecuador.

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Received: 28 April 2014

Accepted: 20 May 2014

Published: 28 May 2014

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Ayala- Varela et al.

Appendix 1

Additional specimens examined

Anolis gemmosus - Ecuador: Carchr. Chilma Bajo, Finca de Anibal Pozo, 0.86397°N, 78.04723°W, 2,022 m, QCAZ

8681-82; Chilma Bajo, Finca de Anibal Pozo, 0.86495 °N, 78.04979'W, 2,071 m, QCAZ 8683; La Centella, 0.89318°N,

78.13471 'W, 1,800-2,400 m, QCAZ 11784; Maldonado, Sendero Ecologico Teldibi, 0.91301 °N, 78.10782'W, 1,477-

l, 635 m, QCAZ 12272, 12278, QCAZ 12279-80, 4360, 4406, 4408; Rio San Pablo, cerca a Chical, 0.90302°N,

78.1 6284 'W, 1 ,399 m, QCAZ 4377, 4382, 4385-86,4388; Rio San Pablo, cerca a Chical, 0.90327°N, 78.1 6201 °W, 1 ,429

m, QCAZ 4393; Rio Verde and Rio Pablo, Rio Estrellita, Guapil, 1 ,428-1 ,466 m, QCAZ 12289, 12294, 12302; Cotopaxi:

1 1 5 km Qeste de Pilalo, 0.928 °S, 79.057 'W, 1 ,500 m, QCAZ 4072; 1 8.2 km de Quillutuha, via a Pucayacu, 0.67843 °S,

79.01 565 'W, 1 ,420 m, QCAZ 8845-49; Alrededores de San Francisco de Las Pampas, 0.42371 °S, 78.96765 °W, 1 ,800

m, QCAZ 1440-47, 2123; Bosque Integral Qtonga , 0.4194 °S, 79.00345°W, 1,720-2,143 m, QCAZ 2758, 2809-10,

3121, 3126-27, 3131, 3133, 3174, 3180-90, 3863-3866, 3869-71 , 3940, 3974-76, 4028-34, 4224-25, 4657, 4663, 4785,

5060, 5063, 5371, 5477-79, 5482-83, 6770-73, 9888, 10424, 10438-39, 10441-42, 10452, 12057, 12060-65, 12067,

12072-73, 12075, 12077-82, Bosque Integral Qtonga, a lo largo del rio Esmeraldas, 0.46333 °S, 79.05027 °W, QCAZ

7281 -89; Bosque Integral Qtonga, alrededores de la estacion, 0.41 933 °S, 79.00336 'W, 1 ,980 m, QCAZ 1 0697 ; Bosque

Integral Qtonga, arriba de la estacion, 0.41 478 °S, 79.00073 'W, QCAZ 3867-68; Bosque Integral Qtonga, orillas del rio

Esmeraldas, 0.41932°S, 78.99396 'W, 1 ,719 m, QCAZ 10393, 10395, 10399; Bosque Integral Qtonga, sendero a la Es-

tacion, 0.41933 °S, 79.00336 °W, 1 ,646 m, QCAZ 10696; Cerca a Naranjito, 0.41944 °S, 79.00333 'W, QCAZ 7825; San

Francisco de Las Pampas, 0.42371 °S, 78.96765 °W, 1 ,600-1 ,800 m, QCAZ 63, 68-70, 72-79, 3134-53, 3155, 3175; Via

a Qtonga, 0.33183 °S, 78.93791 'W, 1,476-1,700 m, QCAZ 8412; Imbabura: 6 de Julio de Cuellaje, 0.4°N, 78.525 °W,

QCAZ 4346-47; 6 de Julio de Cuellaje, 0.401 07 °N, 78.51 81 'W, 1 ,886 m, QCAZ 4349; 6 de Julio de Cuellaje, 0.401 02°N,

78.51 ZZO'W, 1,897 m, QCAZ 4350; 6 de Julio de Cuellaje, punto 8, 0.4°N, 78.525 'W, QCAZ 4348; 6 de Julio de Cuel-

laje, San Antonio, Cordillera deToisan, 0.45803 °N, 78.54722 'W, QCAZ 9450-53; Carretera nueva via a Cuellaje, Sector

de Santa Clara, Reserva Alto Choco, 0.37603°N, 78.45857°W, 2,062 m, QCAZ 4352-54; La Mina, Junin, 0.2754 °N,

78.6603 °W, 1,715 m, QCAZ 3071; Manduriaco, 0.277 °N, 78.873 °W, 1,330 m, QCAZ 5328; Manduriacu, 7.5 km NE of

Bellavista, 0.31006°N, 78.85757'W, 1,177-1,227 m, QCAZ 11606, 12305-314; 12322, 12324, 12326, 12328, 12331;

Reserva Siempre Verde, NE de Cotacachi, 0.37167°N, 78.421 86 °W, 2,468 m, QCAZ 8837; Reserva Alto Choco, Santa

Rosa, 0.36939 °N, 78.44942'W, 2,109 m, QCAZ 7330-31 ; Pichincha: 1-2 km oeste de Tandayapa, 0.004 °S, 78.663 °W,

2,000 m, QCAZ 2070-71 ; 2.9 km de Tandayapa, 0.00952 °S, 78.65698 'W, 1 ,820 m, QCAZ 406-10; 5 km E Tandayapa,

0.02°S, 78.651 °W, 1,975 m, QCAZ 2066-69; A orillas del Rio Chisinche, en la carretera a Conchacato, 0.448°S,

78.76423 'W, 1,693 m, QCAZ 6884-89; 30 km E de Santo Domingo, hacia la Reserva de Bosque Integral Qtonga,

0.3884 °S, 78.92995 'W, QCAZ 9769-70; 9775; Bosque Protector Mindo - Nambillo, refugio, 0.106°S, 78.687°W, 1,700

m, QCAZ 2910; Cooperative El Porvenir, finca El Cedral, 0.114°N, 78.56993°W, 2,297 m, QCAZ 10501-502; Desviacion

a Mindo, 1-5 km de la interseccion hacia abajo, 0.02853°S, 78.75861 °W, 1,661 m, QCAZ 9724-31; Estacion Cientifica

Rio Guajalito, 0.22676 °S, 78.82171 °W, 1,791-1,814 m, QCAZ 1330, 1333, 1500, 1645, 2682-84, 2786, 2813, 2815-16,

3040-45, 3056-57, 3373, 3385, 4123-25, 4210, 4214, 6413-14, 8859, 8864-65, 9974, 11404, 12088-101; Las Tolas,

0.7281 8 °N, 78.77792 'W, 1,200-1,600 m, QCAZ 11848-49; Manuel Cornejo Astorga (Tandapi), frente a la planta de

agua potable "El Placer" via a Conchacato, 0.42471 °S, 78.78905 'W, 1,500 m, QCAZ 6882; Manuel Cornejo Astorga

(Tandapi), via Atenas a 5 km de la carretera principal, 0.40625 °S, 78.83621 °W, 1 ,671 m, QCAZ 5365-70; Mindo, 1 ,342-

l, 560 m, QCAZ 12350-53, 12356, 12358, 12365, 12370, 12375-76; Mindo Biology Station, 0.07805°S, 78.73194 °W,

QCAZ 7518-20, 7522; Mindo, camino entre Mariposas de Mindo y Mindo Garden, 0.06753°S, 78.7535 'W, 1,361 m,

QCAZ 6851-53, 6858; Mindo Garden, 4 km de Mindo, 0.06901 °S, 78.801 66 °W, QCAZ 2787; Mindo, El Monte, Road

to Mindo Garden, 0.07805 °S, 78.7319°W, QCAZ 7521; Mindo, Sachatamia Lodge, 0.02638 °S, 78.75944 °W, 1,700 m,

QCAZ 11857-59; Nanegalito, Finca El Cedral, 0.1141 °N, 78.57007'W, 2,272 m, QCAZ 9462-63; Pachijal, via Nanegali-

to-Los Bancos, 0.13°S, 78.72644 'W, 1,741 m, QCAZ 5494-500; Palmeras, 0.244 °S, 78.794 °W, 1,800 m, QCAZ 871,

881-83,1351-52, 2244, 3004-06; Recinto Chiriboga, Estacion La Favorite , 0.21307°S, 78.78421 °W, 1,680 m, QCAZ

5383-84; Reserva Ecologica Bosque Nublado "Santa Lucia," 0.11928°N, 78.59647°W, 1,624-1,927 m, QCAZ 10664,

11850-52, 11888-93,11897, 11899; Tandayapa, 0.00591 °N, 78.67455'W, 1,670 m, QCAZ 4086. Locality in error. Pich-

incha, San Antonio de Pichincha, 0.00905 °S, 78.44581 °W, QCAZ 724.

Anolis otongae - Ecuador: Cotopaxi: Alrededores de San Francisco de Las Pampas, 0.42371 °S, 78.96765 'W, 1,800

m, QCAZ 2128; Bosque Integral Qtonga , 0.41944 °S, 79.00333 'W, 1,900-2,300 m, QCAZ 1721 , 2050-52, 3129, 3706,

3796, 3872-73, 4025, 4661, 5481, 6219, 11790-91, 12035, 12056, 12058, 12070-71; Los Libres, QCAZ 2781; Penas

Coloradas, 0.52343°S, 79.05908°W, QCAZ 1696; Pichincha: La Victoria, 0.47747°S, 79.05336'W, 2,104 m, QCAZ

6394-96.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (23)

May 2014 | Volume 8 | Number 1 | e76

A new species ofAnolis lizard from western Ecuador

Fernando Ay ala- Varela is the director of the herpetology collection at the Pontificia Universidad Catolica

del Ecuador in Quito. He received his diploma at the Pontificia Universidad Catolica del Ecuador, Quito in

2004. He has been interested in herpetology since childhood and has dedicated a lot of time studying the

lizards of Ecuador, specifically the taxonomy and ecology ofAnolis species. His current research interests

include reproductive biology and ecology of lizards and snakes in Ecuador.

Diana Troya-Rodnguez received a B.Sc. in Biology from Pontificia Universidad Catolica del Ecuador

(PUCE) in 2013. As a student, she joined the Museo de Zoologia QCAZ, Pontificia Universidad Catolica

del Ecuador in Quito, where she developed a great interest in reptiles. She has been studying anole lizards

for the last four years. Eor her undergraduate thesis, Diana worked on the “Comparative phylogeography

of two sympatric species of Anolis (Squamata: Iguanidae) and the impact of global warming on their dis-

tribution.”

Xiomara Talero-Rodnguez is an undergraduate biology student at Pontificia Universidad Catolica del

Ecuador in Quito. She joined Museo de Zoologia QCAZ last year and has been helping with several anole

lizard projects ever since. She is currently interested in studying ecology and behavior of anoles.

Omar Torres-Carvajal graduated in Biological Sciences from Pontificia Universidad Catolica del Ecua-

dor (PUCE) in 1998, and in 2001 received a Master’s degree in Ecology and Evolutionary Biology from

the University of Kansas under the supervision of Dr. Einda Trueb. In 2005 he received a Ph.D. degree

from the same institution with the thesis entitled “Phylogenetic systematics of South American lizards

of the genus Stenocercus (Squamata: Iguania).” Between 2006-2008 he was a postdoctoral fellow at the

Smithsonian Institution, National Museum of Natural History, Washington DC, USA, working under the

supervision of Dr. Kevin de Queiroz. He is currently Curator of Reptiles at the Zoology Museum QCAZ of

PUCE and an Associate Professor at the Department of Biology in the same institution. He has published

more than 30 scientific papers on taxonomy, systematics and biogeography of South American reptiles,

with emphasis on lizards. He is mainly interested in the theory and practice of phylogenetic systematics,

particularly as they relate to the evolutionary biology of lizards.

In accordance with the International Code of Zoological Nomenclature new rules and regulations (ICZN 2012), we have deposited this paper in publicly accessible institutional libraries.

The new species described herein has been registered in ZooBank (Polaszek 2005a, b), the official online registration system for the ICZN. The ZooBank publication LSID (Life Science

Identifier) for the new species described here can be viewed through any standard web browser by appending the LSID to the prefix “http://zoobank.org/”. The LSID for this publication

is: urn:lsid:zoobank.org:pub:61380956-FlAC-46C0-84F3-ClED545C46DC.

Separate print-only edition of paper(s) (reprint) are available upon request as a print-on-demand service. Please inquire by sending a request to: Amphibian & Reptile Conservation

(amphibian-reptile-conservation.org; arc.publisher@gmail.com).

Amphibian & Reptile Conservation is a Content Partner with the Encyclopedia of Life (EOL); http:///www.eol.org/ and submits information about new species to the EOL freely.

Digital archiving of this paper are found at the following institutions: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Ernst Mayr Library, Museum of Comparative Zool-

ogy, Harvard University, Cambridge, Massachusetts (USA); Elorida Museum of Natural History, Gainesville, Elorida (USA).

Complete journal archiving is found at: ZenScientist (http://www.zenscientist.com/index.php/filedrawer); Florida Museum of Natural History, Gainesville, Florida (USA).

Citations

ICZN. 2012. Amendment of Articles 8,9,10,21 and 78 of the International Code of Zoological Nomenclature to expand and refine methods of publication. Zootaxa 3450: 1-7.

Polaszek A et al. 2005a. Commentary: A universal register for animal names. Nature 437 : 477.

Polaszek A et al. 2005b. ZooBank: The open-access register for zoological taxonomy: Technical Discussion Paper. Bulletin of Zoological Nomenclature 62(4): 210-220.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (24)

May 2014 | Volume 8 | Number 1 | e76

of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License,

which permits unrestricted use for non-commercial and education purposes only provided

the original author and source are credited. The official publication credit source: Amphib-

ian & Reptile Conservation at: amphibian-reptile-conservation.org

Reproduction and spawning behavior in the frog,

Engystomops pustulatus (Shreve 1941)

^Santiago R. Ron, ^’^Andrea E. Narvaez, and ^Giovanna E. Romero

^Museo de Zoologia, Escuela de Biologia, Pontificia Universidad Catolica del Ecuador, Av. 12 de Octubre y Roca, Aptdo. 17-01-2184, Quito,

ECUADOR ^La Trobe University, Department of Zoology, Bundoora VIC 3086, AUSTRALIA ^Museo Ecuatoriano de Ciencias Naturales, Herbario

Nacional del Ecuador, Av. Rio Coca E6-115 e Is la Fernandina, Quito, ECUADOR

Amphibian & Reptiie Conservation

[Special Section] 8(1): 25-32.

Abstract— The study of reproductive strategies is central to understand the demography of

populations and the energetic relationships of the species with their ecosystem. Documenting the

reproductive natural history of the species is pressing in groups, like amphibians, that are threatened

with extinction at a global scale. Herein, we describe the reproductive ecology and spawning

behavior of the leptodactylid frog Engystomops pustulatus. In addition, we report observations

that suggest the existence of an alternative mating strategy. Our results show that reproduction

in E. pustulatus is characterized by high maternal investment (15% egg mass relative to body

mass). We found evidence of size-assortative mating with a tendency of larger females to mate with

larger males. Clutch size was correlated with female weight, female condition and male size. Larger

females showed a tendency to lay larger foam nests and larger nests contained more eggs. At

reproductive choruses, there was a male-biased operational sex ratio, indicative of high variance in

male reproductive success. We observed an amplectant couple spawning while an additional male

was embedded in the foam. We hypothesize that this behavior is evidence of an alternative mating

strategy where a small non-amplectant male attempts to fertilize the eggs that are extruded by the

amplectant female.

Resumen. — El estudio de las estrategias reproductivas es fundamental para entender la demografia

de las poblaciones y las relaciones energeticas de las especies con su ecosistema. Documentar

la historia natural reproductiva de las especies es apremiante en grupos, como los anfibios, que

estan amenazados con extincion a nivel mondial. Aqui, describimos la ecologia reproductiva

y el comportamiento de anidacion en la rana leptodactilida Engystomops pustulatus. Ademas,

reportamos observaciones que sugieren la existencia de una estrategia reproductiva alterna.

Nuestros resultados indican que la reproduccion en E. pustulatus esta caracterizada por una alta

inversion energetics de la hembra (15% de masa de huevos en relacion a la masa corporal). Se

evidencia que el apareamiento es selective con respecto al tamaho, con una tendencia de hembras

grandes a aparearse con machos grandes. El tamaho de la puesta estuvo correlacionado con el peso

de la hembra, la condicion de hembra y el tamaho del macho. Las hembras mas grandes mostraron

una tendencia de poner nidos de espuma mas grandes y los nidos mas grandes tuvieron un mayor

numero de huevos. En cores reproductivos, hubo una tasa sexual operativa sesgada hacia los

machos, lo que indica una alta varianza en el exito reproductive de los machos. Se observe una

pareja en amplexus construyento un nido mientras un macho adicional estaba incrustado en el

nido de espuma. Hipotetizamos que este comportamiento evidencia una estrategia de apareamiento

alterna en la que un macho pequeho intents fertilizar huevos puestos por una hembra en amplexus

con otro macho.

Key words. Alternative mating strategy, clutch size, clutch piracy, fertilization rates, nesting behavior, testis size

Citation: Ron SR, Narvaez AE, Romero GE. 2014. Reproduction and spawning behavior in the frog, Engystomops pustulatus (Shreve 1941). Amphibian

& Reptile Conservation 8(1) [Special Section]; 25-32 (e79).

Correspondence. Emails: ^ santiago.r.ron@gmail.com (Corresponding author), ^aenarvaezg@gmail.com,

^giaromev@gmail. com

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (25)

August 2014 | Volume 8 | Number 1 | e79

Ron et al.

Introduction

Understanding the natural history of reproduction is

essential to characterize the ecological niche and the

survival prospects of amphibians. Acquiring a better

understanding of amphibian reproduction will assist con-

servation efforts in the vertebrate class with the higher

number of species threatened with extinction (Chanson

et al. 2008).

The deposition of eggs in foam nests characterizes

the reproduction of most species of the Neotropical fam-

ily Leptodactylidae, which has 201 species distributed

from southern Texas to southern Chile (Frost 2014). In

most species, males call in choruses that are visited by

receptive females, which then actively choose a mate

(Ryan 1985). Amplectant pairs build foam nests where

hundreds of eggs are laid and fertilized (Crump 1974;

Heyer 1969; Ryan 1985). The foam is formed when the

male kicks the jelly surrounding the eggs while the fe-

male discharges them. The foam may protect the eggs

from dehydration and/or predation (Duellman and Tmeb

1994; Menin and Giaretta 2003) or from excessive heat

(Gorzula 1977).

Foam nests may facilitate multiple paternity by retain-

ing sperm (Kusano et al. 1991). There is a high propor-

tion of foam-nesting species among known cases of mul-

timale spawning in anurans, eight out of 15 (Byrne and

Roberts 1999; Kaminsky 1997; Prado and Haddad 2003).

Although several reproductive characteristics of Lepto-

dactylidae should favor multiple male mating strategies,

there are only two documented cases, Leptodactylus

chaquensis and L. podicipinus (Prado and Haddad 2003).

The paucity of records may be partly due to lack of stud-

ies. Although several leptodactylid species are abundant

and live even in urban areas, little is known about its bi-

ology beyond brief accounts of its systematics and mor-

phology. Such is the case of the widely distributed and

abundant Engystomops pustulatus (Ron and Read 2012).

Herein, we describe the reproductive natural history

of Engystomops pustulatus including fertilization rates,

testis size, clutch size, and relative egg mass to explore

factors that influence mate choice and reproductive out-

put. We also describe its spawning behavior with obser-

vations suggest the existence of a secondary male mating

strategy.

Materials and Methods

Study site and species

Engystomops pustulatus inhabits dry shrub, deciduous

forest, and lowland moist forest below 300 m in western

Ecuador. It can be relatively common during the rainy

season, when they reproduce. They are explosive breed-

ers that congregate around temporal pools. Males call

from the water and amplectant pairs build foam nests to

deposit their eggs (Ron and Read 2012). Engystomops

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (26)

pustulatus should not be confused with E. pustulosus, a

Central American species that has been a model for stud-

ies of behavioral ecology (e.g., Ryan 2005). For clarity,

hereafter, we refer to E. pustulosus exclusively as “Tun-

gara frog.”

Operational sex ratio (e.g., the number of males rela-

tive to the number of females in breeding aggregations)

in Engystomops pustulatus was assessed in western

Ecuador at three localities: Reserva Cerro Blanco, (W

80.0214°, S 2.0264°, Provincia del Guayas; 19 March

2003), Patricia Pilar (Provincia Los Rios; 21 Eebmary

2002), and the town of La Mana (Provincia de Cotopaxi;

28 December 2003). Reproductive output, nest size and

size assortment were evaluated in La Mana (W 79.265°,

S 0.943°, elevation 160 m) between 28 December 2003

and 08 February 2004 and Patricia Pilar (W 79.3707°, S

0.5372°, elevation 200 m) between 23 January and 20

April 2008 during the rainy season. At La Mana and Pa-

tricia Pilar, the vegetation is Evergreen Lowland Moist

Forest (as deflned by Sierra et al. 1999). Most of the for-

est in the region has been converted to pastures and agri-

cultural lands. Field observations took place after dusk,

between 19:00 and 3:00 h. Breeding occurred in small

temporary ponds on the streets of the town. Some sites

were under dim artificial light (street poles).

Fertilization rates and nest size

We estimated fertilization rates from amplectant pairs

collected from the field. The amplectant pairs were placed

in individual circular plastic containers (10 cm diameter)

with water depth of one cm. Most pairs made a nest after

few hours. Three or four days later, we washed the foam

with a solution of chlorine and water and counted the

number of hatched and undeveloped eggs (as described

by Ryan 1983). We used this proportion as a proxy for

fertilization rates. This methodology does not allow dis-

criminating between undeveloped eggs as result of egg

unviability or failed fertilization. Therefore, our method-

ology may slightly underestimate fertilization rates.

To estimate nest size, we measured (with digital cali-

pers, to the nearest 0.01 mm) the length of the longest

axis, width at the widest point perpendicular to the lon-

gest axis, and height of all nests laid in the containers. We

estimated nest volume with the formula of Vi ellipsoid:

V = —abc

12

where a, b, and c are the length, width, and height, re-

spectively. The measurements were taken while the nests

were <1 day old.

Adult size and egg mass

Sex was determined by the presence of nuptial pads, vo-

cal sac folds, and/or by gonad inspection. Snout-vent

length (SVL) was measured with Fowler digital calipers

(nearest 0.01 mm). Body mass was measured in the field

August 2014 | Volume 8 | Number 1 | e79

Reproduction and spawning behavior in Engystomops pustulatus

(before and after oviposition in females) with a digital

balance (nearest 0.1 g). Relative egg mass (maternal in-

vestment) was calculated as 1 — the ratio (female mass

after oviposition/female mass before oviposition).

After being kept in the plastic containers to allow

spawning, females were euthanized by immersion in

chloretone, fixed in 10% formalin, and preserved in 70%

ethanol. Egg mass and body mass were measured after

preservation in females that did not spawn. Each female

was weighted on a digital balance (to the nearest 0.001

g), after removing excess ethanol. Then, the remaining

egg masses (including immature eggs and jelly) were

removed from the abdomen and weighted. Relative egg

mass was calculated by dividing total egg mass by non-

gravid female mass. Estimates of relative egg mass could

be influenced by preservation in ethanol. Therefore, com-

parisons with relative egg mass in non-preserved nesting

females should be interpreted with caution. All preserved

specimens are deposited at the amphibian collection of

the Zoology Museum of Pontificia Universidad Catolica

del Ecuador.

Reproductive behavior

Behavioral observations were carried out at male cho-

ruses in La Mana, Ecuador. Spawning behavior was

described from of a single nesting event at La Mana.

Spawning was recorded in the field under infrared light

with a digital camcorder SONY TRV70. The complete

video is available at AmphibiaWeb (http://amphibiaweb.

org).

Statistical analyses

Eor normally distributed variables, we tested the sig-

nificance of relationship between them using linear re-

gression ANOVAs; for non-normal variables, we tested

relationship with Spearman’s rank correlations. Differ-

ences between groups were tested with t-tests (assuming

non-equal variances). Statistical tests were implemented

in software IMP v.5.1 (SAS Institute, 2003).

Results

Reproductive output, fertilization rates, and

nest size

Among 77 nests, the mean number of eggs was 320 (SD

= 142.6, range 0-747). The average percentage of unfer-

tilized eggs was 1.89% (SD = 3.3, 0-19.1, n = 46); Pig.

lA); ~l/5 of the nests had a fertilization rate of 100%.

Snout- vent length difference between both parents was

not correlated with the number of unfertilized eggs

(Spearman’s Rho = 0.098, P = 0.524) or the proportion

of unfertilized eggs (Rho = 0.145, P = 0.341).

Mean nest volume was 37.0 cm3 (SD = 14.4, range

2.8-85.6, n = lA). Nest volume is correlated to the

number of eggs (larger nests have more eggs; Table

1, Pig. 2A) and female size (larger females lay larger

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (27)

-3 -2 -1 0 1 2 3 4 5 6

Pair SVL difference (mm)

25 26 27 28 29 30 31 32 33 34 35 36 37

SVL female (mm)

Fig. 1. Size and fecundity rates for amplectant pairs of Engysto-

mops pustulatus. After collected in amplexus in the field, pairs

were left in plastic containers where they could spawn. (A) Pro-

portion of unfertilized eggs among pairs that successfully built

a nest, (B) Female vs. male snout-vent length (SVL) with linear

regression and 95% confidence intervals (dashed lines).

nests; Table 1, Pig. 2B). A multiple regression of num-

ber of eggs, female SVL, and male SVL explains 25%

of the variation in nest volume {F = 7.51, df = 66, P

< 0.001). However, only number of eggs is signifi-

cant for the regression model (F = 18.18, P < 0.001).

Number of eggs was significantly correlated with

male SVL (Pig. 2C) but not with female SVL. Number

of eggs was correlated with female mass before and after

oviposition and female condition (Table 1).

Non-spawning females had large masses of eggs in

their abdomens (mean relative egg mass = 0.354, SD

= 0.138, range 0.129-0.621, n = 13). Average maternal

investment for spawning females was 15.2% of body

weight (SD = 7.77, 1.8-39.4, n = 42).

Size assortment and spawning

We found size-assortative mating as male and female

size of amplectant pairs was correlated (ANOVA’s F =

24.1, P < 0.001, R^ = 0.176; Pig. IB). Overall, females

were significantly larger than their mates (n= 115; mean

female SVL = 31.0 mm, SD = 1.9, range 25.3-36.5; male

August 2014 | Volume 8 | Number 1 | e79

Ron et al.

0 10 20 30 40 50 60 70 80 90

Nest volume (cm3)

Female SVL (mm)

Male SVL (mm)

Fig. 2. Bivariate plots for (A) nest volume vs. number of eggs,

(B) females size vs. nest volume, and (C) male size vs. num-

ber of eggs in Engystomops pustulatus. Linear regressions with

95% confidence intervals (dashed lines), determination coef-

ficients (R^), and ANOVA’s P values are shown.

SVL = 28.5, SD = 1.3, 25.2-32.3; paired-f = 14.7, df =

104, P < 0.001). However, in 10 pairs (8.6%) the male

was larger. Mean SVL difference between amplectant

male and female was 2.5 mm (SD = 1.74, range -2. 6-6.7,

n = 105).

Reproductive behavior

Males began calling immediately after dusk. They called

while floating in temporary ponds with water <10 cm

deep. Male density at some choruses was high, result-

ing in some males calling a few centimeters away from

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (28)

each other. Males defended calling sites and aggressive

interactions ensued if another male approached within

a radius of <10 cm. Aggressive behavior consisted of

mew-like vocalizations and attempts to clasp the rival

male. Amplexus and egg deposition occurred at the same

ponds where choruses were calling. Amplexus is axillary.

Operational sex ratio at choruses was male-biased.

During a survey at La Mana, we recorded 14 males but

only one female; at Cerro Blanco, the ratio was 3:1 (n =

16 individuals); at Patricia Pilar, the ratio was 8.5:1 (n =

19). The average ratio is 8.5:1 (n = 3 surveys).

Spawning behavior . — Nests were built while in am-

plexus, on shallow water, next to vegetation or muddy

banks. The following description is based on an amplec-

tant pair found on 04 February 2004 at -1:00 AM (male

QCAZ 26672, SVL = 26.2 mm, hereafter referred as

a-male; female QCAZ 26671, SVL = 31.7 mm) building

a foam nest. At the beginning of the observation, the nest

already had a diameter >50 mm. The male remained in

amplexus until the couple left the nest (50 minutes later).

To form the foam, the male kicked the egg mass while

they were being extruded from the female’s vent. Kick-

ing occurred in regular bursts with intervening periods

during which the couple was inunobile. In a typical burst

cycle, the male’s legs move downward, presumably to

place his feet next to the female vent. Then, the male’s

feet move up until they reach the posterior end of his

dorsum. At that moment, usually one or two eggs become

visible in the jelly matrix between the feet. This is fol-

lowed by a series of -20 rapid kicks on which his legs

become partly extended backward and then distended

forward until reaching the posterior end of his dorsum.

During these kicks, his legs move simultaneously but in

opposite directions (forward-backward) and feet momen-

tarily touch medially. The burst ends with to 2-A forceful

kicks on which his legs are nearly completely extended

posterolaterally, partly removing the foam that lies im-

mediately behind the couple. Each male burst seems to

be triggered by an abdominal movement of the female.

Each burst of kicking lasted on average 4.64 s (SD =

0.53, range 2.13-6.22, n = 215); the intervening inuno-

bile periods lasted 9.25 s (SD = 12.15, range 0.12-119,

n = 215). Total duration of bursts was 16’30” during 50’

of observation. The duration of each burst and the num-

ber of bursts decreased during the second half of the se-

quence (Fig. 3).

Multimale nesting behavior . — Multimale spawn-

ing was only observed once, during the spawning event

described in the previous section (male QCAZ 26672,

female QCAZ 26671). From the beginning of the obser-

vation, a peripheral adult male (QCAZ 26673; hereafter

referred as p-male) was sitting on the nest edge, directly

opposite to the nesting couple and with the posterior Vi of

its body embedded in the foam (Fig. 4). On at least flve

occasions its body moved slightly from side to side in se-

quences that lasted 3-4 s (Fig. 3). The movements were

always in concert with the kicking bursts of the a-male.

August 2014 | Volume 8 | Number 1 | e79

Reproduction and spawning behavior in Engystomops pustulatus

Table 1. Pearson’s correlation coefficients and ANOVA’s P values for linear regressions. Body condition is defined

as the residuals between SVL and mass. SVL = snout-vent length.

Variable 1

Variable 2

R2

n

P

Nest volume

Female size (SVL)

0.082

lA

0.013*

Nest volume

Female mass (before oviposition)

0.020

56

0.287

Nest volume

Male size (SVL)

0.026

lA

0.168

Nest volume

Male condition

<0.001

lA

0.796

Nest volume

Number of eggs

0.241

70

<0.001*

Number of eggs

Male size (SVL)

0.051

76

0.049*

Number of eggs

Female size (SVL)

0.027

76

0.151

Number of eggs

Female mass (before oviposition)

0.111

62

0.008*

Number of eggs

Female mass (after oviposition)

0.128

58

0.006*

Number of eggs

Female condition

0.125

62

0.005*

Number of eggs

Male condition

0.011

76

0.352

* Significant at P <0.05

Burst no.

10

^ 6

Z3

JD

N- 4

o ^

6

2 ! 2

0

Fig. 3. Spawning of Engystomops pustulatus nesting couple

(QCAZ 26671-72) and P-male (QCAZ 26673). Above: dura-

tion of kicking bursts. Below: number of bursts per minute; as-

terisks indicate P-male movements in the foam. Measurements

are shown in sequence from the beginning of the observation

until the couple left the nest. See text for details.

Most likely, the movements were generated by kicking

bursts of the p-male legs (hidden below the foam). He

left 23 min later, apparently following an amplectant

couple (not collected) that approached at a distance of 10

cm from the nest (see below).

The p-male (SVL = 25.3 mm) was one of the small-

est in the population. Out of 49 calling males measured

during the same season, only three were smaller (mean

SVL = 27.55 mm, SD = 1.23); out of 59 males found in

amplexus, only one was smaller (mean SVL = 27.9 mm,

SD = 1.20). Assuming a normal SVL distribution, the

probability of drawing a male with equal or lower SVL

by chance is 0.020 (z-score = -2.058). On a sample of

seven males including the p-male, mean testes mass was

0.47% of total body mass (range 0.24-0.70%; mean body

mass = 1.59 g, SD = 0.28). Contrary to our expectations,

the p-male had the proportionally smallest testes.

Discussion

Clutch size, fertilization success, and parental

investment

Number of eggs/clutch in Engystomops pustulatus is

-37% higher than in the tungara frog (Ryan 1985). In

several anurans, clutch size is significantly correlated to

body size (e.g.. Crump 1974; Ryan 1985; Wells 2007). In

E. pustulatus, such a relationship was significant for fe-

male condition and gravid and non-gravid female mass.

However, the relationship was not significant for female

SVL. Interestingly, we also found a significant correla-

tion between number of eggs and male SVL suggesting

that larger males have a higher reproductive success.

This correlation could not be explained by indirect cor-

relations with the other measured variables because they

were either uncorrelated with male SVL (e.g., nest vol-

ume) or uncorrelated with number of eggs (e.g., female

SVL).

Female SVL and number of eggs are correlated with

nest volume. Nest foam also results from intense male

physical activity. However, we were unable to find a re-

lationship between nest volume and either male size or

male body condition (Table 1).

We found size assortative mating as large females

have a tendency to mate with large males. An adaptive

explanation for size assortative mating states that it in-

creases fertilization rates because it results in female and

male vents being closer during amplexus (Licht 1976).

Evidence for this scenario has been reported for the Tun-

gara frog (Ryan 1985). Engystomops pustulatus lacks

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August 2014 | Volume 8 | Number 1 | e79

Ron et al.

Fig. 4. Engystomops pustulatus nesting couple (QCAZ 26671-

72) and P-male (QCAZ 26673). The couple builds the foam

nest as the male kicks the egg masses extruded by the female.

Frame from video (infra-red recording). See text for details.

Fig. 5. Relationship (in log space) for body and testis mass

among 11 species of Leptodactylinae frogs. Except for Engys-

tomops pustulatus, data is from Prado and Haddad (2003).

Open circles indicate species on which multimale spawning

has been reported. Note that E. pustulatus, in which multi-male

spawning apparently occurs, also has larger testis than other

Leptodactylinae.

that relationship as demonstrated by couples with large

differences in size {2-A mm) showing high fertilization

rates (Fig. lA). The lack of influence of size difference

on fertilization may be explained by our observation of

spawning behavior because the male uses his feet to drag

the eggs from the female’s underside to his own vent.

Therefore, the relative position of male and female vents

may have a minor influence in the relative position of

eggs and released sperm. Fertilization rates are generally

high (more than 98% on average) suggesting that size

differences between male and female have little influence

in individual fitness. Similar results have been reported

in other explosive breeding anurans like Lithobates syl-

vaticus (Howard and Kluge 1985) and Anaxyrus cogna-

tus (Krupa 1988).

Size assortative mating could also result from non-

adaptive interactions. If small males mating with large

females are more easily displaced than large males mat-

ing with large females, a size correlation will result

(e.g., Howard and Kluge 1985). This mechanism seems

unlikely in Engystomops. During our fieldwork with E.

pustulatus and with other species of Engystomops in the

Choco and the Amazon region, we never saw unmated

males attempting to displace amplectant males. Attempts

were rare in E. pustulosus and all of them were unsuc-

cessful (Ryan 1985). Therefore, an explanation for size

assortative mating in E. pustulatus and its sister species,

E. puyango (reported by Ron et al. 2010) is pending.

Reproductive investment (or effort) is a measure of

the allocation of energy in reproduction relative to total

energy (Pianka 2011). Theory predicts that a high repro-

ductive investment should be more adaptive if females

are unlikely to survive to another reproduction event

(Williams 1966). Our estimate of mean reproductive in-

vestment for Engystomops pustulatus (15.2%; egg mass

relative to body mass) is relatively high in comparison

to other anurans. For example. Crump (1974) and Prado

and Haddad (2005) report investments ranging from 3.1

to 18.2% for 34 Neotropical species (including nine lep-

todactylids). The investment of E. pustulatus, however,

is not the highest recorded for an anuran. For example,

the myobatrachid Crinia signifera invests 25.9% of the

gravid female mass in each spawning event (Lemckert

and Shine 1993). This high investment was interpreted as

resulting from a low probability of survival to additional

spawning events (Lemckert and Shine 1993). Similarly,

we hypothesize that the observed large investment in E.

pustulatus could result from low survival rates.

Nesting behavior

Overall, nest building behavior was similar to that re-

ported for the Tungara frog (Dalgetty and Kennedy

2010; Heyer and Rand 1977) and Physalaemus ephip-

pifer (Hodl 1990). The kicking bursts observed in E.

pustulatus are comparable to the “rotational movements”

described in P. ephippifer except that the legs seem to

extend further backwards in E. pustulatus (compared to

figure 5 in Hodl 1990).

Nest building is an energetically costly task (Ryan

1985) and the observed decrease in the frequency of

kicking bursts towards the end of spawning (Fig. 3) was

also reported in the tungara frog (Ryan 1985) and Phy-

salaemus ephippifer (Hodl 1990). As in Leptodactylus

labyrinthicus, the Tungara frog, and P. ephippifer, kick-

ing bursts seemed to be triggered by a female abdominal

movement (Heyer and Rand 1977; Hodl 1990; Silva et

al. 2005). The movie quality did not allow us to deter-

mine whether the decrease in burst frequency was male

or female-driven in E. pustulatus.

Multimale mating behavior

Our observation of more than one male spawning with a

female during oviposition suggests that multiple pater-

nity and alternative reproductive strategies may exist in

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August 2014 | Volume 8 | Number 1 | e79

Reproduction and spawning behavior in Engystomops pustulatus

Engystomops pustulatus. Although the P-male was not in

amplexus, its movements were similar and in synchrony

with those of the amplectant male, suggesting that it was

attempting to fertilize eggs (clutch piracy). A similar re-

productive behavior (with synchronic leg movements)

has been reported in Leptodactylus chaquensis although

with up to seven males in addition to the amplectant male

(Prado and Haddad 2003). Egg fertilization by periph-

eral non-amplectant males has also been demonstrated

in Chiromantis xerampelina, a foam-nesting rhacophorid

(Jennions and Passmore 1993).

The evolution of multimale spawning should be fa-

cilitated in reproductive systems where: (1) the opera-

tional sex ratio is strongly male biased, (2) fertilization is

external, (3) fecundity is high, and (4) eggs are spatially

aggregated (Byrne and Roberts 2004; Shuster and Wade

2003). All this characteristics are part of the reproduc-

tion of E. pustulatus. Therefore, the occurrence of multi-

male spawning was probable. As previously reported in

the Tungara frog (Ryan 1983), our data suggests that an

individual male is frequently unable to fertilize all the

eggs of a clutch, even in the absence of sperm competi-

tion. Although the presence of unfertilized eggs suggests

the potential for fitness gain of a p-male sneaking into

the nest of an amplectant pair, the proportion of unfertil-

ized eggs was typically low (1.89% on average). Higher

fitness gains for the p-male may result from sperm com-

petition.

We could not determine the frequency of multimale

spawning in the population. We observed monoandrous

spawning frequently and multimale spawning was only

recorded once, suggesting that it is relatively infrequent.

This is consistent with observations across a variety of

taxa showing that p-male strategies exist at a low fre-

quency in natural populations (Shuster and Wade 2003;

but see Byrne 2002; Jennions and Passmore 1993). The

low number of reports of multimale spawning among

leptodactylids is surprising because the characteristics of

the reproductive system of Leptodactylidae should favor

the evolution of secondary male mating strategies. The

paucity of known cases may be, at least partly, a sam-

pling artifact because the reproductive behavior has been

described in only few species.

Acknowledgments. — Fieldwork in 2003 and 2004

was funded by NSF IRCEB grant 0078150 to D. Can-

natella. Fieldwork in 2008 was funded by a DGA grant

from Pontificia Universidad Catolica del Ecuador to S.

R. Ron. The Ecuadorian Ministerio de Ambiente pro-

vided research and collection permits 004-IC-FAU-DPF,

and 006-IC-FAU-DBAP/MA. Fieldwork at la Mana was

assisted by F. P. Ayala, M. A. Guerra, and S. Padilla and

at Patricia Pilar by A. Argoti, P. Arias, F. Camacho, I.

Narvaez and A. Teran. Jose R. Ron, G. M. Melo, and

R. Valdivieso provided logistic support in Quito. Help-

ful comments for the manuscript were provided by X.

E. Bernal.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (31)

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Received: 16 May 2014

Accepted: 22 July 2014

Published: 08 August 2014

Santiago R. Ron is the curator of amphibians and professor at the Pontificia Universidad Cat61ica del Ecuador

in Quito. His research focuses on the evolution and diversity of Neotropical amphibians and the evolution of

animal communication and sexual selection. In the area of conservation biology, Santiago is interested in the

study of amphibian extinctions in the Andes. He is a founding member of the Ecuadorian Academy of Sciences.

Andrea E. Narvaez is a Ph.D. student at La Trobe University (Australia) and holds a Master’s degree in

Integrative, Evolutionary Biology and Infectious Diseases from the Universite Eran9ois Rabelais de Tours

^ liW (Erance). She is currently investigating the evolution of visual signaling of Ecuadorian anoles. Her research

focus includes ecology and animal behavior, mainly to evaluate the evolution of communication systems,

sexual and natural selection. She is interested in the use of quantitative tools to measure animal behavior and

has experience working with a variety of animals (frogs, crickets, lizards).

Giovanna E. Romero is a Research Associate at Museo Ecuatoriano de Ciencias Naturales, Botany Section

(QCNE). She holds a bachelor degree in biological sciences from Pontificia Universidad Catblica del Ecuador

and a Master’s degree in Plant Biology from the University of Texas in Austin. She has worked for many years

in the Galapagos Islands and has a deep knowledge of their flora and fauna. She has been collaborating with

QCNE since 2012. She is interested in taxonomy and digital curation of ferns and lycophytes.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (32)

August 2014 | Volume 8 | Number 1 | e79

Amphibian & Reptiie Conservation

8(1) [Special Section]: 33-44.

the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported

License, which permits unrestricted use for non-commercial and education purposes only

provided the original author and source are credited. The official publication credit source:

Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org

High prevalence of Batrachochytrium dendrobatidis in an

Andean frog community (Reserva Las Gralarias, Ecuador)

^Juan M. Guayasamin, ^Mngela Maria Mendoza, "^Ana V. Longo,

"^Kelly R. Zamudio, and ^^Elisa Bonaccorso

^Centro de Investigacion de la Biodiversidad y Cambio Climdtico (BioCamb), Universidad Tecnologica Indoamerica, Calle Machala y Sabanilla,

Quito, ECUADOR ^Laboratorio de Macroecologia, Centro de Investigaciones en Ecosistemas, Universidad Nacional Autonoma de MEXICO ^Grupo

de Investigacion en Ecologia y Conservacion Neotropical, SAMANEA Eundacion de Apoyo Educative e Investigative, COLOMBIA "^Department of

Ecology and Evolutionary Biology, Cornell University, Ithaca, New York, USA ^Biodiversity Institute, University of Kansas, Lawrence, Kansas, USA

Abstract— Wfe report patterns of infection of Batrachochytrium dendrobatidis {Bd) in a cloud forest

amphibian community in the Andean Western Cordillera of Ecuador (Reserva Las Gralarias). Data

were obtained during the rainy seasons of two consecutive years, using qPCR (year 2012) and end-

point PCR (year 2013). We show that average Bd prevalence in this amphibian community is high

(2012: 35-49%; 2013: 14-32%), but found no evidence of population declines or that Ed is negatively

affecting host populations. We found a significant correlation between Ed prevalence and taxonomy,

reproductive mode, and habitat, but no correlation between Ed infection intensity and the same three

variables. Contrary to our expectations, frog species with aquatic reproductive modes (glassfrogs,

treefrogs) showed lower Ed prevalence than direct-developing frogs {Pristimantis spp.). Although

further monitoring is needed to determine long-term population trends, our two-year dataset on

disease and population size support the hypothesis that frogs are tolerant to infection, a condition

that could potentially have resulted from exposure to previous Ed epidemic outbreaks.

Resumen. — En este estudio reportamos dates sobre los patrones de infeccion de Batrachochytrium

dendrobatidis {Bd) en una comunidad de anfibios en la Cordillera Occidental de los Andes del

Ecuador (Reserva Las Gralarias). Los dates fueron obtenidos durante la estacion Iluviosa en dos

ahos consecutivos, utilizando qPCR (aho 2012) y PCR de punto final (aho 2013). Los resultados

muestran una alta prevalencia de Eden la comunidad (2012: 35-49%; 2013: 14-32%); sin embargo, no

se encontro evidencia de disminuciones poblacionales o de que Ed este afectando negativamente a

las especies de anfibios. Existe una relacion significativa entre la prevalencia de Edy la taxonomia,

modo reproductive y habitat de los anfibios, pero no hubo correlacion entre la intensidad de

infeccion de Ed y las mismas tres variables. Contrario a nuestras predicciones, las especies de

anuros con larvas acuaticas (ranas de cristal, ranas arboreas) presentaron prevalencias de Ed mas

bajas que los anuros de desarrollo directo {Pristimantis spp.). A pesar de que se requiere de un

monitoreo continue para determinar las dinamicas poblacionales a largo plazo, los dates obtenidos

hasta el memento apoyan un escenario donde las especies de ranas de la Reserva Las Gralarias

parecen tolerar la infeccion de Ed, una condicion posiblemente adquirida mediante la exposicion a

brotes epidemicos previos.

Key words. Chytridiomycosis, emerging disease, amphibian declines, Andes, conservation

Palabras claves. Quitridiomicosis, enfermedad emergente, declinacion de anfibios, Andes, conservacion

Citation: Guayasamin JM, Mendoza AM, Longo AV, Zamudio KR, Bonaccorso E. 2014. High prevalence of Batrachochytrium dendrobatidis in an Andean

frog community (Reserva Las Gralarias, Ecuador). Amphibian & Reptiie Conservation 8(1) [Special Section]: 33-44 (e81).

Correspondence. Emails: ^jmguayasamin® gmail.com (Corresponding author).

August 2014 | Volume 8 | Number 1 | e81

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (33)

Guayasamin et al.

Introduction

A third of global amphibian species are threatened with

extinction (Stuart et al. 2004; Wake and Vredenburg

2008) and, most concerning, numerous local popula-

tion declines and extinctions have occurred in relatively

pristine areas, where anthropogenic habitat destruction is

low (Lips 1998, 1999; Drost and Fellers 1996; La Marca

et al. 2005). In the last two decades, several studies have

attributed anuran mass mortality events to the emergence

of the pathogenic fungus Batrachochytrium dendroba-

tidis (Bd), a pathogen with widespread geographic and

ecological distribution (Berger et al. 1998; Daszak et al.

1999, 2003; Lips et al. 2006; Becker and Zamudio 2011;

Rodriguez et al. 2014). Alternative explanations to am-

phibian declines add a role to global warming and tem-

perature variability (Pounds et al. 2006; Rohr and Raff el

2010; Menendez-Guerrero and Graham 2013).

Batrachochytrium dendrobatidis infects the keratin-

ized skin of amphibians and disrupts the regulatory func-

tioning of the integument (Berger et al. 1998; Voyles et

al. 2009). Infection inhibits host inunune responses in

some species (Fites et al. 2013) and in severe cases of

infection, electrolyte depletion and osmotic imbalance

may lead to mortality (Voyles et al. 2007, 2009). How-

ever, not all amphibian species are equally susceptible to

the pathogen. For example, at Santa Fe, Panama, Bd has

caused declines or local extinctions of most anurans in

the original community, but six species of frogs and toads

remain abundant, despite being infected by the fungus

(Lips et al. 2006). In laboratory challenge experiments,

amphibian mortality rates range from 0% to 100%, de-

pending on the species, host age, pathogen genotype, and

dosage (Berger et al. 2005a; Daszak et al. 2004; Longo et

al. 2014). The reasons for host differences in susceptibil-

ity include immunogenic variation (Ellison et al. 2014;

Savage et al. 2014), microhabitat use (Kriger and Hero

2007; Griindler et al. 2012), association with water as

embryos, tadpoles, or adults (Lips et al. 2003), and host

thermoregulatory behavior (Richards-Zawacki 2010).

Because Bd transmission may happen through frog-frog

contact, or through motile zoospore movement from one

host to another, frogs and toads that spend more time in

water are expected to have higher exposure and suscep-

tibility to infection than species that are primarily terres-

trial (i.e, direct developers; Lips et al. 2003; Kriger and

Hero 2007).

In this study, we report data on infection patterns of

Bd obtained during the rainy seasons of two consecu-

tive years in the amphibian community of Reserva Las

Gralarias, a cloud forest site in the Andean Western Cor-

dillera of Ecuador. We found that Bd prevalence in all

amphibian species is high, but found no evidence that

Bd is negatively affecting amphibians (i.e., no apparent

population declines, or records of clinical signs of chy-

tridiomycosis). We report on infection prevalence and

intensity dynamics for the two-year period, and examine

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (34)

patterns of Bd infection in species that vary in their tax-

onomy, reproductive mode, and habitat.

Materials and Methods

Study site: The study was conducted at Reserva Las

Gralarias (0°0L S, 78°44’ W; 1822-2400 m), a private

reserve covering an area of 1,063 acres (425 ha) located

on the Pacific slopes of the Andes, Pichincha Province,

Mindo Parish, Ecuador. The study site has an eleva-

tional range of 1,825-2,400 m and includes primary and

secondary forest, regenerating pasture, and numerous

ephemeral and permanent streams and creeks (Hutter and

Guayasanfin 2012).

Amphibian taxonomy: For generic and suprageneric clas-

sification, we follow the taxonomic proposals of Hedges

et al. (2008), Guayasamin et al. (2009), and Faivovich et

al. (2005), Pyron and Wiens (2011), as sununarized in

Frost (2014).

Amphibian richness and abundance: During the rainy

seasons of 2012 (23 January-29 March) and 2013 (14

March-22 April), we sampled trails of Reserva Las

Gralarias during the night, including most of its habitat

heterogeneity and elevational gradient, to record the spe-

cies richness of the reserve. We placed eight transects,

each with an area of 500 x 4 m (Appendix 1), to maxi-

mize species detection and to obtain a baseline dataset

on population size and Bd prevalence. Each transect was

sampled by two people for 3-4 hours during the night

(generally starting at 8 pm); temperatures during sam-

pling varied between 11-15 °C. All detected amphibians

were, when possible, photographed. Calling males were

also reported and identified with the aid of photographic

and acoustic guides (Arteaga et al. 2013; Centro Jambatu

2011-2014). We used a Student’s t-test to quantify differ-

ences in population sizes in transects that were sampled

multiple times during the rainy season of 2012 and 2013

(Lucy’s Creek and Kathy’s Creek); the normality of spe-

cies abundance was assessed using a Shapiro-Wilk Test.

Diagnosis of Batrachochytrium dendrobatidis: We

swabbed the ventral regions of all amphibians captured

in our survey, following the standard procedures in Hyatt

et al. (2007; Fig. 1); dry swabs were stored in -4 °C until

analysis. Testing for Bd was carried out using Real-Time

PGR (q-PCR) for samples obtained during 2012 and end-

point Polymerase Chain Reaction (PCR) for samples ob-

tained in 2013; the use of these two methods was contin-

gent on access to q-PCR (available during 2012). In both

cases, DNA extractions were carried out using guanidin-

ium thiocyanate. For samples obtained during 2012, we

used a 1:10 dilution of the extract as template in Taqman

q-PCR assays for the detection of Bd (Boyle et al. 2004).

This assay uses BJ-specific primers ITS 1-3 Chytr and

5.8S Chytr, in addition to the fluorescently-labeled probe

Ch)4r MGB2, and amplifies the ITS-1 fragment of the

August 2014 | Volume 8 | Number 1 | e81

Batrachochytrium dendrobatidis in an Andean frog community

Fig. 1. Swab sample obtained from Centrolene heloderma at

Reserva Las Gralarias, Ecuador.

Bd genome at the junction of the ITS-1 and 5.8S regions.

We used a standard curve that included 1000, 100, 10,

1, and 0.1 zoospore genome equivalents, and followed

qPCR conditions described in Boyle et al. (2004). For

samples obtained during 2013, Bd presence was tested

using the internal transcribed spacer regions (ITS-1, ITS-

2) primers Bdla (5’-CAGTGTGCCATATGTCACG-3’)

and Bd2a (5’-CATGGTTCATATCTGTCCAG-3’) de-

veloped by Annis et al. (2004); the presence/absence of

Bd was determined via the visualization of the amplified

band in agarose gel electrophoresis. The two methods to

detect Bd have different sensitivities; therefore, direct

comparisons of Bd prevalence between years should be

considered with caution. However, family and habitat

correlates with infection status should not be biased by

detection method, and qPCR offers the additional advan-

tage of quantifying infection intensity (load).

Prevalence and correlates of Batrachochytrium den-

drobatidis in amphibians: We estimated prevalence of

Bd within each anuran species as the number of frogs

that tested positive for Bd, divided by the total number of

sampled frogs for that particular species in a given year.

We estimated the 95% confidence interval for preva-

lence in each species, (Wilson 1927; Newcomb 1998).

We modeled Bd presence or absence in each individual

by using a logistic regression. We tested for possible as-

sociations of Bd prevalence with the following variables:

habitat (terrestrial, riparian, lentic), reproductive mode

(aquatic, terrestrial), and taxonomy (family). Statistical

significance of results was assessed with a chi-square

test.

Infection intensity of Batrachochytrium dendrobati-

dis and correlates in amphibians: We tested for possible

associations of Bd intensity (measured as zoospore ge-

nomic equivalents) with the following variables: habitat

(terrestrial, riparian, lentic), reproductive mode (aquatic,

terrestrial), and taxonomy (family, genus, species; Ap-

pendix 2). Given the strong right skew of infection load,

we used the non-parametric Kruskal- Wallis test. All sta-

tistical analyses were performed using R v. 2.15.3 (R

CoreTeam 2012).

Results

Species richness and abundance: During the two sam-

pling periods, we recorded a total of 2,450 individuals

of 28 species (Appendix 2). The abundance of species at

Lucy’s Creek and Kathy’s Creek is summarized in Tables

1 and 2. Because most taxa were scarce, we restricted

the comparisons between years to relatively abundant

species (glassfrogs). Abundances of glassfrogs at Lucy’s

Creek and Kathy’s Creek were not significantly different

between years (Tables 1, 2).

Prevalence of Batrachochytrium dendrobatidis in

amphibians: Swabs of 320 frogs were tested for Bd, and

O

Fig. 2. Significant Bd infection differences in amphibians according to reproductive modes, habitat use, and taxonomy. P values are

reported for 2012 and 2013; significance is noted by ** (p < 0.01) and *** (p < 0.001).

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Guayasamin et al.

Table 1. Abundance of amphibian species at Lucy’s Creek, Reserva Las Gralarias. Abundances are presented as minimum-maxi-

mum, followed, in parenthesis, by mean + standard error. The Student’s t-test was performed only in species with normally distrib-

uted abundances.

Lucy’s Creek

Mest (p)

Population trend

Year

2012

2013

Number of nights sampled

10

6

Family/Species

Centrolenidae

Centrolene lynchi

5 - 20 ( 13 . 1 + 5 . 13 )

2-18 ( 9.0 + 6 . 54 )

0.184

No difference

Centrolene peristictum

4-35 ( 20.7 + 11 . 68 )

6 - 26 ( 15.2 + 7 . 00 )

0.314

No difference

Nymphargus grandisonae

Hylidae

1 - 10 ( 6.2 + 3 . 12 )

0-8 ( 3.8 + 2 . 93 )

0.155

No difference

Hyloscirtus alytolylax

Craugastoridae

3-7 ( 4.3 + 1 . 42 )

0 - 6 ( 3.3 + 2 . 16 )

0.295

No difference

Pristimantis achatinus

0-2 ( 0.3 + 0 . 67 )

0-1 ( 0.2 + 0 . 41 )

—

Pristimantis appendiculatus

0-2 ( 0.8 + 0 . 92 )

0-1 ( 0.5 + 0 . 55 )

—

Pristimantis calcarulatus

0-2 ( 0.2 + 0 . 63 )

0-1 ( 0.2 + 0 . 41 )

—

Pristimantis eremitus

0

0-2 ( 0.5 + 0 . 84 )

—

Pristimantis eugeniae

0

0-2 ( 0.3 + 0 . 82 )

—

Pristimantis illotus

0-1 ( 0 . 1 + 0 . 32 )

0

—

Pristimantis parvillus

0-1 ( 0 . 1 + 0 . 32 )

0

—

Pristimantis sobetes

0

0-2 ( 0.3 + 0 . 82 )

—

Pristimantis w-nigrum

0-2 ( 0.6 + 0 . 84 )

0-2 ( 0.7 + 0 . 82 )

—

approximately a third of those were positive. In samples

from 2012, prevalence of Bd was relatively high, with

42% of all frogs testing positive for Bd infection. Dur-

ing 2013, Bd prevalence was 22%. Differences in preva-

lence between the two years are likely caused by detec-

tion method. Most species infected in 2012 carried low

Bd loads as determined by qPCR; the highest Bd load

obtained was in Centrolene ballux with 22.5 genomic

equivalents. Prevalence per species per year is summa-

rized in Table 3.

The logistic regression shows a significant relation-

ship (p < 0.001) of Bd infection with species reproduc-

tive mode, habitat, and taxonomy (Fig. 2). Frogs with

a terrestrial reproductive mode (direct developers; i.e.,

genus Pristimantis; see Duellman and Trueb 1986) have

a higher Bd prevalence than amphibians with aquatic re-

production (i.e., glassfrogs and treefrogs). Frog species

that are dependent on riverine habitats for reproduction

show significantly less infection than anurans that use

terrestrial or lentic habitats for reproduction (p < 0.001).

Also, species in the Centrolenidae family (glassfrogs)

show a lower Bd prevalence than species in Craugastori-

dae and Hylidae (Table 3). Although, Bd prevalence dur-

ing 2012 was significantly higher than in 2013 (probably

as a result of higher sensitivity of qPCR), we found no

significant interaction among sampling year and repro-

ductive mode, habitat, or taxonomy.

Infection intensity of Batrachochytrium dendrobati-

dis and correlates in amphibians: We found no relation-

ship between Bd infection intensity {Bd load, year 2012)

and taxonomy, reproductive mode, or habitat.

Discussion

Our results show a relatively high mean prevalence of

Bd (36%) across both years in the Andean frog concunu-

nity of Reserva Las Gralarias (see Hossack et al. 2010 for

comparison). From a total of 20 species analyzed, only

three (Nymphargus griffithsi, Pristimantis illotus, and P.

pteridophilus) tested negative for Bd\ however, sample

sizes for non-infected species were low (5, 1, and 3 in-

dividuals, respectively). Infected species included frogs

with very different reproductive modes, including taxa

with terrestrial direct development {Pristimantis spp.),

species that deposit eggs in ponds {Dendropsophus car-

nifex), and others that place their eggs on vegetation from

where hatching tadpoles drop into streams {Centrolene

spp., Nymphargus spp., Hyloscirtus spp.).

Because Bd is an aquatic pathogen (Berger et al.

2005b) we expected amphibian species with aquatic re-

productive modes to show higher infection prevalence

(Lips et al. 2005; Brem and Lips 2008). In fact, the most

dramatic amphibian declines and extinctions in the Andes

have occurred in species with aquatic larvae (La Marca

et al. 2005; Bustamante et al. 2005; Merino-Viteri et al.

2005; Coloma et al. 2010). Our results indicate, surpris-

ingly, a higher Bd prevalence in frogs with a terrestrial

reproductive mode {Pristimantis spp.) than in those that

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Batrachochytrium dendrobatidis in an Andean frog community

Table 2. Abundance of amphibian species at Kathy’s Creek, Reserva Las Gralarias. Abundances are presented as minimum-maxi-

mum, followed, in parenthesis, by mean + standard error. The Student’s t-test was performed only in species with normally distrib-

uted abundances.

Kathy’s Creek

f-test

Population trend

Year

2012

2013

Number of nights sampled

10

5

Family/Species

Centrolenidae

Centrolene ballux

5-37 ( 22.7 + 11 . 6 )

3-25 ( 11.4 + 8 . 67 )

0.078

No difference

Centrolene peristictum

0-5 ( 2.1 + 1 . 66 )

0-5 ( 2 . 0 + 1 . 87 )

0.918

No difference

Nymphargus grandisonae

0 - 7 ( 3.7 + 2 . 21 )

0-6 ( 2.2 + 2 . 28 )

0.242

No difference

Nymphargus griffithsi

0-8 ( 2.3 + 2 . 26 )

0-3 ( 1 . 4 + 1 . 34 )

—

Nymphargus lasgralarias

Hylidae

3-28 ( 19.4 + 8 . 54 )

7-28 ( 15.0 + 8 . 69 )

0.366

No difference

Hyloscirtus alytolylax

Craugastoridae

0-1 ( 0 . 1 + 0 . 32 )

0-1 ( 0.4 + 0 . 59 )

—

Pristimantis achatinus

0-3 ( 0.3 + 0 . 95 )

0-1 ( 0.2 + 0 . 45 )

—

Pristimantis appendiculatus

0 - 7 ( 1.2 + 2 . 10 )

0-1 ( 0.4 + 0 . 59 )

—

Pristimantis calcarulatus

0-3 ( 1 . 1 + 0 . 74 )

0-3 ( 1 . 0 + 1 . 23 )

—

Pristimantis eremitus

0-1 ( 0 . 1 + 0 . 32 )

0-1 ( 0.2 + 0 . 45 )

—

Pristimantis eugeniae

0-1 ( 0 . 1 + 0 . 32 )

1-2 ( 0.8 + 0 . 84 )

—

Pristimantis sobetes

0-1 ( 0.2 + 0 . 42 )

0-2 ( 0.4 + 0 . 89 )

—

Pristimantis w-nigrum

0-1 ( 0 . 1 + 0 . 32 )

0-1 ( 0.4 + 0 . 59 )

—

reproduce in water (mainly glassfrogs; Centrolene spp.,

Nymphargus spp.). This finding supports the idea that

even terrestrial breeders may serve as reservoirs for the

pathogen in diverse amphibian communities (Longo et

al. 2013). Higher prevalence in terrestrial frogs requires

that Bd zoospores survive in terrestrial habitats. John-

son and Speare (2003) indicated that Bd can survive in

moist soil for up to three months. Cloud forests in west-

ern Ecuador typically have near constant rain and high

levels of humidity during the rainy season (Hutter and

Guayasamin 2012; Arteaga et al. 2013), and this may ex-

tend zoospore survival in terrestrial environments at Las

Gralarias. Higher Bd prevalence in terrestrial frogs com-

pared to that in frogs with aquatic reproduction might

also be related to intrinsic differences in, for example, the

efficacy of immune responses (Rosenblum et al. 2009;

Woodhams et al. 2007) or differences in anuran skin mi-

crobiota (Flechas et al. 2012).

Our results also show that Bd prevalence is signifi-

cantly associated with taxonomy (i.e., family). Thus,

glassfrogs (family Centrolenidae) might have innnune

responses or skin microbiota that work as better barri-

ers to the pathogen than those in terrestrial (i.e., Pristi-

mantis) frogs. The strong correlation of prevalence with

taxonomy, habitat, and reproductive mode (Appendix 2)

indicates that further studies need to focus on the specific

effects of each of these factors; in other words, phytog-

eny (and taxonomy) correlates with reproductive mode

and habitat use.

A second surprising finding of our study is that, al-

though prevalence of Bd is high in most anuran species,

we did not observe any sign of population declines or

abrupt crashes, nor have we found dead or sick frogs dur-

ing four years of intensive fieldwork (2010-2014; JMG

pers. obs.). Thus, this frog connnunity persists with an

endemic pathogen and with relatively low loads (less

than 10 zoospores. Table 3). The apparent increased re-

sistance or tolerance of amphibians from Reserva Las

Gralarias to Bd infection may be explained by one or

several of the following mechanisms: (i) amphibian in-

nate and/or acquired defense mechanisms (Savage and

Zamudio 2012; Woodhams et al. 2007); (ii) skin bacte-

rial connnensals with anti-fungal properties (Harris et al.

2006); (iii) behavioral and ecological factors that reduce

the likelihood of infection and disease (e.g., microhabitat

selection, reproductive mode; Lips et al. 2003; Rowley

and Alford, 2007), and/or (iv) variation in Bd virulence

(Berger et al. 2005a; Fisher et al. 2009). Our amphib-

ian monitoring took place during the rainy season when

most species are active. Therefore, future studies should

determine whether or not this apparent tolerance to Bd

is stable through longer periods of time or if it fluctuates

depending on environmental variables influencing host

innnunity, behavior, microbiota, or pathogenicity.

After the emergence of an infectious disease, surviv-

ing hosts can evolve tolerance or resistance (Retallick et

al. 2004; Savage and Zamudio 2011). The earliest known

record of Bd in Ecuador is in 1980, in the Harlequin frog

Atelopus bomolochos (Ron and Merino-Viteri 2000),

a species that is now probably extinct (Coloma et al.

2014). If Bd reached and spread in Ecuador during the

early 1980s (Ron et al. 2003; Lips et al. 2008), we hy-

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Guayasamin et al.

Table 3. Prevalence of Batrachochytrium dendrobatidis (Bd) in amphibians at Reserva Las Gralarias, Ecuador, during the rainy

seasons of 2012 and 2013. Bd prevalence for each species is followed, in parenthesis, by a 95% confidence interval. Bd load sum-

mary data includes only samples that tested positive for Bd.

2012

2013

N

Positive Bd

Prevaience

Bd Load

(mean ± sd)

N

Positive Bd

Prevaience

Family: Centrolenidae

Centrolene ballux

17

8

47%

(24-71%)

6.5 + 10.7

9

2

22%

(39-59%)

Centrolene heloderma

6

1

17%

(1-63%)

0.6

1

0

0%

(0-95%)

Centrolene lynchi

6

1

17%

(1-63%)

-

5

1

20%

(1-70%)

Centrolene peristictum

21

6

29%

(12-52%)

2.1 + 1.5

16

3

19%

(5-46%)

Nymphargus grandisonae

21

5

24%

(9-48%)

4.7

-

Nymphargus griffithsi

3

0

0%

(1-69%)

-

2

0

0%

(0-80%)

Nymphargus lasgralarias

16

6

38%

(16-64%)

6.5 + 1.5

10

2

20%

(4-56%)

Family: Hylidae

Dendropsophus carnifex

10

5

50%

(20-80%)

-

Hyloscirtus alytolylax

9

8

89%

(51-99%)

2.4 + 2.4

7

2

29%

(5-70%)

Family: Craugastoridae

Pristimantis achatinus

7

4

57%

(20-88%)

-

Pristimantis appendiculatus

23

10

44%

(24-65%)

1.9 + 2.4

-

Pristimantis calcarulatus

15

2

13%

(2-42%)

1.1

15

1

7%

(4-34%)

Pristimantis eremitus

4

100%

(40-100%)

0.9 + 0.2

4

2

50%

(9-91%)

Pristimantis eugeniae

18

12

66%

(41-86%)

2.5

2

1

50%

(3-97%)

Pristimantis hectus

8

2

25%

(4-64%)

-

14

4

29%

(10-58%)

Pristimantis illotus

-

1

0

0%

(0-95%)

Pristimantis parvillus

9

4

44%

(15-77%)

-

Pristimantis sobetes

8

3

38%

(10-74%)

-

9

3

33%

(9-69%)

Pristimantis pteridophilus

-

3

0

0%

(0-69%)

Pristimantis w-nigrum

21

13

62%

(39-81%)

1.5 + 0.7

-

TOTAL

222

94

42%

(35-49%)

88

19

22%

(14-32%)

pothesize that many of the population declines observed

in the country at that time (e.g., Coloma 1995, 2002;

Coloma et al. 2000; Ron et al. 2003; Bustamante et al.

2005; La Marca et al. 2005; Lips et al. 2008; Coloma et

al. 2010) could be attributable to chytridiomycosis. Thus,

it is probable that most Andean amphibian communities

have been exposed to Bd for more than three decades and

that current sampling finds remnant species that are tol-

erant to Bd while the susceptible species are already ex-

tinct. Under this scenario, selection should have favored

the persistence of amphibian species or specific popula-

tions that have developed defenses against Bd; therefore.

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Batrachochytrium dendrobatidis in an Andean frog community

changing host composition of these communities right

after pathogen emergence. We acknowledge, however,

that this is a working hypothesis which assumptions

depend on our knowledge of the historical distribution

of the chytrid. For example, if new data shows that Bd

was present in the Ecuadorian Andes before amphibian

declines were noticed, such piece of information would

support the endemic pathogen hypothesis, which states

that environmental changes triggered Bd outbreaks (Ra-

chowicz et al. 2005).

Reserva Las Gralarias is one of the most studied and

species-rich area in the cloud forest of the tropical An-

des, containing numerous species considered endangered

by the lUCN (2014; see Appendix 2). However, the

community (and surrounding areas) lacks at least three

groups of species that were conspicuous in Ecuadorian

cloud forests: marsupial frogs {Gastrotheca plumbea,

G. guentheri), harlequin frogs {Atelopus longirostris, A.

mindoensis), and dendrobatid frogs (Hyloxalus lehmani,

H. maquipucuna) (Coloma et al. 2011-2014; Arteaga et

et al. al. 2013). Marsupial and harlequin frogs are par-

ticularly susceptible to Bd (Lips et al. 2003; Elechas et al.

2012; Ellison et al. 2014; DiRenzo et al. 2014) and are

the primary species that suffered population declines and

extinctions in Ecuador (Lips et al. 2002; La Marca 2005)

even in pristine areas. The absence of these lineages at

Reserva Las Gralarias supports to the hypothesis that this

is a post-decline amphibian community. Understanding

the long-term effects of pathogens (eg., chytrid) and tem-

perature variability in such a community is essential for

the continued effective management of endangered spe-

cies in the Andean cloud forests.

Considering Ecuador’s high diversity of amphibian

species, life history modes, and evolutionary history, our

study provides a baseline to study the evolution of de-

fense strategies against Bd. We reconnnend further re-

search to determine the mechanisms driving the observed

differences in pathogen exposure among hosts differing

in reproductive modes, habitat, and taxonomy.

Acknowledgments. — Previous versions of this article

greatly benefited from the reviews of Luis A. Coloma,

and five anonymous reviewers. This study was funded

by the lUCN Save-Our-Species (SOS) program and Uni-

versidad Tecnologica Indoamerica, through the project

“Conservation of Endangered Species in the Choco Bio-

geographic Zone: Integrating habitat management, bio-

logical monitoring, and community outreach.” SOS is a

joint initiative of lUCN, the Global Environment Eacility

and the World Bank; its objective is to ensure the long-

term survival of threatened species and their habitats.

AMM’s research was supported by a scholarship from

the program “Becas Mixtas del Consejo Nacional de

Ciencia y Tecnologfa, CONACYT,” Mexico. Idea Wild

kindly granted equipment for AMM’s field work. Reser-

va Las Gralarias (Jane A. Lyons) provided invaluable lo-

gistic support throughout the project. Special thanks to

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (39)

Henry Imba, Italo G. Tapia, Lucas Bustamante, and Jai-

me Garcia for assistance during fieldwork. JMG thanks

AA for the one-day delay in Miami, where the final ver-

sion of the ms was finished. Diana Flores provided as-

sistance with molecular diagnosis of Bd. Special thanks

to John Kelly for his help in statistical analyses. Research

permits were issued by the Ministerio de Ambiente, No

05-2013-IC-FAU-DPAP-MA.

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Received: 19 June 2014

Accepted: 04 August 2014

Published: 28 August 2014

Appendix 1. Transects sampled at Reserva Las Gralarias. Each transect has an area of 500 x 4 m. Latitude and longitude are in

decimal degrees.

Transect

Elevation (m)

Latitude

Longitude

Habitat

Lucy’s creek

1822-1858

At start: -0.00492

At end: -0.00342

At start: -78.73344

At end: -78.74051

Riverine vegetation along creek

Kathy’s creek

2041-2066

At start: -0.01696

At end: -0.0156

At start: -78.7314

At end: -78.73386

Riverine vegetation along creek

Santa Rosa river

1884-1882

At start: -0.0133

At end: -0.01054

At start: -78.72368

At end: -78.7211

Riverine vegetation along river

Waterfall trail

1897-2107

At start: -0.0135

At end: -0.01379

At start: -78.72461

At end: -78.7269

Primary and secondary terra firme forest

Five-Frog creek

2141-2156

At start: -0.03166

At end: -0.03098

At start: -78.70421

At end: -78.70853

Riverine vegetation along creek

Osoverde & Guarumo trail

2141-2156

At start: -0.03166

At end: -0.03098

At start: -78.70421

At end: -78.70853

Primary and secondary terra firme forest

Puma trail

1923-2031

At start: -0.00954

At end: -0.00708

At start: -78.7346

At end: -78.73662

Primary and secondary terra firme forest

TKA trail

2192-2216

At start: -0.0275

At end: -0.02516

At start: -78.70477

At end: -78.70353

Primary and secondary terra firme forest

Peccary trail

1803-1896

At start: -0.00750

At end: -0.0076

At start: -78.72635

At end: -78.72862

Primary and secondary terra firme forest

Appendix 2. Amphibians at Reserva Las Gralarias, with corresponding lUCN (2014) conservation status. The list includes three

potential new species (Pristimantis sp. 1, Pristimantis sp. 2, and Pristimantis sp. 3). Reproductive modes are sensu Haddad and

Prado (2005).

Species

Reproductive mode

Habitat for reproduction

Conservation status

Family: Centrolenidae (7 spp.)

Mode 25: Eggs hatching into

exotrophic tadpoles that drop in

lotic water

Centrolene ballux

Mode 25

Vegetation along fast-flowing

streams

Critically Endangered

Centrolene heloderma

Mode 25

Vegetation along fast-flowing

streams

Critically Endangered

Centrolene lynchi

Mode 25

Vegetation along fast-flowing

streams

Endangered

Centrolene peristictum

Mode 25

Vegetation along fast-flowing

streams

Vulnerable

Nymphargus griffithsi

Mode 25

Vegetation along fast-flowing

streams

Vulnerable

Nymphargus grandisonae

Mode 25

Vegetation along fast-flowing

streams

Least Concern

Nymphargus lasgralarias

Mode 25

Vegetation along fast-flowing

streams

Data Deficient

August 2014 | Volume 8 | Number 1 | e81

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Batrachochytrium dendrobatidis in an Andean frog community

Appendix 2 (continued). Amphibians at Reserva Las Gralarias, with corresponding lUCN (2014) conservation status. The list

includes three potential new species (Pristimantis sp. 1, Pristimantis sp. 2, and Pristimantis sp. 3). Reproductive modes are sensu

Haddad and Prado (2005).

Species

Reproductive mode

Habitat for reproduction

Conservation status

Family: Craugastoridae (16

spp.)

Mode 23: Direct development of

terrestrial eggs

Pristimantis achatinus

Mode 23

Terrestrial, mainly in pastures and

modified environments

Least Concern

Pristimantis appendiculatus

Mode 23

Terrestrial, mainly primary and

secondary forests

Least Concern

Pristimantis calcarulatus

Mode 23

Terrestrial, mainly primary and

secondary forests

Vulnerable

Pristimantis crenunguis

Mode 23

Terrestrial, mainly primary and

secondary forests

Endangered

Pristimantis eremitus

Mode 23

Terrestrial, mainly primary and

secondary forests

Vulnerable

Pristimantis eugeniae

Mode 23

Terrestrial, mainly primary and

secondary forests

Endangered

Pristimantis hectus

Mode 23

Terrestrial, mainly primary and

secondary forests

Data deficient

Pristimantis illotus

Mode 23

Terrestrial, mainly primary and

secondary forests

Near Threatened

Pristimantis parvillus

Mode 23

Terrestrial, mainly primary and

secondary forests

Least Concern

Pristimantis pteridophilus

Mode 23

Terrestrial, mainly primary and

secondary forests

Endangered

Pristimantis sobetes

Mode 23

Terrestrial, mainly primary and

secondary forests

Endangered

Pristimantis verecundus

Mode 23

Terrestrial, mainly primary and

secondary forests

Vulnerable

Pristimantis w-nigrum

Mode 23

Terrestrial, mainly primary and

secondary forests

Least Concern

Pristimantis sp. 1

Mode 23

Terrestrial, mainly primary and

secondary forests

Not evaluated

Pristimantis sp. 2

Mode 23

Terrestrial, mainly primary and

secondary forests

Not evaluated

Pristimantis sp. 3

Mode 23

Terrestrial, mainly primary and

secondary forests

Not evaluated

Family: Hylidae (3 spp)

Dendropsophus carnifex

Mode 1 : Eggs and exotrophic

tadpoles in lentic water

Ponds

Least Concern

Hyloscirtus alytolylax

Mode 25

Vegetation along fast-flowing

streams

Near Threatened

Hyloscirtus criptico

Mode 25

Vegetation along fast-flowing

streams

Not evaluated

Family: Caeciliidae (1 sp.)

Caecilia buckleyi

Family: Rhinatrematidae (1 sp.)

Unknown

Not evaluated

Epicrionops bicolor

Unknown

Least Concern

August 2014 | Volume 8 | Number 1 | e81

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Guayasamin et al.

Juan M. Guayasamin is head of the Centro de Investigacion de la Biodiversidad y Cambio Climatico (Bio-

Camb) and Professor at Universidad Tecnologica Indoamerica, Quito, Ecuador. He obtained his Master's de-

gree and Ph.D. in Ecology and Evolutionary Biology (University of Kansas, EEUU). His research includes am-

phibian phylogenetic systematics, taxonomy, biogeography, ecology, and conservation biology {Photographed

by Lucas Bustamante).

Angela M. Mendoza is a biologist from Universidad del Valle (Colombia) with a Master’s in biological sci-

ences at the Universidad Nacional Autonoma de Mexico (UNAM). She is a research assistant in the Conserva-

tion Genetics Laboratory at the Alexander von Humboldt Institute, Colombia. Her main interest is to apply

molecular tools in ecology and conservation, with emphasis in terrestrial vertebrates, mainly Neotropical am-

phibians {Photographed by Angela M. Mendoza).

Ana V. Longo is a doctoral student in the Department of Ecology and Evolutionary Biology at Cornell Uni-

versity, Ithaca, New York. Her main interests are amphibian disease ecology and evolution. Her research aims

to understand the mechanisms underlying seasonal and ontogenetic patterns of disease susceptibility in direct-

developing frogs {Photographed by Alberto L. Lopez-Torres).

Kelly R. Zamudio is a Professor in the Department of Ecology and Evolutionary Biology at Cornell Univer-

sity. She received her B.A. from UC Berkeley in Zoology in 1991, and her Ph.D. from University of Washing-

ton, Seattle, in 1996. Her research focuses on the origin and maintenance of vertebrate biodiversity (especially

reptiles and amphibians). Her lab integrates field research in population biology, demography, and habitat

change with lab research on the genomic underpinnings of population diversification, speciation, and conserva-

tion. {Photographed by Karen Lips).

Elisa Bonaccorso is a biologist, Ph.D. in Ecology and Evolutionary Biology (University of Kansas, EEUU),

and Licenciada en Biologia (Universidad Simon Bolivar, Venezuela). Her research is broad and includes mo-

lecular systematics, ecology of wildlife diseases, and conservation biology {Photographed by Juan M. Guayas-

amin).

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (44)

August 2014 | Volume 8 | Number 1 | e81

Anolis podocarpus. Photo by A. Almenddriz.

September 2014 | Volume 8 | Number 1

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (45)

e82

Amphibian & Reptiie Conservation

8(1) [Special Section]: 45-64.

terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported

License, which permits unrestricted use for non-commercial and education purposes only

provided the original author and source are credited. The official publication credit source:

Amphibian & Reptile Conservation at: amphibian-reptile-conservation.org

Overview of the herpetofauna of the unexplored

Cordillera del Condor of Ecuador

^Ana Almendariz, ^John E. Simmons, ^ ^*Jorge Brito, and ^’^Jorge Vaca-Guerrero

^Instituto de Ciencias Biologicas, Escuela Politecnica Nacional, Casilla 17-01-2759, Quito, ECUADOR ^Museologica, 128 Burnside Street,

Bellefonte, Pennsylvania 16823 USA

Abstract. — ^The Cordillera del Condor is an area rich in unique vegetation assemblages and endemic

faunal elements; the herpetofauna is especially diverse, particularly the anurans. The montane

forest and sandstone tepuis, located atop large andesite and quartz formations, provide a variety

of habitats and microhabitats in which the herpetofauna finds food, shelter, and reproductive sites,

such as terrestrial and arboreal bromeliads and a soil type termed “bamba” that is covered with

mosses and roots. Information compiled from publications and recent studies has revealed the

presence of 120 species of amphibians and 59 species of reptiles, including 41 probable new species

(36 amphibians and five reptiles) in the genera Centrolene, Dendrobates, Pristimantis, Lynchius,

Chiasmocieis, Boiitogiossa, Anoiis, Erythroiamprus, Tantilia, and Dipsas.

Resumen. — La Cordillera del Condor es un area rica en formaciones vegetales unicas y elementos

faum'sticos endemicos; presenta una singular diversidad herpetofaunistica, particularmente de la

anurofauna. Los bosques montanos y los de “tepuy,” asentados sobre piedras grandes de andesita

y cuarzo crean variedad de habitats y microhabitats, en donde la herpetofauna encuentra alimento,

refugio y lugares para la reproduccion, como por ejempio las bromelias terrestres y arboreas y

un suelo denominado “bamba” que esta cubierto de musgos y raices. La informacion recopilada

de material publicado y de los estudios realizados en los ultimos ahos revela la presencia de 120

especies de anfibios y 59 especies de reptiles. Los resultados incluyen 41 especies posiblemente

nuevas (36 anfibios y cinco reptiles) de los generos: Centroiene, Dendrobates, Pristimantis,

Lynchius, Chismocieis, Boiitogiossa, Anoiis, Erythroiamprus, Tantiiia, y Dipsas.

Key words. Ecuador, Cordillera del Condor, amphibian, reptile

Citation: Almendariz A, Simmons JE, Brito J, Vaca-Guerrero J. 2014. Overview of the herpetofauna of the unexplored Cordillera del Condor of Ecuador.

Amphibian & Reptile Conservation 8(1 ) [Special Section]: 45-64 (e82).

Introduction

The fauna of Ecuador, in general, has not been extensive-

ly studied, despite a notable increase in research activ-

ity in recent years (Albuja et al. 2012). In particular, the

Cordillera del Condor region, in southern Ecuador along

the border with Pern (Figure 1), is a very poorly known

area. The purpose of this paper is to suncnnarize and re-

view herpetofaunal studies of the Cordillera del Condor

region. Studies of the avian and mammalian fauna have

been published elsewhere (e.g., Albuja and Patterson

1996; Brito and Arguero 2012; Freile et al. 2014).

The long-running border conflicts between Ecuador

and Pern and the difficulty in accessing the region have

maintained the ecosystems of the Cordillera del Condor

almost intact. It has only been since the end of the con-

flicts known as the Pasquisha War (which ended in Feb-

mary 1981) and the Alto Cenepa War (which ended in

Febmary 1995), that roads into the area have begun to

open, which has resulted in incipient colonization and an

awakened interest in mineral prospection in the region.

Nevertheless, there are still some parts of the Cordillera

del Condor that remain unaltered.

The Cordillera del Condor is part of a biologically di-

verse, discontinuous, sub-Andean cordillera that has sev-

eral characteristics that distinguish it from the rest of the

Andes. Whereas the main Andes are of metamorphic and

igneous origin, the Cordillera del Condor is sedimentary,

composed largely of limestone and sandstone (Schul-

enberg and Awbrey 1997). The region is dominated by

*Current address: Museo Ecuatoriano de Ciencias Maturates, Rumi-

pamba y Avenida de los Shyris, Quito, ECUADOR

Correspondence. Emails: ^ ana. almendariz® epn.edu.ee, ^simmons.johne® gmail.com (Corresponding author, John E. Sim-

mons), ^jorgeyakuma® yahoo.es, "^gheovak® hotmail.com

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (46) September 2014 | Volume 8 | Number 1 | e82

Almendariz et al.

i

77-D'O-W

■—

CD

_|5

^Hivnpabhokn

COLOMBIA

ECUADOR

1 1

ECUAI>0|SE

6 fKta r Mr 1 d Of

1 1 PEHU

yiadiriizA

tA

■k

^in rungirtzi PERU

0 25

50

100

-

TS-o'™

T7-o'o'W

Fig. 1. Map of the Cordillera del Condor region.

geologically complex mountains topped with sandstone

plateaus at elevations of 300 m to almost 3,000 m that

support habitats similar to the sandstone mountains of

the Guyana Shield (Figure 2); many of the plateaus have

vegetation similar to that of the tepuis. Due to its loca-

tion just northeast of the Huancabamba Depression, the

lowest point in the Andes (Duellman 1999), the Cordil-

lera del Condor receives moisture from both the Atlantic

and the Pacific sides of the Andes — moisture from the

Western slopes of the Andes as well as moisture moving

east across the Amazon basin drops over the Cordillera

del Condor, providing frequent, year-round precipitation

(Schulenberg and Awbrey 1997).

The first systematic botanical studies of the region

were carried out in 1990 and 1991 in the Rfo Nangaritza

basin (in the southern region of the Cordillera del Con-

dor), under the auspices of the Proyecto Promobot and

the Tratado de Cooperacion Amazonica, with the partici-

pation of both Ecuadorian and foreign scientists. These

explorations were limited to areas below about 1,700 m

Fig. 2. Alto Paquisha, 2,400 m. Photo by A. Almendariz.

in altitude. In 1993, A. H. Gentry collected plants on one

of the highest points of the mountain range (2,100 m)

as part of a Rapid Assessment Program (RAP) survey,

organized by Conservation International (Cl, a non-gov-

ernmental organization). Gentry found that the vegeta-

tion structure and families of plants were similar to those

of the sandstone tepuis of the Guiana Highlands (Schul-

enberg and Awbrey 1997). In recent years, D. Neill and

his collaborators have surveyed the flora at several points

in the cordillera, publishing descriptions of new species

and studying the environmental heterogeneity associated

with variable types of soils (Neill 2005; Neill and Asan-

za 2012; Neill and Ulloa 2011; Riina et al. 2014; Ulloa

et al. 2012). The diversity of plant assemblages on the

sandstone plateaus produces a variety of microhabitats

that provide food, shelter, and reproductive sites for the

herpetofauna, particularly terrestrial and arboreal brome-

liads; the “bamba” soils of many of these tepuis is thickly

covered with mosses and roots, and serves to Alter the

tannins that darken the turbid water in creeks and streams

(Figure 3).

According to the ecosystem classification for conti-

nental Ecuador (Ministerio del Ambiente del Ecuador

2012), the following ecosystems have been identified in

the Cordillera del Condor:

• Evergreen piedmont forest in the Condor-Kutuku

ranges

• Evergreen forest on the sandstone plateaus of the

Condor range in the lower Ecuadorian Amazon

Fig. 3. Vegetation in the interior of a tepui forest. Photo by A.

Almendariz.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (47)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

• Lower montane evergreen forest in the Condor-

Kutuku ranges

• Evergreen piedmont forest on the sandstone mesas

of the Condor-Kutuku ranges

• Evergreen lower montane forests on the sandstone

mesas of the Condor-Kutuku ranges

• Montane humid shrub in the Condor range

• Evergreen montane forests on the sandstone mesas

of the Condor-Kutuku ranges

• Montane humid shrub with herbaceous rosette

thickets (herbazales) in the Condor range

Materials and Methods

Herpetological surveys of the region have been few and

limited (Figure 1). The information presented below is

drawn from an extensive survey of the literature and

recent field work. The majority of the studies have em-

ployed the Rapid Ecological Assessment strategy or RAP

developed by Cl (Sayre et al. 2002), in habitats where the

presence of herpetofaunal elements was anticipated.

The northern zone of the Cordillera del Condor is

known from three studies. The first was carried out in

1972 in conjunction with a privately funded orchid col-

lecting expedition (accompanied by personnel from the

Missouri Botanical Garden and the University of Kansas

Museum of Natural History), at elevations of 870-2,000

m at the headwaters of the Rio Piuntza, Rio Chuchum-

bleza, Rio Numpatacaimi, and Rio Santa Agueda in Mo-

rona Santiago Province (Duellman and Simmons 1988).

The second survey was a RAP assessment conducted by

Cl, the Escuela Politecnica Nacional, Fundacion Fedima,

and the Universidad Nacional Mayor de San Marcos.

The areas surveyed included the Ecuadorian flank of the

Cordillera del Condor (Coangos and Achupallas in Mo-

rona Santiago Province [Figure 4], Miazi and Shaimi in

Zamora Chinchipe Province). The Peruvian flank of the

Cordillera del Condor was surveyed at the base of Cerro

Machinaza, Alfonso Ugarte-PV3, Falso Paquisha-PV22,

and Puesto de Vigilancia Comainas. Subsequently, a

third survey was carried out by Fundacion Natura (FN

2000) to establish the Parque El Condor, which inven-

Fig. 4. Achupallas sector, 2,100 m. Photo by A. Almenddriz.

toried the Comunidad Numpatakaime and confluence of

the Rio Tsuirim and the Rio Coangos.

Another survey, conducted as part of the Proyecto Paz

y Conservacion Binacional en la Cordillera del Condor

Ecuador-Peru by the Organizacion Intemacional de las

Maderas Tropicales, Conservation International, Fun-

dacion Natura, and the Institute Nacional de Recursos

Naturales (INRENA) in 2005 (Organizacion Intemacio-

nal de las Maderas Tropicales and Fundacion Natura y

Conservacion Intemacional 2005), collected data from

several localities in the southern sector on the Ecuadorian

flank of the Cordillera del Condor, including Condor Mi-

rador and Herradura. The corresponding Pemvian flank

survey was focused on the Zona Reservada Santiago Co-

maina.

Between March 2008 and July 2012, the Escuela Poli-

tecnica Nacional team, under a contract with the Cardno-

Entrix Corporation, carried out 16 expeditions to survey

the herpetofauna of Alto Manchinaza. In 2009, a Cl RAP

survey was conducted by personnel from the Pontificia

Universidad Catolica del Ecuador, Louisiana State Uni-

versity, and Fundacion Ecologica Arcoiris, with support

from Secretaria Nacional de Ciencia y Tecnologia del

Ecuador (SENACYT) of the tepuyes of the upper basin

of the Rio Nangaritza (Guayasamm et al. 2011). In 2012,

the Fundacion Naturaleza y Cultura Intemacional and the

Universidad Estatal Amazonica organized an expedition

to Cerro Plateado (Figure 5), the southern point of the

Cordillera del Condor, which included researchers from

the Escuela Politecnica Nacional.

Results

The 1972 survey of the northern zone of the Cordillera

del Condor resulted in the capture of 30 species, includ-

ing nine new species (e.g., Duellman and Simmons 1988,

Lynch 1974, 1976, 1979; Lynch and Duellman 1980).

Specimens and additional records from this survey are

deposited in the Biodiversity Research Institute at the

University of Kansas, along with additional related spec-

imens accounting for 47 species total (Reynolds 1997;

Schulenberg and Awbrey 1997).

Fig. 5. Peak of Cerrro Plateado, 2,900 m. Photo by V. Carvajal.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (48)

September 2014 | Volume 8 | Number 1 | e82

Almendariz et al.

Fig. 6. Pristimantis sp. Photo by A. Almendariz.

Fig. 7. Centrolene condor. Photo by A. Almendariz.

Fig. 8. Excidobates condor. Photo by A. Almendariz.

Fig. 9. Enyalioides rubrigularis (female). Photo by A. Fig. 10. Hyloscirtus condor. Photo by J. Brito.

Almendariz.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (49)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

The second survey of the northern Zone of the Cordil-

lera del Condor recorded a total of 34 species — 27 am-

phibians and seven reptiles (Almendariz 1997a, 1997b).

Although the survey was conducted under adverse en-

vironmental conditions, geographic range extensions for

several species were recorded. The Peruvian flank sur-

veys recorded 58 species (35 anurans and 23 reptiles);

the data from the Peruvian surveys was collected by per-

sonnel from the Museo de Historia Natural of the Univer-

sidad Nacional Mayor de San Marcos in 1987 (Reynolds

and Icochea 1997a, 1997b) and expanded the ranges of

two species for Peru, Rhinella festae and Hemiphractus

bubalus. The Parque El Condor survey lists a total of 36

species (22 anurans, eight lizards, and six snakes), in-

cluding nine species new to the Cordillera del Condor

herpetofauna, and makes reference to the difficulty of

identifying some of the material. The report summarizes

information known up to the year 2000, and lists a total

of 95 species for the region (56 amphibians and 39 rep-

tiles).

The survey of the southern sector of the Cordillera del

C6ndor collected specimens that were not identified to

the species level of the genus Pristimantis (Figure 6), and

a glass frog provisionally identified as Centrolene cf. cro-

ceopodes that in 2008 was named as Centrolene condor

(Cisneros-Heredia and Morales-Mite 2008; Figure 7).

The surveys of Alto Manchinaza recorded 70 species

of amphibians and 43 species of reptiles (Almendariz et

al. in prep.). The results of these surveys revealed the

Fig. 11. Herpetofaunal assemblages and endemics from the

Cordillera del Condor.

ua UO

AMPHSBIANS RtPHlLES

□ TdIhI N*5ppcl^ M Pntpntialty Nfw Speaps

Fig. 12. Documented and predicted species diversity in the

Cordillera del Condor.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (50)

presence of several little known or new species, includ-

ing a new species of highland poison dart frog, Excido-

bates condor (Almendariz et al. 2012; Figure 8) and geo-

graphic range extensions for Phyllomedusa ecuatoriana,

Centrolene condor, Chimerella mariaelenae, Hyloxalus

mystax, and Enyalioides rubrigularis (Figure 9), among

others. In addition, ecological data and information on

reproduction, vocalizations, and other aspects of the life

history for these species was collected (Almendariz and

Batallas 2012a, 2012b; Batallas and Brito 2014; Brito et

al. in prep.); at least 28 probably new species from differ-

ent genera {Centrolene, Bolitoglossa, Pristimantis, Ano-

lis, Atractus, Erythrolamprus, Tantilla, and Dipsas) were

obtained, which are in the process of being described.

This project included environmental education and com-

munity outreach work (Almendariz 2012).

The 2009 Pontificia Universidad Catolica del Ecua-

dor survey recorded 27 species of amphibians and 17

reptiles, including a new species of anuran, Pristiman-

tis minimus (Teran- Valdez and Guayasamm 2010). This

project included the publication of a field guide to plants

and animals of the tepuis of Nangaritza (Almendariz

2010; Freile et al. 2010).

The 2012 expedition to Cerro Plateado (Almendariz

and Brito 2013) recorded 19 species. Of these, 14 were

anurans and salamanders, including nine species of the

family Craugastoridae (most were members of the genus

Pristimantis). Based on the ecology of the area, it is as-

sumed that more species occur at this locality, including

members of the family Centrolenidae. A new species of

torrent frog, Hyloscirtus condor (Figure 10), was de-

scribed based on specimens obtained on this expedition

(Almendariz et al. 2014), and at least eight new species

in the genera Lynchius, Pristimantis, and Bolitoglossa

were obtained that will be described in the future.

Discussion

The Cordillera del Condor is of particular importance

due to its high biodiversity and the presence of several

unique ecosystems (e.g., the sandstone formations simi-

lar to tepuis). The Condor region, with its diverse range

of habitats, contains numerous species that correspond to

three faunal components: (1) Amazonian lowlands; (2)

eastern flanks of the Andes, and (3) an endemic fauna

limited to the southern part of Ecuador (Duellman and

Lynch 1988). As shown in Figure 11, the Baja Amazonia

herpetofaunal assemblage contains more reptile species

(63%) than amphibian species (28%). In the herpetofau-

nal assemblages associated with the eastern slopes of the

Andes there are slightly more amphibian species (34%

and 32%). The percentages relative to the endemic com-

ponent of the Cordillera del Condor are notably higher in

amphibians than in reptiles (41% and 7%).

A summary of the herpetofaunal diversity of the

region is provided in Tables 1 and 2; a comparison of

known species diversity and predicted species diversity

September 2014 | Volume 8 | Number 1 | e82

Almendariz et al.

Fig. 13. Lynchius sp. Photo by J. Brito.

Fig. 15. Cercosaura dicra. Photo by G. Gallardo.

Fig. 14. Pristimantis muscosus. Photo by A. Almendariz.

Fig. 16. Erythrolamprus sp. Photo by A. Almendariz.

Fig. 17. Tantilla sp. Photo by A. Almendariz.

Fig. 18. Anolis podocarpus. Photo by A. Almendariz.

for the region is provided in Figure 12. Some of the more

distinctive species found in the region include frogs of

the genera Lynchius (Craugastoridae; Figure 13) and

Pristimantis (Craugastoridae; Figure 14), the gynmoph-

thalmid lizard Cercosaura dicra (Figure 15), and the

colubrid snakes Erythrolamprus (Figure 16) and Tantilla

(Figure 17).

During the last five years, the following species have

been described based on material from the Cordillera del

Condor: Enyaliodes rubrigularis (Torres-Carvajal et al.

2009; Figure 9), Anolis podocarpus (Ay ala- Varela and

Torres-Carvajal 2010; Figure 18), Pristimantis minimus

(Teran- Valdez and Guayasanun 2010), Excidobates con-

dor (Almendariz et al. 2012; Figure 8), Hyloscirtus con-

dor (Almendariz et al. 2014; Figure 10), and Siphlophis

ayauma (Sheehy et al. 2014). The work has expanded the

known geographic distribution of Anolis soini (Ayala-

Varela et al. 2011; Figure 19) and revealed new distribu-

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (51)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

tion and natural history information for two other species

of the genera Centrolene and Hyloxalus (Almendariz and

Batallas 2012a, 2012b). It is also noteworthy that several

new species are in the process of being described in the

genera Pristimantis and Chiasmocleis (Almendariz et al.

in prep.). A detailed publication about the herpetofauna

of the Condor region is in preparation (Almendariz et al.

in prep.).

Based on information published in the most recent

studies conducted in the Cordillera del Condor, there are

a total of 120 amphibian species (11 families, 31 gen-

era), and 59 reptile species (nine families, 28 genera), not

including those found at elevations below 850 m (Fig-

ure 20). In addition, based on the specimens discussed

above, the area contains approximately 41 potentially

new species (36 amphibians and five reptiles; see Figure

12). These numbers indicate that the region has signifi-

cant endemic diversity (see Table 1, Table 2, and Figure

11 ).

Conclusion

Within the Cordillera del Condor, four areas protected by

the Sistema Nacional de Areas Protegidas have been es-

tablished: (1) Reserva Biologica El Condor; (2) Reserva

Biologica El Quimi; (3) Reserva Biologica Cerro Platea-

do; and (4) Refugio de Vida Silvestre El Zarza. On the

eastern flank, the Peruvian government has concentrated

its efforts to create Parque Nacional Ichigkat Muja-Cor-

dillera del Condor (SERNANP 2012). Nevertheless, the

ecosystems in the Cordillera del Condor are threatened

by imminent human colonization and settlement, the in-

troduction of agriculture and livestock, and mining; the

latter activity poses the greatest threat to the conserva-

tion of the tepui-like forests and the health of the aquatic

ecosystems, which are the reproductive habitats of many

species of anurans, including hylids and centrolenids. On

the other hand, the fact the amphibians of the Andean re-

gion have limited distribution makes them susceptible to

extinction, and in some cases, the protection of their hab-

itat does not improve their chance of survival (Guayas-

amin et al. 2011). This situation warrants intensified

research and conservation studies of these vertebrates,

especially in little explored areas as in the case of the

Cordillera del Condor. The preliminary results of surveys

of Alto Machinaza and Cerro Plateado have revealed the

presence of possibly new species in these areas, indicat-

ing that future interventions in these areas should comply

strictly with the measures to protect ecosystems, environ-

mental mitigation, and management plans.

Acknowledgments. — We thank the Kinross and Card-

no-Entrix Corporation and their administrative and field

staff for use of facilities to carry out recent held stud-

ies, and Eundacion Naturaleza y Cultura Internacional

and David Neill of the Universidad Estatal Amazonica

for the invitation to participate in the expedition to Cerro

Fig. 19. Anolis soini. Photo by J. Vaca G.

Fig. 20. Familial, generic, and specific diversity of amphibians

and reptiles in the Cordillera del Condor.

Plateado. Thanks also to Bruce MacBryde and the late

Milan D. Eiske for the opportunity to participate in the

1972 expedition into the Cordillera del Condor.

Literature Cited

Albuja L, Almendariz A, Barriga R, Montalvo ED, Ca-

ceres E, Roman JS. 2012. Fauna de Vertebrados del

Ecuador. Instituto de Ciencias Biologicas, Escuela

Politecnica Nacional, Quito Ecuador.

Albuja L, Patterson B. 1996. A new species of northern

shrew-opossum (Paucituberculata: Caenolestidae)

from the Cordillera del Condor, Ecuador. Journal of

Mammalogy 77: 41-53.

Almendariz A. 1997a. Reptiles and amphibians of the

Cordillera del Condor. Pp. 80-82 In: The Cordillera

del Condor Region of Ecuador and Peru: A Biologi-

cal Assessment. Editors, Schulenberg T, Awbrey K.

RAP Working Papers number 7, Conservation Inter-

national, Washington, D.C., USA. 234 p. Available:

https://library.conservation.org/Published%20Docu-

ments/2009/07%20RAP%20Working%20Papers.pdf

[Accessed: 30 August 2014].

Almendariz A. 1997b. Amphibian and reptile species

recorded in the Northern and Western Cordillera del

Condor. Pp. 199-201 In: The Cordillera del Con-

dor Region of Ecuador and Peru: A Biological As-

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (52)

September 2014 | Volume 8 | Number 1 | e82

Table 1. Species of amphibians recorded from the Cordillera del Condor.

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Herpetofauna of the Cordillera del Condor of Ecuador

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1988

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olds &

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1997

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mendariz

1997

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (56)

September 2014 | Volume 8 | Number 1 | e82

Almendariz et al

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (57)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

Rio

Puintza

Duellman

& Lynch

1988

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msnm

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olds &

Icochea

1997

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msnm

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RAP 7

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mendariz

1997

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msnm

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atkaim

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Tsuirim y

Coangos

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dacion

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2000

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natza

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Paz y

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cion, 2005

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (58)

September 2014 | Volume 8 | Number 1 | e82

Table 2 . Species of amphibians recorded from the Cordillera del Condor.

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (59)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (60)

September 2014 | Volume 8 | Number 1 | e82

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Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (61)

September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

sessment. Editors, Schulenberg T, Awbrey K. RAP

Working Papers number 7, Conservation Interna-

tional, Washington, D.C., USA. 234 p. Available:

https://library.conservation.org/Published%20Docu-

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Received: 19 May 2014

Accepted: 20 August 2014

Published: 20 September 2014

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September 2014 | Volume 8 | Number 1 | e82

Herpetofauna of the Cordillera del Condor of Ecuador

Ana Almendariz is a researcher and the Curator of Herpetology at the Institute of Biological Sciences at the

Escuela Politecnica Nacional in Quito, Ecuador. A native of Quito, Almendariz holds an undergraduate degree

in biology and a master’s degree in conservation and management of natural resources. She conducts research

on amphibians and reptiles throughout Ecuador and has published extensively on her research.

John E. Simmons is president of Museologica consulting, and teaches museum studies for Kent State Uni-

versity, Juniata College, and the Universidad Nacional de Colombia. Simmons has an undergraduate degree in

systematics and ecology and a master’s degree in historical administration and museum studies, and previously

was collections manager at the California Academy of Sciences and the Biodiversity Research Institute at the

University of Kansas.

Jorge Brito is a mammal and amphibian researcher at the Museo Ecuatoriano de Ciencias Naturales in Quito,

Ecuador. He has an undergraduate degree in biology from the Universidad Central del Ecuador; his research

interests are focused on amphibians and terrestrial micromammals. He has published several contributions on

these species principally from southeastern Ecuador.

Jorge Vaca-Guerrero is a Junior Investigator at the Institute de Ciencias Biologicas of the Escuela Politec-

nica Nacional in Quito, Ecuador. He has an undergraduate degree in biology from the Universidad Central del

Ecuador, and experience in studies of the herpetofauna of the Eastern region of Ecuador. His principle area of

interest is the evolution of reptiles, particularly vipers.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (64)

September 2014 | Volume 8 | Number 1 | e82

under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs 3.0

Unported License, which permits unrestricted use for non-commercial and education pur-

poses only provided the original author and source are credited. The official publication

credit source: Amphibian Reptile Conservation at: amphibian-reptile-conservation.org

Amphibian & Reptiie Conservation

8(1) [Special Section]: 65-75.

Herpetofaunal community of a high canopy tank bromeliad

{Aechmea zebrina) in the Yasuni Biosphere Reserve of

Amazonian Ecuador, with comments on the use of

“arboreai” in the herpetologicai iiterature

^’^Shawn F. McCracken and ^’^Michael R. J. Forstner

^Department of Biology, Texas State University, San Marcos, Texas, USA

Abstract . — ^Tank bromeliads provide microhabitat that supports a high diversity of organisms in

the harsh environment of tropical forest canopies. Most studies of organisms occupying tank

bromeliads have focused on invertebrates found within bromeliads near or at ground level. Few

investigations of vertebrate communities utilizing this keystone resource are available. We describe

the amphibian and reptile community occupying the high canopy tank bromeliad, Aechmea zebrina,

in lowland rainforest of the Yasum' Biosphere Reserve in the Amazon Basin of Ecuador. We used

single-rope climbing techniques to sample a total of 160 A. zebrina bromeliads from 32 trees, at

heights of 18.3 to 45.5 m above ground. We collected 10 metamorphosed anuran species, one gecko,

one snake, and observed two species of lizard within bromeliads. Summary statistics for a suite

of environmental factors associated with herpetofauna in A. zebrina bromeliads are reported. We

estimated the density of anurans occupying A. zebrina communities and contrast these estimates

with anuran densities from tropical forest floor anuran studies. Finally, we discuss the use of the

term “arboreal” within the herpetologicai literature, and make recommendations for terminology

used to describe the vertical space occupied by a species or assemblage.

Key words. Amphibian, anuran, epiphyte, forest, microhabitat, rainforest, reptile

Citation: McCracken SF, Forstner MRJ. 2014. Herpetofaunal community of a high canopy tank bromeliad {Aechmea zebrina) in the Yasuni Biosphere

Reserve of Amazonian Ecuador, with comments on the use of “arboreal” in the herpetologicai literature. Amphibian & Reptile Conservation 8(1) [Special

Section]: 65-75 (e83).

Introduction

Forest canopies provide habitat for approximately 50%

of terrestrial species, yet there are few studies specific

to canopy herpetofauna (Stewart 1985; Vitt and Zani

1996; Kays and Allison 2001; Guayasamin et al. 2006;

McCracken and Forstner 2008; Lowman and Schowal-

ter 2012; Scheffers et al. 2013; McCracken and Forstner

2014). Basic ecological knowledge of arboreality (tree-

living) and utilization of high canopy microhabitats by

amphibians and reptiles remain depauperate in the litera-

ture (Moffett 2000; Kays and Allison 2001; Lehr et al.

2007). A canopy microhabitat frequently used by herpe-

tofauna in tropical forests are epiphytes, and in particular

epiphytic tank bromeliads that are phytotelms capable of

holding relatively large volumes of water (Lowman and

Rinker 2004; McCracken and Forstner 2008). In lowland

Neotropical rainforest, canopy tank bromeliads typically

reside in the overstory and emergent canopy trees at ver-

tical heights of 5^5 -f meters with ~5 to >150 individuals

on a single tree (McCracken and Forstner 2006). These

arboreal bromeliad conununities create a three-dimen-

sional “wetland in the sky” that have been estimated to

impound up to 50,000 liters of water per hectare (Kitch-

ing 2000; McCracken and Forstner 2006). Tank bromeli-

ads function as a “keystone resource” in the harsh forest

canopy environment where the atmosphere meets and in-

teracts with 90% of Earth’s terrestrial biomass; providing

a climate-buffered refuge, water source, and food source

for canopy herpetofauna (Nadkami 1994; Ozanne et al.

2003; Cardeliis and Chazdon 2005).

Kays and Allison (2001) found only 4% of 752 arti-

cles published between 1988 and 1998 on tropical forest

arboreal vertebrates focused on reptiles and amphibians.

Many species of herpetofauna are described as being

arboreal regardless of whether they are restricted to the

vertical stratum a few centimeters to a few meters above

ground, or solely inhabit the high forest canopy at 20 or

more meters vertical height (Chaparro et al. 2007; Mc-

Cracken et al. 2007; Guayasamin and Funk 2009). Forest

structure is associated with vertical partitioning or strati-

fication of the component plant community (e.g., trees,

shrubs, lianas) and accentuates vertical patterns followed

by other organisms (Moffett 2000; Lowman and Rinker

Correspondence. Emails: ^smccracken® txstate.edu (Corresponding author); ^ mf@txstate.edu

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October 2014 | Volume 8 | Number 1 | e83

McCracken and Forstner

clouds

Roads

Trails

Tree sampled

Yasuni National

Park

20

■ Kilometers

Fig. 1. (A) Map of South America with Ecuador (shaded light blue) and Yasuni National Park (solid dark green) highlighted. The

Amazon ecoregion is outlined with light green line. (B) Northeastern section of Yasuni National Park (light gray line) and surround-

ing region where trees were sampled forAechmea zebrina bromeliads within the vicinity of the Tiputini Biodiversity Station - Uni-

versidad San Francisco de Quito (TBS) and the Yasuni Research Station - Pontificia Universidad Catolica del Ecuador (YRS). (C)

Detail of TBS where trees were sampled for A. zebrina bromeliads. Note: Map is modified from Figure 2 in McCracken and Forstner

(2014) and used under the Creative Commons Attribution license.

2004). Spatial patterns of forest cohabitants, such as tank

bromeliads and their inhabitants, are likewise strongly

influenced by forest structure as a result of the funda-

mental organization of resources and space (Lowman

and Rinker 2004). Identifying the vertical space occupied

by a particular amphibian or reptile species in its given

habitat will allow greater insight to their ecological role

in the system.

Herein, we describe amphibians and reptiles occupy-

ing the high canopy tank bromeliad, Aechmea zebrina, in

lowland rainforest of the Yasum Biosphere Reserve in the

Amazon Basin of Ecuador. We report a suite of environ-

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (66)

October 2014 | Volume 8 | Number 1 | e83

Herpetofaunal community of a high canopy tank bromeliad

Fig. 2. (A) A downward vertical view {in situ) of Aechmea zebrina (foreground center left, and at lower elevation in upper right

and center right) and a cluster of Aechmea tessmannii (center, with one in hloom) hromeliads in the tree canopy from ~34 m. (B) A

community of A. zebrina hromeliads at ~38 m (in situ). (C) An A. zebrina hromeliad (ex situ) inside screen tent being measured and

prepared for dismantling, collected from ~44 m in the canopy. Notice the more upright leaves and reddish color because of increased

sun exposure due to high canopy location.

mental factors associated with herpetofauna in A. zebrina

hromeliads. We estimate the density of anurans occupy-

ing mean A. zebrina community sizes in two tree size

classes, representative of our shortest and tallest trees in

the study. We then compared these with anuran densities

from tropical forest floor anuran studies by calculating

the two-dimensional area (m^) of the tree crowns for the

two tree size classes. In completing our review, we feel

it is important to discuss the use of the term “arboreal”

within the herpetological literature and make recommen-

dations for the incorporation of additional terminology

to provide a more informative description of the vertical

space utilized by a species or assemblage.

Materials and Methods

The study was conducted in the northwestern portion of

the Yasum Biosphere Reserve (Yasum) located in Orel-

lana Province, Ecuador. The reserve includes Yasum Na-

tional Park, Waorani Ethnic Reserve, and their respective

buffer and transition zones (Einer et al. 2009). Yasunf is

part of the Napo Moist Eorest terrestrial ecoregion cover-

ing approximately 1 .7 million ha of the upper Amazon

Basin (Einer et al. 2009; Bass et al. 2010). Yasum has an

elevation range of 190-400 m above sea level; the north-

western region averages 2,425-3,145 nun of rainfall per

year with no less than 100 mm per month, temperature

averages 25 °C (15 °-38 °C), and humidity averages 88%

(Blandin 1976; Duellman 1978; Balslev et al. 1987; Bass

et al. 2010). Yasum holds world record species diversity

for several taxa, including the highest documented land-

scape scale (lowland tropical rainforest) herpetofauna di-

versity with 150 species of amphibians and 121 species of

reptiles (Bass et al. 2010). Collections were made in the

vicinity of two research stations, the Tiputini Biodiver-

sity Station (TBS) (0°38’ 14”S, 76°08’60”W) operated by

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October 2014 I Volume 8 I Number 1 I e83

McCracken and Forstner

Fig. 3. A collection of anurans collected from Aechmea zebrina bromeliads. (A) Pristimantis aureolineatus hiding in leaf axil, and

(B) on a leaf of A. zebrina. (C) Pristimantis waoranii emerging from leaf axil, and (D) on a leaf of A. zebrina. (E) Ranitomeya

ventrimaculata and (F) Scinax ruber collected from A. zebrina bromeliads.

the Universidad San Francisco de Quito and the Yasum

Research Station (YRS) (0°40’27”S, 76°23’51”W) oper-

ated by the Pontificia Universidad Catolica del Ecuador

(Fig. 1). Tiputini Biodiversity Station is only accessible

by river and surrounded by undisturbed primary lowland

rainforest, and YRS is located approximately 27 km west

on an oil pipeline road (Maxus road) that has been expe-

riencing forest disturbance within its vicinity but is still

surrounded by large tracts of undisturbed forest. Sam-

pling of A. zebrina bromeliads took place between 0800

and 1800 hours from April to November of 2008.

We focused our sampling on a single large epiphytic

tank bromeliad species, Aechmea zebrina, that is native

to the Amazon regions of Ecuador and southeastern Co-

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October 2014 I Volume 8 I Number 1 I e83

Herpetofaunal community of a high canopy tank bromeliad

lombia (Smith 1953). Aechmea zebrina occupy vertical

heights of approximately 18^5+ m in the overstory and

emergent canopy trees, and range between 1 to >150 in-

dividuals on a single host tree (SFM, unpublished data).

The leaves are upright and arranged in a spiral with their

leaf axils tightly overlapping to form water-holding res-

ervoirs (Fig. 2). These cavities provide a critical refuge

and food source for invertebrate and vertebrate species in

the harsh canopy climate (Nadkami 1994).

Sampling methodology for A. zebrina bromeliads fol-

lowed our previously published methods (McCracken

and Forstner 2008). Single-rope technique (SRT) was

used to climb trees for canopy access, and five brome-

liads were collected haphazardly from each tree at esti-

mated even vertical intervals between one another (Perry

1978). Before each bromeliad removal, we checked for

active amphibians or reptiles, we recorded the bromeli-

ads elevation, measured the air temperature adjacent to

the bromeliad, and the temperature and pH of water held

in one of the outer leaf axils. Ideally, when the bromeliad

is disturbed the response of most animals is a retreat into

the bromeliads leaf bracts and thus prevents loss of speci-

mens (McCracken and Forstner 2008). Bromeliads were

removed and sealed in a 55-gallon (208 L) plastic bag and

then lowered to the ground. After bromeliad collections

we counted the number of A. zebrina inhabiting the tree

and measured tree height. Bromeliads were transported

back to camp where we processed them in a screened tent

to prevent escape of animals. We first poured all water

from the bromeliads through a 1 mm sieve to separate

arthropods, leaf litter, and detritus. We then measured

the water volume with a graduated cylinder and the pH

of the homogenized solution. We counted the number of

mature leaves (used as a size metric) and measured the

height of bromeliads to nearest centimeter (from base of

plant to highest vertical leaf tip). Bromeliads were then

dismantled leaf-by-leaf to collect all herpetofauna.

We identified and counted all metamorphosed an-

urans and reptiles to species level for each bromeliad.

Larval anurans were also collected and counted, with the

majority identified to genus or species. In an attempt to

better identify larval anurans we maintained individual

tadpoles outdoors in 12 oz. plastic cups with water and

detritus collected from bromeliads. Once tadpoles began

to metamorphose the cups were covered with window

screen to prevent escape. Upon sufficient development

to allow identification the froglets were euthanized and

preserved. All herpetofauna were handled and preserved

following the guidelines compiled by the American So-

ciety of Ichthyologists and Herpetologists (ASIH), and

in compliance to the rules overseen by the Texas State

University Animal Care and Use Connnittee (Permit #:

0721-0530-7, 05-05C38ADFDB, and 06-01C694AF).

Additionally, we report the herpetofauna species ob-

served active amongst A. zebrina bromeliads but not

collected. We calculated summary statistics of recorded

habitat variables for each species and report raw data for

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (69)

Fig. 4. The Banded cat-eyed snake, Leptodeira annulata, col-

lected in an Aechmea zebrina bromeliad at 43.5 m above the

forest floor.

singletons and doubletons. Summary statistics were cal-

culated for recorded habitat variables across all bromeli-

ads sampled, bromeliads occupied by >1 metamorphosed

anurans, and bromeliads not occupied by anurans.

We then compared an estimated number of anuran

individuals in A. zebrina bromeliads per 100 m^ of tree

crown area to other published work of tropical frog as-

semblages collected at or near ground level. No other

studies were available to provide canopy estimates. Mean

anuran abundance per tree was calculated by taking the

mean number of metamorphosed anurans per A. zebrina

bromeliad (x = 0.6) and multiplying by the mean number

of bromeliads per tree (x = 66). Based on tree crown di-

ameter measurements by Asner et al. (2002) in lowland

rainforest of eastern Amazonia we calculated the number

of anurans per 100 m^ of a typical tree crown area for

the two largest tree size classes. The two largest classes

had mean tree heights of 25.3 m (Dominant) and 46.1 m

(Super dominant) with a mean crown diameter of 11.6

m and 19.9 m, respectively. Mean tree crown diameters

were used to calculate the area of a circle. These two tree

size classes were used as we did not measure individual

crown diameters and consider these two as representative

of the shortest (28 m) and tallest (49 m) trees in our study.

We then divided the mean number of anurans per tree

October 2014 I Volume 8 I Number 1 I e83

Water temperature (*C) Bromcliad height (cm) Tree height (m)

McCracken and Forstner

A, zehrina bromdiad host tree height

^ “

O

CTi "

o

1 1 1

Absent Present All Trees

Metamorphosed Anurans

A. zebritia brometiad elevation above

forest floor

1 1 \

Absent Present All Trees

Metamorphosed Anurans

Number of A. zi%riiui bromeliads per tree

o

(N -

A. zebritia bromcLiad heiglit

« e

t

o

O =

o

w

o

1 1 1

Absent Present All Trees

Metamorphosed Anurans

A, zebrina bromebad leaf number

Absent

Present All Trees

Metamorphosed Anurans

A. z^hritui bromeliad water volume

£

0

1

Absent

Present All Trees

Metamorphosed Anurans

A. zebrina bromeliad vrater temperature

A. zebrina bromeliad water pH

0

1 1 1

Absent Present All Trees

Metamorphosed Anurans

A, zebrina bromeliad water pH (post)

Absent

Present

All Trees

Metamorphosed Anurans

Air temperature at^. zebrina bromeliad

collection site

Fig. 5. Box plots of recorded habitat variables for

Aechmea zebrina bromeliads collected from all trees,

bromeliads with >1 metamorphosed anuran, and bro-

meliads absent of anurans. Asterisks represent the

mean, open circles are outliers, horizontal line inside

box is the median, top and bottom lines of the rectan-

gle are the 3rd and 1st quartiles (Q3 and Ql), respec-

tively, and the top and bottom whiskers are maximum

and minimum values excluding outliers, respectfully.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (70)

October 2014 I Volume 8 I Number 1 I e83

Herpetofaunal community of a high canopy tank bromeliad

in our study by the tree size class crown area calculated

from Asner et al. (2002) and multiplied by 100 to gener-

ate an estimated density of individuals per 100 m^.

All calculations and statistics based on counts of

metamorphosed anurans collected (not larval anurans)

and conducted in the R statistical software (version 3.0.1)

(R Development Core Team 2013).

Results

We sampled five bromeliads from each of 32 trees for a

total of 160 A. zebrina bromeliads sampled. We collected

10 metamorphosed anuran species (Fig. 3), one gecko,

one snake (Fig. 4), and two species of lizard were ob-

served amongst bromeliad leaves but not collected (Ta-

ble 1). A total of 95 metamorphosed anurans (x = 0.6 per

bromeliad) were collected from 56 of the 160 bromeliads

(35%) sampled. Between one and five individuals (x =

1.7), and up to two species were observed in single A. ze-

brina bromeliads occupied by metamorphosed anurans.

The species found together include (number of brome-

liads with species together): Pristimantis aureolineatus

and P. waoranii (7), P waoranii and P acuminatus (1),

P. waoranii and P orphnolaimus (1), P. aureolineatus

and Ranitomeya ventrimaculata (1). We also collected

a minimum of four larval amphibian species from the

water-filled leaf axils of A. zebrina bromeliads includ-

ing Osteocephalus fuscifacies, O. planiceps, Ranitomeya

variabilis, and R. ventrimaculatus. A total of 27 1 larval

anurans were collected from 35 of the 160 bromeliads

(21.9%) sampled, with 14 of the 35 larval occupied bro-

meliads (40%) also occupied by >1 metamorphosed an-

urans. Osteocephalus spp. tadpoles account for 60.5% {n

= 1 64) of confirmed species identifications for all larval

anurans, and these were collected from five bromeliads.

A single O. fuscifacies and a single O. planiceps (both

adults) were each found in separate bromeliads with lar-

vae of same species (identified after rearing). The gecko,

Thecadactylus solimoensis (formerly T. rapicauda), was

found in an A. zebrina bromeliad amongst the outer leaf

axils at 31.5 m above the forest floor in a tree 46.0 m

tall (Bergmann and Russell 2007). The Banded cat-eyed

snake, Leptodeira annulata, was found in a central leaf

axil of an A. zebrina bromeliad at 43.5 m above the for-

est floor in a tree 45.5 m tall (Fig. 4). Anolis transversalis

was observed twice amongst the leaves of A. zebrina bro-

meliads during collections; once on a bromeliad at ~27

m above the forest floor (36 m tall tree) and in another

tree at ~35 m above the forest floor (41 m tall tree). A

single male Anolis ortonii was observed displaying his

dewlap on an outer leaf of an A. zebrina bromeliad at

~20 m above the forest floor in a tree 28 m tall. Summary

statistics for all species reported in Table 1.

Trees sampled for A. zebrina bromeliads were 28 to

49 m in height (x = 40.4 m + 5.5, n = 32), and 28 to 49

m in height (x = 40.2 m + 5.8, n = 27) for trees with >1

bromeliad occupied by metamorphosed anurans. Aech-

mea zebrina bromeliads were collected at above ground

elevations of 18.3 to 44.5 m (x = 32.9 m + 5.6, n = 160),

and bromeliads occupied by >1 metamorphosed anurans

occurred at elevations of 20.5 to 44.5 m (x = 32.1 m +

6.3, n = 56). The number of A. zebrina bromeliads per

host tree was 19 to 150 individuals (x = 66 + 40, n =

32), and 19 to 150 individuals (x = 63 + 38, n = 27) for

trees with >1 bromeliads occupied by metamorphosed

anurans. Aechmea zebrina bromeliads were 45 to 126

cm in height (x = 75 + 14, n = 160), and 51 to 125 cm

in height (x = 78 + 15, w = 56) for bromeliads occupied

by >1 metamorphosed anurans. The number of mature

leaves per A. zebrina was 14 to 46 (x = 28 + 6, w = 160),

and 17 to 43 (x = 29 + 6, w = 56) for bromeliads occupied

Table 1. Amphibians and reptiles collected or observed within Aechmea zebrina bromeliads. For each species the

number observed, height range (bromeliad in tree), and mean height are provided. Only metamorphed anurans at time

of collection included.

Species

Number observed

Height range (m)

Mean height (m)

Osteocephalus fuscifacies

3

24 . 3 - 28.1

25.6

Osteocephalus planiceps

1

31.5

-

Osteocephalus taurinus

1

30.6

-

Pristimantis acuminatus

1

40.4

-

Pristimantis aureolineatus

36

22 - 44.5

35.7

Pristimantis orphnolaimus

2

31 . 5 - 38.3

34.9

Pristimantis waoranii

35

21 . 2 - 43.9

31.9

Ranitomeya ventrimaculata

1

36.5

-

Ranitomeya variabilis

9

25 . 7 - 35.2

30.9

Scinax ruber

6

33 . 8-35

34.8

Anolis ortonii

1

20

-

Anolis transversalis

2

27-35

31

Thecadactylus solimoensis

1

31.5

-

Leptodeira annulata

1

43.5

-

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (71)

October 2014 I Volume 8 I Number 1 I e83

McCracken and Forstner

by >1 metamorphosed anurans. The water volume of A.

zebrina bromeliads was 42 to 3645 mL (x = 1343 + 656,

n = 160), and 355 to 3645 mL (x = 1428 + 726, n = 56)

for bromeliads oecupied by >1 metamorphosed anurans.

Water temperature within an outer leaf axil of A. zebrina

bromeliads at time of collection was 22.3 to 32.3 °C (x =

26.2 + 2.1, n = 160), and 22.6 to 31.2 °C (x = 26.2 + 1.9,

n = 56) for bromeliads occupied by >1 metamorphosed

anurans. Water pH within an outer leaf axil of A. zebrina

bromeliads at time of collection was 2.82 to 6.34 (x =

4.18 + 0.66, n = 160), and 3.22 to 6.34 (x = 4.34 + 0.73,

n = 56) for bromeliads occupied by >1 metamorphosed

anurans. Water pH of sieved homogenized water for each

A. zebrina bromeliad was 3.14 to 6.08 (x = 4.44 + 0.53,

n = 160), and 3.60 to 6.08 (x = 4.48 + 0.55, n = 56) for

bromeliads occupied by >1 metamorphosed anurans. Air

temperature adjacent to bromeliads at time of collection

was 21.1 to 34.6 °C (x = 27.8 + 2.8, n = 160), and 21.1 to

33.5 °C (x = 27.6 + 2.7, n = 56) for bromeliads occupied

by >1 metamorphosed anurans. Summary statistics for

bromeliads absent of anurans are contrasted with those

given above in Fig. 5.

By taking the mean number of metamorphosed an-

urans per bromeliad (x = 0.6) and multiplying by the

mean number of A. zebrina bromeliads per tree (x = 66),

we calculated an estimated mean of 39.6 metamorphosed

anurans occupying the A. zebrina bromeliads of an av-

erage tree in our study. The Dominant class tree crown

area from Asner et al. (2002) was 105.7 m^ (25.3 m tall)

with a calculated 37.5 anurans per 100 m^, and the Super

dominant class tree crown area was 311 m^ (46.1 m tall)

with a calculated 12.7 anurans per 100 m^.

Discussion

Our study identified 14 species of herpetofauna (10 an-

urans and four reptiles) utilizing the tank bromeliad A^c/^-

mea zebrina as habitat in the high canopy environment of

the northwestern Amazon Basin. A range of 1-5 meta-

morphosed anurans per bromeliad, with up to two spe-

cies occupying a single bromeliad, were detected in over

one-third of the bromeliads sampled. The observation of

larval, metamorphs, and adults of Osteocephalus fuscifa-

cies confirm that this species is a phytotelm breeder as

proposed by Jungfer et al. (2013). The observation of lar-

val, metamorphs, and an Sidult Ranitomeya ventrimacula-

ta confirm that this species does deposit tadpoles in high

canopy bromeliads as proposed by Brown et al. (2011).

Our detection of the gecko Thecadactylus solimoensis at

3 1 .5 m vertical height within the leaf axil of an A. zebrina

bromeliad confirms this species use of bromeliads in the

high canopy (Vitt and Zani 1997; Bergmann and Russell

2007). Our observation of the snake Leptodeira annulata

within the leaf axils of an A. zebrina bromeliad at 43.5

m in the canopy is the highest recorded vertical height to

our knowledge; L. annulata is described as terrestrial to

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (72)

semi-arboreal with a previous maximum observed ver-

tical height of 6 m above ground (Duellman 1978; Vitt

1996; Kacoliris 2006; Avila and Morals 2007).

In McCracken and Forstner (2014) we analyzed the

habitat data for differences among forest disturbance

treatments and found no differences in habitat variables

between treatments and no relationships between habitat

variables and anuran occupancy or abundance. Also, we

found differences between forest disturbance treatments

for anuran abundance and occupancy; but report the

summary statistics of the habitat data here as a resource

characterizing the habitat occupied by canopy tank bro-

meliad dwelling herpetofauna. Of particular interest in

this study was the mildly acidic mean water pH (4.18 in

situ in leaf axils, 4.34 in sieved homogenized water) in A.

zebrina bromeliads; as this is within the range reported

to affect development of embryonic and larval anurans

(Beattie and Tyler-Jones 1992). However, bromeliads are

a known breeding site for amphibians and we observed

an abundance of aquatic invertebrates and larval anurans

in our collections (Benzing 2000).

Using the two largest tree size classes of Amazonian

trees from Asner et al. (2002) as representative crown

area for the shortest (28 m) and tallest (49 m) trees in

our study, we calculated an estimate of 12.7-37.5 an-

urans per 100 m^ of crown area for an average tree in

our study. We consider this estimated range of canopy

anuran density to be conservative because 1) the height

of trees for the tree size classes used from Asner et al.

(2002) are shorter than our shortest and tallest trees; 2)

it is calculated on the two-dimensional space of the tree

crown and does not include the vertical space occupied

by a tree; 3) anurans were only collected from A. zebrina

bromeliads and not other available habitat; and 4) the

mean anuran abundance per tree in our study was used

for calculations of both tree size classes, not accounting

for the range of tree heights and number of bromeliads

per tree. Regardless of these constraints, the estimated

high anuran density of 37.5 anurans/100 m^ is the great-

est of any reported density for tropical frog assemblages

from comparable studies (e.g., 36.1 anurans/100 m2

at La Selva, Costa Rica [Lieberman 1986]; 15.5 an-

urans/100 m^ at Rio Llullapichis, Peru [Toft 1980]; see

also Allmon 1991 and Rocha et al. 2007 for compiled

sites comparison). The low estimate of 12.7 anurans/100

m^ is still amongst the highest densities of reported stud-

ies, particularly in South America (Allmon 1991; Rocha

et al. 2007). A limitation of this comparison is that these

studies rely on the method of quadrat surveys for density

calculations, where the majority of observed anurans are

going to be leaf-litter inhabitants or those that are within

arms reach (~2 m vertical height). Achieving a more ac-

curate canopy anuran density will require research sam-

pling all available canopy microhabitats and recording

crown measurements for all sampled trees.

Within the herpetological community the use of the

term “arboreal” has deviated from its recognized defini-

October 2014 I Volume 8 I Number 1 I e83

Herpetofaunal community of a high canopy tank bromeliad

tion of “inhabiting or frequenting trees” and taken on a

broader meaning in reference to vertical habitat use by am-

phibians and reptiles to simply mean living above ground

level (Merriam-Webster.com. 2014. Merriam-Webster

Dictionary. Available from http://www.merriam-webster.

com [Accessed 27 April 2014]). While this definition

suffices to distinguish these species (arboreal) from those

occupying fossorial and ground level habitat, it does not

adequately clarify the above ground vertical space uti-

lized by a particular species. As an example, Doan (2003)

reports the visual encounter survey (VES) method as the

best way to sample for arboreal herpetofauna in rainfor-

ests. The VES method only allows the researcher access

to habitat within arms reach (~2 m vertical height) and

fails entirely at observing animals within the other ~40-i-

m of vertical habitat above in many rainforests. Arboreal

herpetofauna may occur at vertical heights between >0 m

to 88 m on vegetation and/or trees; simply referring to a

species as arboreal provides no information about its oc-

cupied vertical range (Spickler et al. 2006). To alleviate

confusion and accurately represent the vertical space oc-

cupied by a species or assemblage we propose two alter-

natives to be used separately or preferably together. Eirst,

basic descriptors delineating vertical zones for a defined

forest type could be used to accompany “arboreal” (e.g.,

“arboreal within the understory” where “understory” has

been defined as “near-ground nondominant vegetation”)

(Dial et al. 2004). Second, authors should specify vertical

height ranges when describing or discussing “arboreal”

anurans (e.g., “the arboreal frog Pristimantis waoranii is

found in the overstory at 20.5^4 m” where “overstory”

has been defined as “high, dominant foliage”) (Dial et

al. 2004). Providing vertical range data or descriptions

is critical to understanding the many aspects of natural

history for a species.

Conclusion

The canopy of tropical forests are among the most

species-rich terrestrial habitats on Earth, yet remain a

relatively unexplored biotic frontier (Basset et al. 2003;

Lowman and Schowalter 2012). Our research has shown

the tank bromeliad Aechmea zebrina to support a di-

verse and abundant herpetofauna community in the harsh

equatorial tree canopy environment of the Yasum Bio-

sphere Reserve in the Amazon Basin of Ecuador. Addi-

tionally, our canopy work has contributed to the descrip-

tion of two new species of bromeliad-inhabiting anurans

{Pristimantis aureolineatus [Guayasamin et al. 2006]

and P. waoranii [McCracken et al. 2007]), the detection

of Batrachochytrium dendrobatidis (Chytrid fungus) on

anurans from the forest floor to the canopy in Amazo-

nia (McCracken et al. 2009), and identified the use of

high canopy bromeliads by the anuran Scinax ruber (Mc-

Cracken and Eorstner 2014). While canopy surveys of

tank bromeliads are labor intensive, they provide a very

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (73)

effective technique for collecting data on canopy inhabit-

ing organisms and associated microhabitat factors.

Our estimates of canopy anuran densities, based on

collections from a single species of bromeliad, demon-

strate the potential ecological importance and current

lack of knowledge on the canopy herpetofauna compo-

nent in tropical systems. Typical inventories of herpeto-

fauna in tropical forests are conducted at ground level (~2

m vertical height stratum) where microclimatic variables

are more stable (Guayasamin et al. 2006). Sampling such

shallow strata within the strongly vertical structure of

these forests has likely served to bias metrics of herpeto-

fauna assemblages by focusing on a narrow environmen-

tal space and neglecting the large available habitat above

into the canopy (Guayasamin et al. 2006; Scheffers et al.

2014). Euture inventory studies should routinely include

canopy surveys to properly represent the herpetofauna of

forested habitat.

Use of the term “arboreal” in the herpetofauna litera-

ture does not adequately deflne the vertical range of a

species or assemblage. This serves to limit compilation

and synthesis from the literature for the ecology of many

of these tropical reptiles and amphibians. Our proposed

amendments to accompany the description of arboreality

in herpetofauna functionally serve to give scale and pro-

vide a better understanding of the vertical habitat utilized

by a species or assemblage. As research on canopy her-

petofauna continues to expand, knowledge of the vertical

space occupied will be essential to answering hypothe-

sis-driven research questions and enacting sufficient con-

servation measures to protect all species.

Acknowledgments. — SEM sincerely thanks all the

fieldwork assistants who have contributed to this work

over the years. We thank all the staff at the Tiputini Bio-

diversity Station - Universidad San Erancisco de Quito

and the Yasuni Research Station - Pontificia Universi-

dad Catblica del Ecuador. We also thank the Waorani and

Kichwa peoples who allowed us to conduct fieldwork

in their territories. Thank you to Bejat McCracken for

everything, but especially the photography. Thank you

to Jerad Tullis in the Department of Geography at Texas

State University who constructed the mosaic satellite im-

age in Eigure 1. Lastly, we thank all our funding sources:

National Science Eoundation (Graduate Research Eel-

lowship Program and a GK-12 grant No. 0742306), Tex-

as State University - Department of Biology, the TAD-

POLE Organization, Sigma Xi - The Scientific Research

Society, Texas Academy of Science, and The Explorer’s

Club. This work was conducted under permit numbers

006-IC-EA-PNY-RSO and 012-IC-EA-PNY-RSO issued

by the Ministerio del Ambiente, Ecuador.

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ogy and behavior of Eleutherodactylus aureolineatus

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Received: 01 May 2014

Accepted: 08 August 2014

Published: 30 October 2014

B Shawn F. McCracken is a Research Assistant Professor at Texas State University in San Marcos, Texas, USA.

He received his B.A. in biology and a Ph.D. in aquatic resources at Texas State University. He is the founder

and executive director of the TADPOLE Organization. His research interests include the conservation, ecol-

S9F ogy, and systematics of amphibians; with an emphasis on the effects of anthropogenic disturbance to amphib-

ian diversity and abundance in tropical rainforests. His current research focuses on tbe effects of deforestation

to canopy inhabiting herpetofauna and microclimate in Amazonian Ecuador, with a concentration on epiphytic

canopy tank bromeliads. In the USA, he conducts research on the endangered Houston toad (Anaxyrus hous-

tonensis) and the state threatened Texas tortoise (Gopherus berlandieri).

Michael R. J. Forstner is a Professor in Biology at Texas State University, and the Alexander-Stone Chair

in Genetics. He has a B.S. from Southwest Texas State University, M.S. from Sul Ross State University, and

a Ph.D. from Texas A&M University. He has broad interests in the effective conservation of rare taxa, par-

ticularly reptiles and amphibians. The students and colleagues working with him seek to provide genetic and

ecological data relevant to those conservation efforts.

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (75)

October 2014 I Volume 8 I Number 1 I e83

Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits unrestricted use

for non-commercial and education purposes only, in any medium, provided the original author and the official and

authorized publication sources are recognized and properly credited. The official and authorized publication credit

sources, which will be duly enforced, are as follows: official journal title Amphibian & Reptile Conservation-, of-

ficial journal website <amphibian-reptile-conservation.org>.

Amphibian & Reptiie Conservation

8(1) [Special Section]: 76-88.

A new species of Phoiidoboius (Squamata:

Gymnophthalmidae) from the Andes of southern Ecuador

^Omar Torres-Carvajal, ^Pablo J. Venegas, ^Simon E. Lobos, "^Paola Mafla-Endara,

and ^Pedro M. Sales Nunes

de Zoologia, Escuela de Ciencias Bioldgicas, Pontificia Universidad Catolica del Ecuador, Avenida 12 de Octubre 1076y Roca, Apartado

17-01-2184, Quito, ECUADOR ^Division de Herpetologia-Centro de Ornitologia y Biodiversidad (CORBIDI), Santa Rita N°105 36 Of. 202, Urb.

Huertos de San Antonio, Surco, Lima, PERILED epartamento de Ciencias Naturales, Universidad Tecnica Particular de Loja, San Cayetano Alto s/n

C.P 11 01 608, Loja, ECUADOR ^Universidade Federal de Pernambuco, Centro de Ciencias Bioldgicas, Departamento de Zoologia, Av. Professor

Moraes Rego, s/n. Cidade Universitdria CEP 50670-901, Recife, PE, BRAZIL

Abstract . — ^We describe a new species of Phoiidoboius lizard from the Amazonian slopes of the

Andes of southern Ecuador. Among other characters, the new species differs from other species of

Phoiidoboius in having a distinct diagonal white stripe extending from the fourth genial scale to the

fore limb. We present a phylogeny based on mitochondrial DNA sequence data as additional evidence

supporting delimitation of the new species, which is sister to all other species of Phoiidoboius.

Our phylogeny further supports the south-to-north speciation hypothesis proposed for other lizard

clades from the northern Andes.

Key words. Clade Phoiidoboius, DNA, lizard, phylogeny. South America, systematics

Citation: Torres-Carvajal O, Venegas PJ, Lobos SE, Mafla-Endara P, Nunes PMS. 2014. A new species of Phoiidoboius (Squamata: Gymnophthalmidae)

from the Andes of southern Ecuador. Amphibian & Reptile Conservation 8(1) [Special Section]: 76-88 (e84).

Introduction

The gynmophthalmid lizard clade Phoiidoboius was

recently defined by Torres-Carvajal and Mafla-Endara

(2013) as the largest crown clade conimnmg Phoiidoboius

montium Peters, 1863, but not Macropholidus ruthveni

Noble, 1921. This phylogenetic definition (de Queiroz

and Gauthier 1994) is based on a phylogenetic tree ob-

tained from analyses of mitochondrial DNA nucleotide

sequence data (Torres-Carvajal and Mafla-Endara 2013),

and is in conflict with previous non-phylogenetic defini-

tions of both Phoiidoboius and Macropholidus (Monta-

nucci 1973; Reeder 1996) based on morphological data.

As defined by Torres-Carvajal and Mafla-Endara (2013),

Phoiidoboius contains four species — P. affinis, P mac-

brydei, P montium, and P. prefrontalis. Contrary to pre-

vious taxonomic arrangements (Montanucci 1973; Reed-

er 1996), “P.” annectens was shown to be part of the

clade (traditionally ranked as a genus) Macropholidus.

In addition, Torres-Carvajal and Mafla-Endara (2013)

concluded that the controversial generic allocation of P.

anomalus from southern Peru (Montanucci 1973; Reeder

1996) still remains to be established.

Phoiidoboius lizards occur between 1,800 and 4,100

m along the southern part of the northern Andes (i.e.,

Ecuador and southern Colombia). Only one species, P.

macbrydei, occurs also in the Huancabamba Depression

in extreme southern Ecuador and possibly northern Peru.

Herein, we describe a new species of Phoiidoboius from

the Andes in southern Ecuador using data on morphology

and color pattern. We also present molecular evidence

supporting recognition of the new species by performing

phylogenetic analyses of nucleotide sequence data.

Methods

Morphological data: Type specimens and additional

specimens examined (Appendix 1) were deposited in the

herpetological collection at Museo de Zoologia, Ponti-

ficia Universidad Catolica del Ecuador, Quito (QCAZ).

The following measurements were taken with a digital

caliper and recorded to the nearest 0.1 mm, except for

tail length, which was taken with a ruler and recorded

to the nearest millimeter: head length (HE), head width

(HW), shank length (ShL), axilla-groin distance (AGD),

snout-vent length (SVL), and tail length (TL). Sex was

determined by dissection or by noting the presence of

everted hemipenes. We follow the terminology of Reeder

(1996) for description of the holotype and scale counts.

Data for other species of Phoiidoboius were taken from

Montanucci (1973).

The left hemipenis of two type specimens of the new

species (QCAZ 4998 and 4999) were prepared following

the procedures of Manzani and Abe (1988), as modified

by Pesantes (1994) and Zaher (1999), where the retractor

Correspondence. ^ omartorcar@gmail.com (Corresponding author); ^sancarranca@yahoo.es ; ^lobossimon@gmail.com;

"^paola. mmafen@gmail. com; ^pedro. nunes@gmail. com

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (76) November 2014 | Volume 8 | Number 1 | e84

Torres-Carvajal et al.

Fig. 1. Holotype (QCAZ 4998; SVL = 45.52 mm) of Pholidobolus hillisi sp. nov. in dorsal (A) and ventral (B) views. Photographs

by OTC.

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November 2014 I Volume 8 I Number 1 I e84

A new Pholidobolus from Ecuador

muscle is manually separated and the everted organ is

filled with stained petroleum jelly and paraffin. In addi-

tion, the hemipenial calcareous structures were stained

in an alcoholic solution of Alizarin Red, following the

adaptation of the procedures of Uzzell (1973) proposed

by Nunes et al. (2012). Description of the hemipenes fol-

lows the terminology of Dowling and Savage (1960),

Savage (1997), Myers and Donnelly (2001, 2008), and

Nunes et al. (2012).

DNA sequence data: Total genomic DNA was di-

gested and extracted from liver or muscle tissue using

a guanidinium isothiocyanate extraction protocol. Tissue

samples were first mixed with Proteinase K and a lysis

buffer and digested overnight prior to extraction. DNA

samples were quantified using a Nanodrop® ND-1000

(NanoDrop Technologies, Inc), re- suspended and diluted

to 25 ng/ul in ddH20 prior to amplification.

Using primers and amplification protocols from the

literature (Pellegrino et al. 2001; Torres-Carvajal and

Mafla-Endara 2013) we obtained 1,573 nucleotides (nt)

representing mitochondrial genes 12S (344 nt), 16S (549

nt), and ND4 (680 nt) from three individuals of the new

species described herein (GenBank accession numbers

KP090167-KP090175).

Chronophylogenetic analyses: We added the three

sequences generated in this study to the mtDNA dataset

of Torres-Carvajal and Mafla-Endara (2013). Editing,

assembly, and alignment of sequences were performed

with Geneious ProTM 5.3 (Biomatters Ltd. 2010). Genes

were combined into a single dataset with three partitions,

one per gene. The model of evolution for each partition

was obtained in jModeltest 2 (Darriba et al. 2012) under

the Akaike information criterion. Chronophylogenetic

analyses were performed in Beast 2.1.3 (Bouckaert et al.

2014) as described in Torres-Carvajal and Mafla-Endara

(2013), except that we performed four independent 108

generation runs with random starting trees, sampling ev-

ery 10,000 generations. The resultant 36,000 trees were

used to calculate posterior probabilities (PP) for each bi-

partition in a maximum clade credibility tree in TreeAn-

notator 2.1.2 (Rambaut and Drummond 2014).

Systematics: The taxonomic conclusions of this study

are based on the observation of morphological features

and color pattern, as well as inferred phylogenetic rela-

tionships. We consider this information as species de-

limitation criteria following a general lineage or unified

species concept (de Queiroz 1998, 2007).

Pholidobolus hillisi sp. nov.

urn:lsid:zoobank.org:act:EB5A9DDD-742C-456F-B5C9-6E57EDEEE698

Proposed standard English name: Cuilanes of Hillis

Proposed standard Spanish name: Cuilanes de Hillis

Amphib. Reptile Conserv. | amphibian-reptile-conservation.org (78)

Holotype: QCAZ 4998 (Figs. 1, 2), adult male, Ecua-

dor, Provincia Zamora-Chinchipe, near San Francisco

Research Station on Loja-Zamora road, 3°57’57”S,

79°4’45”W, WGS84, 1,840 m,21 July 2012, collected by

Santiago R. Ron, Andres Merino, Fernando Ayala, Teresa

Camacho, and Martin Cohen.

Paratypes (5): ECUADOR: Provincia Zamora-

Chinchipe: QCAZ 4999 (adult male), 5000 (juvenile

female), same data as holotype; QCAZ 6840 (adult fe-

male), 6842 (adult female), 6844 (adult male), San Fran-

cisco Research Station, 3°58’14”S, 79°4’41”W, WGS84,

1,840 m, 29 October 2004, 9 June 2005, and 29 Septem-

ber 2005, respectively, collected by Kristin Roos, Alban

Pfeiffer, Andy Fries, Ulf Soltau, and Florian Werner.

Diagnosis: Pholidobolus hillisi is unique among spe-

cies of Pholidobolus in having a distinct diagonal white

stripe on each side of the chin, extending from the fourth

genial to the fore limb (Fig. 3). It further differs from all

species of Pholidobolus, except P. affinis, in having three

supraoculars (two in P. macbrydei, P montium, and P

prefrontalis). Pholidobolus affinis differs from the new

species by having flanks with black reticulations on a

reddish orange ground color (flanks brown in P. hillisi'.

Fig. 4).

The new species also can be distinguished from P.

montium and P. macbrydei by the presence of prefrontal

scales (absent in the last two species). While P. hillisi

shares with P. affinis and P. prefrontalis the presence of

prefrontal scales, it differs from them in having a dark

brown dorsum with a conspicuous light brown vertebral

stripe (dorsum pale brown without a vertebral stripe in P.

affinis and P. prefrontalis'. Fig. 4). Furthermore, P. hillisi

has fewer dorsal scales in transverse rows (28-31) than

P. affinis (45-55), P. montium (35-50), P. prefrontalis

(37^6), and P. macbrydei (31^3).

Pholidobolus hillisi shares with all other recognized

species of Pholidobolus the absence of a single trans-

parent palpebral disc and the presence of a ventrolateral

fold between fore and hind limbs. These characters dis-

tinguish members of Pholidobolus from members of its

sister clade Macropholidus (Torres-Carvajal and Mafla-

Endara 2013).

Characterization: (1) Three supraoculars, anterior-

most larger than posterior one; (2) prefrontals present;

(3) femoral pores present in both sexes; (4) two to five

opaque lower eyelid scales; (5) scales on dorsal surface

of neck striated, becoming keeled from fore limbs to tail;

(6) two or four rows of lateral granules at midbody; (7)

28-31 dorsal scales between occipital and posterior mar-

gin of hind limb; (8) lateral body fold present; (9) keeled

ventrolateral scales on each side absent; (10) dorsum

dark brown with a conspicuous narrow, pale brown, ver-

tebral stripe that becomes grayish brown towards the tail;

(11) labial stripe white; (12) sides of body dark brown;

November 2014 I Volume 8 I Number 1 I e84

Torres-Carvajal et al.

Fig. 2. Head of the holotype (QCAZ 4998) of Pholidobolus hillisi sp. nov. in dorsal (A), lateral (B), and ventral (C) views. Photo-

graphs by OTC.

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November 2014 I Volume 8 I Number 1 I e84

A new Pholidobolus from Ecuador

Fig. 3. Head of five speeies of Pholidobolus in ventral view. (A) P. affinis', (B) P. hillisi sp. nov.; (C) P. macbryder, (D) P montiunr,

(E) P prefrontalis. Photographs by OTC.

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November 2014 I Volume 8 I Number 1 I e84

Torres-Carvajal et al.

(13) white stripe along fore limb present; (14) a distinct

diagonal white stripe on each side of the chin, extending

from the fourth genial to the fore limb; (15) adult males

with red flecks and ocelli (black with white center) dorsal

to insertion of fore and hind lim bs.

Description of hoiotype: Adult male (QCAZ 4998);

snout- vent length 45.52 mm; tail length 104 mm; dorsal

and lateral head scales juxtaposed, finely wrinkled; ros-

tral hexagonal, 2.09 times as wide as high; frontonasal

pentagonal, wider than long, laterally in contact with na-

sal, smaller than frontal; prefrontals pentagonal, nearly

as wide as long, with medial suture, laterally in contact

with loreal and first superciliary; frontal hexagonal, lon-

ger than wide, slightly wider anteriorly, in contact with

the prefrontals and supraoculars I and II on each side;

frontoparietals pentagonal, longer than wide, with me-

dial suture, each in contact laterally with supraoculars II

and III; interparietal roughly hexagonal, lateral borders

parallel to each other; parietals slightly smaller than in-

terparietal, tetragonal and positioned anterolaterally to

interparietal, each in contact laterally with supraocular

III and dorsalmost postocular; postparietals three, medial

scale smaller than laterals; supralabials seven, fourth lon-

gest and below the center of eye; infralabials five, fourth

below the center of eye; temporals enlarged, irregularly

hexagonal, juxtaposed, smooth; two large supratemporal

scales, smooth; nasal divided, irregularly pentagonal,

longer than wide, in contact with rostral anteriorly, first

and second supralabials ventrally, frontonasal dorsally,

loreal posterodorsally and frenocular posteroventrally;

nostril on ventral aspect of nasal, directed lateroposteri-

orly, piercing nasal suture; loreal rectangular; frenocular

enlarged, in contact with nasal, separating loreal from

supralabials; supraoculars three, with the first being the

largest; four elongate superciliaries, first one enlarged,

in contact with loreal; palpebral disk divided into two

scales, pigmented; suboculars three, elongated and simi-

lar in size; three postoculars, medial one smaller than the

others; ear opening vertically oval, without denticulate

margins; tympanum recessed into a shallow auditory me-

atus; mental semicircular, wider than long; postmental

pentagonal, slightly wider than long, followed posteri-

orly by four pairs of genials, the anterior two in contact

medially and the posterior two separated by postgenials;

all genials in contact with infralabials; gulars imbricate,

smooth, widened in two longitudinal rows; gular fold

incomplete; posterior row of gulars (collar) with four

scales, the medial two distinctly widened.

Scales on nape similar in size to dorsals, except for

the anteriormost that are widened; scales on sides of neck

small and granular; dorsal scales elongated, imbricate,

arranged in transverse rows; scales on dorsal surface of

neck striated, becoming keeled from fore limbs to the

tail; number of dorsal scales between occipital and poste-

rior margin of hind limbs 28; dorsal scale rows in a trans-

verse line at midbody 30; one row of smooth, enlarged

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ventrolateral scales on each side; dorsals separated from

ventrals by three rows of small scales at the level of the

13th row of ventrals; lateral body fold present; ventrals

smooth, wider than long, arranged in 20 transverse rows

between the collar fold and preanals; six ventral scales in

a transverse row at midbody; subcaudals smooth; limbs

overlap when adpressed against body; axillary region

composed of granular scales; scales on dorsal surface of

fore limb striated, imbricate; scales on ventral surface of

fore limb granular; two thick, smooth thenar scales; su-

pradigitals (left/right) 3/3 on finger I, 6/6 on II, 8/8 on III,

9/9 on IV, 6/6 on V; supradigitals 3/3 on toe I, 6/6 on II,

9/9 on III, 11/12 on IV, 8/8 on V; subdigital lamellae of

fore limb single, 5/5 on finger I, 8/9 on II, 13/13 on III,

14/14 on IV, 8/9 on V; subdigital lamellae on toes I and

II single, on toe III paired on the distal half, on toe IV all

paired, on toe V paired at the base; number of subdigi-

tal lamellae (pairs when applicable) 6/5 on toe I, 9/9 on

II, 13/14 on III, 19/20 on IV, 12/12 on V; groin region

with small, imbricate scales; scales on dorsal surface of

hind limbs striated and imbricated; scales on ventral sur-

face of hind limbs smooth; scales on posterior surface

of hind limbs granular; six femoral pores on each leg;

preanal pores absent; cloacal plate paired, bordered by

four scales anteriorly, of which the two medialmost are

enlarged.

Measurements (mm) and proportions of the hoiotype:

HL 12.6; HW 9.3; ShL 5.2; AGD 24.6; TL/SVL 1.72;

HL/SVL0.25; HW/SVL0.18; ShL/SVLO.lO; AGD/SVL

0.48.

Hemipenial morphology (Fig. 5): Both organs ex-

tend along approximately nine millimeters in length. The

lobes of the organs are fully everted and each hemipenis

is fully expanded.

The hemipenial body is roughly conical in shape,

with the base distinctly thinner than the rest of the organ,

ending in two small lobes with apical folds in the apex.

The sulcus spermaticus is central in position, originat-

ing at the base of the organ, which bears a fleshy fold

partially overlapping the sulcus spermaticus. From this

point on, the sulcus proceeds in a straight line towards

the lobes, and acquires an S-shape at the first third of the

body. The sulcus becomes broader at halfway the length

of the hemipenial body, and returns to its regular width

at the apical region; it gets divided in two branches at the

lobular crotch. Just before the crotch, the central region

of the sulcus bears a tiny fleshy fold, which is not part of

the sulcus division. From this point on, the two branch-

es of the sulcus run on the medial regions of the lobes

among conspicuous lobular folds. The sulcate face of the

hemipenial body presents two nude areas, parallel to the

sulcus spermaticus, which run throughout the hemipenial

body, getting thinner and encircling the base of the lobes.

The lateral and asulcate faces of the hemipenial body

are ornamented with 28-30 rows of roughly equidistant

flounces with calcareous spinules. The first four rows are

November 2014 I Volume 8 I Number 1 I e84

A new Pholidobolus from Ecuador

Fig. 4. Five species of Pholidobolus from Ecuador. (A) P. affinis',

sp. nov. Photographs by OTC (A, B, C, D) and S. R. Ron (E).

straight, with a large series of spinules on the central as-

pect of the asulcate face, and small isolated series of 5-6

spinules bordering the nude areas parallel to the sulcus

spermaticus. A V-shaped nude area at the central asulcate

face of the body separates the remaining flounces. The

flfth and sixth flounces are also interrupted laterally by

an extension of the basal nude area. From the seventh to

the apical-most one, the flounces cross the lateral aspects

of the organ from the sulcate to the asulcate face, initially

in roughly straight lines, gradually assuming chevron-

shapes and getting reduced in length towards the apex

of the organ.

The region between the asulcate and the lateral sur-

faces is marked by a conspicuous unevenness forming a

bulge, which is shared by closely related species, such as

Macropholidus annectens, M. huancabambae, M. ruth-

(B) P. macbrydei; (C) P montiunr, (D) P prefrontalis; (E) P hillisi

veni, Pholidobolus affinis, P macbrydei, P montium, and

P prefrontalis (Nunes, 2011).

Color of holotype in preservative: Dorsal back-

ground uniformly dark brown with a narrow light brown

vertebral stripe extending from occiput onto tail; ver-

tebral stripe slightly wider anteriorly; dorsal surface of

head light brown medially (rostral, frontonasal, prefron-

tals, frontal and frontoparietals) and dark brown laterally

(including supraoculars); white supralabial longitudinal

stripe extending from first supralabial to fore limb; lateral

aspect of neck dark brown with a dorsolateral light brown

stripe that extends posteriorly along the flanks to the hind

limbs; ventrolateral aspect of head and neck with a lon-

gitudinal white stripe extending posteriorly from fourth

genial to insertion of fore limb and then laterally along

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November 2014 I Volume 8 I Number 1 I e84

Torres-Carvajal et al.

Fig. 5. Left hemipenis of Pholidobolus hillisi sp. nov. (QCAZ 4999) in sulcate (left), lateral (middle), and asulcate (right) views.

Photographs by P Nunes.

upper arm; fore limbs with scattered ocelli (black with

white center); flanks grayish brown with two dorsolateral

stripes, the dorsal one light brown and the ventral one

dark brown; tail light brown dorsally and dark brown on

the sides; two and three well-defined, small ocelli (black

with white center) dorsal to insertion of fore and hind

limbs, respectively; ventral surface of head gray, with

dirty cream genials and scattered brown marks; chest,

belly and ventral surface of limbs and tail dark gray.

Variation: Measurements and scale counts of Pholi-

dobolus hillisi are presented in Table 1. Superciliaries

usually four, five in QCAZ 6840; supralabials usually

seven (eight of left side of specimen QCAZ 6840). Rows

of lateral granules at midbody two (QCAZ 4999, 6844) to

four (QCAZ 6842). Three specimens including the holo-

type, with a ventrolateral row of smooth enlarged scales

(QCAZ 4999, 6840). Specimen QCAZ 6842 has a tiny

scale separating the cloacal scales posteriorly; all four

scales bordering the cloacal plate anteriorly are similar

in size in two specimens (QCAZ 4999, 6844), whereas

the lateralmost scales overlap the cloacal scales in one

specimen (QCAZ 6840).

No variation was observed in color pattern in preser-

vative among adult males. They can be distinguished

from females by the presence of ocelli and pale flecks

around insertion of fore and hind limbs. Moreover, the

characteristic diagonal white stripe on each side of the

chin that extends from the fourth genial to the forearm is

more conspicuous in males than in females. Females are

larger (maximum SVL 55.7 mm, n=3) than males (maxi-

mum SVL 51.1 mm, n=3).

Coloration in life of an adult male paratype (QCAZ

4999) was similar to the holotype’s coloration in pre-

servative described above, except that specimen QCAZ

4999 had small red flecks both at insertion of fore limbs

Table 1. Sexual variation in lepidosis and measurements of Pholidobolus hillisi sp. nov. Range followed by mean + standard devia-

tion are given.

Character

Males (n=3)

Females (n=3)

Dorsal scales between occipital and posterior margin of hind limb

28-30 (29+1)

29-31 (30+1)

Dorsal scale rows in a transverse line at midbody

27-34 (30.33+3.51)

29-35 (31+3.46)

Ventral scales between collar fold and preanals

18-20 (20.33+1.15)

18-19 (18.67+0.58)

Ventral scale rows in a transverse line at midbody

6-7 (6.67+0.58)

6

Subdigital lamellae on Finger IV

14-15 (14.33+43.0)

13-15 (13.67+1.15)

Subdigital lamellae on Toe IV

19-20 (19.33+0.58)

19

Femoral pores

5-8 (6.33+1.52)

2-5 (3.5) (n=2)

Maximum SVL

51.1

55.7

TL/SVL

1.86 (n=l)

1.84-2.14(1.99) (n=2)

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A new Pholidobolus from Ecuador

0.15 0.10 0.05 0

Neusticurus rudls

Placosoma glabellum

Bachia flavescens

Cercosaura quadrilineata

Potamites ecpieopus

Proctoporus bolivianus

Riama cashcaensis

Macropholidus ruthveni

M. annectens

M. huancabambae

Pholidobolus hillisi sp. nov.

P hillisi sp. nov.

P. hillisi sp. nov.

P macbrydei

P. macbrydei

P. pre frontalis

P pre frontalis

P. montium

P montium

P affinis

P. affinis

Fig. 6. Maximum clade credibility tree inferred from the analysis of a dataset eontaining three mitochondrial genes under uneor-

related, log normally distributed rates; branch lengths are in substitutions per site. Posterior probability values are shown above

branches; asterisks correspond to values of 1.

extending onto sides of neck and at insertion of hind

limbs extending onto base of tail. In addition, the lateral

white stripe that starts on first supralabial extends further

posteriorly along flanks in specimen QCAZ 4999 (Fig.

4).

Phylogenetic relationships: The maximum clade

credibility tree resulting from the chronophylogenetic

analysis supports inclusion of the new species within the

Pholidobolus clade (Torres-Carvajal and Mafla-Endara

2013) with strong support (PP = 0.96; Fig. 6). Phyloge-

netic relationships among other species of Pholidobolus

and species of Macropholidus are identical to those ob-

tained by Torres-Carvajal and Mafla-Endara (2013).

Macropholidus ruthveni is sister (PP = 0.99) to a clade

containing both M. annectens and M. huancabambae (PP

= 1). Pholidobolus macbrydei is sister (PP = 0.91) to a

clade with the three remaining species of Pholidobolus;

the latter clade included P prefrontalis as sister (PP =

0.99) to a clade containing P. affinis and P. montium as

sister taxa (PP = 0.99). In contrast to the results reported

by Torres-Carvajal and Mafla-Endara (2013), the chrono-

phylogenetic tree inferred in this paper suggests that the

diversification of the clades Macropholidus and Pholi-

dobolus occurred at about the same time (Fig. 6).

Distribution and ecology: Pholidobolus hillisi inhab-

its low montane forests in the eastern slopes of the Andes

of southern Ecuador. This area represents a weather di-

vide between the humid Amazon and the dry Inter- Ande-

an regions (Beck et al. 2008). The new species is known

from Provincia Zamora-Chinchipe, at 1,840 m (Fig. 7),

in the deep valley of the Zamora river. The only gym-

71- w »-V¥ n-w

ii-W w Jl- w ta-ff Tf-'^

Fig. 7. Distribution of Pholidobolus in Ecuador. P. affinis (white

circles); P. macbrydei (blue circles); P. montium (green circles);

P. prefrontalis (orange circles); P hillisi sp. nov (red circle).

nophthalmid species known to occur in sympatry with

P. hillisi is Alopoglossus buckleyi, although P macbry-

dei is parapatrically distributed (Fig. 7). Two specimens

(QCAZ 4998, 4999) were found under logs and rocks

next to the Zamora river between 1 130 hrs and 1145 hrs.

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November 2014 I Volume 8 I Number 1 I e84

Torres-Carvajal et al.

whereas another specimen (QCAZ 5000) was basking

on a rock next to the road at 1200 hrs. Other specimens

(QCAZ 6840, 6842, 6844) were found and captured by a

domestic cat around the San Francisco Research Station

in pasture with interspersed shrubs.

Etymology: The specific epithet hillisi is a noun in the

genitive case and is a patronym for David M. Hillis, who

has had a great impact in the development of the field of

molecular systematics (e.g., Hillis et al. 1996). In par-

ticular, he published a classic paper on evolutionary ge-

netics of Pholidobolus lizards, where he compared some

phylogenetic tree reconstruction techniques and empha-

sized the importance of phylogenetics in biogeography

(Hillis 1985).

Remarks: The Andes of southern Ecuador and northern

Peru between 4°S and 7°S consist of relatively low-ele-

vation mountains that create a mixture of environments.

This region, known as the Huancabamba Depression, has

long been recognized as a major biogeographic barrier

for Andean organisms (e.g., Cadle 1991; Duellman 1979;

Vuilleumier 1969). Although all species of Pholidobolus,

except P. macbrydei, are restricted to the southern part

of the northern Andes (i.e., Ecuador and southern Co-

lombia), the new species described herein occurs on the

northern limit of the Huancabamba Depression.

The Huancabamba Depression seems to have in-

fluenced the radiation of several Andean lizard clades,

such as Stenocercus, Riama, Macropholidus, and Pholi-

dobolus (Doan 2003; Torres-Carvajal 2007; Torres-Car-

vajal and Mafla-Endara 2013). Except for Macropholi-

dus, these clades have diversified along the northern

Andes, suggesting that common geological or climatic

events have influenced these radiations. The phyloge-

netic tree presented in this paper further supports the idea

of a south-to-north sequence of speciation events (Doan

2003; Torres-Carvajal 2007) which is congruent with the

recent south-to-north uplift of the northern Andes (Simp-

son 1979; Aleman and Ramos 2000).

Acknowledgments. — We thank Santiago R. Ron for

photographs and Andrea Varela for assembling some

of the figures. Special thanks to Tiffany Doan and an

anonymous reviewer for their valuable comments. OTC

received funds from Secretaria de Educacion Superior,

Ciencia, Tecnologia e Innovacion (SENESCYT). PMSN

is grateful to Funda9ao de Amparo a Pesquisa do Estado

de Sao Paulo (FAPESP) for financial support (Grant #

2012/00492-8). Specimens were collected under collec-

tion permit 001-11 IC-FAU-DNB/MA issued by Ministe-

rio de Ambiente del Ecuador.

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Received: 10 September 2014

Accepted: 31 October 2014

Published: 12 November 2014

Additional specimens examined

P/70//C/060/US aff/n/s.— ECUADOR: Provincia Chimborazo: Colta, r41’56”S, 78°46’25”W, 3,215 m, QCAZ 9899-01 ; Sicalpa, 1°42’18”S,

78°46’32”W, 3,212 m, QCAZ 11887. Provincia Cotopaxi: Cutuchi river, San Miguel de Salcedo, 1°2’9”S, 78°35’53”W, 2,640 m, QCAZ

9641 . Provincia Tungurahua: 6 km N Mocha to 400 m Panamerican Highway, 1°22’1”S, 78°39’16”W, 3,205 m, QCAZ 9895-97; Ambato

surroundings, 1°14’59,8”S, 78°37’33”W, QCAZ 9340-73, 9375-9443; Chamisa on road to Guadalupe, r21’44”S, 78°30’39”W, 2,348

m, QCAZ 7266; Cotalo on path to Mucubi Community, 1°25’46”S, 78°31’3”W, 2,626 m, QCAZ 9839; Patate, 1°18’42”S, 78°30’36”W,

2,199 m, QCAZ 9847-50; Poatug Hamlet, Aya Samana, 1°16’58”S, 78°29’29”W, 2,573 m, QCAZ 10005, 10008, 10011-13, 10016,

10018; Poatug Hamlet, Terremoto, 1°16’23”S, 78°29’29”W, 2,547 m QCAZ 9997-10000, 10002-10004; San Miguelito on path to Pil-

laro, ri3’12”S, 78°31’31”W, 2,689 m, QCAZ 9844; San Miguelito on path to Teran, 1°12’58”S, 78°31’42”W, 2,741 m, QCAZ 9843.

Pholidobolus macbrydei.— ECUADOR. Provincia Azuay: 10 km S Cutchil, 3°8’2”S, 78°48’47”W, 2,900 m, QCAZ 823-24;1.2 km E Qs-

orancho, 2°46’8”S, 78°38’10”W, 2,390 m, QCAZ 826; 6.2 km S Cutchil, 3°6’32”S, 78°48’4”W, 2,800 m, QCAZ 827; 20 km NE Cuenca,

2°51’0”S, 78°51’14”W, QCAZ 1359; 7 km Sigsig, 2°59’56”S, 78°48’25”W, 2,890 m, QCAZ 1537; 6 km S Qha, 3°29’49”S, 79°9’47”W,

QCAZ 3658; 20 km Cuenca-EI Cajas, 2°46’39”S, 79°10’12”W, 3,508 m, QCAZ 9932-34, 9936-38, 10020; Cochapamba, 2°47’50”S,

79°24’56”W, 3,548 m, QCAZ 10133-35; Cochapata, 3°25’47”S, 79°3’35”W, 3,074 m, QCAZ 12605-07; Cuenca, Cuenca-Azoguez

Panamerican Highway 2°53’43”S, 78°57’30”W, 2,486 m, QCAZ 6985; El Cajas National Park, path to PatuI Community, 2°44’28”S,

79°14’5”W, 4,092 m, QCAZ 8010-11; El Cajas National Park, PatuI river, 2°41’37”S, 79°13’56”W, 3,610 m, QCAZ 8893; El Cajas Na-

tional Park, Zhurcay river, 3°2’30”S, 79°12’56”W, 3,766 m, QCAZ 8900-01; El Cajas National Park, 2°42’21”S, 79°13’32”W, 3,600 m,

QCAZ 8946; El Capo, 2°46’43”S, 79°14’43”W, 4,100 m, QCAZ 4997; Giron, San Gregorio Community, Quinsacocha paramo, 3°6’22”S,

79°13’4”W, 3,242 m, QCAZ 8510-11; Giron, San Gregorio Community, Quinsacocha paramo, 3°2’30”S, 79°12’56”W, 3,766 m, QCAZ

8894-99, 8902-05, 8907; Giron, San Gregorio Community, Quinsacocha paramo, 3°2’30”S, 79°12’57”W, 3,766 m, QCAZ 8906; Guablid,

2°46’30”S, 78°4r51”W, 2,453 m, QCAZ 991 3-1 7, 9919-20, 9940-41; Gualaceo-Limon road, 2°56’53”S, 78°42’43”, 3,110 m, QCAZ 819-

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22; Gualaceo-Limon road, 8.1 km O Azuay-Morona Santiago border, 2°57’50”S, 78°427”W, 3,140 m, QCAZ 825; Gualaceo, 2°52’56”S,

78°46’31”W, 2,298 m, QCAZ 9606; Gualaceo-Plan de Milagro road, 2°54’35”S, 78°44’4”W, 2,624 m, QCAZ 10875; Las Tres Cruces,

2°46’30”S, 79°14”53”W, QCAZ 4136; Maylas, Gualaceo-Macas road, 2°58’25”S, 78°41’41”W, 3,100 m, QCAZ 7269; Mazan Protected

Forest, 2°52’29”S, 79°7’26”W, 2,700 m, QCAZ 1296-97; Mazan Protected Forest, 2°52’31”S, 79°7’45”W, 3,189 m, QCAZ 8008, 8013;

Qna-La Paz road, 3°22’42”S, 79°11’20”W, 2,969 m, QCAZ 6031; Patacocha hill, 3°7’16”S, 79°3’54”W, 3,340 m, QCAZ 6144; Pucara,

Tres Chorreras, 3°12’49”S, 79°28’3”W, QCAZ 11038; Quinoas river, 3°5’14”S, 79°16’40”W, 3,200 m, QCAZ 1564-66; San Antonio,

2°51’40”S, 79°22’43”W, 2,943 m, QCAZ 9668; San Vicente-Cruz path, 2°47’43”S, 78°42’53”W, 3,044 m, QCAZ 11416-17, 11420;

Sigsig, 3°7’46”S, 78°48’14”W, 2,969 m, QCAZ 5605-08; Sigsig road, 3°3’17”S, 78°47’19”W, 2,574 m, QCAZ 9605; Tarqui, 3°0’57”S,

79°2’40”W, 2,627 m, QCAZ 8512. Provincia Cahar: Canar, 2°33’39”S, 78°55’51”W, QCAZ 9947; Culebrillas, 2°25’35”S, 78°52’12”W,

4,000 m, QCAZ 1349; Guallicanga ravine, 2°25’56”S, 78°54’8”W, 3,960 m, QCAZ 10048-49; Guallicanga river, 2°28’24”S, 78°58’22”W,

3,048 m, QCAZ 10051-52; Ingapirca, 2°32’43”S, 78°52’28”W, 3,400 m, QCAZ 1551; Juncal, 2°28’24”S, 78°58’22”W, 3,048 m, QCAZ

10050; Mazar Protected Forest, 2°32’48”S, 78°41’54”W, QCAZ 7376-84, 7883; Mazar Reserve, La Libertad, 2°32’45”S, 78°41’46”W,

2,842 m, QCAZ 10970-72. Provincia Chimborazo: Alao, 10 km Fluamboya, 1°52’22”S, 78°29’51”W, 3,200 m, QCAZ 1567-68; Atillo

Grande, Magdalena lake, 2°11’15”S, 78°30’25”W, 3,556 m, QCAZ 9214; Atillo Grande, Frutatian lake, 2°12’57”S, 78°30’5”W, 3,700 m,

QCAZ 9216-18; Culebrillas, Sangay National Park, 1°57’39”S, 78°25’55”W, 3,345 m, QCAZ 9612; Pungala, Eten Community, Timbo,

1°55’45”S, 78°32’14”W, 3,408 m, QCAZ 9616-21 ; Pungala, Melan Community, 1°52’30”S, 78°32’52”W, 3,564 m, QCAZ 9626-29, 9631;

Qzogoche, 2°22’7”S, 78°41’20”W, 4,040 m, QCAZ 6006-07; Shulata, 2°20’22”S, 78°50’36”W, 3,228 m, QCAZ 5597-9;. Provincia El

Qro: Guanazan, 3°26’24”S, 79°29’13”W, 2,638 m, QCAZ 7891, 7894. Provincia Loja: 17.1 km S Saraguro, 3°43’45”S, 79°15’53”W,

3,150 m, QCAZ 828; 26 km N Loja, Fluashapamba Native Forest, 3°39’30”S, 79°16’20”W, 2,894 m, QCAZ 8651; Cordillera of Lagunil-

las, Jimbura, 4°49’1”S, 79°21’43”W, 3,600 m, QCAZ 3785; Cordillera of Lagunillas, Jimbura, 4°37’42”S, 79°27’49”W, 3,450 m, QCAZ

6145-47; Fierro Urco, 3°42’38”S, 79°18’18”W, 3,439 m, QCAZ 6949-50; Gurudel, 3°39’22”S, 79°9’47”W, 3,100 m, QCAZ 5078-79;

Jimbura, Jimbura lake, 4°42’32”S, 79°26’48”W, 3,036 m, QCAZ 6945-48; Jimbura, path to Jimbura lake, 4°42’34”S, 79°26’8”W, 3348

m, QCAZ 10054-62; Military antenna, Saraguro, 3°40’46”S, 79°14’16”W, 3,190 m, QCAZ 3673-75, 9632; San Lucas, 3°43’55”S,

79°15’38”W, 2,470 m, QCAZ 2861; Saraguro, 3°37’13”S, 79°14’9”W, 3,100 m, QCAZ 3606, 3754; Urdaneta, 3°36’6”S, 79°12’31”W,

QCAZ 201 9. Provincia Tungurahua: Poatug Flamlet, El Corral, 1°16’21”S, 78°28’5”W, 3,468 m, QCAZ 8047, 9995-96. Provincia Zamo-

ra Chinchipe: Loja-Podocarpus National Park road, 3°59’44”S, 79°8’28”W, 2,776 m, QCAZ 10870-71; Valladolid, Podocarpus National

Park, 4°29’3”S, 79°8’56”W, 1 ,800 m, QCAZ 3743.

Pholidobolus montium. — ECUADQR: Provincia Cotopaxi: 2 km S Chugchilan on road to Quilotoa, 0°48’24”S, 78°56’11”W, 2,917 m,

QCAZ 8056-58; Latacunga, 0°52’27”S, 78°38’26”W, 2,857 m, QCAZ 873-74, 1411-12, 9642; Mulalo, 0°46’35”S, 78°34’40”W, 3,030

m, QCAZ 9639; San Juan de Paste Calle, 0°45’4”S, 78°38’51”W, 1,956 m, QCAZ 8053-54; South llliniza, 0°39’43”S, 78°42’40”W,

3,400 m, QCAZ 858-59, 1454. Provincia Imbabura: Atuntaqui, 0°19’59”N, 78°12’50”W, QCAZ 855; Cotacahi, Peribuela, Cuicocha

Lake, Cotacachi-Cayapas Reserve, 0°17’34”N, 78°21’5”W, 3,082 m, QCAZ 9683, 9685-86; 0°23’4”N, 78°15’25”W, 2,900 m, QCAZ

6137, 6139; Cotacachi-Cayapas Reserve, Jose Marla Yerovi Islets, 0°18’20”N, 78°2T41”W, 3,093 m, QCAZ 10959-60; El Juncal,

0°26’6”N, 77°57’58”W, QCAZ 6451 . Provincia Pichincha: 16 km W Chillogallo, Quito-Chiriboga road, 0°17’46”S, 78°39’30”W, 3,100 m,

QCAZ 797; 5 km E Pifo-Papallacta road, 0°15’3”S, 78°17’58”W, 2,800 m, QCAZ 1107-08; Alambi, 0°1’59”S, 78°34’26”W, 2,727-3,800

m, QCAZ 9691; AlangasI, 0°18’24”S, 78°24’40”W, QCAZ 1453, 1469; Amaguaha, Hacienda San Ignacio, 0°22’22”S, 78°30’14”W,

QCAZ 1463-64, 5275; Calacall, Simon Bolivar Street, uphill through secondary road, 0°T1”N, 78°30’49”W, 3,001 m, QCAZ 11674,

11676, 11678-79; Calacall Stadium, 0°0’0,3”S, 78°30’38”W, 2,833 m, QCAZ 11682; Carretas, 0°6’25”S, 78°26’46”W, QCAZ 875;

Chillogallo, 0°16’48”S, 78°33’25”W, QCAZ 840-43; Cumbaya, La Primavera, 0°12’6”S, 78°25’40”W, QCAZ 7248; Guayllabamba,

0°3’23”S, 78°20’26”W, QCAZ 7905; Inga, 5.5 km SE La Merced, 0°17’51”S, 78°20’52”W, 2,798 m, QCAZ 5278; Lloa, 0°14’52”S,

78°34’33”W, QCAZ 4109; Lloa Stadium, 0°14’39”S, 78°35’12”W, 3,059 m, QCAZ 11661; Loreto, road to Molinuco, Central Stadium,

0°23’4”S, 78°24’30”W, 2,844 m, QCAZ 11663; Machachi, 0°29’50”S, 78°32’25”W, QCAZ 844-48, 1374-77, 1462; Machachi, The

Tesalia Springs Company S.A. surroundings, 0°30’27”S, 78°33’57”W, 2,900 m, QCAZ 1465-67, 830-31, 833, 860-61, 1459-61; None,

0°4’42”S, 78°34’24”W, 2,843 m, QCAZ 11653-55; None School, 0°4’4”S, 78°34’35”W, 2,754 m, QCAZ 11656-58; Pasochoa, 0°26’24”S,

78°30’15”W, 2,850 m, QCAZ 1451-52; Pomasqui, 0°3’3”S, 78°27’21”W, QCAZ 862-68; Pululahua Volcano, 0°2’34”N, 78°30’15”W,

QCAZ 1450, 1520; Quito, Bellavista, 0°11’21”S, 78°28’35”W, QCAZ 1099; Quito, Chillogallo, 0°16’26”S, 78°33’23”W, QCAZ 8967; Qui-

to, Itchimbla, 0°13’21”S, 78°29’56”W, QCAZ 834, 1455-58, 1643, 2843; Quito, Garden of the Pontificia Universidad Catolica del Ecua-

dor (PUCE), 0°12’33”S, 78°29’28”W, 2,800 m, QCAZ 856-57, 7032, 1295, 2853; Quito, Parque Metropolitano, 0°10’35”S, 78°27’40”W,

QCAZ 4051; Quito, Universidad Central del Ecuador, 0°1T59”S, 78°30’19”W, 2,800 m, QCAZ 3727; Rio Guajalito Protected Forest,

0°13’44”S, 78°48’22”W, QCAZ 1338-39; San Antonio de Pichincha, 0°0’33”S, 78°26’45”W, QCAZ 580-81 , 790-92, 849, 1119-20, 1368,

1393, 2220, 2223, 2653; Tababela, International Airport, 0°6’21”S, 78°21’4”W, QCAZ 8046, 9044, 10064, 10974-76; Quito, Tumbaco,

0°12’34”S, 78°24’2”W, QCAZ 1113-14; Uyumbicho, 0°22’59”S, 78°31’6”W, QCAZ 870.

Pholidobolus prefrontalis . — ECUADQR: Provincia Chimborazo: AlausI, 2°1T54”S, 78°50’42”W, 2359 m, QCAZ 9907-9911; Tixan,

2°9’22”S, 78°48’3”W, 2,908 m, QCAZ 9951-54.