ELUCIDATING THE RELATIONSHIPS WITHIN THE LIMNONECTES KUHLII SPECIES COMPLEX (AMPHIBIA: ANURA: DICROGLOSSIDAE)

by

David S. McLeod M.S. University of Nebraska-Lincoln, 1999 B.S.Ed. Emporia State University, 1995

Submitted to the Department of Ecology and Evolutionary Biology and the Faculty of the Graduate School of The University of Kansas in partial fulfillment of the requirements for the degree of Doctor of Philosophy

Linda Trueb Chair

Rafe M. Brown

Edward O. Wiley

Leonard Krishtalka

David Tell Committee Members

Date Defended: 28 April 2010

The Dissertation Committee for David S. McLeod certifies that this is the approved version of the following dissertation:

ELUCIDATING THE RELATIONSHIPS WITHIN THE LIMNONECTES KUHLII SPECIES COMPLEX (AMPHIBIA: ANURA: DICROGLOSSIDAE)

Dr. Linda Trueb Chair

Date Approved: 28 April 2010

i TABLE OF CONTENTS

TABLE OF CONTENTS………………………………………………………………………………ii

ABSTRACT AND INTRODUCTION…………………………………………………………………..iii

ACKNOWLEDGMENTS……………………………………………………………………………...v

CHAPTER I. The Limnonectes kuhlii Complex: Diversity Hidden in Plain Sight…………….....1.1

CHAPTER II. Of Least Concern? Systematics of a Cryptic Species Complex: Limnonectes kuhlii (Amphibia: Anura: Dicroglossidae) ………………………....2.1

FIGURES……………………………………….………………………………………..2.33

TABLES…………………………………………………………………………………2.36

APPENDIX I……………………………………………………………………………..2.38

APPENDIX II………………………………………………………………………….....2.47

CHAPTER III. Limnonectes kuhlii (Amphibia: Anura: Dicroglossidae): Identity Complex or a Complex Identity? ……………………………………….3.1

FIGURES………………………………………………………………………………...3.31

TABLES…………………………………………………………………………………3.35

APPENDIX I……………………………………………………………………………..3.38

APPENDIX II…………………………………………………………………………….3.60

APPENDIX III…………………………………………………………………………...3.65

APPENDIX IV…………………………………………………………………………...3.69

CHAPTER IV. A new species of big-headed, fanged dicroglossine frog (Genus Limnonectes) from Thailand. …………………………………………...4.26

APPENDIX I……………………………………………………………………………..4.45

APPENDIX II…………………………………………………………………………….4.46

ABSTRACT AND INTRODUCTION

In a period in which all life on Earth faces considerable threats and pressures, it is especially significant that many recent studies have demonstrated that complexes of multiple, unidentified, cryptic species often reside in what was once through to be a single biological form.

This is particularly true of amphibians, which tend to exhibit both conservative morphological evolution and frequent convergent evolution. Molecular phylogenetic tools have provided a useful means by which species complexes can be explored and the diversity hidden within them revealed.

This study considers a group of widely distributed Southeast Asian anurans that, until recently, has been referred to Limnonectes kuhlii Tschudi (1838). Populations of these frogs are remarkably similar morphologically and any observed physical differences have not been treated as informative for intra- or interspecific delimitation to date. Through a detailed population-level sampling of molecular (mtDNA: 12S–16S gene regions) and morphological (external and osteological characters) data, I demonstrate that what has long been considered a single species is, instead, a complex of more than 24 unique evolutionary lineages. These compose four major clades that can be defined biogeographically, and each exhibits high levels of geographic endemism. Additionally, I employ the L. kuhlii Complex as a model system to demonstrate that putative species defined by genetic data can be identified and corroborated through phylogenetic analysis of diverse morphological data. Results from this study reveal multiple instances in which sympatric/syntopic lineages are not each other’s closest relatives. The phylogenetic hypothesis presented here, in combination with an evaluation of morphological characters, creates a framework within which this previously hidden biological diversity now can be identified and described by taxonomists. Ultimately, this work suggests that we have grossly

iii underestimated the biodiversity of the Old World tropics and reinforces an urgent need for effective conservation policies and practices.

This study contains four components, each addressing particular aspects of the

Limnonectes kuhlii Complex. Chapter 1 is a systematic review of L. kuhlii and the L. kuhlii

Complex. Chapter 2 is a molecular phylogenetic analysis of the L. kuhlii Complex. Chapter 3 considers this species complex as a case study, evaluating both molecular and morphological evidence in a phylogenetic context for the purpose of species delimitation. Chapter 4, the description of L. megastomias, stands as an example of the diversity that has been subsumed within L. kuhlii sensu lato. Chapters 2 and 4 were published during the course of my dissertation work, the description of L. megastomias being the first work in this series.

iv ACKNOWLEDGEMENTS

I am deeply indebted to the many people who have made this work possible during my time at KU. I would, however, be remiss if I did not mention—first, and foremost—the four women who have been most instrumental in this: First, my family (Heather, Jadelin, and

Adeline McLeod) has supported me in every conceivable way during my Ph.D. program. From financial support to raising tadpoles in the “jungle lab” in Thailand, my wife and daughters have been my team. It was on a beach in Thailand that Heather and I decided to return to the US from our work in Taiwan so that I could pursue my Ph.D. It was also in Thailand that I lived for a year, with Heather and Jadelin joining me for a semester to work with me in the field—a remarkable year that I wouldn’t trade for anything. Adeline hasn’t been to the tropics yet, but has been out “herping” and will, no doubt grow to become just as much a herpetologist as her sister. Thank you for family. Thank you for laughter. Thank you for your support of this crazy adventure. I can’t wait to see what comes next.

The other significant woman is Dr. Linda Trueb. Linda was gracious enough to take me on as a graduate student—even though I came to her wanting to study amphibian ecology in

Southeast Asia (neither of which are her particular areas of expertise). She has patiently listened to me as I’ve tried to buck the system and make definitive statements about the future (whether my studies, career path, or otherwise) only later to come back and say “OK, Linda, you were right.” Through Linda, I’ve discovered the remarkable world of amphibian morphology and the pleasures of ‘discovering’ structural elements that, at first sight, are marveled at, and then queried for their purposes. Thank you, Linda.

v My parents, grandparents, sister, and uncle—who, during my formative years endured escaped snakes, pickled frogs, frantic phone calls, and turtles in the freezer—never ceased to encourage me in my pursuit of this crazy path. Thank you for your patience, your faith, and your support. My entire family has been exceptionally supportive during this time, whether in watching the kids, lending a hand, or simply providing a place to get away. Thanks for going above and beyond!

My cohort of fellow students has been instrumental in my Ph.D. experience. Drs. Juan

Guayasamin and Elisa Bonacorso, and future doctors Charles Linkem, Jamie Oaks, and Cameron

Siler have each played significant roles as peer-teachers. From them I have learned much about the science of systematics and the functional aspects of the tools required for this study. Charles and Jamie, in particular helped me through the learning curve as I have tackled phylogenetic analyses for the first time. I have learned much from you and with out your help I would not have gotten to this point. Thank you for your patience. A special word of thanks is due to Elisa, for a timely conversation that helped motivate me to get over the change in dissertation topics and dive in to the projects at hand. Perhaps, Elisa, I should say that this work on the

Limnonectes kuhlii Complex is your fault!

Dr. Robert F. Inger and Tan Fui Lian (Field Museum of Natural History) served as unofficial advisors to me. During my visits to Chicago, they shared their knowledge of these curious frogs with me, generously shared specimens and data, and provided me with many fine hours of conversation. I came to KU prior to Dr. Rafe Brown’s arrival. At that time people questioned why I did, as no one was working in Southeast Asia at KU. Rafe has added a tremendous new dimension to herpetology at KU. Thank you, Rafe, for your friendship, input and insight. Dr. David Blackburn arrived at near the end of my Ph.D. program but immediately

vi became a friend, a sounding board, and a source of inspiration. Thank you for your ideas, your willingness to read the roughest of manuscripts, and for helping me see beyond my myopic view of my research. Dr. Kumthorn Thirakhupt, Dr. Anchalee Aophol, and Dr. Wichase Khonsue opened their labs at Chulalongkorn University to me, befriended me, and helped me navigate a new culture and the governmental systems of permits in Thailand. Taksin Artchawakom and the staff at SERS were gracious hosts and surrogate family during my fieldwork in Thailand.

Several undergraduate students have assisted me during my Ph.D. work: Kyle Hesed was an outstanding field companion during our year in Thailand. Bethany Blackmon sorted and staged countless tadpoles. Colin Husted volunteered to do molecular work. Ben Crary took on the onerous task of sorting gut contents (for fun). Stephani Horner has proved to be an invaluable asset during my final year, measuring hundreds of frogs and entering data into spreadsheets (not to mention taking on her own independent projects with the “kuhlii” frogs). I don’t pay you enough, Steph. I’m doubling your salary effective immediately.

Finally, this work would not have been possible without financial support from The

University of Kansas Biodiversity Institute and the Department of Ecology and Evolutionary

Biology. Through Panorama small grants and other funding, the BI and EEB generously supported me at all stages of this research. The David L. Boren Fellowship through the NSEP program funded my year in Thailand, as did a grant from the USGS. This work was facilitated by permits granted from the National Research Council of Thailand.

O JEHOVA

Quam ampla sunt Tua Opera! Quam sapienter Ea fecisti! Quam plena est Terra possessione Tua!

C. Linnaeus Systema Naturae, 10th ed., 1758 vii CHAPTER I

THE LIMNONECTES KUHLII COMPLEX: DIVERSITY HIDDEN IN PLAIN SIGHT

“Until you know what grows and lives in a particular place, and recognize its position in the

biosphere, you can neither exploit nor conserve those biological resources properly.”

D. Galloway

Quoted in Pain (1988:49)

Historically, taxonomists have relied primarily on morphological characteristics to diagnose species. Such work is facilitated by the presence of observable, discrete features that distinguish two closely related taxa. Taxonomic progress is hindered when the organisms examined are morphologically indistinguishable from one another. Such confounding phenotypic similarity can be the result of phylogenetic relatedness, where genetic divergence is accompanied by low rates of morphological evolution or where changes have occurred along ecological or behavioral axes. Alternatively, morphological similarity may be attributed to convergent evolution. Such imperceptibly different organisms are often regarded as “cryptic species.” In a phylogenetic context, cryptic species are those distinct taxa (two or more) that are hidden under a single name (Bickford et al., 2007; Pfenninger and Schwenk, 2007). The concept of cryptic species is not a new one, with terms such as “dual species” and “biological species” being applied to morphologically similar, almost identical, species as early as 1886 and 1892, respectively (Borkin et al., 2004). Mayr (1963) considered sibling species to be those that are reproductively isolated, but in which there are only slight or imperceptible morphological

1.1 differences. In the literature, the terms “cryptic species” and “sibling species” often have been treated as synonyms. The distinction between these two concepts can be made on the basis of relationship, in which sibling species are closely related taxa, and the term cryptic species can applied to any indistinguishable taxa, irrespective of relatedness (Lukhtanov and Shapoval,

2008).

During the last two decades there has been a dramatic increase (>60%) in the number of amphibian species being described (Amphibiaweb, 2010; Hanken, 1999; Köhler et al., 2005;

Vieites et al., 2009). Many of these descriptions have resulted from the re-examination of currently recognized species, particularly those that occur over wide geographic ranges, wherein multiple cryptic species have been uncovered from within nominative taxa (e.g., Bain et al.,

2003; Fouquet et al., 2007; Stuart et al., 2006). Recognition of these cryptic species is critical if we are to estimate accurately true biodiversity and make informed conservation decisions.

Moreover, should we fail to recognize this hidden diversity we risk overlooking mechanisms of evolution (e.g., diversification without morphological change) and the potential contributions of these organisms to the economy of the biosphere (Bickford et al., 2007; Vieites et al., 2009).

Throughout East and Southeast Asia there exists a group of morphologically similar anurans that, for the past 170+ years, has been referred to Limnonectes kuhlii Tschudi (1838).

When encountered in the field, these frogs are often quite abundant along forest-stream environments (Inger, 1966; Iskandar, 1998; Pope, 1931, pers. obs.) and, as such, are considered a species of Least Concern by the IUCN (2008). In part because of the ease of collecting them, frogs assigned to this species have been included in many phylogenetic studies (Che et al., 2007;

Chen et al., 2005; Delorme et al., 2004; Emerson and Berrigan, 1993; Emerson et al., 2000;

1.2

Evans et al., 2003; Frost et al., 2006; Marmayou et al., 2000; Matsui et al., 2010; McLeod, 2008, in press; Vences et al., 2000; Zhang et al., 2005). A review of the literature reveals that numerous authors have noted some degree of morphological variation between among populations of these anurans (e.g., condition of nuptial pads in males; relative finger lengths; tuberculation of the hind limbs; internarial distances; widths of temporal and interorbital stripes), but not enough to warrant taxonomic changes (Berry, 1975; Boulenger, 1920; Inger, 1966;

Iskandar, 1998; Malkmus et al., 2002; Pope, 1931; Taylor, 1962).

Based on molecular data, several authors suggested that Limnonectes kuhlii represented a cryptic species complex (Emerson et al., 2000; Evans et al., 2003; McLeod, 2008). Recently,

McLeod (in prep., in press) demonstrated that the concept of Limnonectes kuhlii sensu lato as a single evolutionary lineage must be rejected on the basis of overwhelming evidence from molecular and morphological data. A conservative estimate is that the L. kuhlii Complex contains at least 24 unique evolutionary lineages (McLeod, in prep.). Several of these have been recognized previously as distinct species, including L. asperatus (Inger, Boeadi, and Taufik,

1996), L. fragilis (Liu and Hu in Liu, Hu, Fei, and Huang, 1973), L. fujianensis Ye and Fei,

1994, L. bannaensis Ye, Fei, and Jiang, 2007, L. megastomias McLeod, 2008, and L. namiyei

(Stejneger, 1901). There remain at least 18 unnamed lineages within the L. kuhlii Complex.

Following the definitions of Vieites et al. (2009), 13 of these are considered Unconfirmed

Candidate Species (UCS) based on molecular data alone, and 5 as Confirmed Candidate Species

(CCS) with corroborating support from morphological data (McLeod, in prep.).

In light of the undescribed diversity within this cryptic species complex recognized by

McLeod (in prep., in press), the goal of this study is to construct a framework within which a

1.3 series of taxonomic descriptions of the unnamed lineages of the Limonectes kuhlii Complex can be erected, and in so doing, provide a systematic review of L. kuhlii. The junior synonyms are reviewed, and brief discussion is given to the specific, generic and subgeneric taxonomic history of L. kuhlii to establish a stable taxonomy.

Systematic Review of Limnonectes kuhlii (Tschudi, 1838)

Synonyms:

Rana kuhlii Tschudi, 1838

Rana palmata Tschudi, 1838

Rana conspicillata Günther, 1872

Nyctibatrachus sinensis Peters, 1882

Rana paradoxa Mocquard, 1890

Non-synonymous references

Rana corrugata—Taylor, 1934: Specimens from “Chieng Mai, Siam” [Chiang Mai

Province, Thailand] were referred to R. corrugata incorrectly by Taylor. Rana

corrugata is a junior synonym of Lankanectes corrugatus. The members of the L.

kuhlii Complex from Chiang Mai should be referred to “Lineage 12” (McLeod, in

prep., in press) which was considered by McLeod (in prep.) to be a CCS. Despite

their distant evolutionary relationship (Delorme et al., 2004), there is a remarkable

amount of phenotypic similarity between these species (pers. obs.).

Rana (Rana) kuhli—Boulenger, 1920; Van Kampen, 1923

Dicroglossus kuhlii—Deckert, 1938

1.4

Rana (Limnonectes) kuhlii—Dubois, 1981 by implication (Frost, 2009).

Dicroglossus kuhli—Manthey and Denzer, 1982

Limnonectes (Limnonectes) kuhlii—Dubois, 1987 "1986."

Limnonectes kuhlii—Fei, Ye, and Huang, 1990

Discussion of synonyms and their status

“I have concluded that in a previous existence you must have been a dreadful sinner. How else

to explain the taxonomic mess you have got yourself into?”

R. F. Inger

Pers. comm.

Limnonectes kuhlii is the generotype of the dicroglossine genus Limnonectes Fitzinger,

1843 that comprises 55 currently recognized species (AmphibiaWeb, 2010). Whereas Duméril and Bibron (1841) often are credited with authorship, Tschudi (1838) is recognized as having priority based on an earlier valid, albeit uninformatively brief, description of this frog: “Rana

Kuhlii unterscheidet sich von allen übrigen durch den kürzern und gedrängtern Kopf und die starker entwickelten Schwimmhäute an den Füssen; sie ist auch auf Java” [Rana Kuhlii differs from all others by the shorter and more compact head and the strongly developed webbing on the feet; it is also in Java.]

It should be noted that a review of the literature reveals references to Hermann Schlegel as the authority for Rana kuhlii (Bourret, 1942; Duméril and Bibron, 1841; Tschudi, 1838; Van

Kampen, 1923; and etc.). To my knowledge, only Bourret (1942) provided a date (1833) and

1.5 page number (50) as a citation for this work. Other authors, including Tschudi (1838), simply referred to Schlegel as the authority, but provided no citation. Schlegel was working at the

Rijksmuseum van Natuurlijke Histoire (RMNH) [now the National Museum of Natural History,

Netherlands] in Leiden and may have applied informal names (as shelf labels) to specimens in the Museum (K. Adler, A. Bauer, D. Frost, pers. comm.). Therefore, it is possible that both

Tschudi and Duméril and Bibron used these “shelf names” in their descriptive works, crediting

Schlegel as the authority. Bourret’s (1943) reference to Schlegel (1833: p. 50) contains no mention of Rana kuhlii, nor is there any other reference in the Reptile volume of Fauna Japonica to this frog. Consequently there is no evidence that would support the authority or priority of

Schlegel over Tschudi.

Some confusion surrounds the number and identities of specimens that were examined in the earliest descriptions of Limnonectes kuhlii (as Rana kuhlii) because a holotype was not designated, no illustrations were provided, and collection localities were specified only as “Java”

(Duméril and Bibron, 1841; Tschudi, 1838). In an apparent contradiction, Duméril and Bibron

(1841) reported examining different numbers of specimens for their description of L. kuhlii.

“Nous ne pouvons cependant pas assure que cela soit un des caractères de l'espèce, car elle ne nous est connue que par le seul individu que nous avons maintenant sous les yeux et dont l'état de conservation est loin d'être parfait.” [We cannot, however, be sure that this is a trait of the species, because we have only seen it in the one specimen that we are currently describing, which has not been perfectly preserved.] “Sur les six individus don’t nous venons de faire la description, cinq proviennent d’un envoi addressé de Java au muséum par M. Diard, le sixième a

été recueilli aux Célèbes par MM. Quoy et Gaymard.” [Of the six specimens that we used for

1.6 our description, five have been sent from the museum in Java by Mr. Diard, the sixth was collected in the Celebes by Mr. Quoy and Mr. Gaymard.]. It is known that Tschudi worked at both Leiden and Paris museums (Tschudi, 1838: p. 5) and that both currently hold syntypes of L. kuhlii. Tschudi (1838) provided only a reference to “Mus. Lund.” [= RMNH], but these are attributed specifically to Rana palmata (p. 80). Furthermore, based on examination of the

MNHNP catalog record, it seems that the single specimen in Paris may have been an exchanged specimen from Leiden. Thus, it seems impossible to determine at this point the identity of the specific type material used by Tschudi for his description with absolute certainty. Guibé (1950) designated a single specimen from the Muséum Nationale d’Histoire Naturelle in Paris (MNHNP

4469) as the holotype [lectotype by implication (Frost, 2009)]. Two syntypes are in the National

Museum of Natural History, Leiden (Gassó Miracle et al., 2007). Frost (2009) questioned the status of the MNHNP specimen citing commentary by Hoomoed (Frost, 1985) who indicated that, in recognition of the shift in priority to Tschudi, a change in the status of type material was required. Considering all evidence available, it would seem that the two type specimens in

Leiden most likely represent the type material of Tschudi, but it is possible (depending on the date of acquisition) that the Paris specimen may have been of the same series and that Guibé’s designation as lectotype is valid.

To complicate matters further, evidence from recent molecular analyses of haplotypes from Java suggest that there may be two distinct evolutionary lineages on the island (McLeod, in prep.). Thus, with only “Java” as a type locality, it may be impossible to identify which, of three possible type specimens, represents true Limnonectes kuhlii. Examination of the type materials from both the RMNH and MNHNP reveals that there are morphological differences between

1.7 these specimens. For example, the ratio of the -narial distance to snout–vent length (SVL) is greater in the Leiden specimens (9%) than in the Paris specimen (7%), and the ratio of odontoid length to SVL in males is smaller in the Leiden specimen (5%) than in the Paris specimen (8%).

The poor state of preservation, particularly of the Paris specimen, obscures useful morphological features (e.g., tubercles on the hind limbs and body) and precludes definitive redescriptions of these individuals. Whereas these data alone are insufficient for justifying taxonomic decisions, they do indicate that caution should be exercised when making changes to the type status of

Javan L. kuhlii. With the identity of the type material of L. kuhlii in question and the substantial number of undescribed species within the L. kuhlii Complex, it may take another 170 years to emerge from this taxonomic quagmire.

Rana palmata Tschudi, 1838 is a junior synonym of Rana kuhlii Tschudi, 1838 (Frost,

2009). Tschudi attributes the name to Kuhl but provides no diagnosis for R. palmata other than that of R. kuhlii. Because this name was published first as a junior synonym it is unavailable because it does not conform to articles 11d and 16b of the ICZN. It is worth noting that Bourret

(1943:p. 42) provided a semi-complete citation “1824. Letters sur les Reptiles de Java. Bull.

Univ. Sci., 2 pp. 83, 341.”‡ However, upon examination of these letters, it is clear that Kuhl made no mention of this anuran, and certainly did not provide a description or illustration of it.

‡ full citations: (1) Kuhl, H. 1824. Sur les reptiles de Java. Extrait d’une letter addressée de Java en Hollande, par M. Kuhl, datée de Pjihorjavour au pied du Pangerango, le 18 juillet 1821. — Bulletin des sciences naturelles et de géoloie. Deuxième section du Bulletin universel des sciences et de l’industrie 2:79–83.

(2) Kuhl, H. 1824. Seconde letter de M. Kuhl sur les reptiles de l’île de Java. Buitenzorg, le 8 août 1821. — Bulletin des sciences naturelles et de géoloie. Deuxième section du Bulletin universel des sciences et de l’industrie 2: 370–371.

1.8

These citations seem to be referencing either the collections of Kuhl, or were misapplied by

Tschudi and subsequent authors. Nevertheless, this name is unavailable because it is a nomen oblitum, and, more importantly, it is preoccupied by Rana palmata Bonnaterre, 1789 (Frost,

2009).

Rana conspicillata was described by Günther (1872) from specimens collected around

Matang, Sarawak, Malaysian Borneo. Günther (1874) subsequently placed this taxon into synonymy with L. kuhlii, but noted that “it is very singular that in some specimens the web is much more emarginated than in the typical form.” Based on recent molecular and osteological data from topotypes (McLeod, in prep.), it seems that this member of the Limnonectes kuhlii

Complex merits further study and redescription. This junior synonym is an available name, which could be resurrected and applied to this anuran if it is recognized as a valid species.

Nyctibatrachus sinensis was described from a single female specimen from “Mons Lofau

(Provincia Canton)” [Lofau Mountains, Guangdong Province, China] by Peters (1882). This species was placed in synonymy with Limnonectes kuhlii by Boulenger (1887) and with

Quasipaa spinosa (David 1875) by Liu and Hu (1961). Dubois (1987) considered N. sinensis incertae sedis within the subgenus Limnonectes. The type specimen (ZMB 10373) can no longer be located at the Museum fur Naturkunde, Berlin (ZMB) (M. O. Rödel, pers. comm.). Three species of Limnonectes currently are known to occur in China—L. bannaensis, L. fragilis

(Hainan endemic), and L. fujianensis (Zhang et al., 2009). Given the morphological similarities of female L. kuhlii, L. bannaensis, L. fujianensis, and Q. spinosa (pers. obs.), and in the absence of the type specimen, the placement of this taxon remains incertae sedis. Furthermore, in the absence of the type material, it would be impossible to place the type specimen of N. sinensis

1.9 into one of the L. kuhlii Complex groups on the basis of the description by Peters and the type locality (even if it were possible to rule out conspecificity with Q. spinosa), because it is conceivable that both L. bannaensis and L. fujianensis may occur in Guangdong Province.

Rana paradoxa was described by Mocquard (1890) from a collection of six specimens made at “Kina Balu” [Mount Kinabalu, Sabah, Malaysian Borneo]. Personal communication with A. Ohler corroborates the comments of Guibé (1950) regarding the deposition of five syntypes at the MNHNP (MNHN 1889.0223–.0225, .0245–.0246). This species was placed in synonymy with Limnonectes kuhlii by Boulenger (1891). It has been speculated that frogs of the

L. kuhlii Complex on Mt. Kinabalu may represent a unique evolutionary lineage (Malkmus et al.,

2002). Based on molecular data, McLeod (in press) recognized the haplotype samples from Mt.

Kinabalu as an UCS. Additional evidence from personal examination of the syntypes and whole voucher specimens that were collected recently suggests that two separate species occur on Mt.

Kinabalu (unpubl. data) and that one of these will be referred to this junior synonym. Rana paradoxa is a primary homonym of Rana paradoxa Linnaeus, 1758 [Pseudis paradoxa] (Frost,

2009).

Generic and Subgeneric Placements of Limnonectes kuhlii

Limnonectes kuhlii is variously placed in the subgenera Rana (Boulenger, 1920; Bourret,

1942), and Limnonectes (Dubois, 1981), and in the genera Rana (Tschudi, 1838) by original description, Dicroglossus (Deckert, 1938), and Limnonectes (Dubois, 1987). Despite disputes over the classifications of Dubois (1992, 2005) and Frost et al. (2006) (Emerson and Berrigan,

1993; Inger, 1996; 1999; Pauly et al., 2009; Scott, 2005; Wiens, 2007), the generic and sub-

1.10 generic placement of L. kuhlii has remained stable. However, Inger (1996) criticized Dubois’

(1987, 1992) diagnoses of both the genus and subgenus Limnonectes based on the use of pleisiomorphic characters for the genus and an absence of characters for the subgenus.

Dubois (1981) identified a Limnonectes kuhlii species group as equivalent to Boulenger’s

(1920) Rana kuhlianae and comprising “Rana corrugata,” “Rana kuhlii,” and “Rana laticeps.”

Boulenger (1920:7) diagnosed this group with “Tympanum hidden or but feebly distinct; tips of toes dilated into more or less distinct discs; male without vocal sacs, without enlargement of the fore limb, with large tooth-like prominences in the front of the lower jaw; nasal bones in contact with each other; omosternal style forked at the base.” Given our current understanding of the phylogenetic relationships of these frogs (Delorme et al., 2004; Emerson et al., 2000; Evans et al., 2003; McLeod, in press), it is clear that Boulenger’s (1920) and thus Dubois’ (1987, 1992) groups are based more on bauplan than they are ancestor-descendant relationships.

Taxonomic Conclusions

I adopt the recommendation of Dubois (1981, 1987) in recognizing Limnonectes as a valid genus, the monophyly of which has been supported by morphological and molecular evidence (Emerson and Berrigan, 1993; Emerson et al., 2000; Evans et al., 2003; McLeod, in press; Scott, 2005). I recognize the Limnonectes kuhlii Complex as monophyletic based on molecular data (McLeod, in prep., in press), expand it to include Limnonectes kuhlii, L. asperatus, L. bannaensis, L. fragilis, L. fujianensis, and L. namiyei. In addition, I consider the

Limnonectes kuhlii Complex to include the unnamed putative species (UCS and CCS) of

McLeod (in prep..).

1.11

Diagnosis of the Limnonectes kuhlii Complex

A highly aquatic frog, usually found in small lotic systems (streams and rivulettes) in hills and mountains, occasionally in lentic habitats (ponds); strong sexual dimorphism, head larger in males than in females, lacking vocal sac (present in L. namiyei), males with nuptial pads (absent in L. kuhlii from Java), odontoids on lower jaw larger in males than females; head longer than wide; tympanum covered with skin rendering this structure invisible or partially visible; dark bar across interorbital area, light-bordered behind; webbing of toes extends to disc; In an era of modern systematics, it is generally recognized that phylogenetic history, and not necessarily a list of diagnostic features, is what defines monophyletic groups (Hillis, 2007). Moreover, recognizing that the characters we use to diagnose a taxon will most likely remain valid only until we reexamine relationships and propose new hypotheses, it may seem futile to even attempt to identify discrete diagnostic characters. However, identifying these diagnostic characters is a worthwhile and practical endeavor towards our goal of understanding the fullness of Earth’s biodiversity. Taxonomic characters, whether physical (macroscopic, microscopic, or molecular) or behavioral (Quicke, 1993), allow us to identify known diversity, and discover unrecognized diversity. When the characters we employ do not match an organism in hand we are forced to further consider the issue of identity. In this case it led to population-level sampling, a careful examination of molecular and morphological features, and a re-definition of Limnonectes kuhlii as a cryptic species complex. Furthermore, it has resulted in the identification of distinct lineages of these anurans occurring in syntopy, the discovery of interesting patterns of endemism, and the recognition of the L. kuhlii complex as another model system for understanding biogeographic patterns in Southeast Asia. Nevertheless, it is conceivable that in the near future, closer examination of materials from Java will force us, yet again, to reevaluate the identity of the Javan “kuhlii” and determine which lineage represents true L. kuhlii.

1.12

Acknowledgements

I am deeply indebted to A. Bauer, K. Adler, D. Frost, R. F. Inger, and D. Fauntin for sharing their knowledge of taxonomy, the ICZN, and historical literature. D. Blackburn, W. E.

Duellman, and L. Trueb provided constructive comments on early drafts of this manuscript. I am especially thankful for the materials and commentary provided to me by A. Bauer and D. Frost that allowed me to complete this manuscript in a timely manner.

1.13

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1.20

CHAPTER II

OF LEAST CONCERN? SYSTEMATICS OF A CRYPTIC SPECIES COMPLEX: LIMNONECTES KUHLII

(AMPHIBIA: ANURA: DICROGLOSSIDAE)

Abstract

Several recent studies have demonstrated that a tremendous amount of biological diversity can be masked by phenotypic similarity in a cryptic species complex. It has been speculated that the widely distributed and relatively common Southeast Asian frog Limnonectes kuhlii represents a complex of multiple species. The phylogeny within the Limnonectes kuhlii

Complex is estimated in this study based on approximately 2400 base pairs of mtDNA data

(tRNAPhe, 12S, tRNAVal, and 16S genes) from 244 individuals representing multiple populations from throughout the known distribution of this anuran. Analyses are conducted using parsimony, maximum likelihood and Bayesian methods. The results suggest that what has been recognized historically as a single species is a complex of more than 22 distinct evolutionary lineages, 16 of which are currently subsumed under the nominal Limnonectes kuhlii. Several cases of sympatric lineages were detected, and in all cases co-occuring lineages were not each other’s closest relatives.

Key Words: cryptic species; diversity; Limnonectes kuhlii; South East Asia; species complex

1. Introduction

In the past two decades, increased attention has been given to the need for conservation measures that protect populations of amphibians across the globe. Amphibian populations face numerous threats to their survival, including, but not limited to, habitat loss, environmental

2.1

degradation, competition with invasive species, pathogens and disease, and a variety of anthropogenic factors (Collins and Storfer, 2003; Mendelson et al., 2006). Because amphibians are relatively sensitive indicators of environmental health and climate change, the research focus on their conservation priorities is not misplaced. The IUCN Red List report (2008) indicates that

32% of the 6433 recognized species of amphibians are threatened or extinct. It is anticipated that this number will only continue to increase with time. In contrast, 43% are “known not to be threatened” and, therefore, are considered species of Least Concern (IUCN et al., 2008).

But suppose that some of the taxa categorized as “known not to be threatened” on the basis of widespread distributions, local abundance, and tolerance to disturbance do not represent a single species but, instead, are collectives of several cryptic species. Cryptic species are genetically distinct, but morphologically similar, taxa that are currently, or were historically, classified as a single species. Cryptic species are neither taxonomically nor biogeographically unique, but instead are common among many taxa and found in all parts of the globe (Pfenninger and Schwenk, 2007). The application of molecular phylogenetic techniques has permitted fine- scale examination of species-level differences. In the last decade, numerous studies have uncovered cryptic diversity in nominal species of insects, mammals, fungi, and other organisms with the use of molecular data (Bickford et al., 2007; Mayer et al., 2007; Murray et al., 2008).

Several recent studies have revealed cryptic diversity among species of Southeast Asian anurans

(Evans et al., 2003; Inger et al., 2009; Stuart et al., 2006). Stuart et al. (2006) even suggest that there may not be any single widespread species of forest-dwelling anuran in SE Asia, and that cryptic lineages occurring in sympatry may be the rule rather than the exception. Furthermore,

Stuart et al. (2006) and Inger et al. (2009) found that when cryptic lineages occur in sympatry, they frequently are not sister lineages of one another.

2.2

The type species of the dicroglossine genus Limnonectes is the widely distributed frog,

Limnonectes kuhlii Tschudi (1838) (Figure 1). Limnonectes Fitzinger 1843 comprises 55 currently recognized species (Amphibiaweb, 2009) found throughout east and southeast Asia; these anurans are characterized by fanglike odontoid processes on the lower jaw, male-biased size dimorphism, and a great amount of phenotypic similarity (Emerson et al., 2000). Numerous authors have included Limnonectes kuhlii in phylogenetic analyses at the level of class (Frost et al., 2006), family (Che et al., 2007; Chen et al., 2005; Delorme et al., 2004; Emerson and

Berrigan, 1993; Marmayou et al., 2000; Vences et al., 2000) and genus (Emerson et al., 2000;

Evans et al., 2003; Zhang et al., 2005). Several of these studies have incorporated representatives of L. kuhlii from different populations and demonstrated that multiple distinct evolutionary lineages are contained within the nominal species (Emerson et al., 2000; Evans et al., 2003;

Zhang et al., 2005). McLeod (2008) proposed a preliminary phylogeny of the L. kuhlii Complex based on molecular data (mtDNA) and recognized a new, large-bodied species (L. megastomias) from within nominal L. kuhlii based on both molecular and morphological evidence.

In most cases, Limnonectes kuhlii is distinguished from its congeners (Che et al., 2007;

Emerson et al., 2000; Evans et al., 2003; Frost et al., 2006; Jiang and Zhou, 2005; Zhang et al.,

2005; Zhang et al., 2009) by the indistinct (or hidden) tympanum and fully webbed toes (Inger,

1966; Taylor, 1962; Tschudi, 1838). Examination of whole, preserved voucher specimens from across the distribution of L. kuhlii reveals more phenotypic variation among populations than formerly acknowledged. There are morphological characters that vary across populations, and variation within populations from a single locality. Other authors have also noted morphological differences in populations of L. kuhlii. Boulenger (1920) indicated that although he observed great intraspecific variation in Limnonectes kuhlii, he was unable to find characters that defined

2.3

“geographic races.” Taylor (1962) alluded to the fact that northern and southern populations in

Thailand present different degrees of rugosity on the thigh and leg, whereas the southern Thai and Malaysian specimens are more rugose than others. These differences are obvious when comparing illustrations from Taylor (1962) showing L. kuhlii from northern Thailand, and specimens from Peninsular Malaysia (Berry, 1975). Inger (1966) also reported differences in the size and density of tubercles on the hind limbs of specimens from Sabah and Sarawak, but he did not consider these geographic differences sufficient to warrant a taxonomic change.

In light of the frequency with which Limnonectes kuhlii is incorporated into larger phylogenetic analyses, it not trivial that this generotypic species is actually a species complex.

Are the samples of L. kuhlii being used in analyses actually “real” L. kuhlii? What is the identity of true L. kuhlii? What are the implications of representing L. kuhlii, or Limnonectes, with a species that is not L. kuhlii? To address these and related issues, a comprehensive assessment of the diversity within this species complex, couched within a phylogenetic context is required. To date, no single work has attempted such a study of the L. kuhlii Complex.

Molecular tools were used to investigate a nominal taxon that currently is recognized as a single, widespread species and categorized as an IUCN species of Least Concern. The objectives of this study are to: (1) delineate the extent of genetic variation occurring across the species geographic distribution; (2) create a phylogenetic framework based on molecular data that will permit a thorough examination of morphological variation in these frogs (McLeod, in prep); (3) test the hypothesis that sympatric species are not sister lineages; and (4) evaluate the conservation implications of a species complex characterized by high degrees of endemism being recognized as single widespread species.

2.4

2. Materials and Methods

2.1 Taxon Sampling

Based on work by Evans et al. (2003) and others (Che et al., 2007; Emerson et al., 2000;

Frost et al., 2006; Zhang et al., 2005), species in the genera Occidozyga, Fejervarya,

Hoplobatrachus, and Paa were used as outgroups, and species in the genus Limnonectes formed the ingroup (Appendix 1). New mitochondrial sequences were obtained from 244 individuals of the L. kuhlii Complex (Appendix 1). Multiple individuals (10 when possible) were sequenced from each population. Additionally, partial and complete sequences were obtained from

GenBank for 12 individuals of ingroup and outgroup taxa, and 55 sequences of 27 Limnonectes species and 6 outgroup taxa from Evans et al. (2003) were obtained from the author (Appendix

1). Museum codes correspond to those of Leviton et al. (1985) and Leviton and Gibbs (1988), with the addition of FRIM for Forest Research Institute, Malaysia, THNHM for Thailand

Natural History Museum, and ZRC for Zoological Reference Collection, Raffles Museum of

Biodiversity Research, National University of Singapore.

2.2 Molecular Data

The gene order of the approximately 2400 bp of mitochondrial DNA region sequenced

(5'–3') is tRNAPhe, 12S ribosomal DNA (rDNA), tRNAVal, and16S rDNA. For samples in this study, the average sequence length is 1907 bp (max = 2446 bp; min = 671 bp). These mitochondrial DNA data were chosen for use in this study to take advantage of abundant comparative material already available from previous studies (Che et al., 2007; Emerson et al.,

2000; Evans et al., 2003; Frost et al., 2006; Jiang and Zhou, 2005; Zhang et al., 2005; Zhang et al., 2009). Muscle or liver tissue samples stored in 95% EtOH or salt buffers were subsampled,

2.5

and DNA was extracted using the guanidine thiocyanate method of Esselstyn et al. (2008).

Primers used to amplify the target DNA fragments using the polymerase chain reaction (PCR) were taken from Evans et al. (2003). The following thermal cycler profile was used for 12S–16S amplification: 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for

70 s, and a final extension phase at 72°C for 7 min. Amplified products were visualized on 1.0% agarose gels. Clean single bands of the target product were purified with 1 µL of a 20% diluted solution of ExoSAP-IT (US78201, Amersham Biosciences, Piscataway, NJ) on the following thermal cycler profile: 31 min at 37°C, followed by 15 min at 80°C. Cycle sequencing reactions were run using ABI Prism BigDye Terminator chemistry (Ver. 3.1; Applied Biosystems, Foster

City, CA) and purified with Sephadex Medium (NC9406038, Amersham Biosciences,

Piscataway, NJ) in Centri-Sep 96 spin plates (CS-961, Princeton Separations, Princeton, NJ). All samples were sequenced in both forward and reverse directions. Purified products were analyzed with an ABI Prism 3130xl Genetic Analyzer (Applied Biosystems). Consensus gene sequences were vetted and edited using Sequencher 4.8 (Gene Codes Corp., Ann Arbor, MI). All new sequences from this study have been deposited in Genbank (see Appendix 1 for specimen data and accession numbers).

2.3 Phylogenetic Analysis

An initial alignment was produced in Muscle v3.7 (Edgar, 2004), and manual adjustments were made in Se-Al V 2.0a11 (Rambut, 2007). Phylogenetic inference was performed using maximum likelihood, maximum parsimony, and Bayesian methods. The total dataset consisted of 311 taxa and 2614 characters. Evaluation of uncorrected pair-wise distances was conducted with MEGA v4.0 (Tamura et al., 2007).

2.6

Evans et al. (2003) excluded a 34 base-pair region from the Asian and Sunda Shelf

Limnonectes because homology in this region was difficult to assess, but included it for the

Philippines and Sulawesi species. I conducted analyses with and without this same region for all taxa in the reduced dataset. Ultimately, exclusion of these characters had no effect on the topology of the tree, but exclusion did yield higher support for some branches. The results discussed below reflect analyses of a dataset of 2578 characters in which base pairs from the alignment region 2262–2297 were excluded.

The best fitting models of sequence evolution were determined using the Akaike

Information Criterion (AIC) in Modeltest v3.7 (Posada and Crandall, 1998) under both partitioned and non-partitioned schemes. Maximum likelihood and Bayesian analyses were conducted with and without the data partitioned by gene. Likelihood values of best trees from partitioned and non-partitioned analyses were compared under AIC, AICc, and BIC model criteria to determine whether partitioning yielded a better estimation of phylogeny.

Maximum likelihood and Bayesian analyses were run under the models selected by

Modeltest (Posada and Crandall, 1998). Maximum likelihood analyses were conducted using

RAxML v7.0.4 (Stamatakis, 2006) with 100 replicate best tree inferences. Clade support was assessed with 1000 bootstrap pseudoreplicates. The dataset was reduced to facilitate computation of parameters during Bayesian and parsimony analyses. Identical haplotypes were eliminated and no more than four individuals per population were included. Thus, the final, reduced dataset for Bayesian and Parsimony analyses contained 94 taxa and 2611 characters. Maximum parsimony heuristic searches were executed in PAUP* v4.0b10 (Swofford, 2002) with tree bisection-reconnection (TBR) branch swapping and 1000 random taxon replicates. All characters were weighted equally, and gaps were treated as missing data. Bayesian analyses were

2.7

conducted using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). Four independent analyses were run with four Metropolis-coupled Markov chains (MCMC) each. All Markov chains were run for 5 million generations, with sampling every 1000 generations. The output files were examined in Tracer v1.4 (Rambaut and Drummond, 2007) to determine the number of generations to exclude as burn-in and to ensure that all parameters converged. Additionally, Are

We There Yet (Wilgenbusch et al., 2004) was used to ensure that the multiple runs converged and that sampling was sufficient. The sump and sumt commands were executed in MrBayes, summing over the multiple convergent runs.

To estimate relative timing of divergence events, Beast version 1.5.3 (Drummond and

Rambaut, 2007) was used to produce an ultrametric tree and erect 95% confidence intervals for node heights. No dates were placed on the tree because reliable calibration points are not available for these anurans. The same dataset and partitioning scheme was used as the Bayesian analysis. A relaxed clock model (Drummond et al., 2006) with uncorrelated rates drawn from a lognormal distribution was selected. Two MCMC runs were performed, each running 20 million generations and logging parameters every 2000 generations. A burnin of 10% was selected after likelihood scores parameters were inspected in TRACER for stability. The ultrametric tree was generated from the combined tree files of the two MCMC runs.

2.4 Species Concept

I employ the General Lineage Concept of species (de Queiroz, 1998, 1999) as being the same as the Evolutionary Species Concept (Simpson, 1961; Wiley, 1978). A molecular estimate of phylogenetic relationships to guide species delimitation, diagnosis, and identification of relevant comparisons for species diagnoses is utilized. I consider as distinct species those

2.8

lineages that are morphologically and genetically distinct, and for which the hypothesis of conspecificity can be confidently rejected by analyses of both morphological and genetic data.

Recently, there has been renewed interest in, and discourse related to, the delineation of species boundaries using both tree-based and non–tree-based methods (e.g., Monaghan et al.,

2009; Sites and Marshall, 2003; Vieites et al., 2009; Wiens and Penkrot, 2002). Though it should not be used as the sole criterion for delimiting species, uncorrected pair-wise divergences of the mtDNA may be useful to identify candidate species (Fouquet et al., 2007; Vences et al.,

2005a; Vences et al., 2005b; Vieites et al., 2009). I diagnosed clades of haplotypes based on a threshold of 7% uncorrected pair-wise sequence divergence. Haplotype clades were assumed to represent allopatric populations. If the sympatry (or syntopy) of haplotypes was known, then decisions for clade designation were based additionally on the identification of morphological variation in voucher specimens (McLeod, unpubl. data). The use of a 7% threshold of sequence divergence was considered conservative compared to other amphibian-oriented systematics studies that used values of 5% (Fouquet et al., 2007) and 3% or less (Ron et al., 2006; Vieites et al., 2009). Because this work is based solely on analyses of molecular data, it is not prudent to make taxonomic changes (i.e., identify new species) based only on the results of this study. To facilitate the evaluation and discussion of phylogenetic relationships, I have maintained previously published taxonomies and have referred to undescribed monophyletic groups of frogs in the Limnonectes kuhlii Complex as numbered “Lineages” in the text and figures. These

“Lineages” correspond to the “Candidate Species” of Vences et al. (2005a; 2005b) and, more specifically, to the “Unconfirmed Candidate Species” of Vieites (2009).

3. Results

2.9

The first 480 bp (tRNAPhe and part of 12S genes) proved difficult to sequence successfully for a number of individuals; consequently, some specimens lack data for this region.

Of the 2578 included characters, 1037 were constant, 273 were variable, and 1268 were parsimony informative. When the dataset was considered without partitions, the best-fit model for the data was the general time reversible (GTR) model, with a proportion of invariable sites (I) and a parameter for variation in rates among sites (Γ). Under a by-gene partitioning scheme, the best-fit model for the 12S and 16S gene regions as GTR + I + Γ, the Hasegawa-Kishino-Yano

(HKY) + Γ model for tRNAVal, and the Kimura 1980 + I model for the tRNAPhe gene region.

Evaluation of likelihood scores under both partitioned and non-partitioned schemes revealed that under AIC, AICc, and BIC model criteria, partitioned data provided a better estimation of the phylogeny. Based on this, results presented below are those from partitioned datasets.

3.1 Sequence Divergence.

All taxa in the dataset were considered for evaluation of mean pair-wise sequence divergence in the 12S–16S mtDNA gene region. When all haplotypes from the Limnonectes kuhlii Complex were considered collectively, the mean intraclade pair-wise sequence divergence was 10.9%. Similarly, all non-kuhlii Complex haplotypes had a collective mean intraclade sequence divergence of 12.7%. The L. kuhlii Complex lineages (viz., haplotype clades), had mean intraclade pair-wise sequence divergence values ranging from 0.1–4.4% (Table 1). Mean pair-wise sequence divergences between haplotype clades in the L. kuhlii Complex ranged from

6.5–18.8%.

3.2 Phylogenetic Analyses

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Parsimony-based inference recovered three equally most parsimonious trees (Length =

7350; Consistency Index = 0.301; Retention Index = 0.656). Partitioned maximum-likelihood analysis yielded the single best scoring tree (–ln = 45170.19) presented in a simplified form in

Figure 2. An expanded version of the same tree, showing all haplotype terminals, is presented in

Appendix 2. The partitioned Bayesian analyses yielded a consensus tree with a negative harmonic mean likelihood of 34618.28, which was summed from four independent runs. All parameters converged in all four runs. Burn-in was estimated at 200,000 generations, leaving a posterior distribution estimated from 19,204 trees per run. All three analyses produced essentially the same tree topology with the few exceptions discussed below and support values for all analyses are presented in Figure 2. Relative age and timing of divergence among the four major geographical clades of L. kuhlii complex frogs (Clades A–D) is shown with node bars representing 95% confidence intervals in Figure 3.

3.3 Relationships

There is weak support for some deep nodes in the preferred tree (Figure 2). There is, however, strong support for the nodes of interest within the Limnonectes kuhlii Complex.

Haplotypes from specimens currently referred to L. kuhlii (Lineages 1, 2, 4–6, 9, 11–18, 20–22) form a paraphyletic assemblage with respect to L. fragilis (Lineage 3), L. fujianensis (Lineage 7),

L. bannaensis (Lineage 8), L. megastomias (Lineage10), and L. asperatus (Lineage 19). The clade composed of non–L. kuhlii Limnonectes species (Clades E1–E4) is the sister taxon to the well-supported clade containing all members of the L. kuhlii Complex. The L. kuhlii Complex comprises four major geographic clades (Clades A–D) consisting of 22 distinct lineages. Clades

A, C and D are recovered with robust support in all three analyses, and Clade B is recovered with

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moderate support. Clade A comprises two Sunda Shelf lineages (Lineages 1–2), contains the type locality for L. kuhlii (Java), and is the sister taxon to the rest of the L. kuhlii lineages in the complex. The Sunda Shelf clade is most closely related to a second SE Asian clade (Clade B) comprising lineages from Cambodia, Lao People’s Democratic Republic (PDR), and the Hainan endemic L. fragilis (Lineages 3–5). All Bornean taxa (Lineages 14–22) form Clade D which is the sister taxon to the SE Asia assemblage containing Lineages 6–13 (Clade C).

Relationships among species of Limnonectes identified by Evans et al. (2003) also are supported in this analysis, with minor differences. Most notably, in Evans et al. (2003), all L. cf. kuhlii samples were nested within the Limnonectes clade (but with weak support). In this study, the L. kuhlii Complex (inclusive of those species that have been described from within L. kuhlii) is the sister taxon to the clade containing all other Limnonectes species. Additionally, in Evans et al. (2003), L. leporinus is shown with weak support in the Bayesian and maximum likelihood analyses to be most closely related to the Philippines clade containing L. finchi, L. palavensis, and L. parvus (Clade E2 in this study). Here, L. leporinus is shown (also with weak support) to be most closely related to the Philippines and Sulawesi clade (Clade E3 in this study).

Evaluating multiple samples of mitochondrial DNA from populations of Limnonectes kuhlii revealed several instances of deeply divergent lineages occurring in sympatry. In each case, these lineages are not each other’s closest relatives. At one locality in Vietnam (Ha Giang

Province), two lineages occur syntopically. One population can be identified as L. bannaensis

(Lineage 8), and the other, Lineage 13. Similarly, in Phongsaly Province, Lao PDR, two lineages occur in syntopy—one being L. bannaensis and the other Lineage 12. In Malaysian Borneo, there seem to be three instances of sympatry. In Sabah, Lineages 16, 17, and 20 co-occur in

Sipitang District, and Lineages 15 and 21 in Kota Marudu District. In Sarawak, Lineages 16 and

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18 occur sympatrically in Bintulu District. Morphological evidence suggests that this pattern of co-occuring cryptic lineages is more common in Borneo than elsewhere (McLeod, unpubl. data).

4. Discussion

4.1 Genetic Diversity and Taxonomic Implications

Taxonomic decisions usually are made on the basis of recognizable morphological characters. Two taxa once placed under the nominal species Limnonectes kuhlii have been elevated to specific status on the basis of morphology alone—L. bannaensis (Ye et al., 2007) and

L. fujianensis (Ye and Fei, 1994). Speciation, however, is not always accompanied by recognizable phenotypic change (Bickford et al., 2007). In the case of the L. kuhlii Complex of frogs, there is morphological conservation which, combined with our historical reliance on mensural and descriptive characters, apparently has led us to underestimate the diversity in these frogs for more than 170 years. This study presents 16 distinct, unnamed, evolutionary lineages

(29% of the known Limnonectes diversity), in addition to another 6 currently recognized species with respect to which true L. kuhlii is not monophyletic. Clearly, the concept of L. kuhlii being a single widespread species must be rejected.

Two patterns are seen in comparisons of the uncorrected mean pair-wise distances. First, there is nearly as much genetic diversity within the L. kuhlii Complex as there is within all of

Limnonectes, in which mean pair-wise sequence divergences are 10.9% and 12.7%, respectively.

Second, the genetic diversity within the L. kuhlii Complex is comparable to the interspecific and intraspecific diversity found among other anurans. For example, Vences et al. (2005b) found that among species of the family Mantellidae from Madagasgar, inter- and intra-specific genetic distances ranged 1–16.5% and 0–5.1%, respectively. In this study, sequence divergences

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between lineages (viz., interspecific) ranged 6.5–18.8%, and within lineage divergences (viz. intraspecific) ranged from 0.1–4.4% (Table 1). It is worth noting that these metrics of diversity are higher than those in other phylogenetic and taxonomic studies of amphibians. Ron et al.

(2006) found that the distances between pairs of unambiguously recognized Engystomops species that ranged from 2.9–4.1%. The use of mtDNA for species delimitation is controversial

(but see Wiens and Penkrot, 2002), and employing genetic distances in species delimitation should be done in tandem with other evidence such as morphological differences, and/or reproductive isolation (Vieites et al., 2009). Nevertheless, the evidence presented here from genetic data alone strongly suggests that there are probably numerous undescribed species hidden within the L. kuhlii Complex.

A growing concern among ecologists and other field biologists is that attempts to resolve cryptic species groups with molecular evidence will yield species that cannot be identified in the field. Certainly, the use of molecular evidence alone to justify nomenclatural changes could create an environment of taxonomic chaos for field workers faced with sympatric (or worse, syntopic) species that are morphologically indistinguishable. Fortunately, there is evidence

(Inger et al., 2009; Stuart et al., 2006) that a molecular phylogeny can serve as a reliable sorting tool when specimens (live or preserved) are examined a posteriori, and that morphological characters once considered “individual variation,” in fact can be reliable apomorphies for species recognition. The intent of this study is not to propose taxonomic changes to the lineages that constitute the L. kuhlii Complex, but rather to create a framework for a detailed morphological study of these frogs. Ultimately, the combination of molecular and morphological data should elucidate the full extent of the diversity within this group and permit appropriate application of names to the evolutionary lineages (viz., species) recognized here.

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4.2 Lineage Diversity

The results of these analyses recover four major clades of Limnonectes kuhlii complex diversity (Figure 2). Based on the overlap in 95% confidence intervals of node age (Figure 3) it would seem that these four clades (Clades A–D) might have diverged at approximately the same time. This coincidence raises the possibility of a single historical event influencing these splits such as a change in sea levels or a substantial change in climate that severely fragmented habitats. When the diversity of the L. kuhlii Complex is considered in a geopolitical framework, the results of these analyses suggest that most countries sampled actually contain two or more distinct evolutionary lineages of L. kuhlii Complex frogs (Table 2). In most cases, these lineages are not each other’s closest relative. Furthermore, most countries also seem to be home to at least one endemic L. kuhlii Complex lineage (Table 2). Without further sampling, however, the respective ranges of these lineages remains unknown.

Though no specific locality is provided by Tschudi (1838) or Duméril and Bibron (1841),

Java is designated by both of the latter references as the place of origin of the type material for

Limnonectes kuhlii. Based on the results of this study, only one lineage occurs on the island.

Taxonomically, this is significant because it restricts the identity of L. kuhlii to a Javan endemic rather than a widely distributed SE Asian species. Javan L. kuhlii is the sister lineage to an unnamed lineage from western Sumatra (Lineage 2). Examination of museum specimens suggest that at least one other morphologically distinct lineage occurs in Sumatra (McLeod, unpubl. data), but additional molecular work is needed before its relationship to other L. kuhlii

Complex members is understood.

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Zhang et al. (2005) considered Limnonectes fujianensis from China as the sister taxon to

L. kuhlii from Taiwan, and a genetically remote population allied to L. kuhlii from Yunnan as the sister to the L. fujianensis + Taiwanese L. kuhlii clade. The Yunnan specimens of Zhang et al.

(2005) were subsequently described as L. bannaensis (Ye et al., 2007). Thus, at least three species of Limnonectes currently are known to occur in China—L. bannaensis, L. fragilis

(Hainan endemic), and L. fujianensis (Zhang et al., 2009). For the purposes of this study, five samples each of Taiwanese L. kuhlii from two allopatric populations on the island (Nanto and

Taoyuan counties) were analyzed. Placement in the phylogeny and low pair-wise divergence values (0.0–3.5%) from this study support the conspecific status of the Taiwanese populations with mainland L. fujianensis. The results of this study are also concordant with the relationships proposed by Che et al. (2007), who used specimens only from mainland China. It is worth noting that in Che et al. (2007) and Che et al. (2009), specimens called L. kuhlii (from China and

Vietnam) probably should be identified as L. bannaensis.

The known distribution of Limnonectes bannaensis includes Yunnan, Guangdong, and

Guangxi provinces in southern China (Ye et al., 2007; Zhang et al., 2005). The results of this study extend this distribution and reveal that L. bannaensis is distributed throughout much of north and central Vietnam, and eastern Lao PDR. In northern Vietnam, there is at least one locality where L. bannaensis occurs syntopically with Lineage 13 on a single stream system.

There is a considerable difference in body size for members of these two lineages. For example, sexually mature (gravid) females of Lineage 13 are noticeably smaller (mean snout–vent length

= 40.0 mm; n = 2) than those of L. bannaensis (mean snout–vent length = 65.9 mm, range =

54.0–87.4 mm; n = 12). There is, however, enough morphological similarity between these two

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lineages that members of Lineage 13 could easily be mistaken for juveniles of L. bannaensis. At present, Lineage 13 is known only from this one locality.

Two lineages occur in Myanmar. One of these, along with populations from Lao PDR and northern Thailand comprise a haplotype clade (Lineage 12). In Myanmar, this lineage seems to be geographically isolated on the Shan (eastern) Plateau, separated from the other Myanmar lineage (9) by the central Irrawady River Valley to the west and a montane valley to the north.

There are reports of Limnonectes kuhlii from the Assam, Meghalaya, and Arunachal Pradesh regions of India (Chanda, 2002; Dutta, 1997), although the actual identity of these frogs is questionable (van Dijk et al., 2004). Based on these data from Myanmar, it is possible that L. kuhlii frogs of northeastern India are closely allied to those of Lineage 9.

Three lineages occur in Lao PDR, none of which is the closest relative of the other.

Lineage 5 seems to be endemic to Laos and is the sister taxon to Lineage 4, a Cambodian endemic. Though weakly supported in maximum likelihood and Bayesian analyses, Lineages 4 and 5 form the sister clade to Lineage 3, the Hainan Island endemic, Limnonectes fragilis. These three lineages are of interest as they occur near the base of L. kuhlii Complex phylogeny and are not members of the larger SE Asian clade (i.e., Lineages 6–13).

In Thailand, Limnonectes kuhlii is distributed along the mountainous western edge of the

Kingdom from north to south, including the central portion of the Thai-Malay Peninsula (Chan- ard, 2003). Two samples used here from Chiang Mai belong to Lineage 12, composed also of populations in Myanmar and Laos PDR. Samples from western and peninsular Thailand are lacking from this study, and thus, phylogenetic placement of L. kuhlii Complex frogs from this region is unknown. McLeod (2008) recognized L. megastomias (Lineage 10) from populations in Nakhon Ratchasima, Sa Kaeo, and Loei provinces in Thailand. Molecular data presented by

2.17

McLeod (2008) included samples from only Nakhon Ratchasima and Sa Kaeo provinces. In this study, additional sequence data from populations of L. megastomias corroborated the identity of

Nakhon Ratchasima and Sa Kaeo populations, and placed the Loei population (Lineage 11) as sister taxon to L. megastomias. In a pattern seen throughout this study, the mean uncorrected pair-wise sequence distances are quite low (0.3% within both lineages), and the inter-lineage divergence is relatively high at 7.4%. Additional examination of morphological characters will be necessary to differentiate these two phenotypically similar lineages.

Molecular samples from three populations in peninsular Malaysia were available for this study, but voucher specimens were not. While conservatively treated as one lineage in this study, pair-wise distances between the Genting Highlands population and the other two are relatively high (~7.5%), but low between Temengor Forest Reserve and Sungai Logging Camp populations (1.3%). These results suggest that more work is necessary to elucidate the diversity among these frogs. Inger and Voris (2001) argued that among stream breeding frogs such as L. kuhlii, genetic divergence values should be lower for Peninsular Malaysia/Sumatra or Peninsular

Malaysia/Borneo pairs of populations than for Sumatra/Borneo pairs. Results from this study

(Table 1) do not support this hypothesis, but instead show that deep divergences exist between

Peninsular Malaysia/Sumatra lineages (16.6%), Peninsular Malaysia/Borneo lineages (14.5–

17.6%), and Sumatra/Borneo lineages (13.4–17.5%).

No single geopolitical area rivals the island of Borneo for the amount of Limnonectes kuhlii diversity. Inger and Tan (1996) cited personal communication with D. Iskandar that

Bornean L. kuhlii are not conspecific with Javan L. kuhlii and that there may be multiple species on the island. Results of the current analysis reveals that at least eight endemic lineages occur on

Borneo, all of which are deeply divergent (range: 15.2–18.8%) from Javan L. kuhlii.

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Morphological examination of preserved museum specimens indicates that several more unique lineages from Malaysian Borneo (Sabah and Sarawak) will be uncovered with the addition of molecular sequence data (McLeod, unpubl. data). Much of Indonesian Borneo (Kalimantan) has not been sampled; therefore, it is possible that this half of the island contains as much or more diversity than found in Brunei and Malaysian Borneo.

Zhang et al. (2005) grouped populations from Borneo with Limnonectes asperatus, a result corroborated by this study. The results of the present study show that all Bornean taxa sampled constitute a monophyletic assemblage of Lineages 14–22. Limnonectes asperatus

(Lineage 19) belongs to the clade containing Lineages 19–22, which is the sister taxon to clade comprising Lineages 14–18. Though morphologically distinct (Inger et al., 1996), specimens of asperatus have been misidentified as L. kuhlii in research collections (McLeod, pers. obs.), so it is not surprising to find this species nested deeply within the tree of the L. kuhlii Complex. For the purposes of this study, the haplotypes that constitute Lineage 19 are being treated as a single evolutionary unit, but with caution. Though there is relatively low sequence divergence (1.8–

6.6%) between haplotype samples in this clade, it is clear, upon examination of the paratypes of

L. asperatus and specimens from the Natunas Islands (Indonesia), that significant morphological differences exist between these populations (McLeod, unpubl. data). Unfortunately, the voucher specimens associated with the Kalimantan (Indonesian Borneo), and Sarawak (Malaysian

Borneo) samples that constitute Lineage 19 in this phylogenetic hypothesis were unavailable for morphological examination. Therefore, it is not possible to make conclusive statements about their identity or distribution at present. Further examination of morphological and molecular data may reveal that these samples represent additional, unique lineages closely related to L. asperatus.

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5. Conclusions

Results of this study support the hypothesis of Stuart et al. (2006) that there may not be any single widespread species of forest-dwelling frog in SE Asia. Whereas historically,

Limnonectes kuhlii has been considered a single, widespread species, the data presented here suggest that nominal L. kuhlii is in fact a complex of cryptic species. This finding is particularly significant because L. kuhlii is the generotypic species for the dicroglossine genus Limnonectes and is a frequently used taxon in evolutionary studies (Appendix 2). Evidence from this study indicates that “true” L. kuhlii is known only from the type locality, Java. Additionally, samples of non-Javan L. kuhlii used in other studies (Appendix 2) can be identified as L. fujianensis

(Emerson et al., 2000), L. bannaensis (Bossuyt and Milinkovitch, 2000; Frost et al., 2006; Zhang et al., 2005), and as representatives of the unnamed lineages constituting Lineages 12 (Vences et al., 2000), Lineage 21 (Emerson et al., 2000), and Lineage 22 (Evans et al., 2002) from this study

(Appendix 2). It is possible that including a single sample (or even a few samples) of non-Javan

L. kuhlii in phylogenetic studies may misrepresent relationships within the L. kuhlii Complex or even within the genus Limnonectes. For example, Evans et al. (2003), presented “cf kuhlii 2” from Taiwan as the sister taxon to the clade containing L. laticeps, L. gyldenstolpei, L. microdiscus, and L. kardasani. All Taiwanese “kuhlii” included in this study are probably conspecific with mainland China L. fujianensis. The laticeps-kardasani clade of Evans et al.

(2003) was recovered in this study, but none of the L. kuhlii Complex samples (including L. fujianensis) was sister to this clade. This may be a result of the increased taxon sampling in this study.

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There is much more species diversity within the Limnonectes kuhlii Complex than has been demonstrated by other recent studies. These data suggest that there are at least 16 currently unrecognized unique and independent evolutionary lineages of frogs of the L. kuhlii Complex.

In Laos, Vietnam, and Borneo, this hidden diversity is compounded by the presence of sympatric

(and syntopic) lineages. Interestingly, this is concordant with the findings of Stuart (2006) and

Inger et al. (2009) where, in cases of sympatry, co-occurring lineages were not each other’s closest relatives. Nevertheless, to avoid creating an environment of confusion and frustration, taxonomic changes are deferred until a comprehensive study integrating genetic and morphological data can be completed.

The recovery of sympatric/syntopic cryptic lineages in this study resulted from moderate population-level sampling (ideally, 10 specimens per locality). Consider, for example, the four haplotypes that comprise Lineage 13. These four specimens and >20 individuals of L. bannaensis were collected during at least two expeditions to northern Vietnam. The last expedition was conducted with the sole purpose of recovering these small-bodied L. kuhlii

Complex frogs. This would suggest that Lineage 13 is not only endemic to Vietnam, but may also be rare and in need of conservation measures. Furthermore, this diversity would have likely gone undetected had only one or two specimens been collected from the field or sampled for this study.

The recognition of sympatric lineages also presents several questions that require additional research efforts to be adequately understood. How are these sympatric/syntopic populations partitioning ecological resources? What are the barriers to reproduction that would result in relatively high levels of genetic divergence? What historical events may have led to the diversification, dispersal, and establishment of two related lineages in a given area? How much

2.21

more biological diversity would be revealed if phylogenetic studies routinely expanded sampled multiple individuals and multiple populations?

Given the rapid loss of SE Asian forests and the tremendous threat this poses to the persistence of forest biota (Sodhi et al., 2009), particularly for range-restricted species, the recognition of the diversity of the Limnonectes kuhlii Complex is particularly relevant to making informed conservation and management decisions. This study demonstrates that each country examined is home to multiple lineages of L. kuhlii frogs that dwell in forest streams. Most countries contain at least one endemic L. kuhlii Complex lineage, and some (e.g., Malaysian

Borneo) contain multiple endemic lineages. In light of these results, the designation of IUCN categories such as “Least Concern” should be carefully considered when being applied to widely distributed “species” such as L. kuhlii. Without examination of these potential reservoirs of undescribed cryptic diversity, we may unknowingly be watching rare or endemic species being pressed towards extinction. Whereas biological entities (anurans in this case) do not recognize geopolitical boundaries, governmental and non-governmental decision makers do. It is therefore important to be able to document and highlight the biodiversity of a particular country or region, especially when one considers the tremendous amount of genetic diversity that can be hidden in a group such as L. kuhlii.

It is clear that even the picture of cryptic diversity within Limnonectes kuhlii presented here is far from complete. In particular, there are significant portions of the range of the L. kuhlii

Complex that have not been well sampled or sampled at all. This paucity of sampling hampers our ability to understand the systematics of the L. kuhlii Complex and the biogeographic relationships among members of this group. Results of this work, however, can provide

2.22

phylogenetic context for future molecular, morphological, ecological, biogeographic, and behavioral studies.

Finally, several issues related to the maintenance of museum collections are raised by a study such as this. First, without the field collection of high-quality tissue samples from hundreds of individual frogs, this study would not have been possible. Moreover, it is clear that if multiple samples (both tissue and whole preserved frogs) from a given population had not been available for study, existence of sympatric and syntopic cryptic species would have likely been missed. Without the practice of depositing tissues along with whole voucher specimens in accessible museum collections, it would be most difficult to go beyond what is presented herein.

The ability to match a whole voucher specimen with a tissue sample permits checking the molecular results against morphology. Without morphology, unwarranted and confounding taxonomic changes might be made based solely on molecular data, or conversely, real and recognizable diversity might be missed. Thus, this study, and others like it, can stand as evidence for the necessity of governmental and institutional policies that support and encourage the collection and preservation of quality scientific data through standardized field techniques and collections management strategies.

Acknowledgments

I am deeply indebted to C. Linkem, J. Oaks, D. Blackburn, E. Bonnacorso, J.

Guayasamin, and the lab group of R. Brown for their patience and expertise that have contributed greatly to this project. A. Larson and two anonymous reviewers provided useful comments on this manuscript. I am particularly fortunate to have spent numerous hours with R.

F. Inger and Tan Fui Lian discussing their insights and ideas about these frogs and their

2.23

experiences with them. Thanks to L. Trueb, and H. McLeod for their unwavering support during this process. Funding for this project was provided through the David L. Boren fellowship,

USGS, and the University of Kansas Biodiversity Institute Panorama funds. A. Ohler (MNHP),

Chou W.H. (NMNS), J. van Egmond, J. Vindum (CAS), T. LaDuc (TNHC), J. McGuire (MVZ), Nguyen T. (VNMN), H.Voris (FMNH), A. Resetar (FMNH), and C. Austin (LSU) facilitated loans of preserved specimens from their respective institutions.

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2.32

Figure 1. Distribution of Limnonectes kuhlii Complex samples used in this study. Numbered circles correspond to informal taxonomic designations (lineages) in this study. Where two or more lineages occur in sympatry, lines are drawn to the location and all lineages present at that locality are indicated.

2.33

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$ 6*"3(#('))6"&8

70 89 .93 # L;@Q,><;006"!7"'8 B .90 * * " G;,0DHI006*&7*'8

* D/.<.EF=;-03;=;CE<;006)&7)$8 ) 6+:("%&%'(')6)#7()8 * 98 48 * * ( 1"%%"&%'('))6((7!##8 - 98 ' 3C;.@;-006!#"7%&"8 C * 99 !& 54 2&3"'/-2("'))6%&*7%$"8 * .92 * !! 9:;<=;.>006%$*7%#$8 42 * !% KL0M.>,?:<.;006%##7%"(8 .99 60 .88 !$ O

Figure 2. Relationships among frogs of the genus Limnonectes revealing diversity within the L. kuhlii complex. Simplified phylogram based on a maximum likelihood analysis of mitochondrial DNA sequences (~2400 bp, 12S–16S). Numbers above branches are nonparameteric bootstrap support values from Maximum Likelihood analysis; numbers below branches are Bayesian posterior probabilities. “*” indicates 100% likelihood bootstrap and Bayesian posterior probability support. “-” indicates a branch that does not appear in the Bayesian consensus tree. Country abbreviations as follows: BN = Brunei, ID = Indonesia, MY = Malaysia. Shaded symbols indicate the relationships between known examples of sympatric/syntopic lineages. Numbers in brackets refer to taxon ID numbers in Appendix 1. 2.34

Figure 3. Relative timing of divergence estimated on an ultrametric tree generated from the reduced data set (94 taxa). Bars along nodes indicate 95% C.I. of node heights of genetic splits of Limnonectes kuhlii Complex taxa and congeners. Grey vertical bar shows possible coincidence of four splitting events involving the geographic clades of L. kuhlii Complex taxa.

2.35

22

0.001

clade

- 21 0.006 0.071

intra 20 0.033 0.079 0.076

19 0.040 0.093 0.092 0.094 represented by

18 0.035 0.126 0.127 0.123 0.131

17 alyasia. 0.001 0.071 0.109 0.112 0.120 0.114 which are

ld type represent

16 0.008 0.076 0.091 0.105 0.110 0.110 0.115

15 0.007 0.092 0.096 0.123 0.117 0.118 0.126 0.131

14 .104 0.006 0.100 0.085 0.085 0.101 0.096 0.100 0 0.109 and its allies, . Values in bo

13 0.001 0.144 0.155 0.140 0.148 0.164 0.140 0.149 0.152 0.145

clades L. kuhlii 12

0.018 0.065 0.138 0.147 0.140 0.144 0.161 0.133 0.139 0.141 0.138

11 0.003 0.071 0.078 0.137 0.151 0.137 0.147 0.161 0.135 0.140 0.144 0.143

excludes 10 0.003 0.074 0.069 0.079 0.146 0.161 0.148 0.153 0.171 0.140 0.143 0.146 0.143

9 0.015 0.102 0.093 0.092 0.089 0.138 0.150 0.143 0.141 0.164 0.135 0.144 0.146 0.141 ” clade

8 0.019 0.099 0.098 0.102 0.090 0.100 0.131 0.141 0.137 0.139 0.166 0.135 0.135 0.141 0.137

7 0.014 0.092 0.108 0.118 0.115 0.110 0.113 0.141 0.145 0.142 0.144 0.168 0.137 0.140 0.143 0.142

6 Limnonectes 0.043 0.133 0.126 0.128 0.140 0.138 0.135 0.141 0.145 0.161 0.155 0.154 0.176 0.150 0.153 0.159 0.153

5 0.002 0.163 0.147 0.136 0.140 0.142 0.137 0.136 0.141 0.142 0.162 0.145 0.156 0.169 0.138 0.147 0.147 0.150

1

4 0.003 0.078 0.161 0.147 0.135 0.14 0.149 0.141 0.137 0.143 0.142 0.167 0.145 0.148 0.175 0.139 0.144 0.151 0.152

3 122 — wise divergence values within and between 0.100 0.104 0. 0.117 0.104 0.107 0.102 0.121 0.118 0.116 0.106 0.119 0.116 0.118 0.128 0.109 0.102 0.104 0.109 -

4

06 2 0.0 0.113 0.149 0.141 0.166 0.142 0.136 0.143 0.147 0.145 0.145 0.145 0.13 0.148 0.137 0.154 0.175 0.136 0.138 0.144 0.143

8

1 0.044 0.114 0.125 0.156 0.150 0.170 0.161 0.147 0.159 0.162 0.154 0.160 0.169 0.152 0.156 0.156 0.162 0.18 0.155 0.156 0.163 0.155

LIM 0.127 0.158 0.159 0.132 0.154 0.155 0.169 0.159 0.152 0.155 0.156 0.155 0.151 0.162 0.162 0.160 0.158 0.163 0.183 0.161 0.157 0.168 0.163

OUT 0.195 0.207 0.215 0.209 0.180 0.206 0.210 0.218 0.215 0.206 0.207 0.211 0.206 0.209 0.217 0.212 0.224 0.219 0.216 0.237 0.207 0.210 0.220 0.217

# 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 Uncorrected mean pair

names and numbers. Country abbreviations as follows: BN = Brunei, IN = Indonesia, MY = M

s

china china

(Java) eo F (BN) F eo CLADE NAME Table 1. divergences. For the purpose of this table, the “ lineage Outgroups Limnonecte kuhlii Sumatra fragilis Cambodia Laos Malay. Pen. fujianensis bannaensis Myanmar megastomias Thailand NC Indo Vietnam (MY) A Borneo (MY) B Borneo (MY) C Borneo (MY) D Borneo (MY) E Borneo asperatus Born (MY) G Borneo (IN) H Borneo 2.36

Table 2. Distribution of clades within geopolitical units. Without further sampling, total numbers of lineages (endemic and not) are considered tentative.

Geopolitical Unit Lineage numbers Endemics Total Borneo 14, 15, 16, 17, 18, 19, 20, 21, 22 9 9 Kalimantan (IN) 19, 22 1 2 Sabah (MY) 14, 15, 16, 20, 21 3 5 Sarawak (MY) 16, 18, 20 1 3 Brunei 20 1 Cambodia 4 1 1 China 3, 7, 8 1 3 Java (IN) 1 1 1 Laos PDR 5, 8, 12 1 2 Natunas Isl. (IN) 19 1 Myanmar 9, 12 1 2 Peninsular MY 6 1 Sumatra (IN) 2 1 1 Thailand 10, 11, 12 1 3 Vietnam 8, 13 1 2

2.37 APPENDIX I SPECIMENS EXAMINED Museum codes correspond to those of Leviton et al. (1985) and Leviton and Gibbs (1988), with the addition of: BSI-FS for Biotic Survey and Inventory Field Series (deposited at MVZ); CUMZA for Chulalongkorn University Museum of Zoology Amphibian Collection; DSM for D. McLeod field series (deposited at KU).

GPS Museum GenBank ID # Species General Locality Specific Locality coordinates First Publication Voucher Accession no. N/S E

Outgroups SBE 072’ 1 Occidozyga laevis Malaysia Selangor Dist. U66138, U66139 Emerson et al., 2000 deposited in Mun. Los Baños, University of Phil., Luzon Isl., 2 Hoplobatrachus rugulosus Barangay Batong 14.155 121.235 PNMMalaysia 7827 AY313685 Evans et al., 2003 Laguna Prov. Malake, Mt. Makiling AF241244, 3 Fejervarya limnocharis China, Sichuan Hogya Xian, Bing Ling FMNH 267579 Evans et al., 2003 AF261262 Phil., Luzon Isl., Mun. Tayabas, Barangay 4 Fejervarya vittigera 14.050 121.541 PNM 7826 AY313683 Evans et al., 2003 Quezon Prov. Lao

5 Paa robertingeri China, Sichuan Hejiang SCUM0405169 DQ458244 Che et al., 2007

6 Paa boulengeri China, Sichuan Mt. Emei SCUM37989 DQ458243.1 Che et al., 2007

Limnonectes Phil., Mindoro Isl., Mun. San Teodoro, 7 L. cf. acanthi Oriental Mindoro 13.438 121.067 TNHC 54922 U66120 U66121 Evans et al., 2003 Tamaraw Falls Prov. Phil., Palawan Isl., Mun. of Puero Princesa, 8 L. acanthi 9.806 118.686 PNM 7604 AY313722 Evans et al., 2003 Palawan Prov. Barangay Irawan Indo., Sulawesi Isl., Desa Cikoro, Mt. 9 L. arathooni Sulawesi Selatan TNHC 59087 AY313744 Evans et al., 2003 Lompobatang Prov. Malaysia 10 L. cf. blythii 1 Endau Rompin N.P. SBE 062’ U55263, U55270 Evans et al., 2003 (Peninsular) Malibou Anai, Anai 11 L. cf blythii 2 Indonesia, Sumatra U66130, U66131 Evans et al., 2003 Valley Phil., Mindanao Isl., Mun. Monkayo, Mt. 12 L. ferneri Davao del Norte 7.971 126.297 CMNH 5572 Pasian Prov. Phil., Mindanao Isl., Mun. Monkayo, Mt. 13 L. ferneri Davao del Norte 7.971 126.297 CMNH 5573 Pasian Prov. Malaysia, Borneo 14 L. finchi Sipitang Dist. FMNH242870 U55264, U55271 Evans et al., 2003 Isl., Sabah State

15 L. grunniens Indo., Haruku Isl. Saparua Deposited in MZB U66124, U66125 Evans et al., 2003 Phuluang Wildlife AF183123, 16 L. gyldenstolpei Thailand, Loei PWRC002 Evans et al., 2003 Research Center AF183124 Indo., Sulawesi Isl., 17 L. modestus Klabat Mt. 1.490 124.842 AMNH 167138 Evans et al., 2003 Sulawesi Utara Prov. Indonesia, Sulawesi, 18 L. modestus Gorontalo TNHC 59710 AY313749 Evans et al., 2003 Sulawesi Utara Prov. Indo., Borneo Isl, 19 L. ibanorum Kalimantan Barat Bentuang N.P. FMNH 251721 U66122, U66123 Evans et al., 2003 Prov. Malaysia, Borneo 20 L. ingeri Belaga Dist. FMNH 251722 U55268, U55275 Evans et al., 2003 Isl., Sarawak State

21 L. kardasani Indo., Lombok Isl. LSUMZ 81722 AY313693 Evans et al., 2003 Selangor Dist., Gombak SBE 071’ AF183125, 22 L. laticeps Malaysia Evans et al., 2003 Field Study Center deposited at the AF183126 Indo., Borneo Isl., University of 23 L. leporinus Kalimantan Timor Near Kutai N. P. 0.532 117.465 AMNHMalaysia 167165 AY313691 Evans et al., 2003 Prov. Lahad Datu Dist., Malaysia, Borneo 24 L. leporinus Danum Valley Research FMNH 230212 U55262, U55269 Evans et al., 2003 Isl., Sabah State Center Phil. Samar Isl., Mun. Bagakay, Bagakay AF183129, 25 L. leytensis 11.437 124.367 USNM 222546 Evans et al., 2003 Samar Prov. Mines AF183130 Phil., Mindanao Isl., Mun. Malagos, Davao 26 L. leytensis Davao del Norte RMB 3788 Evans et al., 2003 City Prov. Phil., Luzon Isl., 27 L. macrocephalus Cagayan River Valley 17.500 121.750 FSO 54563 U66116 U66117 Evans et al., 2003 Cagayan Prov.

2.38 Phil., Luzon Isl., 28 L. macrocephalus Mun. Tiwi, Mt. Malinao RMB 3804 Evans et al., 2003 Albay Prov. Indo., Java Isl, Java 29 L. macrodon Tarogong, Garut FMNH 257159 Y66132, U66133 Evans et al., 2003 Barat Prov. Phil., Samar Isl. Mun. Bagakay, Bagakay 30 L. magnus 11.437 124.367 USNM 534311 U66118, U66119 Evans et al., 2003 Samar Prov. Mines Malaysia, Borneo Gunung Buda, near Mulu 31 L. malesianus 4.494 119.767 Evans et al., 2003 Isl., Sarwawk Prov. N.P.

32 L. microdiscus Indo., Java Isl. Sukabumi LSUMZ 81739 AY313688 Evans et al., 2003 Indo., Sulawesi Isl., 33 L. microtympanum Sulawesi Selatan Barruo 4.494 119.767 AMNH167146 Evans et al., 2003 Prov. Indo., Sulawesi Isl., 34 L. microtympanum Sulawesi Selatan Barruo 4.494 119.767 AMNH16176?? AY313743 Evans et al., 2003 Prov.

35 L. heinrichi Sulwesi, Indonesia Simoro, Biromaru 1.260 119.970 LSU84215 Evans et al., 2003

36 L. heinrichi Sulwesi, Indonesia Lemo, NW Sulawesi 0.441 119.983 AMNH16136 Evans et al., 2003 Malaysia, Boreno 37 L. palavensis Lahad Dist. FMNH 2330800 U55266, U55273 Evans et al., 2003 Isl., Sabah State

38 L. paramacrodon Brunei Tutong Dist. FMNH 248283 Y55267, U55274 Evans et al., 2003 Phil., Mindanao Isl., Mun. Calinan, Barangay 39 L. parvus Davao del Norte 7.186 125.416 PNM 7447 AY313694 Evans et al., 2003 Malagos Prov.

40 L. woodworthi Phil., Luzon Isl., RMB3970 Evans et al., 2003

41 L. woodworthi Phil., Luzon Isl., RMB3339 Evans et al., 2003 Phil., Negros Isl., 42 L. visayanus Negros Oriental RMB4106 Evans et al., 2003 Prov.

43 L. visayanus Phil., RMB3220 Evans et al., 2003 Limnonectes kuhlii Complex 46 Lineage 1 L. kuhlii Indonesia, Java Isl. Sukabumi -6.924 106.922 MZB amph.6501 AY313687 Evans et al., 2003 AF183137, 47 Lineage 1 L. kuhlii Indonesia, Java Isl. Cibodas, Mt. Gede -6.780 106.947 Deposited in MZB Emerson et al., 2000 AF183138 HM067245 48 Lineage 2 Indonesia, Sumatra Batu Layang -3.464 102.316 RMBR 515

49 Lineage 2 Indonesia, Sumatra Batu Layang -3.464 102.320 RMBR 393 HM067244 SCUMH008 DQ458235 50 Lineage 3 L. fragilis China, Hainan Isl. Mt. Limu 19.135 109.773 Che et al., 2007 Cambodia, Ta Veng Dist., Virachey 51 Lineage 4 14.188 107.293 FMNH 262722 HM067166 Ratanakiri Prov. NP Cambodia, Ta Veng Dist., Virachey 52 Lineage 4 14.188 107.293 FMNH 262723 HM067167 Ratanakiri Prov. NP Cambodia, Ta Veng Dist., Virachey 53 Lineage 4 14.188 107.293 FMNH 262724 HM067168 Ratanakiri Prov. NP Cambodia, Ta Veng Dist., Virachey 54 Lineage 4 14.188 107.293 FMNH 262725 HM067169 Ratanakiri Prov. NP Cambodia, Stung Siem Pang Dist. 55 Lineage 4 14.268 106.629 FMNH 262726 HM067170 Treng Prov Virachey NP Cambodia, Stung Siem Pang Dist. 56 Lineage 4 14.268 106.629 FMNH 262727 HM067171 Treng Prov Virachey NP Cambodia, Stung Siem Pang Dist. 57 Lineage 4 14.268 106.629 FMNH 262728 HM067172 Treng Prov Virachey NP Cambodia, Stung Siem Pang Dist. 58 Lineage 4 14.268 106.629 FMNH 262729 HM067173 Treng Prov Virachey NP Cambodia, Stung Siem Pang Dist. 59 Lineage 4 14.268 106.629 FMNH 262730 HM067174 Treng Prov Virachey NP Kaleum Dist., Xe Sap Lao PDR, Xe Kong 60 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.917 FMNH 258505 HM067146 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 61 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.917 FMNH 258506 HM067147 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 62 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.917 FMNH 258507 HM067148 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 63 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.925 FMNH 258508 HM067149 Prov. Area

2.39 Kaleum Dist., Xe Sap Lao PDR, Xe Kong 64 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.925 FMNH 258509 HM067150 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 65 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.925 FMNH 258510 HM067151 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 66 Lineage 5 Nat. Biodiv. Conserv. 16.009 106.925 FMNH 258511 HM067152 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 67 Lineage 5 Nat. Biodiv. Conserv. 16.069 106.975 FMNH 258512 HM067153 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 68 Lineage 5 Nat. Biodiv. Conserv. 16.069 106.975 FMNH 258513 HM067154 Prov. Area Kaleum Dist., Xe Sap Lao PDR, Xe Kong 69 Lineage 5 Nat. Biodiv. Conserv. 16.069 106.975 FMNH 258514 HM067155 Prov. Area Batang Kali, Genting 70 Lineage 6 Malaysia, Pahang 3.423 101.786 FRIM 1141 HM067200 Highland Temengor Forest 71 Lineage 6 Malaysia, Perak 5.569 101.655 LSUHC7034 HM067230 Reserve Sungai Lembing Logging 72 Lineage 6 Malaysia, Pahang 3.087 103.050 LSUHC5008 HM067229 Camp Sungai Lembing Logging 73 Lineage 6 Malaysia, Pahang 3.087 103.050 LSUHC4922 HM067228 Camp

74 Lineage 7 L. fujianensis Taiwan ROC Taoyuan Co. 24.784 121.281 NMNST 16602 HM067231

75 Lineage 7 L. fujianensis Taiwan ROC Taoyuan Co. 24.784 121.281 NMNST 16603 HM067232

76 Lineage 7 L. fujianensis Taiwan ROC Taoyuan Co. 24.784 121.281 NMNST 16604 HM067233

77 Lineage 7 L. fujianensis Taiwan ROC Taoyuan Co. 24.784 121.281 NMNST 16605 HM067234

78 Lineage 7 L. fujianensis Taiwan ROC Taoyuan Co. 24.784 121.281 NMNST 16606 HM067235

79 Lineage 7 L. fujianensis Taiwan ROC Nanto Co. 23.923 120.890 NMNST 16650 HM067236

80 Lineage 7 L. fujianensis Taiwan ROC Nanto Co. 23.923 120.890 NMNST 16651 HM067237

81 Lineage 7 L. fujianensis Taiwan ROC Nanto Co. 23.923 120.890 NMNST 16652 HM067238

82 Lineage 7 L. fujianensis Taiwan ROC Nanto Co. 23.923 120.890 NMNST 16653 HM067239

83 Lineage 7 L. fujianensis Taiwan ROC Nanto Co. 23.923 120.890 NMNST 16654 HM067240 DQ118518, 84 Lineage 7 L. fujianensis China KIZ YP027 DQ118474

85 Lineage 7 L. fujianensis China, Anhui Prov. NC 007440

86 Lineage 7 L. fujianensis China, Fujian Prov. Mt. Wuyi 27.533 117.400 YNUHU20026017 DQ458234 Che et al., 2007 AF183131, 87 Lineage 7 L. fujianensis Taiwan ROC Wulai FMNH 257133 Emerson et al., 2000 AF183132 Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 88 Lineage 8 L. bannaensis 22.774 104.867 TAO 693 HM067246 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 89 Lineage 8 L. bannaensis 22.774 104.867 TAO 694 HM067247 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 90 Lineage 8 L. bannaensis 22.774 104.867 TAO 695 HM067248 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 91 Lineage 8 L. bannaensis 22.774 104.867 TAO696 HM067249 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 92 Lineage 8 L. bannaensis 22.774 104.867 TAO698 HM067251 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 93 Lineage 8 L. bannaensis 22.774 104.867 TAO700 HM067253 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 94 Lineage 8 L. bannaensis 22.774 104.867 TAO701 HM067254 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo Lineage 8 L. bannaensis 22.774 104.867 TAO 702 HM067255 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 95 Lineage 8 L. bannaensis 22.774 104.867 TAO703 HM067256 Dist., Commune Vietnam, Ha Tinh 96 Lineage 8 L. bannaensis Ke Go Natural Reserve 18.246 105.683 AMNH 106381 HM067268 Prov. Vietnam, Quang 97 Lineage 8 L. bannaensis Minh Hoa Dist. 17.687 105.750 AMNH 106382 HM067269 Binh Prov. Vietnam, Quang 98 Lineage 8 L. bannaensis Minh Hoa Dist. 17.687 105.750 AMNH 106383 HM026270 Binh Prov.

2.40 Vietnam, Ha Tinh Huong Son Dist., Huong 99 Lineage 8 L. bannaensis 18.365 105.221 AMNH 106384 HM067271 Prov. Son Reserve Vietnam, Vihn Phu 100 Lineage 8 L. bannaensis Tam Dao Hill Station 21.551 105.532 AMNH 106430 HM067272 Prov. Vietnam, Ha Giang 101 Lineage 8 L. bannaensis Vi Xuyen Dist. 22.761 104.870 AMNH 106556 HM067273 Prov. Vietnam, Ha Giang 102 Lineage 8 L. bannaensis Yen Minh Dist. 22.908 105.217 AMNH 106557 HM067274 Prov. Vietnam, Ha Giang 103 Lineage 8 L. bannaensis Yen Minh Dist. 22.908 105.223 AMNH 106558 HM067275 Prov. Vietnam, Lao Cai 104 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.373 AMNH 141069 HM067276 Prov. Vietnam, Lao Cai 105 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.383 AMNH 141075 HM067277 Prov. Vietnam, Lao Cai 106 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.383 AMNH 141137 HM067278 Prov. Vietnam, Lao Cai 107 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.379 AMNH 141147 HM067279 Prov. Vietnam, Lao Cai 108 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.379 AMNH 141154 HM067280 Prov. Vietnam, Lao Cai 109 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.379 AMNH 141155 HM067281 Prov. Vietnam, Lao Cai 110 Lineage 8 L. bannaensis Van Ban Dist. 21.923 104.379 AMNH 141156 HM067282 Prov. Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 111 Lineage 8 L. bannaensis 22.771 104.862 TNE-01 HM067257 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 112 Lineage 8 L. bannaensis 22.771 104.850 TNE-03 HM067259 Dist., Commune Vietnam, Ha Giang Bac Quang Dist., Duc 113 Lineage 8 L. bannaensis 22.319 105.033 TNE-04 HM067260 Dist., Xuan Comm. Vietnam, Ha Giang Bac Me Dist., Phieng 114 Lineage 8 L. bannaensis 22.652 105.317 TNE-05 HM067261 Dist., Luong Comm. Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 115 Lineage 8 L. bannaensis 22.773 104.882 TNE-06 HM067262 Dist., Commune Vietnam, Ha Giang Bac Me Dist., Phieng 116 Lineage 8 L. bannaensis 22.652 105.332 TNE-07 HM067263 Dist., Luong Comm. Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 117 Lineage 8 L. bannaensis 22.773 104.882 TNE-08 HM067264 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 118 Lineage 8 L. bannaensis 22.773 104.873 TNE-09 HM067265 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 119 Lineage 8 L. bannaensis 22.771 104.860 TNE-10 HM067266 Dist., Commune China, Guangxi Shiwan Dashang Nature 120 Lineage 8 L. bannaensis 21.846 107.889 KU 311786 HM067225 Province Reserve China, Guangxi Shiwan Dashang Nature 121 Lineage 8 L. bannaensis 21.846 107.889 KU 311788 HM067218 Province Reserve China, Guangxi Shiwan Dashang Nature 122 Lineage 8 L. bannaensis 21.841 107.877 KU 311790 HM067219 Province Reserve China, Guangxi Shiwan Dashang Nature 123 Lineage 8 L. bannaensis 21.846 107.889 KU 311791 HM067220 Province Reserve China, Guangxi Shiwan Dashang Nature 124 Lineage 8 L. bannaensis 21.846 107.889 KU 311792 HM067221 Province Reserve China, Guangxi Shiwan Dashang Nature 125 Lineage 8 L. bannaensis 21.846 107.889 KU 311793 HM067222 Province Reserve China, Guangxi Shiwan Dashang Nature 126 Lineage 8 L. bannaensis 21.846 107.889 KU 311794 HM067223 Province Reserve China, Guangxi Shiwan Dashang Nature 127 Lineage 8 L. bannaensis 21.846 107.889 KU 311795 HM067224 Province Reserve Mengyang Co., AY703869, 128 Lineage 8 L. bannaensis China, Yunnan Prov. 22.000 100.783 ZNAC 21020 Jinghong City AY703856 AY703868, 129 Lineage 8 L. bannaensis China, Yunnan Prov. Mengla Co. 21.467 101.567 ZNAC 21014 AY703855 Vietnam, Quang 130 Lineage 8 L. bannaensis Minh Hoa Dist. 17.450 106.250 AMNH 161202 DQ283370 Frost et al., 2006 Binh Prov. Bossuyt and 131 Lineage 8 L. bannaensis Vietnam VUB 0930 DQ346995 Milinkovich, 2000 Lao PDR, Huaphahn Vieng Tong Dist., Phou 132 Lineage 8 L. bannaensis 20.233 103.267 FMNH 255140 HM067133 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 133 Lineage 8 L. bannaensis 20.233 103.267 FMNH 255141 HM067134 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 134 Lineage 8 L. bannaensis 20.233 103.267 FMNH 255142 HM067135 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 135 Lineage 8 L. bannaensis 20.233 103.267 FMNH 255143 HM067136 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 136 Lineage 8 L. bannaensis 20.233 103.267 FMNH 255144 HM067137 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 137 Lineage 8 L. bannaensis 20.233 103.200 FMNH 255145 HM067138 Prov Louey Nat. Biodiv. Conserv. Area 2.41 Lao PDR, Huaphahn Vieng Tong Dist., Phou 138 Lineage 8 L. bannaensis 20.233 103.200 FMNH 255146 HM067139 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 139 Lineage 8 L. bannaensis 20.233 103.200 FMNH 255147 HM067140 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 140 Lineage 8 L. bannaensis 20.233 103.200 FMNH 255148 HM067141 Prov Louey Nat. Biodiv. Lao PDR, Huaphahn ViengConserv. Tong Area Dist., Phou 141 Lineage 8 L. bannaensis 20.233 103.200 FMNH 255149 HM067142 Prov Louey Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 142 Lineage 8 L. bannaensis 22.094 102.213 FMNH 258519 HM067158 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 143 Lineage 8 L. bannaensis 22.094 102.214 FMNH 258520 HM067159 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 144 Lineage 8 L. bannaensis 22.094 102.214 FMNH 258522 HM067161 Prov. Dendin Nat. Biodiv. Myanmar, Sagaing Conserv.Alaungdaw Area Kathapa 145 Lineage 9 22.300 94.414 CAS 205260 HM067285 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 146 Lineage 9 22.300 94.414 CAS 205261 HM067286 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 147 Lineage 9 22.300 94.414 CAS 205262 HM067287 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 148 Lineage 9 22.300 94.414 CAS 205263 HM067288 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 149 Lineage 9 22.315 94.484 CAS 205280 HM067289 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 150 Lineage 9 22.317 94.470 CAS 208057 HM067290 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 151 Lineage 9 22.309 94.407 CAS 210183 HM067291 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 152 Lineage 9 22.314 94.408 CAS 210186 HM067292 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 153 Lineage 9 22.314 94.408 CAS 210187 HM067293 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 154 Lineage 9 22.314 94.408 CAS 210193 HM067294 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 155 Lineage 9 22.314 94.408 CAS 210194 HM067295 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 156 Lineage 9 22.314 94.408 CAS 210195 HM067296 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 157 Lineage 9 22.314 94.408 CAS 210196 HM067297 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 158 Lineage 9 22.314 94.408 CAS 210197 HM067298 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 159 Lineage 9 22.314 94.408 CAS 210198 HM067299 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 160 Lineage 9 22.314 94.408 CAS 210199 HM067300 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 161 Lineage 9 22.314 94.408 CAS 210200 HM067301 Div. N.P. Myanmar, Sagaing Alaungdaw Kathapa 162 Lineage 9 22.314 94.408 CAS 210203 HM067302 Div. N.P. Myanmar, Sagging 163 Lineage 9 Mon Ywa Dist., AK Park 22.314 94.407 CAS 221717 HM067307 Divisino Myanmar, Sagaing Htamanthi Wildlife 164 Lineage 9 25.322 95.531 CAS 232197 HM067318 Div. Sanctuary Myanmar, Sagaing Htamanthi Wildlife 165 Lineage 9 25.322 95.531 CAS 232198 HM067319 Div. Sanctuary Myanmar, Sagaing Htamanthi Wildlife 166 Lineage 9 25.473 95.624 CAS 232281 HM067320 Div. Sanctuary Pyin OO Lwin Dist., Myanmar, Mandalay 167 Lineage 9 Shwe U Daung Wildlife 22.912 96.101 CAS 216154 HM067303 Div. Sanctuary Myanmar, Chin Min Dat Dist., Nat Ma 168 Lineage 9 21.372 93.976 CAS 219994 HM067304 State Taung N.P. Myanmar, Chin Min Dat Dist., Nat Ma 169 Lineage 9 21.372 93.976 CAS 219995 HM067305 State Taung N.P. Myanmar, Chin Min Dat Dist., Lone 170 Lineage 9 21.172 94.028 CAS 234727 HM067324 State dhone village Myanmar, Chin Min Dat Dist., Hlae Yaw 171 Lineage 9 21.172 94.028 CAS 234728 HM067325 State village Myanmar, Chin Min Dat Dist., Lone 172 Lineage 9 21.515 93.991 CAS 234871 HM067326 State dhone village Myanmar, Chin Min Dat Dist., Ran Long 173 Lineage 9 21.675 93.801 CAS 235006 HM067327 State village Myanmar, Chin Min Dat Dist., Ran Long 174 Lineage 9 21.675 93.801 CAS 235007 HM067328 State village Myanmar, Chin Min Dat Dist., Ran Long 175 Lineage 9 21.675 93.801 CAS 235008 HM067329 State village Myanmar, Chin Min Dat Dist., Ran Long 176 Lineage 9 21.675 93.801 CAS 235009 HM067330 State village 2.42 Myanmar, Chin Min Dat Dist., Ran Long 177 Lineage 9 21.675 93.801 CAS 235010 HM067331 State village Myanmar, Chin Min Dat Dist., Ran Long 178 Lineage 9 21.675 93.801 CAS 235011 HM067332 State village Myanmar, Chin Min Dat Dist., Sawn 179 Lineage 9 21.604 93.937 CAS 235100 HM067333 State Taung village Myanmar, Chin Min Dat Dist., Sawn 180 Lineage 9 21.604 93.937 CAS 235101 HM067334 State Taung village Myanmar, Chin Min Dat Dist., Sawn 181 Lineage 9 21.604 93.937 CAS 235102 HM067335 State Taung village Myanmar, Chin Min Dat Dist., Sawn 182 Lineage 9 21.604 93.937 CAS 235103 HM067336 State Taung village Myanmar, Chin Min Dat Dist., Sawn 183 Lineage 9 21.604 93.937 CAS 235104 HM067337 State Taung village Myanmar, Chin Min Dat Dist., Twei Pa 184 Lineage 9 21.484 94.000 CAS 235132 HM067338 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 185 Lineage 9 21.484 94.000 CAS 235133 HM067339 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 186 Lineage 9 21.484 94.000 CAS 235134 HM067340 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 187 Lineage 9 21.484 94.000 CAS 235135 HM067341 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 188 Lineage 9 21.484 94.000 CAS 235136 HM067342 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 189 Lineage 9 21.484 94.000 CAS 235137 HM067343 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 190 Lineage 9 21.484 94.000 CAS 235138 HM067344 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 191 Lineage 9 21.484 94.000 CAS 235139 HM067345 State Lan village Myanmar, Chin Min Dat Dist., Twei Pa 192 Lineage 9 21.484 94.000 CAS 235140 HM067346 State Lan village Myanmar, Chin Min Dat Dist., Kan pet let 193 Lineage 9 21.186 94.055 CAS 235239 HM067347 State town Myanmar, Chin Min Dat Dist., Kan pet let 194 Lineage 9 21.193 94.050 CAS 235301 HM067348 State town Moe Maik Township, Myanmar, Shan 195 Lineage 9 Shwe u Daung Wildlife 23.090 96.250 CAS 221808 HM067308 State Sanctuary Myanmar, Shan Taunggyi Dist., Ma 196 Lineage 9 20.706 96.513 CAS 231014 State Gawe Reserve Myanmar, Kachin Putao Dist., Nagmung 197 Lineage 9 27.491 97.836 CAS 224555 HM067309 State Township Myanmar, Kachin Putao Dist., Nagmung 198 Lineage 9 27.509 97.834 CAS 224593 HM067310 State Township Myanmar, Kachin Putao Dist., Nagmung 199 Lineage 9 27.526 97.796 CAS 224619 HM067311 State Township Myanmar, Kachin Putao Dist., Nagmung 200 Lineage 9 27.376 97.896 CAS 225180 HM067312 State Township Myitkyina Township, Myanmar, Kachin 201 Lineage 9 Pidaung Wildlife 25.356 97.200 CAS 230364 HM067313 State Sanctuary Myitkyina Township, Myanmar, Kachin 202 Lineage 9 Pidaung Wildlife 25.356 97.200 CAS 230365 HM067314 State Sanctuary Myanmar, Kachin Mohnyin Township, 203 Lineage 9 25.091 96.403 CAS 232919 HM067321 State Hepu village Myanmar, Kachin Mohnyin Township, 204 Lineage 9 25.074 96.392 CAS 232987 HM067322 State Hepu village Myanmar, Kachin Mohnyin Township, 205 Lineage 9 25.094 96.380 CAS 233014 HM067323 State Hepu village Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 206 Lineage 10 L. megastomias 14.106 102.256 FMNH 266220 HM067183 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 207 Lineage 10 L. megastomias 14.106 102.256 FMNH 266221 HM067184 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 208 Lineage 10 L. megastomias 14.106 102.256 FMNH 266222 HM067185 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 209 Lineage 10 L. megastomias 14.106 102.262 FMNH 266223 HM067186 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 210 Lineage 10 L. megastomias 14.106 102.262 FMNH 266224 HM067187 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 211 Lineage 10 L. megastomias 14.106 102.262 FMNH 266225 HM067188 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 212 Lineage 10 L. megastomias 14.106 102.262 FMNH 266226 HM067189 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 213 Lineage 10 L. megastomias 14.106 102.262 FMNH 266227 HM067190 Prov Da NP

2.43 Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 214 Lineage 10 L. megastomias 14.106 102.262 FMNH 266228 HM067191 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 215 Lineage 10 L. megastomias 14.106 102.262 FMNH 266229 HM067192 Prov Da NP Thailand, Sa Kaeo Muang Sa Kaeo, Pang Si 216 Lineage 10 L. megastomias 14.106 102.262 FMNH 266230 HM067193 Prov Da NP Thailand, Nakhon Sakaerat Env. Res. 217 Lineage 10 L. megastomias 14.494 101.883 KU 307760 HM067201 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 218 Lineage 10 L. megastomias 14.494 101.883 KU 307761 HM067202 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 219 Lineage 10 L. megastomias 14.494 101.883 KU 307762 HM067203 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 220 Lineage 10 L. megastomias 14.494 101.871 KU 307763 HM067204 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 221 Lineage 10 L. megastomias 14.494 101.871 KU 307765 HM067205 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 222 Lineage 10 L. megastomias 14.494 101.871 KU 307766 HM067206 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 223 Lineage 10 L. megastomias 14.494 101.871 KU 307767 HM067207 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 224 Lineage 10 L. megastomias 14.494 101.871 KU 307768 HM067208 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 225 Lineage 10 L. megastomias 14.494 101.871 KU 307769 HM067209 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 226 Lineage 10 L. megastomias 14.494 101.871 KU 307770 HM067210 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 227 Lineage 10 L. megastomias 14.494 101.871 KU 307771 HM067211 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 228 Lineage 10 L. megastomias 14.494 101.871 KU 307772 HM067212 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 229 Lineage 10 L. megastomias 14.494 101.871 KU 307773 HM067213 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 230 Lineage 10 L. megastomias 14.494 101.871 KU 307774 HM067214 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 231 Lineage 10 L. megastomias 14.494 101.871 KU 307775 HM067215 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 232 Lineage 10 L. megastomias 14.494 101.871 KU 307776 HM067216 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 233 Lineage 10 L. megastomias 14.494 101.871 KU 307777 HM067217 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 234 Lineage 10 L. megastomias 14.494 101.871 CUMZA 2003.134 HM067350 McLeod, 2008 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 235 Lineage 10 L. megastomias 14.494 101.871 CUMZA 2003.135 HM067351 McLeod, 2008 Ratchasima Prov. Station. Phu Rua Dist., Phu 236 Lineage 11 Thailand, Loei Prov. 17.334 101.500 FMNH 266212 HM067175 Luang Wildlife Sanctuary Phu Rua Dist., Phu 237 Lineage 11 Thailand, Loei Prov. 17.280 101.517 FMNH 266213 HM067176 Luang Wildlife Sanctuary Phu Rua Dist., Phu 238 Lineage 11 Thailand, Loei Prov. 17.280 101.517 FMNH 266214 HM067177 Luang Wildlife Sanctuary Phu Rua Dist., Phu 239 Lineage 11 Thailand, Loei Prov. 17.280 101.526 FMNH 266215 HM067178 Luang Wildlife Sanctuary Phu Rua Dist., Phu 240 Lineage 11 Thailand, Loei Prov. 17.259 101.502 FMNH 266216 HM067179 Luang Wildlife Sanctuary Phu Rua Dist., Phu 241 Lineage 11 Thailand, Loei Prov. 17.259 101.502 FMNH 266217 HM067180 Luang Wildlife Sanctuary Phu Rua Dist., Phu 242 Lineage 11 Thailand, Loei Prov. 17.259 101.502 FMNH 266218 HM067181 Luang Wildlife Sanctuary Phu Rua Dist., Phu 243 Lineage 11 Thailand, Loei Prov. 17.259 101.506 FMNH 266219 HM067182 Luang Wildlife Sanctuary Myanmar, Shan 244 Lineage 12 Kalaw township 20.711 96.487 CAS 221714 HM067306 State Myanmar, Shan Taunggyi Dist., Ma 245 Lineage 12 20.692 96.506 CAS 230947 HM067315 State Gawe Reserve Myanmar, Shan Taunggyi Dist., Ma 246 Lineage 12 20.692 96.506 CAS 230948 HM067316 State Gawe Reserve Myanmar, Shan Taunggyi Dist., Ma 247 Lineage 12 20.692 96.506 CAS 230949 HM067317 State Gawe Reserve Lao PDR, Phongsaly Phongsaly Dist., Phou 248 Lineage 12 22.094 102.213 FMNH 258517 HM067156 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 249 Lineage 12 22.094 102.213 FMNH 258518 HM067157 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 250 Lineage 12 22.094 102.214 FMNH 258521 HM067160 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 251 Lineage 12 22.096 102.231 FMNH 258523 HM067162 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 252 Lineage 12 22.096 102.231 FMNH 258524 HM067163 Prov. Dendin Nat. Biodiv. Conserv. Area 2.44 Lao PDR, Phongsaly Phongsaly Dist., Phou 253 Lineage 12 22.096 102.231 FMNH 258525 HM067164 Prov. Dendin Nat. Biodiv. Lao PDR, Phongsaly PhongsalyConserv. Area Dist., Phou 254 Lineage 12 22.151 102.202 FMNH 258526 HM067165 Prov. Dendin Nat. Biodiv. Lao PDR, Bokeo Conserv. Area AF215209.1, 255 Lineage 12 Ban Tup 20.283 100.700 MNHN 1997.3904 Vences et al., 2000 Prov. AF215415.1 Thailand, Chiang 256 Lineage 12 Mueang Dist. 18.837 98.902 CUMZA 2003.5 HM067353 Mai Thailand, Chiang 257 Lineage 12 Mueang Dist. 18.837 98.902 CUMZA 2003.8 HM067352 Mai Myanmar, Shan 258 Lineage 12 Mine Phyut Town 20.879 99.877 CAS 235470 HM067349 State Vietnam, Ha Giang 259 Lineage 13 Vi Xuyen Dist. 22.761 104.882 AMNH 106355 HM067267 Prov. Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 260 Lineage 13 22.771 104.850 TNE-02 HM067258 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 261 Lineage 13 22.774 104.867 TAO 697 HM067250 Dist., Commune Vietnam, Ha Giang Vi Xuyen Dist., Cao Bo 262 Lineage 13 22.774 104.867 TAO 699 HM067252 Dist., Commune Malaysia, Borneo 263 Lineage 14 Kinabalu NP 6.035 116.547 FMNH 257155 HM067144 Isl., Sabah State Malaysia, Borneo 264 Lineage 14 Kinabalu NP 6.035 116.547 FMNH 257156 HM067145 Isl., Sabah State Malaysia, Borneo 265 Lineage 14 Kinabalu NP 6.035 116.547 FMNH 234378 HM067117 Isl., Sabah State Malaysia, Borneo 266 Lineage 14 Kinabalu NP 6.035 116.547 FMNH 257154 HM067143 Isl., Sabah State Malaysia, Borneo 267 Lineage 14 Kinabalu NP 6.035 116.547 FMNH 234375 HM067116 Isl., Sabah State Malaysia, Borneo Kota Marudu Dist. Marak 268 Lineage 15 6.300 116.700 FMNH 235674 HM067121 Isl., Sabah State Parak Malaysia, Borneo Kota Marudu Dist. Marak 269 Lineage 15 6.300 116.700 FMNH 235677 HM067122 Isl., Sabah State Parak Malaysia, Borneo Sipitang Dist., 270 Lineage 16 4.900 115.700 FMNH 238471 HM067123 Isl., Sabah State Mendolong Malaysia, Borneo Tenom Dist., Crocker 271 Lineage 16 5.217 115.950 FMNH 238517 HM067125 Isl., Sabah State Range NP Malaysia, Borneo 272 Lineage 16 Bintulu Div. 2.907 113.090 FMNH 273417 HM067197 Isl., Sarawak State Malaysia, Borneo Tenom Dist., Crocker 273 Lineage 16 5.217 115.950 FMNH 238615 HM067128 Isl., Sabah State Range NP Malaysia, Borneo Tenom Dist., Crocker 274 Lineage 16 5.217 115.950 FMNH 243627 HM067132 Isl., Sabah State Range NP Malaysia, Borneo Tenom Dist., Crocker 275 Lineage 16 5.217 115.950 FMNH 243619 HM067131 Isl., Sabah State Range NP Malaysia, Borneo Tenom Dist., Crocker 276 Lineage 16 5.217 115.950 FMNH 238661 HM067129 Isl., Sabah State Range NP Malaysia, Borneo Tenom Dist., Crocker 277 Lineage 16 5.217 115.950 FMNH 238534 HM067126 Isl., Sabah State Range NP Malaysia, Borneo 278 Lineage 17 Sipitang Dist. 4.900 115.700 FMNH 238511 HM067124 Isl., Sabah State Malaysia, Borneo 279 Lineage 17 Sipitang Dist. 4.900 115.700 FMNH 243604 HM067130 Isl., Sabah State Malaysia, Borneo 280 Lineage 18 Bintulu Div. 2.907 113.090 FMNH 273423 HM067198 Isl., Sarawak State Malaysia, Borneo 281 Lineage 18 Bintulu Div. 2.907 113.090 FMNH 273428 HM067199 Isl., Sarawak State Malaysia, Borneo 282 Lineage 18 Bintulu Div. 2.656 112.904 FMNH 273405 HM067196 Isl., Sarawak State Malaysia, Borneo 283 Lineage 18 Bintulu Div. 2.656 112.904 FMNH 273342 HM067195 Isl., Sarawak State Malaysia, Borneo 284 Lineage 18 Bintulu Div. 2.656 112.904 FMNH 273341 HM067194 Isl., Sarawak State Indonesia, Serasan 285 Lineage 19 L. asperatus 2.509 109.024 BJE 170 HM067283 Isl. Indonesia, Serasan 286 Lineage 19 L. asperatus 2.509 109.024 BJE 171 HM067284 Isl. Indo., Borneo Isl. AF183127, 287 Lineage 19 L. asperatus Palangkaraya 2.217 112.904 FMNH 252416 Emerson et al., 2000 Kalimantan Selatan AF183128 Indo., Borneo Isl. 288 Lineage 19 L. asperatus Bukit Raya NP -0.604 113.933 RMBR 1193 HM067241 Kalimantan Selatan Indo., Borneo Isl. 289 Lineage 19 L. asperatus Bukit Baka -0.724 112.281 RMBR 707 HM067242 Kalimantan Selatan Indo., Borneo Isl. 290 Lineage 19 L. asperatus Bukit Baka -0.724 112.281 RMBR 761 HM067243 Kalimantan Selatan Malaysia, Borneo Miri Dist., Lambir Hills 291 Lineage 19 L. asperatus 4.198 114.063 LSUHC 4090 HM067227 Isl., Sarawak State NP 2.45 Malaysia, Borneo Sipitang Dist., 292 Lineage 20 4.900 115.700 FMNH 234394 HM067118 Isl., Sabah State Mendolong Malaysia, Borneo Miri Dist., Lambir Hills 293 Lineage 20 4.198 114.063 LSUHC 4089 HM067226 Isl., Sarawak State NP AF183133, 294 Lineage 20 Brunei, Borneo Isl Belait Dist. FMNH 248357 Emerson et al., 2000 AF183134 Malaysia, Borneo Sipitang Dist., 295 Lineage 20 4.900 115.700 FMNH 234395 HM067119 Isl., Sabah State Mendolong Malaysia, Borneo Sipitang Dist., 296 Lineage 20 4.900 115.700 FMNH 238554 HM067127 Isl., Sabah State Mendolong Lahad Datu Dist., Malaysia, Borneo 297 Lineage 21 Danum Valley Research 4.833 117.594 FMNH 230306 HM067112 Isl., Sabah State Center Lahad Datu Dist., Malaysia, Borneo 298 Lineage 21 Danum Valley Research 4.833 117.584 FMNH 230311 HM067113 Isl., Sabah State Center Lahad Datu Dist., Malaysia, Borneo 299 Lineage 21 Danum Valley Research 4.833 117.583 FMNH 230312 HM067114 Isl., Sabah State Center Lahad Datu Dist., Malaysia, Borneo 300 Lineage 21 Danum Valley Research 4.833 117.598 FMNH 230313 HM067115 Isl., Sabah State Center Malaysia, Borneo Kota Marudu Dist. Marak 301 Lineage 21 6.300 116.700 FMNH 235665 HM067120 Isl., Sabah State Parak Lahad Datu Dist., Malaysia, Borneo AF183135, 302 Lineage 21 Danum Valley Research 4.833 117.598 FMNH 230302 Emerson et al., 2000 Isl., Sabah State AF183136 Center Kalimantan, 303 Lineage 22 Kutai N. P 0.532 117.465 AMNH167141 AY313686 Evans et al., 2003 Indonesia Kalimantan, 304 Lineage 22 Kutai N. P 0.532 117.465 AMNH167142 Indonesia

2.46

Appendix 2. Relationships among all Limnonectes haplotypes sampled in this study. Phylogram based on a maximum likelihood analysis of mitochondrial DNA sequences (~2400 bp, 12S–16S). Numbers above branches are nonparameteric bootstrap support values from Maximum Likelihood analysis. Taxa highlighted in grey boxes represent haplotypes previously published and referred to Limnonectes kuhlii. See Appendix 1 for haplotype data and literature citations. 2.47 CHAPTER III

LIMNONECTES KUHLII (AMPHIBIA: ANURA: DICROGLOSSIDAE):

IDENTITY COMPLEX OR A COMPLEX IDENTITY?

Abstract

Using evidence from the phylogenetic analyses of both molecular and morphological data, the Limonectes kuhlii Complex is used as a model system for delimiting the boundaries of cryptic species. Results from analysis of molecular data alone recovers a cryptic species complex consisting of at least 24 distinct evolutionary lineages, several of which are independently corroborated with morphological data. Recognizing that the utility of morphological characters used to diagnose a species may change in light of subsequently recognized cryptic diversity, I re-examine a suite of historically significant external morphological characters and found that none apply universally to all members of this species complex. Examination of osteological characters reveals several elements that may prove to be phylogenetically and diagnostically informative. Collectively, the results of this study create a framework within which the Limnonectes kuhlii Complex diversity can be recognized, diagnosed, and described.

Keywords: cryptic species; diversity; Limnonectes kuhlii; morphology, South East Asia; species complex

3.1

Introduction

Several trends in amphibian biology have emerged during the last decade. In light of rapidly dwindling habitats, human encroachment, and the general loss of biodiversity, one trend has been a marked effort to estimate more accurately the scope anuran diversity and determine the rate at which it is being lost (Stuart et al., 2006; Vieites et al., 2009). A result of this has been a dramatic increase in the number of amphibian species being described (Hanken, 1999;

Kohler et al., 2005; Vieites et al., 2009). Another notable trend is a renewed interest in, and discussion of, the metrics (both tree-based and non-tree based used) to delineate species boundaries (Monaghan et al., 2009; Sites and Marshall, 2003; Vieites et al., 2009; Wiens and

Penkrot, 2002); in many ways this trend is a by-product of the accelerated alpha-taxonomic activity. A growing number of studies have re-examined wide-ranging species and have recovered multiple cryptic species from within the nominal taxon (e.g., Bain et al., 2003;

Fouquet et al., 2007; Stuart et al., 2006). A third trend has been the application of molecular phylogenetic approaches as the primary means of recognizing genetic diversity, with some authors pairing this methodology with traditional approaches to describe diagnostic characters necessary to identify new species (Bain and Truong, 2004; Doan and Castoe, 2003; Inger et al.,

2009). The summary impact of these activities has been (1) the recognition and description of many “cryptic” species; (2) the realization of just how poorly we have underestimated regional and global biodiversity to date; and (3) an increasing refinement of approaches for delimiting species boundaries, especially when using molecular data as a primary data source (e.g, barcoding). Whereas the same trends characterize the broader field of biology and traverse many taxonomic disciplines (Bickford et al., 2007; Pfenninger and Schwenk, 2007), discussion herein is focused on amphibians, and anurans in particular.

3.2

Throughout East and Southeast Asia, one group of frogs that has proven particularly enigmatic to biologists in both field and lab for nearly 200 years. Limnonectes kuhlii Tschudi

(1838) is the type species of the dicroglossid genus Limnonectes Fitzinger (1843), which comprises 55 currently recognized species (Amphibiaweb, 2010). Based on L. kuhlii’s broad geographic distribution and evidence of morphological and molecular diversity, this species has been thought to contain multiple cryptic species (Emerson et al., 2000; Evans et al., 2003;

McLeod, 2008, in press). Recently, Matsui et al. (2010) and McLeod (in press) used molecular phylogenetic analyses of mitochondrial DNA (mtDNA) to highlight the relational complexity of this cryptic species complex. Matsui et al. (2010) used 15 new mtDNA sequences to suggest the placement of Limnonectes namiyei, a Japanese endemic within the Limnonectes kuhlii Complex, and to corroborate the identities of other “kuhlii” samples from Lao People’s Democratic

Republic (Lao PDR) and Taiwan. McLeod (in press) presented a molecular phylogeny of the L. kuhlii Complex using 244 new mtDNA sequences from individuals representing approximately

63 populations and 13 countries across the known distribution of this anuran. Results of McLeod

(in press) corroborated previous phylogenetic treatements of the L. kuhlii Complex (Emerson et al., 2000; Evans et al., 2003; Matsui et al., 2010; Zhang et al., 2005) and further demonstrated that L. kuhlii, which historically had been recognized as a single species, is a complex of more than 22 well supported evolutionary lineages, 16 of which are currently subsumed under the nominal L. kuhlii. Additionally, McLeod (in press) also uncovered several cases of sympatric/syntopic lineages, and in no case were co-occuring lineages each other’s closest relatives.

Despite a number of modern phylogenetic studies incorporating Limnonectes kuhlii

Complex frogs (Che et al., 2007; Chen et al., 2005; Delorme et al., 2004; Emerson et al., 2000;

3.3

Evans et al., 2003; Frost et al., 2006; Marmayou et al., 2000; Vences et al., 2000; Zhang et al.,

2005), only one (Emerson and Berrigan, 1993) has stepped beyond the convenient shelter of molecular data to traverse the morphological landscape in search of evidence for relationships among these frogs and their allies. None has attempted to combine both molecular and morphological data in a phylogenetic context. This may be, in part, because of the lack of distinct morphological character variation among populations of L. kuhlii Complex. Whereas L. kuhlii has been distinguished from its congeners by the indistinct (or hidden) tympanum (see note in Appendix II) and fully webbed toes (Boulenger, 1920; Duméril and Bibron, 1841; Inger,

1966; Taylor, 1962; Tschudi, 1838), there seem, at first glance, to be few obvious characters by which to differentiate one member of the L. kuhlii Complex from another. Only a few morphological characters have been used to diagnose L. kuhlii sensu lato, yet some of these have been noted to vary within and between populations (e.g., condition of nuptial pads in males, relative finger lengths, tuberculation of the hind limbs, differences in internarial distances, and the widths of temporal and interorbital stripes (Berry, 1975; Boulenger, 1920; Inger, 1966;

Iskandar, 1998; Malkmus et al., 2002; Pope, 1931; Taylor, 1962). In many cases, this variability has not been discussed, but can be parsed from a careful review of the literature. Additionally, there has been a long-standing awareness of strong male-biased size dimorphism among frogs of the L. kuhlii Complex frogs (Boulenger, 1920; Inger, 1966; Pope, 1931; Taylor, 1962); however, this feature is considered common to most species of Limnonectes (Emerson et al., 2000).

With respect to the high levels of purported genetic diversity juxtaposed against what seems to be minimal phenotypic diversity, the widely distributed Limnonectes kuhlii Complex provides a unique model system for evaluating cryptic species boundaries using molecular and morphological data. The goals of this study are four. First, the diversity of this complex is

3.4 reassessed with new data. Second, morphological characters are examined via phylogenetic methods to explore whether they provide support for the candidate species recognized by molecular data. Third, traditional morphological characters used to diagnose a species are examined to determine if, once cryptic species are identified, these characters apply to all members of the group. And fourth, a phylogenetic framework is provided for the taxonomic revision of the L. kuhlii Complex (McLeod, in prep).

Materials and methods

Species Concept and Species delimitation

I employ the Evolutionary Species Concept (Simpson, 1961; Wiley, 1978) expanded to the General Lineage Concept of species (de Queiroz, 1998, 1999). A combined approach that includes both morphological and molecular data to estimate phylogenetic relationships is used to guide species delimitation and identification of relevant comparisons for species diagnoses.

Distinct lineages are those populations (1) that are morphologically and genetically distinct, and

(2) for which the hypothesis of conspecificity can be confidently rejected by analyses of both morphological and genetic data.

Whereas the utility of mtDNA in phylogenetic studies of amphibians has been debated

(e.g., Hertwig et al., 2004), and it seems clear that sequence data alone should not be used as the sole criterion for delimiting species, uncorrected pair-wise divergences of the mtDNA may be useful to identify candidate species (Fouquet et al., 2007; Vences et al., 2005a; Vences et al.,

2005b; Vieites et al., 2009). I diagnosed candidate species (highly divergent haplotype clades) based on a threshold of 7% uncorrected pair-wise sequence divergence. Haplotype clades were assumed to represent allopatric populations. If the sympatry (or syntopy) of haplotypes was

3.5 known, then decisions for clade designation were based additionally on the identification of morphological variation in voucher specimens. The use of a 7% threshold of sequence divergence was considered conservative in contrast to other amphibian-oriented systematics studies that used values of 5% (Fouquet et al., 2007) and 3% or less (Ron et al., 2006; Vieites et al., 2009).

The 16 unnamed haplotype clades of McLeod (in press) are equivalent to the “Candidate

Species” of Vences et al. (2005a; 2005b) and Unconfirmed Candidate Species (UCS) of Vieites et al. (2009). Based on the definition of Vieites et al. (2009), recognition of Confirmed

Candidate Species (CCS) requires detectable genetic differentiation to all described species

(though without necessitation of an a priori threshold) and additional concordance with at least one of three character-based criteria: (1) a distinct differentiation in a character that mediates a pre-mating reproductive barrier (e.g., advertisement calls); (2) a diagnostic morphological difference in a character that in the respective group of animals is known to be of low intra- specific variability and of high value to discriminate species (e.g., dermal spines, ridges, tubercles, and etc.); and/or (3) sympatric occurrence without admixture, and with at least one phenotypic character-state difference, which even if subtle, is strictly correlated to the genealogy inferred from a neutral molecular marker.

To facilitate the evaluation and discussion of phylogenetic relationships, I have maintained previously published taxonomies and the lineage numbers applied to the haplotype clades of McLeod (in press) in the text and figures. New haplotype clades resulting from the addition of molecular sequence data from Matsui et al. (2010) to the data set of McLeod (in press), (e.g., Limnonectes namiyei) are designated with numbers 100 and above. The intent of this study is not to propose taxonomic changes to the lineages that constitute the L. kuhlii

3.6

Complex. A separate treatment of taxonomic descriptions and appropriate application of names to the evolutionary lineages (i.e., species) recognized here will be presented elsewhere (McLeod, in prep).

Taxonomic sampling

A total of 320 mtDNA sequences, including 311 samples from McLeod (in press) and 9 samples from Matsui et al. (2010), was used in the molecular analyses of this study. I collected external morphological data from 298 specimens, including all available vouchers used in

McLeod (in press) and 72 additional specimens for which molecular sequence data was unavailable. To examine osteological characters, I obtained or made skeletal preparations of two individuals (one male, one female) from as many of McLeod’s (in press) haplotype clades as specimens were available for. The 36 osteological specimens represent represent 17 taxa of the

Limnonectes kuhlii Complex (27 specimens) and 5 outgroup and congeneric taxa (9 specimens).

Outgroup taxa used are those of McLeod (in press) and were chosen on the basis of previously published phylogenies (Che et al., 2007; Emerson et al., 2000; Frost et al., 2006; Zhang et al.,

2005). A map showing the location of morphological and molecular samples used in this study is presented in Figure 1.

Of the 27 osteological preparations of taxa in the Limnoncectes kuhlii Complex, 16 have unambiguously associated molecular and external morphological data (i.e., all data taken from the same individual). Of the remaining specimens, nine were tentatively assigned to haplotype clades on the basis of collection locality and an examination of external morphology (Table 1).

Though treated tentatively, specimens of Limnonectes namiyei and L. fujianensis are assigned to their respective clades with some measure of confidence based on the endemicity of L. namiyei

3.7 and evidence that Taiwanese “kuhlii” are conspecific with mainland Chinese L. fujianensis

(Matsui et al., 2010; McLeod, in press). Furthermore, based on evidence of tremendous diversity and multiple cases of sympatry/syntopy in Borneo (McLeod, in press), caution was taken in assigning eight specimens from this region to appropriate clades. As a result, two osteological specimens that have no reasonable counterpart in the molecular dataset are referred to by collection locale (Sarawak A and B) and are not included in all analyses.

Morphological data

Specimens used in this study were dried skeletons, cleared-and-double-stained skeletons, and whole alcoholic specimens from the Biodiversity Institute herpetology collections at the

University of Kansas (KU). In addition, specimens were borrowed from the following collections: American Museum of Natural History (AMNH), California Academy of Sciences

(CAS), The Natural History Museum of Chulalongkorn University (CUMZ), Field Museum of

Natural History (FMNH), Forest Research Institute Malaysia (FRIM), La Sierra University

Herpetological Collection (LSUHC), Muséum National d’Histoire Naturelle (MNHN), Museum of Natural Science, Louisiana State University (LSUMZ), National Museum of Natural History,

Netherlands (RMNH), National Science Museum, Thailand (THNHM), National Museum of

Natural Science, Taiwan (NMSM), Texas Natural History Collection (TNHC), Vietnam National

Museum of Nature (VNMN). A table of specimen data for all materials examined is provided in

Appendix IV.

Cleared-and-double-stained specimens were prepared by the author using a modification of the protocol of Taylor and Van Dyke (1985). Dry osteological materials were skeletonized by hand. Morphological observations and illustrations were made with a Meiji Techno® stereo

3.8 dissecting microscope equipped with a camera lucida. Illustrations of one or more representative taxa were prepared to clarify the osteological characters and character states described herein.

Morphological data (osteological + external morphology) were compiled and formatted as a data matrix of 36 taxa and 146 characters. The complete data matrix containing all morphological character states is presented in Appendix III. Osteological data from 36 individuals were coded as discrete variables, many of which are multistate characters. Many of the external morphological characters are represented as continuous variables (measurements).

Some of these data were coded as discrete variables for each individual; others were coded and applied to the entire clade, thereby utilizing larger amounts of data (e.g., mean snout–vent length). Most characters used in this study were taken from Scott (2005); some of Scott’s characters were modified for clarity, and new characters were added for this study (Appendix I).

A table is provided at the end of Appendix I to show the relationship between the characters and states of this study and those of Scott (2005). Scott (2005) discussed her characters and provided chronological references to previous phylogenetic use of the morphological characters used in her study. Scoring of all characters used in this study was done directly from specimens. In cases of unknown character states (e.g., when a damaged specimen lacked a particular element), characters were recorded as “?.” In cases of logical inconsistency or inapplicability (e.g., the state of nuptial pads in female specimens) characters were coded as “–.” In analyses, both “?” and “–” were treated as missing data. All characters were treated as unordered. Because of the rarity of skeletonized or cleared-and-stained material for most taxa, it was not possible to examine more than two specimens (1 male, 1 female) for most species. Two exceptions to this are Limnonectes bannaensis (Lineage 8) and Lineage 12 in which three and four specimens, respectively, were available from different populations.

3.9

Osteological terminology used herein is that of Trueb (1973, 1993). An effort was made to cross-reference terminology to The Amphibian Anatomy Ontology web project (Maglia et al.,

2007). Mineralization is recognized as a disorganized, bubbly textured calcification following

Scott (2005).

Molecular data

Mitochondrial DNA data were selected for use in this study to take advantage of abundant comparative material already available from previous work (Che et al., 2007; Emerson et al., 2000; Evans et al., 2003; Frost et al., 2006; Jiang and Zhou, 2005; Matsui et al., 2010;

Zhang et al., 2005; Zhang et al., 2009). The gene order of the approximately 2400 bp of

Mitochondrial DNA region analyzed (5'–3') is tRNAphe, 12S ribosomal DNA (rDNA), transfer

RNA for Valine, and16S rDNA. For samples in this study, the average sequence length is 1921 bp (max = 2446 bp; min = 698 bp). DNA extraction and sequencing protocols are detailed in

McLeod (in press). Nine exemplars of anurans of the Limnonectes kuhlii Complex from Matsui et al. (2010) that were incorporated into this study from published GenBank files were manually aligned to a matrix of 311 terminals and 2613 molecular characters from McLeod (in press).

Phylogenetic analysis

In all analyses, Occidozyga laevis and Fejervarya limnocharis were used to root the phylogenies. Also included in all analyses were Limnonectes blythii, L. gyldenstolpei and L. laticeps (congeners of the L. kuhlii Complex), which were chosen to serve as phylogenetic

“landmarks” for comparing the broader relationships of this study to others such as Evans et al.

3.10

(2003) or Emerson et al.(2000), both of which included more than just members of the L. kuhlii

Complex in their analyses.

A re-analysis of the molecular data set of McLeod (in press) was performed in light of the nine sequences added to this study from Matsui et al. (2010). A maximum likelihood analysis of

320 terminals was conducted using RAxML v7.0.4 (Stamatakis, 2006) with 100 replicate best tree inferences. Clade support was assessed with 1000 bootstrap pseudoreplicates and was considered significant for bootstrap (BS) values above 70%. Evaluation of uncorrected pairwise distances was conducted with MEGA v4.0 (Tamura et al., 2007).

Parsimony, Bayesian, and maximum likelihood analyses were performed on the morphology data set. Parsimony heuristic searches were executed in PAUP* v4.0b10 (Swofford,

2002) with tree bisection-reconnection (TBR) branch swapping and 10,000 random stepwise addition sequence replicates, saving one tree at each replicate. No limit was imposed on the maximum number of trees to be saved and PAUP* was instructed to increase the maximum by

100 automatically if/when the default value of 100 trees was reached. Summary values (e.g., tree length, consistency index) were reported by PAUP*. Support for individual branches was evaluated using nonparametric bootstrapping (Felsenstein, 1985) in PAUP* with 500 bootstrap pseudoreplicates. Each pseudoreplicate included 10 random-taxon-addition sequence replicates, again using TBR branch swapping and retaining a single tree per replicate. Bootstrap values greater than 70% are considered significant. All characters were considered unordered and equally weighted. Gaps were treated as missing data. Bayesian analyses were conducted using

MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003). Four independent analyses were run, each with four Metropolis-coupled Markov chains (MCMC) each. All Markov chains were run for 5 million generations, with sampling every 1000 generations. The output files were examined in

3.11

Tracer v1.4 (Rambaut and Drummond, 2007) to determine the number of generations to exclude as burn-in and to ensure all parameters converged. Additionally, Are We There Yet

(Wilgenbusch et al., 2004) was used to ensure the multiple runs converged and that sampling was sufficient. The sump and sumt commands were executed in MrBayes, summing over the multiple convergent runs. Clade support was considered significant where Bayesian posterior probabilities (BPP) were greater than 95%. Maximum likelihood analyses were performed using the MK model in Garli (Zwickl, 2006) which accommodates morphological data and constant characters. All defaults were maintained and three independent runs were completed to compare likelihood scores. Support for individual branches was evaluated in Garli with 500 bootstrap pseudoreplicates. Sumtrees (Sukumaran and Holder, 2009) was used to compute a majority rule consensus tree.

Exploration of Morphological Character Data

There are relatively few morphological characters that have been considered to be diagnostic for Limnonectes kuhlii s.l., or that have been discussed in relation to populations of this anuran. A parsimony-based ancestral-state reconstruction was performed in Mesquite

(Maddison and Maddison, 2009) to evaluate the diagnostic utility of these characters across the

L. kuhlii Complex. Morphological characters were traced onto the molecular tree, pruned to 25 terminals corresponding to matching morphological data. In cases in which a haplotype terminal lacked associated unambiguously matching morphological data, character states were approximated from taxa assumed to be closely related, and tentatively assigned to the haplotype clade as described above. To simplify the graphical depiction of character 138 (condition of nuptial pads), coding was changed to reflect the presence or absence of nuptial pads in males

3.12

(females were coded to match males of the same haplotype clade). Coding of character 138 was not changed in any other analysis. Optimization of morphological characters on the pruned molecular tree was performed in PAUP* under both ACCTRAN and DELTRAN assumptions.

Results

Molecular characters

The molecular dataset analyzed comprises 2613 total characters. After deleting a hypervariable portion of the 16S region (following McLeod, in press), 2577 characters were available for analysis. Of these, 1017 were constant, 285 were variable and parsimony- uninformative, and 1275 were parsimony informative. This differs slightly from McLeod (in press), which included 2578 characters —1037 constant, 273 variable and parsimony- uninformative, and 1268 were parsimony-informative. The difference is the result of changes to the alignment after the addition of new sequences for this study. These minor alignment changes did not alter the topology of the tree.

Morphological characters

The morphological dataset includes 137 osteological characters and 9 characters coded from external morphology. Of the 146 morphological characters, 65 are constant, 75 are variable and parsimony-informative, and 6 variable characters are parsimony uninformative.

Molecular Phylogenetic Analyses

Maximum likelihood analysis resulted in the single best scoring tree (–ln = 47412.43) presented in Figure 2. Results of the Maximum Likelihood analysis are congruent with McLeod

3.13

(in press). The monophyly of the Limnonectes kuhlii complex is supported. These results corroborate the recent molecular phylogenetic hypotheses of McLeod (in press) and Matsui et al.

(2010), and present a phylogenetic hypothesis of the relationships of the L. kuhlii Complex, which is composed of four major, well-supported, geographic clades. Clade A comprises two

Sunda-shelf lineages (1 and 2) and is the sister taxon to an Indochinese clade (Clade B) comprising Lineages 3–5 from Hainan Island, Cambodia and Lao PDR, respectively. Clade C consists of Indochinese, Sunda-shelf and offshore island Lineages 6–13 and 100. Clade D comprises all Bornean lineages (19–22 and 101), is the sister taxon to Clade C. Clade C + D is sister to Clade B.

The single “Limnonectes megastomias” sample of Matsui et al. (2010) is here identified as belonging to Lineage 11 (this study), and not of L. megastomias (See Discussion). The single sample from Sarawak of Matsui et al. (2010) is identified as a unique evolutionary lineage

(Lineage 101) on Borneo. Mean pair-wise sequence divergence between Lineage 101 and other clades in the L. kuhlii Complex ranges from 7.2–17.5%. The single sample from Java (Lineage 1 herein) included in Matsui et al. (2010) was found to have an uncorrected pairwise sequence divergence measures of 6.8 and 10% from the two molecular sequences of Javan L. kuhlii included in Evans et al. (2003) and McLeod (in press), whereas the two Evans et al. (2003) samples are 4.4% different from each other. It is worth noting both this study and Evans (2003) provide genetic samples corresponding to whole voucher specimens that are accessible in museum collections. Unfortunately, while Matsui et al. (2010) provide GenBank accession numbers for their molecular sequences, at least four of their sequences (including the phylogenetically and taxonomically important “Java”, “Sarawak”, and “Limnonectes namiyei”

3.14 samples) come from uncataloged or non-vouchered specimens. The inability to verify specimen identification prevents independent corroboration of their results.

Morphological Phylogenetic Analyses

Results of the morphology-only analyses are summarized on a Bayesian consensus tree

(Figure 3). The analysis of morphological characters resulted in 126 equally parsimonious trees

(TL = 368; CI = 0.315; RI = 0.529). The strict consensus tree is poorly resolved, but contains four lineages of the Limnonectes kuhlii Complex; each with high BS support that is congruent with the UCS recognized by molecular data (Lineages 8–10, 13, Limnonectes gyldenstolpei, L. laticeps, Fejervarya limnocharis, and Occidozyga laevis). Monophyly of the L. kuhlii Complex is not supported owing to the placement of L. blythii within the L. kuhlii Complex polytomy.

Maximum likelihood analysis resulted in a single consensus tree (–ln = 1439.56) with a topology resembling those of the trees produced by the parsimony and Bayesian analyses. Six pairs of taxa in the Limnonectes kuhlii Complex were recovered; however, only two (Lineages 8 and 10), had high BS support. All recovered clades were congruent with results from the molecular analysis.

Bayesian analyses resulted in a consensus tree with a negative harmonic mean likelihood of 1472.18, which was summed from four independent runs. All parameters converged in all four runs, examination of which was accomplished both in Tracer and AWTY. Burn-in was estimated at 50,000 generations, resulting in a posterior distribution of 19,804 trees per run. The

Bayesian consensus tree contains only one clade (100 + 101+Sarawak A) that is incongruent with the results of other analyses (Figure 3). Two lineages (8 and 10) of the Limnonectes kuhlii

Complex were recovered with robust BPP support. In addition, Lineages 5, 9, 11–13 and the

3.15 clade consisting of Lineages 4 and 5 were resolved, though only with moderate-to-weak support.

The L. kuhlii Complex is again resolved as non-monophyletic because of the placement of L. blythii, L. gyldenstolpei, L. laticeps, and Occidozyga laevis.

When Bayesian posterior probabilities from the analysis of morphological data were summarized on the pruned molecular tree, moderate support was found for four UCS (Lineages

5, 9, 11, and 13). Although lower, support was found for four other relationships (Lineages 4, 8,

12, and Clade B). No support was found for the relationship between Lineages 7 and 100, or

Clades A, C, and D. These results are summarized in Table 2.

Morphological Character Exploration

Based on previous taxonomic and systematic studies in which the morphology of

Limnonectes kuhlii s.l. was examined, I mapped the following six morphological characters onto the reduced molecular tree (Figure 4): nuptial pads in males (Char. 138), presence of a visible tympanum (Char. 141), relative finger lengths (Char. 144), internarial distance (Char. 143), body size (Char. 145), and sex-biased size dimorphism (Char. 146).

No single character state for the traits examined was found to occur in all lineages of the

Limnonectes kuhlii complex. The morphological data were examined to determine whether potentially diagnostic characters exist for the three geographic clades (B–D) of lineages of the L. kuhlii Complex for which more than one osteological exemplar was available (thereby excluding

Clade A). A summary of characters found to optimize unambiguously on these nodes under both

Acctran and Deltran assumptions is presented in Table 3.

3.16

Discussion

Complex diversity

The re-analysis of molecular data from McLeod (in press) in this study resulted in a relatively well supported phylogeny with the monophyletic L. kuhlii Complex comprising 24 distinct haplotype clades, 18 of which are currently subsumed under the nominal L. kuhlii. As discussed above, these 18 unnamed haplotype clades recognized in the molecular phylogeny can be considered as UCS following the definitions of Vieites et al. (2009).

One new UCS is identified here. Lineage 101 in this study, represents sequence data for a single frog of the Limnonectes kuhlii Complex from Matang, Sarawak (Matsui et al., 2010).

Results of the likelihood analysis of molecular data in this study recovered this sample as a unique lineage that is sister to Lineage 19, which comprises anurans allied to Limnonectes asperatus (McLeod, in press). As noted above, the uncorrected mean pair-wise sequence divergence between the Matang sample and other L. kuhlii Complex is quite high (>7.2%).

Matsui et al. (2010) placed the Matang sample in a sister relationship with a single sample a of a frog of the L. kuhlii Complex from Kinabalu, Sabah (Malaysian Borneo). This is not incorrect in the sense that if only two samples of Bornean anurans of the L. kuhlii Complex are included in an analysis, they will form a sister relationship, but it clearly underestimates the true relationships among the Bornean “kuhlii.” No osteological representative for Lineage 19 was available for this study, and thus the relationship between these two UCS cannot be recovered.

However, a weakly supported relationship between Lineages 101 and Sarawak A is recovered in the Bayesian analysis of morphological data. Additional data are necessary to understand the relationships among the Bornean members of this complex. It is worth noting that the two

3.17

Matang samples in this study are from the type locality of Rana conspicillata, Günther (1872), which was subsequently placed into synonomy with L. kuhlii by Günther (1874).

Limnonectes namiyei (Lineage 100 herein) was considered the sister taxon to L. kuhlii

(based on morphology) by Emerson and Berrigan (1993). As noted above, however, they provided no locality information that would identify the lineage of the L. kuhlii Complex that they sampled. Molecular analysis recovers a strongly supported sister relationship between L. namiyei and L. fujianensis. Morphology-only analyses failed to reconstruct this relationship.

Matsui et al. (2010) provided catalog numbers for tissue samples of L. namiyei, but these samples are tail-clips from tadpoles that were released alive (Matsui, pers. comm.); this effectively eliminates an possibility to verify species identification and confounds efforts to reproduce the results of these authors accurately or independently.

Parsimony, Bayesian, and maximum likelihood analyses of morphological data yielded a poorly resolved, but monophyletic Limnonectes clade. Parsimony analysis resulted in greater support of L. kuhlii Complex relationships than did either maximum likelihood or Bayesian analyses, recovering four well-supported groups of L. kuhlii Complex taxa corresponding to UCS recognized with molecular evidence (Lineages 8–10, and 13). The sister relationship between

Lineages 4 and 5 (viz., Clade B) was recovered with low support, but only in the Bayesian analyses. None of the morphological analyses revealed the two samples of Cambodian Lineage

4 to be each other’s closest relatives. Three exemplars of both Lineages 8 and 12 were included in all analyses; however, in both cases, only two of these were recovered as closest relatives. It is interesting that in the case of Lineage 8, the two samples recovered as sister taxa are from the same collection locality, and the other is from a geographically distant population. In the case of

Lineage 12, the two samples that were recovered with low support are from geographically

3.18 distant populations in Myanmar and Thailand, and the third is from the Myanmar population.

Thus, it seems that we are not simply observing inter-populational variation (in the geographic sense) in this phylogenetic context. Analyses of morphology-alone resulted a non-monophyletic

L. kuhlii Complex because of the placement of species not traditionally allied to L. kuhlii (L. blythii, L. gyldenstolpei, and L. laticeps) within the ingroup. These results and the overall lack of resolution may be an artifact of homoplasy in the morphological data or the result of inadequate taxon and character sampling.

Recognizing Candidate Species

Elevating an Unconfirmed Candidate Species to Confirmed Candidate Species status requires corroborating support from character data (Vieites et al., 2009). Support of haplotype clades (UCS) using BPP values from the morphological analyses provides evidence for the recognition of several CCS using this method. Five UCS of the eight lineages of the L. kuhlii

Complex represented by two or more osteological specimens were found to have moderate

(>85%) BPP support. Interestingly, two currently recognized species within the L. kuhlii

Complex had markedly different levels of support in this analysis. Lineage 10 (L. megastomias) has robust support, yet Lineage 8 (L. bannaensis) is recovered with only 22% BPP support. In part, this may be the consequence of the failure of the three samples of L. bannaensis in the morphological analysis to have been recovered as each other’s closest relatives.

With respect to the recognition of UCS and CCS, it is worth noting here that McLeod

(2008) included morphological data from specimens of Linneage 11 (Loei, Thailand) in the description of the L. megastomias, but no molecular data were available for analysis in that study. Sequence data (mtDNA) from eight of these individuals were added to the analyses of

3.19

McLeod (in press) and also were included in this study. Evaluation of these molecular sequences and subsequent re-examination of morphological data seem to support the recognition of Lineage

11 as a CCS, though the BS and BPP support for this lineage in the morphological analysis is slightly less than significant. Furthermore, based on re-examination of previously published data

(McLeod, 2008), results from this study suggest that L. megastomias (Lineage 10) is restricted to two adjacent areas in Thailand—Nakhon Ratchasima and Sa Kaeo provinces.

Utility of Morphology in a Cryptic Species Complex

Historically, taxonomists have diagnosed species using morphological characters that are sufficient for separating the diversity known to the author at that time. In light of new evidence

(i.e., recognition of new diversity), these characters may be found to no longer have the same degree of utility. Results of this study indicate that those characters used historically to diagnose a species may not have universal applicability once the traditional “species” is recognized as a complex of cryptic species. None of the historically recognized characters examined here can be considered synapomorphies for the L. kuhlii Complex. A few characters seem to support particular lineages or clades, but most likely only when considered in tandem with additional characters will they be diagnostic. The graphic presentation of these historically diagnostic traits on a phylogenetic trees serves as a simple and convenient tool for examining the potential phylogenetic and taxonomic utility of these traits in a species complex (Figure 4). All Characters and states examined for this study are discussed and illustrated in Appendix I. A discussion of the six historically significant diagnostic characters for L. kuhlii s.l. is provided in Appendix II.

3.20

Osteological Exploration

Morphological characters with which to evaluate the members of the L. kuhlii Complex were selected without a priori knowledge of the variability (or lack there of) among taxa, especially with regards to osteological features. The intent was to record all character data, whether variable or not, with the understanding that although invariable characters do not help discriminate between clades, they can be of interest when considering other taxonomic and biological questions.

Character optimization under both ACCTRAN and DELTRAN assumptions recovered multiple osteological characters for each of the geographic Clades B–D. Of these, only eight characters optimized unambiguously under both ACCTRAN and DELTRAN methods (Table 3).

These characters do not appear to be synapomorphies for these clades, but may serve as potentially diagnostic features, particularly when taken in combination.

Taxonomic implications and Conclusions

The results of this study clearly demonstrate the Limnonectes kuhlii Complex is exceptionally diverse, certainly more so than previously expected and potentially more so than any other example of an anuran species complex previously studied. Blackburn (2008) revealed multiple cryptic species of Arthroleptis through a molecular phylogenetic analysis. Using mtDNA, Stuart et al. (2006) recognized 14 species (named and unnamed) from within Rana chalconota (+6 cryptic species) and Odorrana livida (+6 species). Using both mitochondrial and nuclear DNA, Fouquet et al. (2007) identified six and eleven lineages from Sinax rubber and

Rhinella margaritifera, respectively. Using multiple lines of evidence, including morphology,

Bain et al. (2003) described six new species from within Rana choloronota. Based on results

3.21 from this study 24 distinct evolutionary lineages are recovered, 17 of which (Lineages 2, 4–6, 9,

11–18, 20–21, and 101) are recognized as UCS on the basis of molecular data alone.

In phylogenetic analyses of morphology alone, five of the UCS (Lineages 5, 9, 11–13) are recovered, two (Lineages 9 and 13) with high BS support from the parsimony analysis.

Additional evidence for elevation of these five lineages to CCS is provided using BPP support.

These results suggest that morphological characters, used in a phylogenetic context, can recover the same sets of relationships hypothesized in a molecular-only analysis. In this light, morphological characters can help to illuminate real biological diversity—even in a species complex where phenotypic similarity has historically obscured cryptic diversity.

Previous authors who have examined specimens of Limnonectes kuhlii s.l. clearly have been struck by the notion that there is some measure of diversity among these frogs.

Nevertheless, external morphology alone has been insufficient, in most cases, for parsing out species-level differences. This study suggests that examination of osteological materials may yield additional apomorphic traits that may help to further resolve the relationships among the members of the L. kuhlii Complex and diagnose lineages therein.

Whereas there is some measure of convenience in the acquisition and analysis of molecular sequence data, it would seem prudent to caution against the use of molecules alone for implementing taxonomic changes. The addition of morphological data, however, provides practical and tangible evidence to support recognizing of species-level diversity. When morphological characters are combined with molecular data, the probability of interpreting evolutionary relationships correctly would seem to be even greater. In this respect, the framework of this study should make it possible to elucidate even greater diversity in the L. kuhlii Complex as new morphological and molecular samples become available. Diagnoses and

3.22 descriptions of these and additional unnamed candidate species will be presented elsewhere

(McLeod, in prep.)

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Figure 1.—Distribution of Limnonectes kuhlii Complex samples used in this study. Squares represent new localities and samples not included in McLeod (in press). Numbers correspond to haplotype clade designations in this study. Where two or more lineages occur sympatrically, lines are drawn to the location and all lineages present at that locality are indicated.

3.31

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Figure 3.—Bayesian topology resulting from analysis of morphological data. Numbers above branches are nonparametric bootstrap support values from Parsimony and Maximum likelihood analyses. Numbers below branches are Bayesian posterior probabilities. Inset “B” shows an alternate topology resulting from Parsimony and Maximum Likelihood analyses. Numbers in brackets refer to specimen IDs in Appendix 4. “NS” indicates that this clade was not recovered in the Maximum Likelihood analysis.

3.33 A B C D

Universal Character C B

Nuptial Tympanum Pads Condition 2#"&"01 <0*+&1+0;1 ./&"01 :+&1+0;1

IN:SVL 3456 RFL 5476 @A?#1"# 7486 =>,()%?#%)?0-"# %9%86

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Figure 4.—Ancestral state reconstruction of six morphological characters used historically to diagnose Limnonectes kuhlii. Characters were mapped onto the Maximum Likelihood tree shown in Figure 3.1, pruned to reflect only those terminals represented in the morphology-only analyses. This figure illustrates how characters once considered diagnostic for a species may not be diagnostic for a species complex. A hypothetical character with universal applicability to all members of the L. kuhlii Complex is shown at the top of the page. Biogeographic clades A– D are shown at the top of the page for reference. 3.34 Table 1.—For the morphological analyses, 15 terminals were assigned to haplotype clades (recognized with molecular data) to maximize the taxon and character sampling for those morphological specimens in which molecular data were not available.

Identity Morphological sample Fejervarya limnocharis DSM 745 Occidozyga laevis KU 307565 Limnonectes blythii DSM 378 Limnonectes gyldenstolpei DSM 1272 Limnonectes gyldenstolpei DSM 1148 Limnonectes laticeps FMNH 198080 Limnonectes kuhlii Lineage 1 LSU81895 Limnonectes fujianensis Lineage 7 KU 194630 Limnonectes bannaensis Lineage 8 KU 292029 Limnonectes namiyei Lineage 100 CAS 22816 Lineage 12 DSM 527 Lineage 16 FMNH 234404 Lineage 20 FMMH 234421 Lineage 21 FMNH 76353 Lineage 101 FMNH 76385

3.35 Table 2.—Summary of support for molecular haplotype clades using morphological data. Bayesian posterior probabilities (BPP) from the analysis of morphological data were summarized on the molecular tree pruned to 25 terminals for which morphological and molecular data were available. Haplotype BPP Clade Support

4 0.04 5 0.90 8 0.22 9 0.87 10 0.99 11 0.94 12 0.09 13 0.92 Lineage B 0.35 Lineage C 0.00 Lineage D 0.00

3.36 Table 3.—Summary of phylogenetically informative osteological characters for biogeographic clades of Limnonectes kuhlii Complex lineages. Characters were optimized Only those characters that optimized at nodes of interest under both ACCTRAN and DELTRAN criteria are included. Further evaluation of these characters is necessary to determine their utility in diagnosing clades or species.

Clade Character No. CI RI HI

B 33 0.250 0.538 0.750 99 0.154 0.312 0.846 143 0.333 0.400 0.667 146 0.286 0.615 0.714 C 27 0.250 0.786 0.750 36 0.250 0.400 0.750 D 38 0.222 0.500 0.778 59 0.333 0.455 0.667

3.37 APPENDIX I:

MORPHOLOGICAL CHARACTERS EXAMINED

Morphological character descriptions are organized by anatomical region and numbered sequentially. Osteological terminology follows that of Trueb (1973; 1993), with efforts to cross- reference terminology with The Amphibian Anatomy Ontology web project (Maglia et al., 2007). Digits of the manus (fingers) are numbered II–V based on homology, not tradition (Alberch and Gale, 1985). To avoid confusion, however, in the text I have referred to Digit II as the “first finger” and so on. For the purpose of coding, however it is important to recognize that Digit II here is equivalent to the traditionally referenced Digit I. The aim here is not to create unjustified confusion, but to accurately present morphological characters in light of our understanding of evolutionary history. Characters related to a single structure are grouped together, but the order in which characters are presented within a region is arbitrary. Each account provides a description of the different character states and their codings; most characters are illustrated for clarity. For consistency, the presence of a character or state is coded “0” and absence as either “1” or as the last option in a multistate character. Most characters are those of Scott (2005); however, many have been modified for the sake of clarity. Other characters are novel to this study. Where appropriate, accounts include discussion of character treatment by other authors. Illustrations of some characters and character states are included at the end of this appendix (Figures I.1–I.5). In figures, cartilaginous structures are blue, bone is grey, and mineralized cartilage is shown in red.

OSTEOLOGY Skull in Dorsal Aspect (Figure I.1; A) 1. Exostosis, cranial bones: present (0); absent (1).

2. Nasals, shape, dorsal view: large, triangular (0); large, rectangular, square, or round (1); small, narrow slip of bone, triangular or club-shaped (2). (Figure I.1; B)

3. Nasals, medial contact: narrow, separated by less than 2 mm (0); wide, separated by more than 2mm (1); sutured (2). (Figure I.1; C)

4. Nasals, relationship to sphenethmoid: overlapping (0); not overlapping (1).

3.38 5. Squamosal, lengths of zygomatic and otic rami: zygomatic ramus longer than otic ramus (0); zygomatic and otic rami approximately equal in length (1); zygomatic ramus shorter than otic ramus (2).

6. Squamosal, position of zygomatic ramus relative to anterior ramus of pterygoid in dorsal view: does not appear to overlap (0); overlapping (1). (Figure I.1; F–G)

7. Squamosal, length of zygomatic ramus relative to length of orbit: distal end of ramus reaching no farther than the mid-length of orbit (0); distal end of ramus extending anterior to mid-point of orbit (1).

8. Squamosal, otic ramus, relation to stapes (columella): not exending beyond posterior margin of stapes (0); extending beyond posterior margin of stapes.

9. Squamosal, otic plate: present (0); absent (1).

10. Squamosal, otic plate, relationship with prootic: overlapping prootic, reaching posterolateral margin of epiotic eminence (0); overlapping prootic, not reaching epiotic eminence (1). (Figure I.1; F)

11. Crista parotica, nature: cartilaginous (0); mineralized (1).

12. Crista parotica, angle in frontal plane, assessed from position of anterior margin of crista parotica: perpendicular to body axis (0); angled forward (1).

13. Crista parotica, posterolateral process: present (0); absent (1).

14. Crista parotica, posterolateral process, shape: exceedingly short, not more than a slight bulge (0); long, with thin, spurlike projection (1); robust, with thick, blunt projection (2). (Figure I.1; G)

15. Frontoparietal fenestra: present (0); absent (1). The frontoparietal fenestra is defined here as any hole or separation in the dermal bones dorsal to the frontoparietal fontanelle.

16. Frontoparietal fenestra, size: large, exposing more than one-third the width of

frontoparietals and fenestra together, often frontoparietals so slender as to be narrower than fenestra (0); small gap, with each frontoparietal narrow, but wider than fenestra (1); small, a round gap at posterior edge of orbits (2). In frogs of the 3.39 Limnonectes kuhlii Complex, a gap is present in the anterior one third of the frontoparietals, but was not considered to be a fenestra. Further investigation may reveal this to be a result of incomplete fusion in an individual animal or a phylogenetically informative species-level character.

17. Frontoparietals, posteromedial process in adult male: present (0); absent (1). This character also occurs in female frogs of the L. kuhlii Complex, but the processes are always poorly developed, indicating that the degree of development corresponds to the hypertrophied condition of the skull and musculature in males.

18. Frontoparietal, medial ridge in adult males: present (0); absent (1). In many frogs examined of the L. kuhlii Complex, the medial ridge also is present in females. Whereas the lack of development of this character in females results in a ridge that is difficult to see, its presence is easily palpable. The medial ridge is a cartilaginous structure that I found to be variably ossified in different specimens (e.g., cartilaginous in male FMNH 262723, ossified in male KU 307774). The degree of ossification in this structure may be a phylogenetically relevant feature, or simply may be an individual, age-determined trait.

19. Frontoparietal, medial ridge, state of development in adult male: poorly developed, a shallow ridge not reaching the crux of the posteromedial Y-shaped flange (0); well developed, a tall ridge reaching the top of the posteromedial Y-shaped flange (1).

20. Frontoparietals, most anterior points of each element: lateral edges configured into a Y- or V-shaped anterior margin (0); uniform, resulting in a straight or T-shaped anterior margin (1); medial and lateral margins extending beyond the midpoint of each element, to produce a W-shaped anterior margin (2); medial edges configured in an inverted “V” or roof-shaped anterior margin (3); mid-point of each element extending beyond either medial or lateral edges of the element, resulting in an M- shaped anterior margin (4). State 3 may be due to the apparent inward curvature of the anterior section of a poorly ossified frontoparietal. Scott (2005) considered State 4 (Character 70, State 3) as “heart-shaped,” but failed to provide an illustration to clarify her definition. (Figure I.1; B–E)

3.40 21. Frontoparietals, forward projection of anterior margin, relation to neopalatine (and planum antorbitale): does not reach posterior margin of neopalatine (0); reaches level of neopalatine (1).

22. Frontoparietal, shape: rectangular, lateral and medial edges parallel (0); wider anteriorly than posteriorly (1); wider posteriorly than anteriorly (2); approximately an isosceles triangle, with apex of triangle facing laterally and base adpressed to other frontoparietal (3). This character does not consider the posterior expansion of the frontoparietal, but examines only the portion forming the roof of the central brain case.

Skull in Lateral and Frontal Aspects

23. Quadratojugal, maxillary contact: present (0); absent (1).

24. Palatoquadrate, pars articularis, mineralization: present (0); absent (1).

25. Premaxilla, alary process, orientation relative to pars dentalis (longitudinal plane): perpendicular [dorsal] (0); backward [posterodorsal] (1); forward [anterodorsal] (2).

26. Premaxilla, angle of alary process (transverse plane): perpendicular [no lateral deviation] (0); inclined laterally away from midline [lateral deviation] (1).

27. Premaxilla, bifid distal end of alary process: present, medial projection longer than lateral, deep U-shaped concavity separates projections (0); absent (1).

28. Tympanic annulus: present (0); absent (1).

29. Tympanic annulus, development: complete (0); incomplete (1).

30. Tympanic annulus, shape: round (0); pear-shaped, involving squamosal as its dorsal limit, with dorsal section of cartilage fused onto squamosal (1). The presence, development, and shape of the tympanic annulus are not logically independent characters, but it seems reasonable to score them as such for the sake of capturing relevant variation.

31. Pars media plectri of the stapes (columella): present (0); absent (1).

Skull in Ventral Aspect

3.41 32. Sphenethmoid, ventromedial portion: fused (0); paired (1). In the examples of Limnonectes examined in this study, the sphenethmoid is fully fused and mineralized anteriorly, but seems to lack mineralization beneath the cultriform process of the parasphenoid. In all Limnonectes sp. examined the sphenethmoid is scarcely visible dorsally, as the nasals and frontoparietals articulate with one another.

33. Sphenethmoid, extent of forward expansion of mineralized anteroventral portion (antrum pro lobo olfactoria, sensu Gaupp, 1896): ventral sphenethmoid narrow, adpressed to braincase in the region of the planum antorbitale, no additional mineralization extending anteriorly (0); extending beyond the planum antorbitale, covering less than half the palate; (1) extending forward considerably, covering half the palate or more (2).

34. Neopalatine: present (0); absent (1).

35. Neopalatines, development: well developed (0); reduced to thin slivers of bone (1).

36. Neopalatine, medial end of neopalatine, shape: squared (0); pointed (1); slight posterior elongation (2); great posterior elongation (3). (Figure I.2; C–D)

37. Neopalatines, ventral ridge: present (0); absent (1). (Figure I.2; A–B)

38. Neopalatines, ventral ridge, shape in posterior–anterior view: rectangular, tapering slightly at both medial and lateral ends (0); triangular, tapering on lateral end (1); round, slightly raised mound (2). This character seems somewhat plastic. When present, this ridge can be seen along the midline of the neopalatine, extending from the maxillary edge towards (but never reaching) the medial end. There is occasionally bilateral asymmetry in this character.

39. Vomer, anterior process: present (0); absent (1).

40. Vomer, anterior process, anterior attainment of premaxilla-maxilla articulation: present (0); absent (1).

41. Vomer, lateral margin between anterior process and prechoanal process: smooth (0); irregular or weakly serrate (1); coarsely serrate (2); bifid (3).

3.42 42. Vomer, postchoanal process, development: poorly developed, in same plane (horizontal) as prechoanal process and body of vomer (0); strongly developed, vertical or oblique, not in same plane as prechoanal process (1). Pre- and postchoanal processes are here defined as the specific vomerine elements forming the margins of the choanae.

43. Vomer, postchoanal process, fusion with sphenethmoid: present (0); absent (1).

44. Vomer, posterior process: present (0); absent (1). The posterior process of the vomer is here defined as any portion of the vomer that extends posteriorly along the medial margin of the vomer and the beyond the vomerine corpus or dentigerous process.

45. Vomerine teeth: present (0); absent (1). (Figure I.2; E)

46. Vomerine tooth row, length relative to line drawn through axis of dentigerous process and extending to lateral margin of vomer: less than one-third total length

(0); one-third to one-half total length (1) ; greater than one-half total length (2).

47. Maxillary and premaxillary teeth: present (0); absent (1).

48. Premaxilla, pars palatina, medial palatine process, ratio of length to width (measured from the pars palatina) relative to lateral palatine process: less (0); equal (1); greater (2).

49. Maxilla, anteromedial flange of pars palatina: present (0); absent (1).

50. Maxilla, anteromedial flange of pars palatina, anterior process, form: thin, strongly curved anterolaterally, tip of process extends beyond anterior flange (0); robust, weakly curved anterolaterally, tip of process extends beyond anterior flange (1); robust, weakly curved anterolaterally, tip of process does not extend beyond anterior flange (2); thin, oriented medially, tip extends beyond flange [if present] (3). (Figure I.2; A)

51. Pterygoid, anterior ramus, contact with maxilla (in ventral/ventrolateral view): present, adpressed (0); absent, separated by cartilage (1).

52. Pterygoid, anterior ramus, relation to neopalatines and planum antorbitale: greatly separated, reaching only to midorbital level (0); almost in contact or slightly 3.43 overlapping (1); curving medially away from maxilla toward an enlarged, wider planum antorbitale, separated from lateral border of planum antorbitale by a wide gap (2).

53. Pterygoid, medial ramus: long and relatively wide (0); reduced in proportional length, even to rudimentary bumps (1).

54. Mandibular odontoids: present (0); absent (1). The presence of odontoid processes clearly is related to the shape of the medial process of the pars palatina and the anterior process (and flange) of the maxilla—the elements that create a socket in the palate into which the odontoid fits.

55. Mandibular odontoids, composition: formed by dentary (0); formed by both dentary and mentomeckelian bones (1). In all specimens examined of the L. kuhlii Complex, the cartilaginous connection between odontoids and Mentomekelian bones was densely mineralized. This condition, however, should not be mistaken for a case of odontoids developing from the mentomekelian bones, because in this group, odontoids clearly originate from the dentary.

56. Mentomeckelian bone, relative height of medial versus lateral end (anterior aspect): equal (0); less (1).

57. Mentomeckelian bone, lateral process: present (0); absent (1).

58. Mentomeckelian bone, fusion to dentary: present (0); absent (1).

59. Parasphenoid, cultriform process, shape of tip: robust, rounded (0); robust, sharply pointed (1); bifid (2); squared (and serrate) (3); narrow, pointed (4). (Figure I.2; C–E)

60. Parasphenoid, cultriform process, anterior terminus: near anterior margin of orbit, but posterior to levels of neopalatines and planum antorbitale (0); between midlength and anterior two thirds of orbit (1); at level of neopalatines and planum antorbitale (2); anterior to anterior margin of neopalatines and planum antorbitale (3).

61. Parasphenoid, lateral (subotic) alae, in frontal plane, assessed using the anterior margin: perpendicular to body axis [lateral] (0); oriented slightly anterolaterally (1); oriented slightly posterolaterally (2).

3.44 62. Parasphenoid, lateral (subotic) alae, length: moderately long (0); reduced or short (1).

Hyolaryngeal Apparatus

63. Hyoid, hyale, free flange toward jaw just anterior to jaw angle: present (0); absent (1).

64. Hyoid, hyale, anterior horn: long, straight, thin (0); long and usually curled, relatively thick (1); small nipple-shaped knob (2); slightly elongated, but not more than three times its width (3); absent (4).

65. Hyoid, anterolateral (alary) process, shape of stalk: narrow (0); broad (1); absent or so broadly connected to the hyoid plate that no stalk is evident (2).

66. Hyoid, anterolateral (alary) process, angle of stalk: anterior orientation (0); lateral orientation (1).

67. Hyoid, hyoglossal sinus, with/depth ratio: less than 1.1 (0); 1.1–1.5 (1); greater than 1.5 (2). The hyoglossal sinus is the space that described by the anterior margin of the hyoid plate and hyale that flank it laterally. This sinus may be partially occluded by a trans-sinus membrane.

68. Hyoid plate, shape: wide, width greater than length (0); narrow, longer than wide (1); uniform, length equal to width (2).

69. Hyoid plate, posterolateral process: present (0); absent (1).

70. Hyoid plate, posterolateral process, length: long, greater than half the length of posteromedial process (0); short, one-half to one-third the length of posteromedial

process (1); very short, less than one-third the length of posteromedial process (2).

71. Hyoid plate, posterolateral process, shape: straight, acuminate (0); sickle shaped (1).

72. Hyoid plate, posteromedial process (thyrohyals), expanded flange on medial side: present (0); absent (1).

73. Hyoid plate, posteromedial process (thyrohyals), expanded flange on lateral side: present (0); absent (1).

3.45 74. Hyoid plate, posteromedial process (thyrohyals), width of distal epiphysis relative to proximal epiphysis: narrower (0); wider (1); equal (2).

75. Hyoid plate, distance between posteromedial processes relative to width of proximal epiphysis of posteromedial process: narrower (0); wider (1); equal (2).

76. Hyoid plate, posteromedial process, extent of ossification: present within process (0); ossification abuts hyoid plate (1); ossification invades body of hyoid plate (2).

Vertebral Column

77. Presacral vertebrae, number: 7 (0); 8 (1); 9 (2); 10 (3).

78. Presacral vertebrae I, neural arch ossification: medially complete (0); medially incomplete (1). In specimens of Limnonectes sp. examined, the neural arch of Presacral vertebrae I is incomplete (State 0), but in all cases, the cartilaginous bridge is fully mineralized.

79. Presacral vertebrae I, atlantal intercotylar distance relative to width of cotyl: wide, more than one cotyl’s width apart (0); equal (1); narrow; less than one cotyl’s width apart (2).

80. Presacral vertebrae, last presacral vetebra, lengths of transverse processes relative to those of Presacral IV: much shorter (0); approximately equal (1).

81. Presacral vertebrae, last presacral vertebra, orientation of transverse processes in frontal plane: lateral orientation, perpendicular to vertebral column (0); slight anterolateral orientation, approximately 20–30° (1); acute anterolateral orientation, approximately 45° or more.

82. Presacral vertebrae II–IV, neural spines: present (0); absent (1).

83. Presacral vertebrae, last presacral vertebra, condition of centrum: amphicoelous (0); opisthocoelous (1); procoelous (2).

84. Sacral vertebra, fusion with last presacral vertebra: present (0); absent (1).

Pectoral Girdle

3.46 85. Suprascapula, suprascapular cartilage, ossification: limited, only proximal section ossified, forming a Y-shaped flange of mineralization with the cleithrum, with fork facing dorsally (0); extensive, more than one third of blade, forming one rounded, rectangular or triangular flange with cleithrum on the anteroproximal surface (1).

86. Clavicle: present (0); absent (1).

87. Clavicle, medial terminus at midline: present (0); absent (1).

88. Clavicle, appearance in ventral aspect: approximately equal in width along entire length, robust relative to coracoid (i.e., ≥ ½ width of coracoid) (0); approximately equal in width along entire length, but slender relative to coracoid (i.e., < ½ width of coracoid) (1).

89. Clavicle, orientation relative to longitudinal body axis: strongly or slightly bowed, anteromedial orientation greater than 35º (0); straight and approximately perpendicular, with anteromedial orientation being less than 30º (1).

90. Epicoracoid, midline relationship of contralateral componenets: wide overlap (0); slight overlap (1); fusion (2).

91. Coracoid, shape of sternal end: trumpet-shaped (0); T-shaped (1); Y-shaped, with broader sternal end than in State 0, and narrowing substantially toward the glenoid end (2).

92. Coracoid, shape, dilation of sternal end relative to glenoid end: less than 1.3× width of glenoid end (0); more than 1.4× width of glenoid end (1).

93. Omosternum, style, ossification (mineralization): present (0); absent (1).

94. Omosternum, style, shape, posterior bifurcation: present (0); absent (1).

95. Omosternum, style, shape of anterior process: uniformly tapered and thin, its width less than or equal to that of one basal process (0); slightly dilated at anterior end, its width greater than that of one basal process (1); slightly dilated at anterior end, its width equal to or greater than that of one basal process (2).

3.47 96. Omosternum, style, width of posterior gap relative to width of one of the basal processes: gap less than the width of one process (0); gap equal to the width of one posterior process (1); gap greater than the width of one posterior process (2).

97. Mesosternum: present (0); absent (1).

98. Mesosternum, ossification (mineralization): present (0); absent (1).

99. Mesosternum, shape, width of proximal end relative to distal end: approximately equal (0); wider (1).

100. Mesosternum, shape, length relative to width: length less than 2× width (0); length 2–2.9× width (1); length more than 3× width (2). Width of the mesosternum was measured at the narrowest point of constriction.

101. Xiphisternum, shape: large and round (0); approximately X-shaped, wider than long (1); Bi-lobed, wider than long (2); approximately X-shaped, equally as long as wide (3); large,∪-shaped plate (4); rectangular with smooth posterior edge (5); large, anchor shaped (6); narrow and rectangular, divided with two long projections with distal expansions (7); rectangular with strongly serrated posterior margin (8). (Figure I.3)

Pelvic Girdle

102. Postsacral vertebrae, fusion: urostyle present with discrete postsacral vertebrae (0); urostyle present with no separate postsacral vertebrae (1); absent (2).

103. Urostyle, dorsal ridge (or crest, crista dorsalis sensu Gaupp, 1896): present (0); absent (1).

104. Urostyle, dorsal ridge, length relative to body of urostyle: less than half (0); half (1); more than half (2).

105. Urostyle, anterodorsal process at anterior edge of dorsal ridge: present (0); absent— i.e., poorly developed, indistinct to absent (1).

106. Urostyle, transverse process: present (0); absent (1).

107. Ilium, dorsal protuberance: present (0); absent (1).

3.48 108. Ilium, dorsal protuberance, shape and orientation: oval, vertical (0); spike-like or slightly rounded, projecting laterally (1); large spike- or flange-like, not adpressed to shaft (2). Definition of dorsal protuberance follows that of Lynch (1971). It is unclear whether Scott (2005), Clarke (1981), and Lynch (1971) are all using the terms “dorsal protuberance” and “dorsal prominence” in the same manner.

109. Ilium, height of dorsal crest as measured centrally: ≤ 1× height of ilial shaft (0); 1.1–2.0× height of ilial shaft (1); ≥ 2.1× height of ilial shaft (2); absent (3).

110. Ilium, ilial shaft: uniform height (0); ventral flange present resulting in a greater height posteriorly than anteriorly (1). CS 1 is seen only in L. blythii.

111. Sacral diapophyses, expansion (ratio of distal width to proximal width): strongly dilated, ratio = ≥ 2 (0); slightly dilated, ratio = 1.1–1.9 (1); undilated, ratio ≤ 1 (2).

112. Sacral diapophyses, distal ends in lateral aspect: dorsoventrally compressed (0); cylindrical (1).

113. Sacral diapophyses, orientation of anterior margin: posterolateral (0); lateral, perpendicular to vertebral column (1); anterolateral (2).

Limbs

114. Carpal state sensu Laruent and Fabrezi (1990): Type A, 7 elements with Distal Carpalia (DC) 3, 2, and Element Y free (0); Type B, 6 elements, DC 2 fused with Element Y, DC 3 free (1); Type C, 5 elements, DCs 3 and 2 and Element Y fused (2); Type D, 6 elements, DC 2 and Element Y free, DCs 3–5 fused (3); Type E, 5 elements, DC 2 fused to Element Y, DCs 3–5 fused (4); Type F, 4 elements, Element Y and DCs 2–5 fused (5); as in Type D, 6 elements, but incomplete fusion of DC 3 with 4 and 5, suture visible (6).

115. Digits, manus and pes, intercalary element between penultimate and terminal phalanges: present (0); absent (1).

116. Digital subarticular sesamoids of manus: present (0); absent (1).

117. Aponeurosis palmaris sesamoid: present (0); absent (1).

118. Prepollical elements, number: 1 (0); 2 (1); 3 or more (2).

3.49 119. Prepollex, fusion with Metacarpal II: present, forming fighting spike (0); absent (1).

120. Prepollex, distal element, length relative to proximal element: less than twice length (0); more than twice length (1). (Figure I.4; B)

121. Humerus, crista lateralis in mature male: present proximally (0); present distally (1); absent (2).

122. Humerus, crista ventralis: length of flange relative to humeral length: long, about half length of humerus (0); short, about a quarter or third length of humerus (1).

123. Humerus, crista ventralis, shape of distal margin: tapered, gradually grading to bone (0); truncated, ending abruptly (1).

124. Metacarpal of Digit IV in breeding males, distal tuberosity: present (0); absent (1). It is important to note that digits of the manus are numbered II–V based on homology.

125. Metacarpal of Digit II of breeding male, nuptial tuberosity on outer edge: present (0); absent (1).

126. Metacarpal of Digit II in breeding male: uniformly thicker than other metacarpals, lacking spinous projections (0); thicker, with spine-like projections that may or may not penetrate the skin (1); like other metacarpals (2).

127. Manus, Digit IV, terminal phalanx, shape: straight (0); curved ventrally (1).

128. Manus, Digit IV, terminal phalanx, distal tip, shape: knob-like (0); pointed (1); bilobed (2); T-shaped (3); Y-shaped (4).

129. Pes, Digit IV, terminal phalanx, shape: straight (0); curved ventrally (1). (Figure I.5; C)

130. Pes, Digit IV, terminal phalanx, distal tip, shape: knob-like (0); pointed (1); bilobed (2); T-shaped (3); Y-shaped (4). (Figure I.5; B, D)

131. Prehallical elements, number: 1 (0); 2 (1); 3 or more (2).

132. Distal Tarsal 1 (not naviculare): present, independent element, not fused (0); absent, fused, not independent element (1).

133. Distal Tarsals 2 and 3, fusion: present (0); absent (1).

3.50 134. Tarsometatarsal joint, ventromedial surface, sesamoids: present (0); absent(1).

135. Tarsometatarsal joint, ventrolateral surface, sesamoids: none (0); one (1); two (2); three (3).

136. Tarsal sesamoid: present (0); absent (1).

137. Cartilago sesamoides: present (0); absent (1).

External Morphology

138. Breeding males, nuptial excrescences on manus: on Digit II (0); on Digits II and III (1); on Digits II–IV (2); absent (3).

139. Breeding male, vocal sac, evidenced by buccal vocal slits: present (0); absent (1).

140. Mandibular odontoid, size of odontoid relative to head length (HL): small, less than 3% of HL (0); medium, 3–6% of HL (1); large, 6–9% (2), extremely large, more than 9% of HL (3). Odontoid size was determined by subtracting mandibular height from total odontoid height (McLeod, 2008).

141. Tympanic membrane, superficial visibility: indistinct, covered by skin of similar thickness to that on remainder of head (0); distinct, as skin over tympanum is thinner than on remainder of head (1); partly distinct, only a crescent visible (2).

142. Tympanic membrane (tympanum), diameter relative to diameter of eye: less than 50% of eye diameter (ED) (0); 50–99% of ED (1); greater than ED (2).

143. Internarial distance relative to snout–vent length (SVL): 5–6% SVL (0); 6–7% SVL (1); 7–8% SVL (2); 8–9% SVL (3); more than 9% SVL (4).

144. Manus, Digit II, length relative to Digit III when adpressed: shorter (0); equal or longer (1).

145. Body size: very small frogs (0) = 10–39 mm; small (1) = 40–49 mm; medium (2) = 50–59 mm; large (3) = 60–69 mm; exceptionally large (4) = 70+ mm. Mean SVL was coded for each clade, such that size classes are defined as a function of mean SVL for adult males and females combined. Averaging SVL across all adult individuals (as identified by the presence of secondary sexual characters, or ova in females) is considered here to be a conservative method for differentiating between 3.51 clades, and as a way to reduce the effect of male-biased size dimorphism, which is considered a general characteristic for members of the genus Limnonectes (Emerson et al., 2000).

146. Sexual dimorphism, adult female SVL relative to adult male SVL: approximately equal (0); smaller (1); larger (2).

3.52 Literature Cited

Alberch, P., Gale, E. 1985. A Developmental Analysis of an Evolutionary Trend: Digital

Reduction in Amphibians. Evolution 39, 8–23.

Clarke, B. T. 1981. Comparitive Osteology and Evolutionary relationships in the African

Raninane (Anura Ranidae). Italian Journal of Zoology Supp. XV, 285–331.

Emerson, S., Inger, R., Iskandar, D. 2000. Molecular systematics and biogeography of the fanged

frogs of Southeast Asia. Molecular Phylogenetics and Evolution 16, 131–142.

Lynch, J. D. 1971. Evolutionary relationships, osteology and zoogeography of Leptodactyliod

frogs. Univ. of Kansas Publs. Mus. Nat. Hist. 53, 1–238.

Maglia, A. M., Leopold, J. L., Pugener, A. L., Gauch, S. 2007. The Amphibian Anatomical

Ontology web project.

Scott, E. 2005. A phylogeny of ranid frogs (Anura: Ranoidea: Ranidae), based on a simultaneous

analysis of morphological and molecular data. Cladistics 21, 507–574.

Trueb, L. 1973. Bones, frogs, and evolution. In: Vial, J. G. (Ed.) Evolutionary Biology of the

Anurans: Contemporary Research on Major Problems. University of Missouri Press,

Columbia, Missouri, pp. 65–132.

Trueb, L. 1993. Patterns of cranial diversity among the Lissamphibia. . In: Hanken, J., Hall, B.

K. (Eds.), The Skull, Volume 2: Patterns of Structural land Systematic Diversity. Chicago

Press, Chicago, pp. 255–343.

3.53 Table I.1.—Character numbers used by Scott (2005) and this study are shown to facilitate comparison between studies. New characters in this study have no corresponding number in Scott (2005). Characters modified from Scott (2005) may appear multiple times, indicating that her character was split into multiple characters for the sake of clarity in this study.

McLeod Scott McLeod Scott McLeod Scott McLeod Scott 1 61 41 81 3 121 113 2 64 42 40 82 6 122 3 63 43 41 83 18 123 4 62 44 42 84 7 124 115 5 73 45 44 85 8 125 116 6 46 86 126 117 7 47 45 87 127 8 48 46 88 20 128 9 65 49 47 89 21 129 10 65 50 47 90 23 130 11 67 51 48 91 24 131 12 68 52 49 92 25 132 103 13 66 53 50 93 133 14 66 54 94 134 106 15 69 55 52 95 135 107 16 69 56 53 96 136 109 17 57 97 137 110 18 58 54 98 138 132 19 59 56 99 139 137 20 70 60 57 100 140 21 61 59 101 32 141 144 22 62 60 102 142 145 23 75 63 82 103 8 143 24 76 64 83 104 144 25 78 65 84 105 10 145 26 79 66 87 106 11 146 27 67 107 146 28 80 68 91 108 12 29 80 69 93 109 13 30 80 70 94 110 31 81 71 111 14 32 33 72 95 112 15 33 34 73 96 113 16 34 36 74 97 114 102 35 36 75 98 115 36 76 116 105 37 77 117 108 38 78 0 118 112 39 39 79 19 119 111 40 39 80 2 120

3.54

CAS

—

G );

FMNH 20); 234421 (Lin. —

limnocharis F.

CUMZA 2003.237 (

—

E );

L. bannaensis KU 292029 ( — ); C );

L. namiyei Skull in dorsal view. The following specimens are illustrated above: A, D, F above: illustrated are following specimens The view. dorsal in Skull — CAS 22816 ( — Figure I.1 Figure B 12). 221714 (Lin.

3.55

KU

—

B, E 13); 02 (Lin. TNE

—

bars = 5 mm ). Scale

F. limnocharis F. CUMZA 2003.237 ( — ); C ); Skull in ventral view. The following specimens are illustrated above: A, D above: illustrated are following specimens The view. ventral in Skull — O. laevis .2 I Figure Figure 307565 (

3.56

KU CAS9); Java); 221808 (Lin. ). Scale bar = 5 mm. bar ). Scale

L. kuhlii O. laevis ); KU); 307565 ( Xiphisternum in ventral view. The following specimens are illustrated above above illustrated are following specimens The view. ventral in Xiphisternum — L. megastomias .3 I Figure Figure LSU 81895 ( bottom): to top right, to (left 307775 (

3.57

numbered are digits that ). Note

L. megastomias

Manus dorsal view. Specimen illustrated above: KU 307774 ( above: illustrated Specimen Manus view. dorsal — .4 I V based on homology. Scale bar = 5 mm. bar Scale V on homology. based – Figure Figure II

3.58

B, FMNH 128181

–

). Scale bar = 2 mm. bar ). Scale

F. limnocharis F. D, CUMZA 2003.233 ( – Digits of pes in lateral (A, C) and dorsal (B, D) views. Specimens illustrated above: A above: illustrated (B, D) (A,dorsal views. Specimens C) and ofDigits lateral pes in — .5 I Figure Figure C “B”); (Sarawak

3.59 APPENDIX II:

DISCUSSION OF MORPHOLOGICAL CHARACTERS HISTORICALLY USED

TO DIAGNOSE LIMNONECTES KUHLII

Numerous authors have cited the hidden or “indistinct” tympanum (viz., the tympanic annulus as it appears covered with skin; Char. 141 in this study) as one of the key diagnostic features of Limnonectes kuhlii (e.g., Boulenger, 1920; Duméril and Bibron, 1841; Inger, 1966;

Iskandar, 1998; Taylor, 1962). Emerson and Berrigan (1993) considered a hidden tympanum the one unambiguous synapomorphy for the clade comprising L. kuhlii, L. namiyei, and L. laticeps.

Based on the results this study, it is evident that within the L. kuhlii Complex, this character is highly variable. Whereas most specimens examined have a hidden tympanum (Character 141:0), many specimens from Lineages 1, 10, 11, 12, and Limnonectes gyldenstolpei have a visible tympanum (Character 141:1; Fig. 4). Limnonectes gyldenstolpei is sister to L. laticeps (Evans et al., 2003; McLeod, in press).

The lengths of the first and second fingers (Char. 144) often are cited as being equal in length or the first finger longer than the second (Boulenger, 1920; Inger, 1966; Malkmus et al.,

2002; Taylor, 1962). In this study, only 49 of 291 samples (3 in the osteological specimens) were found to have the first finger equal to or longer than the second (144:1). Most Javan specimens exhibit Character State 1, as do many (but not all) representatives of the Bornean lineages. However, three of twenty-one Javan specimens I examined have Character State 0

(i.e., first finger shorter than second), ironically, one of these is the single osteological voucher represented on the tree in Figure 4. This is a prime example where having a single voucher to

3.60 represent the biological diversity in a single species (let alone a species complex) could lead to an underestimation or misrepresentation of real diversity.

Boulenger(1920), Inger (1966), and Malkmus et al. (2002) described male Limnonectes kuhlii as lacking nuptial pads (Char. 138). Pope (1931) noted nuptial pads on the first digit of

Chinese “L. kuhlii.” In this study, nuptial pads were found on 133 of 146 males examined. In most cases (94), the nuptial pad was only on the first digit; however, in 38 males, nuptial pads were on the first and second digits, and in one case, nuptial pads were found on the first three digits of a large specimen of L. megastomias. Interestingly, the only haplotype clade in which all males examined lack nuptial pads is Lineage 1—L. kuhlii from Java.

Inger (1966) used internarial distances (char. 143, this study) to differentiate among

Bornean populations of Limnonectes kuhlii from Sarawak and those from Sabah and Kalimantan.

Although this character varies within and between populations, it may be phylogenetically significant. For example, among the osteological exemplars used here, Character 143:2 is found only in lineages of Clade C.

With regard to body size (Char. 145) and sexual size dimorphism (Char. 146),

Limnonectes kuhlii always is regarded as exemplifying male-biased size dimorphism resulting from the hypertrophied heads of males. Pope (1931) noted that males actually have smaller bodies if one accounts for head size. Here, I have considered body size as a function of mean

SVL for adult males and females combined. Averaging SVL for all adults (as identified by the presence of secondary sexual characters, or ova in females) seems to be a conservative method for differentiating among clades, and as a way to reduce the effect of male-biased size dimorphism, which has been used to characterize the genus Limnonectes (Emerson et al., 2000).

Interestingly, when comparing male and female SVLs, the results of this study demonstrate that

3.61 this male-biased size dimorphism does not hold true for all clades in the Limnonectes kuhlii

Complex. Coding for sexual size dimorphism was determined by examining mean SVL for adults of each sex in a given clade. Depending on the sample size and the proportion of each sex in the sample, coding for this character could be skewed and, therefore, should be treated with some caution. Nevertheless, in some larger samples (e.g., Clade 8, Limnonectes bannaensis), a total of 43 males and 22 females was examined from China, Vietnam, and Lao PDR. Males and females have mean SVL measures of 58.2 mm and 62.3 mm, respectively, indicating that in this lineage, females are larger than males.

Emerson and Berrigan (1993) included karyotype number (2N = 22) and the enlongation of the zygomatic ramus of the squamosal as characters used to unite the Limnonectes kuhlii, L. namiyei, and L. laticeps clade in their study. In the present study, all but two individuals examined of the L. kuhlii Complex possess an elongated zygomatic ramus of the squamosal

(Char. 5:0). After examination of specimens for this study, it is clear that the length of the zygomatic ramus varies among lineages and between sexes (Char. 7), and seems to exhibit a pattern of allometric growth, particularly in males. Karyotype data were not collected for this study, but it is worth noting that the Emerson and Berrigan (1993) provided no specimen number or collection locale for their sample of L. “kuhlii.” This is problematic given the current understanding of the relationships within the L. kuhii Complex because it confounds the re- interpretation of their results. This is especially significant when considering karyotype data, because not all members of the L. kuhlii Complex share the same diploid chromosome number.

Limnonectes namiyei, L. bannaensis, and L. fujianensis share 2N = 22 (Kou et al., 1990;

Kuramoto, 1972; Peng et al., 2005), whereas L. kuhlii from Java is reported to have a 2N = 26 state (Iskandar, 1998), and karyotype data are not available for other members of the L. kuhlii

3.62 complex for comparison. Iskandar (1998) provides neither locality data nor any original citation for karyotype data and thus, it is assumed that the source for these data is from his personal work using a Javan sample of L. kuhlii.

Literature Cited

Boulenger, G.A., 1920. A monograph of the South Asian, Papuan, Melanesian and Australian

frogs of the genus Rana. Records of the Indian Musuem 20, 1–223.

Duméril, A.M.C., Bibron, G., 1841. Erpetétology Général ou Histoire Naturelle complète des

Reptiles. Libraire Encyclopédique de Roret, Paris.

Emerson, S., Berrigan, D., 1993. Systematics of Southeast Asian ranids: multiple origins of

voicelessness in the subgenus Limnonectes (Fitzinger). Herpetologica 49, 22–31.

Emerson, S., Inger, R., Iskandar, D., 2000. Molecular systematics and biogeography of the

fanged frogs of Southeast Asia. Molecular Phylogenetics and Evolution 16, 131–142.

Evans, B.J., Brown, R.M., Mcguire, J.A., Supriatna, J., Andayani, N., Diesmos, A., Iskandar, D.,

Melnick, D.J., Cannatella, D.C., 2003. Phylogenetics of Fanged Frogs: Testing

Biogeographical Hypotheses at the Interface of the Asian and Australian Faunal Zones.

Systematic Biology 52, 794–819.

Inger, R., 1966. The systematics and zoogeography of the amphibia of Borneo. Fieldiana:

Zoology 52, 402.

Iskandar, D.T., 1998. The amphibians of Java and Bali. Research and Development Center for

Biology–LIPI.

3.63 Kou, Z.-t., Li, W.-h., Jin, A.-l., Song, Z., 1990. Rare karyotype of Genus Rana—A preliminary

study on the karyotype and C-Banding of Rana kuhlii Dumeril et Bibron. In E.M Zhao

(ed) From Water onto Land, Chinese Forestry Publishing House, Bejing:, 159–163.

Kuramoto, M., 1972. Karyotypes of the six species of frogs (genus Rana) endemic to the Ryuku

Islands. Caryologia 25, 547–559.

Malkmus, R., Manthey, U., Vogel, G., Hoffmann, P., Kosuch, J., 2002. Amphibians and Reptiles

of Mount Kinabalu (North Borneo). A.R.G. Ganter Verlag Kommanditgesellschaft,

Ruggel.

McLeod, D.S., in press. Of Least Concern? Systematics of a cryptic species complex:

Limnonectes kuhlii (Amphibia; Anura: Dicroglossidae).

Peng, Q., Wang, Y., Xu, J., Tang, X., Zhang, J., Ji, D.-L., Hu, L.-L., Nie, L.-W., 2005.

Karyotype, C-band and Ag-NORs of Limnonectes fujianensis. Chinese Journal of

Zoology 40, 77-78.

Pope, C., 1931. Notes on amphibians from Fukien, Hainan, and other parts of China. Bulletin of

the American Museum of Natural History 61, 397–611.

Taylor, E.H., 1962. The Amphibian Fauna of Thailand. The University of Kansas Science

Bulletin 43, 599.

3.64

39 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

38 Char.

------

1 0 0 2 2 2 1 1 2 1 0 2 1 2 0 0 0 0 1 1 0 0 2 2 2 1 2 2

37 Char.

1 0 0 0 1 0 0 0 0 0 0 1 0 1 1 0 0 1 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0

36 Char.

2 0 0 0 1 3 0 2 2 3 2 2 2 2 2 2 2 0 2 2 2 0 0 0 0 3 1 0 2 2 2 1 1 0 0 0

35 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

34 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

33 Char.

0 0 0 0 0 0 0 0 1 1 1 1 1 1 2 0 1 0 1 0 1 1 0 0 0 0 1 1 1 0 0 1 1 1 1 1

32 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

31 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

30 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

29 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

28 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

27 Char.

1 0 0 1 0 0 0 1 1 1 1 1 1 1 1 1 1 0 1 0 0 1 0 0 0 0 1 1 0 1 0 1 1 0 0 0

26 Char.

1 0 0 1 1 1 1 1 0 1 1 0 1 1 0 0 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0

25 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0

24 Char.

1 1 0 0 0 0 1 0 0 ? ? 0 0 0 ? 1 1 1 0 0 0 0 1 1 1 0 1 1 0 1 0 1 1 1 0 0

23 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

22 Char.

0 0 0 0 0 1 2 0 0 1 1 0 0 0 0 0 1 0 1 2 1 1 2 2 2 2 2 2 2 2 0 1 1 1 0 0

21 Char.

0 1 1 1 1 0 1 1 1 1 0 0 0 0 0 1 1 0 0 0 1 0 1 1 1 0 1 0 0 0 1 1 1 0 1 1

20 Char.

3 3 3 3 3 2 3 3 3 1 1 2 3 3 2 3 3 3 3 3 3 3 3 3 1 1 4 4 4 4 3 3 3 1 2 3

19 Char.

------0 1 0 0 1 1 0 0 0 1 1 1 1 1 0

18 Char.

------0 0 0 1 0 0 0 0 0 0 0 0 1 1 1 0 0 0 0

17 Char.

------0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0

16 Char. ------1 1

Char.

. 15

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1

14 Char.

- - - - 0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0

13 Char.

ATRIX 0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0

Char.

M 12

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 1 1 0 1 0

11 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

10 Char.

- - - - 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1

9 Char. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0

HARACTER 8 Char.

1 1 1 1 1 0 1 1 1 ? ? 1 1 1 1 1 1 0 1 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 0 0

C

7 Char.

0 0 1 0 1 1 0 0 0 1 0 1 0 0 0 0 1 0 1 0 1 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0

6 Char.

0 1 1 0 1 0 1 0 0 0 1 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0

5 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1

4 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

3 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

2 Char.

ORPHOLOGICAL ORPHOLOGICAL 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

Char. M 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

: III PPENDIX A LSU81895 KU307775 KU307774 DSM378 DSM527 KU292029 KU194630 FMNH234404 FMNH234421 FMNH76353 FMNH76385 FMNH258508 FMNH262730 FMNH262723 FMNH258507 FMNH137443 FMNH128181 CAS230949 FMNH266218 FMNH266212 CAS221808 CAS210195 TAO699 TNE02 TAO701 TNE03 DSM745 CUMZA2002.237 KU306935 KU307565 CAS221714 DSM1272 DSM1148 CAS22816 FMNH198080 FMNH198082 3.65

78 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1

77 Char.

1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

76 Char.

2 2 2 2 2 2 1 2 2 ? ? 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 2 2 2 2 2 2 2 2

75 Char.

0 0 0 0 2 1 0 0 1 ? ? 2 0 1 0 0 0 0 0 0 0 0 0 0 1 0 2 0 0 2 2 0 0 0 0 0

74 Char.

0 0 0 0 2 0 0 0 0 ? ? ? 0 2 2 0 0 0 0 ? 0 2 0 0 0 0 0 0 0 ? 0 0 ? ? 0 0

73 Char.

1 1 1 1 1 1 1 1 1 ? ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1

72 Char.

1 1 1 1 1 1 1 1 1 ? ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1

71 Char.

0 1 1 1 1 1 1 1 1 ? ? 1 0 0 0 1 ? 1 0 0 1 1 0 0 1 1 0 0 0 0 1 1 0 1 0 0

70 Char.

0 1 1 0 0 1 0 1 1 ? ? ? 1 1 1 0 1 0 0 ? 0 0 0 1 0 0 1 0 0 0 0 0 ? ? 0 0

69 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

68 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 0 0 0 0 0 ? 0 0

67 Char.

1 0 1 0 1 2 2 0 0 ? ? 0 1 0 1 2 1 2 0 1 0 0 2 2 1 0 1 2 0 1 1 1 2 ? 0 0

66 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 ? 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 ? 0 0

65 Char.

1 2 2 1 2 1 1 1 2 ? ? 1 1 1 1 1 2 1 1 1 1 1 2 2 1 1 1 1 2 2 1 1 1 ? 1 1

64 Char.

2 2 2 2 2 2 2 2 2 ? ? 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 4 2 2 2 2 ? 2 4

63 Char.

1 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? 1 1

62 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

61 Char.

0 0 0 0 0 0 2 0 2 0 0 2 0 0 2 0 2 0 0 0 2 2 0 0 0 2 1 1 0 0 0 0 0 0 2 2

60 Char.

2 3 0 3 0 1 1 0 0 0 0 0 2 2 2 2 2 2 0 0 0 2 2 2 2 0 0 0 0 0 2 2 2 1 2 0

59 Char. 2 1 1 1 3 1 1 2 1 2 2 2 1 1 2 2 1 1 1 1 2 2 2 2 1 1 1 2 4 4 1 2 2 1 1 1

.

58 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

57 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

56 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

55 Char. ONTINUED - - - - 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C Char. 54

. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 0 0 0

53 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

52 Char.

ATRIX 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

Char.

M 51

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

50 Char.

- - 2 1 1 0 3 2 2 2 2 2 2 3 2 3 2 2 2 1 1 1 2 2 2 1 1 1 2 2 3 2 2 2 2 2

49 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0

48 Char.

0 0 0 1 0 0 1 0 0 0 0 0 2 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1

Char. HARACTER 47

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

C

46 Char.

- - 0 1 1 2 1 2 1 2 2 2 2 2 ? 2 2 1 1 1 1 2 2 2 1 2 1 1 1 2 1 ? 1 2 2 ?

45 Char.

0 0 0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0

44 Char.

0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1

43 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

42 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 0 1 0 0

41 Char.

ORPHOLOGICAL ORPHOLOGICAL 1 0 0 2 0 0 1 2 0 0 1 0 1 0 0 2 0 0 0 0 3 3 1 1 1 1 1 1 1 1 0 1 1 1 2 2

Char. M 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

: III PPENDIX A LSU81895 KU307775 KU307774 DSM378 DSM527 KU292029 KU194630 FMNH234404 FMNH234421 FMNH76353 FMNH76385 FMNH258508 FMNH262730 FMNH262723 FMNH258507 FMNH137443 FMNH128181 CAS230949 FMNH266218 FMNH266212 CAS221808 CAS210195 TAO699 TNE02 TAO701 TNE03 DSM745 CUMZA2002.237 KU306935 KU307565 CAS221714 DSM1272 DSM1148 CAS22816 FMNH198080 FMNH198082 3.66

117 Char.

1 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 0 1 1 1 1 1 1

116 Char.

1 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 0 1 0 0 1 1 1

115 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

114 Char.

3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3

113 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

112 Char.

0 1 1 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

111 Char.

2 2 2 2 2 2 2 2 2 2 1 2 2 2 2 2 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 1 2 2

110 Char.

0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

109 Char.

1 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 1 1 1 1 1 1 2 1 2 1 1 1 0 1 1 2 2 2 2 2

108 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

107 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

106 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

105 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

104 Char.

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

103 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

102 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

101 Char.

1 3 ? 2 1 1 3 1 3 ? ? 3 1 1 3 3 ? 2 2 2 2 2 1 1 2 2 4 ? 1 4 2 2 2 1 ? ?

100 Char.

- 1 0 0 1 1 2 1 2 1 2 1 1 1 1 0 1 0 2 1 1 0 0 1 1 0 1 2 0 0 1 2 2 1 ? ?

99 Char.

0 0 0 1 0 1 0 1 1 1 0 2 2 0 2 1 0 1 0 1 2 2 1 1 2 2 1 ? 2 2 0 1 0 1 ? ?

98 Char. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ?

Char.

. 97

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ? ?

96 Char.

2 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 2 2 2 2 2 2 2 2 2 2 0 2 2 1 2 ?

95 Char.

0 1 1 2 2 2 2 0 2 2 2 0 0 0 0 2 2 1 0 0 1 0 0 0 1 2 2 2 2 2 1 2 2 2 0 ?

94 Char. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ONTINUED

93 Char. C 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

.

92 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ? ?

91 Char. 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

ATRIX 90 Char.

2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 ?

M

89 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

88 Char.

0 0 1 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 1 0 0

87 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 ?

86 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

HARACTER

Char.

C 85 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

84 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

83 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

82 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

81 Char.

0 0 0 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0

80 Char. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

ORPHOLOGICAL ORPHOLOGICAL 79 Char. 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 0 0 2 2 2 2 2 2 M

: III PPENDIX LSU81895 KU307775 KU307774 DSM378 DSM527 KU292029 KU194630 FMNH234404 FMNH234421 FMNH76353 FMNH76385 FMNH258508 FMNH262730 FMNH262723 FMNH258507 FMNH137443 FMNH128181 CAS230949 FMNH266218 FMNH266212 CAS221808 CAS210195 TAO699 TNE02 TAO701 TNE03 DSM745 CUMZA2002.237 KU306935 KU307565 CAS221714 DSM1272 DSM1148 CAS22816 FMNH198080 FMNH198082 A 3.67

146 Char.

1 1 1 1 1 2 2 1 1 ? ? 2 2 2 2 ? ? 1 1 1 0 0 0 0 2 2 2 2 2 2 1 1 1 1 0 0

145 Char.

3 4 4 4 2 2 1 2 2 0 ? 2 2 2 2 ? ? 2 3 3 2 2 1 1 2 2 1 0 0 0 2 3 0 4 ? ?

144 Char.

0 0 0 0 0 0 0 ? ? ? ? 0 0 0 1 ? ? 0 0 0 0 0 0 0 0 1 0 0 0 0 1 0 0 0 ? ?

143 Char.

4 2 1 1 2 2 2 ? ? ? ? 4 3 3 3 ? ? 2 2 2 3 2 4 2 3 3 2 2 1 2 2 4 4 2 ? ?

142 Char.

------1 ? ? ? ? ? ? 1 1 1 1 1 2 1 ? ?

141 Char.

0 1 1 1 1 1 1 ? ? ? ? 1 1 1 1 ? ? 0 0 0 1 1 1 1 1 1 0 0 1 1 1 0 0 1 1 1

140 Char.

------? 1 2 2 ? ? ? ? 2 2 ? ? 2 2 2 1 2 2 1 ? ?

139 Char.

------1 1 1 1 1 ? ? 1 1 ? 1 1 1 1 1 0 1 1 0 1

138 Char.

------1 ? 1 ? ? ? ? 0 0 ? 0 1 1 0 1 0 ? ?

137 Char. 1 1 1 1 ? 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1

. 136 Char.

0 0 0 0 ? 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

135 Char.

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 2 1 1 0 0 0 0 0 0

134 Char.

1 1 1 1 1 1 1 1 1 ? ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1

133 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ONTINUED

Char. C 132

0 0 0 0 0 0 0 0 0 ? ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 .

131 Char.

2 2 2 2 2 2 2 2 2 ? ? 2 2 2 2 2 2 ? 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

130 Char.

0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 0 0 0

ATRIX

129 Char.

0 0 0 0 0 0 0 0 0 0 ? 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 M

128 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0

127 Char.

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

126 Char.

------2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

125 Char.

------

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 HARACTER

Char.

C 124

------1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

123 Char.

1 1 1 0 1 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 1 0 0 0 0 1 1 1 0 1 1 1 1 0 0

122 Char.

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 0 0 1 1 1 1 1 1 1 1 1 1

121 Char.

------2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2

120 Char.

- - 1 0 0 0 0 0 0 0 0 ? ? 0 1 0 0 0 0 ? 0 0 0 ? 0 0 0 0 1 0 1 0 0 0 0 0

119 Char. 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

ORPHOLOGICAL ORPHOLOGICAL 118 Char. 1 1 1 1 1 1 1 1 1 1 ? 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 M

: III PPENDIX LSU81895 KU307775 KU307774 DSM378 DSM527 KU292029 KU194630 FMNH234404 FMNH234421 FMNH76353 FMNH76385 FMNH258508 FMNH262730 FMNH262723 FMNH258507 FMNH137443 FMNH128181 CAS230949 FMNH266218 FMNH266212 CAS221808 CAS210195 TAO699 TNE02 TAO701 TNE03 DSM745 CUMZA2002.237 KU306935 KU307565 CAS221714 DSM1272 DSM1148 CAS22816 FMNH198080 FMNH198082 A 3.68 APPENDIX IV SPECIMENS EXAMINED Museum codes correspond to those of Leviton et al. (1985) and Leviton and Gibbs (1988), with the addition of: BOR for University of Malaysia Sabah Borneensis Collection; BSI-FS for Biotic Survey and Inventory Field Series (deposited at MVZ); CUMZA for Chulalongkorn University Museum of Zoology Amphibian Collection; DSM for D. McLeod field series (deposited at KU); GMU for Gadjah Mada University, Biological Faculty; KUHE: Kyoto University, Human and Environmental Studies;N: Nikolai Orlov tissue collection, Zoological Institute, Russian Academy of Sciences. Specimen data in this table are from all osteological specimens examined, and those from which morphological data were taken but which do not appear in McLeod (in press). No voucher specimens were examined from the Matsui et al. (2010) study. All Matsui et al. (2010) samples are indicated with ‡. Only data for specimens used in the molecular analysis and prepared as cleared and stained or skeletal materials are presented here. Data for Those specimens represented by osteological material in the morphology analyses are indicated with †.

GPS coordinates Museum GenBank ID # Species General Locality Specific Locality N/S E Voucher Accession no. Outgroups 1 †L. blythi Thailand, Surat Thani Prov. Phanom Dist. 8.90683 98.527 CUMZA2002.206 2 †L. laticeps Malaysia, Selangor Prov. Bukit Lanjan 3.18333 101.617 FMNH198080 3 †L. laticeps Malaysia, Selangor Prov. Bukit Lanjan 3.18333 101.617 FMNH198082

4 Thailand, Nakhon Sakaerat Env. Res. 3.18333 101.617 DSM1148 †L. gyldenstolpei Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 5 3.18333 101.617 DSM1272 †L. gyldenstolpei Ratchasima Prov. Station.

6 Thailand, Surat Thani Prov. Koh Samui Isl., Hat Mae 9.56733 100.01 CUMZA2003.233 †F. limnocharis Nam

Koh Samui Isl., Hat Mae 7 Thailand, Surat Thani Prov. 9.56733 100.01 CUMZA2002.237 †F. limnocharis Nam

8 Phil., Panay Isl., Antique Mun., of San Remigio, 10.8127 122.165 KU306935 †O. laevis Prov. Barangay Aningalan

Phil., Luzon Isl., Quezon Mun., of Polillo, 9 14.7526 121.968 KU307565 †O. laevis Prov. Barangay Pinaglubayan Limnonectes

10 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.83936 106.928 BSI-FS 0026 Kadudampit, Sukabumi

West Java Prov., 11 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.83936 106.928 BSI-FS 0027 Kadudampit, Sukabumi

12 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.83936 106.928 BSI-FS 0028 Kadudampit, Sukabumi

West Java Prov., 13 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.83936 106.928 BSI-FS 0029 Kadudampit, Sukabumi

14 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.83936 106.928 BSI-FS 0030 Kadudampit, Sukabumi

15 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.83936 106.928 BSI-FS 0031 Kadudampit, Sukabumi

16 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.83936 106.928 BSI-FS 0032 Kadudampit, Sukabumi

17 Lineage 1 L. kuhlii Indonesia, Java Isl. West Java Prov., -6.84279 106.926 BSI-FS 0067 Kadudampit, Sukabumi

West Java Prov., 18 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.84279 106.926 BSI-FS 0068 Kadudampit, Sukabumi

West Java Prov., 19 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.84279 106.926 BSI-FS 0069 Kadudampit, Sukabumi

3.69 West Java Prov., 20 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.84279 106.926 BSI-FS 0081 Kadudampit, Sukabumi

West Java Prov., 21 Lineage 1 L. kuhlii Indonesia, Java Isl. -6.84279 106.926 BSI-FS 0082 Kadudampit, Sukabumi 22 Lineage 1 L. kuhlii Indonesia, Java Isl. Tjibodes FMNH173498 23 Lineage 1 L. kuhlii Indonesia, Java Isl. Tjibodes FMNH173499 24 Lineage 1 L. kuhlii Indonesia, Java Isl. Tjibodes FMNH173500 Leiden 4297a 25 Lineage 1 L. kuhlii Indonesia, Java Isl. (Syntype)

Leiden 4297b 26 Lineage 1 L. kuhlii Indonesia, Java Isl. (Syntype)

West Java prov., 27 †Lineage 1 L. kuhlii Indonesia, Java Isl. Kadudampit, Gede -6.76729 106.934 LSUMZ 81895 Panagrago

MNHN 4469 28 Lineage 1 L. kuhlii Indonesia, Java Isl. (Syntype)

West Java prov., 29 Lineage 1 L. kuhlii Indonesia, Java Isl. Kadudampit, Gede -6.76729 106.934 TNHC 059826 Panagrago

West Java prov., 30 Lineage 1 L. kuhlii Indonesia, Java Isl. Kadudampit, Gede -6.76729 106.934 TNHC 059829 Panagrago

GMU 31 Indonesia, Java Isl. Central Java, Purwerojo -7.7243 110.013 AB526316 ‡Lineage 1 L. kuhlii unnumbered

West Sumatra Prov., 32 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Harau -0.09806 100.729 FMNH266608 River

West Sumatra Prov., 33 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Harau -0.09806 100.729 FMNH266610 River

West Sumatra Prov., 34 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266612 waterfall

West Sumatra Prov., 35 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266615 waterfall

West Sumatra Prov., 36 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266616 waterfall

West Sumatra Prov., 37 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266618 waterfall

West Sumatra Prov., 38 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266619 waterfall

West Sumatra Prov., 39 Lineage 2 Indonesia, Sumatra Isl. Payakumbuh, Bunta -0.29806 100.799 FMNH266621 waterfall

West Sumatra Prov. 40 Lineage 2 Indonesia, Sumatra Isl. Lubuk Selasih, Tarusan -1.20694 100.874 FMNH266630 waterfall

West Sumatra Prov. 41 Lineage 2 Indonesia, Sumatra Isl. Lubuk Selasih, Tarusan -1.20694 100.874 FMNH266631 waterfall

West Sumatra Prov. 42 Lineage 2 Indonesia, Sumatra Isl. Lubuk Selasih, Andaleh -1.17194 100.874 FMNH266636 waterfall

3.70 West Sumatra Prov. 43 Lineage 2 Indonesia, Sumatra Isl. Lubuk Selasih, Andaleh -1.17194 100.874 FMNH266637 waterfall Ta Veng Dist., Virachey 44 Lineage 4 Cambodia, Ratanakiri Prov. 14.188 107.293 FMNH 262723 HM067167 † NP Cambodia, Stung Treng Siem Pang Dist. 45 Lineage 4 14.268 106.629 FMNH 262730 HM067174 † Prov Virachey NP Kaleum Dist., Xe Sap 46 †Lineage 5 Lao PDR, Xe Kong Prov. Nat. Biodiv. Conserv. 16.009 106.917 FMNH 258507 HM067148 Area Kaleum Dist., Xe Sap 47 †Lineage 5 Lao PDR, Xe Kong Prov. Nat. Biodiv. Conserv. 16.009 106.925 FMNH 258508 HM067149 Area 48 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Yan Liao 25.0742 121.526 KU193332 49 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Yan Liao 25.0742 121.526 KU193333 50 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Yan Liao 25.0742 121.526 KU193334 51 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Yan Liao 25.0742 121.526 KU193335 52 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Yan Liao 25.0742 121.526 KU193336 53 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Muzha 24.9962 121.571 KU194628 54 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Muzha 24.9962 121.571 KU194629 55 †Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Muzha 24.9962 121.571 KU194630 56 Lineage 7 L. fujianensis Taiwan, ROC Taipei Co., Muzha 24.9962 121.571 KU194631

57 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22614 Nago

Okinawa Prefecture, 58 Lineage 100 L. namiyei Japan, Okinawajima Island, 26.5915 127.977 CAS 22622 Nago

59 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22810 Nago

60 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22811 Nago

Okinawa Prefecture, 61 Lineage 100 L. namiyei Japan, Okinawajima Island, 26.5915 127.977 CAS 22812 Nago

62 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22813 Nago

63 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22814 Nago

64 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22815 Nago

65 Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22816 †Lineage 100 L. namiyei Nago

66 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22817 Nago

67 Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa Prefecture, 26.5915 127.977 CAS 22818 Nago 68 ‡Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa KUHE L0809191 AB526309 69 ‡Lineage 100 L. namiyei Japan, Okinawajima Island, Okinawa KUHE L0809192 AB526310

70 Lineage 8 L. bannaensis China,Guangxi Prov. Jing Xin Co., Prov. 23.0696 105.585 KU292023 Nature reserve

71 Lineage 8 L. bannaensis China,Guangxi Prov. Jing Xin Co., Prov. 23.0696 105.585 KU292024 Nature reserve

72 Lineage 8 L. bannaensis China,Guangxi Prov. Jing Xin Co., Prov. 23.0696 105.585 KU292025 Nature reserve

73 Lineage 8 L. bannaensis China,Guangxi Prov. Jing Xin Co., Prov. 23.0696 105.585 KU292026 Nature reserve

Jing Xin Co., Prov. 74 Lineage 8 L. bannaensis China,Guangxi Prov. 23.0696 105.585 KU292027 Nature reserve

75 Lineage 8 L. bannaensis China,Guangxi Prov. Jing Xin Co., Prov. 23.0696 105.585 KU292028 Nature reserve

3.71 Jing Xin Co., Prov. 76 China,Guangxi Prov. 23.0696 105.585 KU292029 †Lineage 8 L. bannaensis Nature reserve

Shiwan Dashang Nature 77 Lineage 8 L. bannaensis China, Guanxi Province 21.846 107.889 KU311787 Reserve

Shiwan Dashang Nature 78 Lineage 8 L. bannaensis China, Guanxi Province 21.846 107.889 KU311789 Reserve

Shiwan Dashang Nature 79 Lineage 8 L. bannaensis China, Guanxi Province 21.846 107.889 KU311796 Reserve

Vi Xuyen Dist., Cao Bo 80 Lineage 8 L. bannaensis Vietnam, Ha Giang Dist. 22.774 104.867 TAO702 Commune

Vi Xuyen Dist., Cao Bo 81 Lineage 8 L. bannaensis Vietnam, Ha Giang Dist. 22.774 104.867 TAO936 Commune

82 Lineage 8 L. bannaensis Vietnam, Ha Giang Dist. Vi Xuyen Dist., Cao Bo 22.774 104.867 TAO937 Commune 83 ‡Lineage 8 L. bannaensis Vietnam, Ha Tinh Prov. Rao An N24606 AB526319 Lao PDR, Xieng Khouang 84 Phonsavan 19.4496 103.176 KUHE 40652 AB526320 ‡Lineage 8 L. bannaensis Prov.

85 Lao PDR, Xieng Khouang Phonsavan 19.4496 103.176 KUHE 40656 AB526321 ‡Lineage 8 L. bannaensis Prov.

Vi Xuyen Dist., Cao Bo 86 †Lineage 8 L. bannaensis Vietnam, Ha Giang Dist., 22.774 104.867 TAO701 HM067254 Commune Vi Xuyen Dist., Cao Bo 87 †Lineage 8 L. bannaensis Vietnam, Ha Giang Dist., 22.771 104.850 TNE-03 HM067259 Commune Moe Maik Township, 88 †Lineage 9 Myanmar, Shan State Shwe u Daung Wildlife 23.090 96.250 CAS 221808 HM067308 Sanctuary Alaungdaw Kathapa 89 †Lineage 9 Myanmar, Sagaing Div. 22.314 94.408 CAS 210195 HM067296 N.P. Thailand, Nakhon Sakaerat Env. Res. 90 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3241 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 91 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3242 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 92 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3243 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 93 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3244 Ratchasima Prov. Station.

94 Lineage 10 L. megastomias Thailand, Nakhon Sakaerat Env. Res. 14.494 101.883 CUMZ(A)3245 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 95 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3246 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 96 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3247 Ratchasima Prov. Station.

97 Lineage 10 L. megastomias Thailand, Nakhon Sakaerat Env. Res. 14.494 101.883 CUMZ(A)3248 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 98 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3249 Ratchasima Prov. Station.

Thailand, Nakhon Sakaerat Env. Res. 99 Lineage 10 L. megastomias 14.494 101.883 CUMZ(A)3250 Ratchasima Prov. Station.

Muang Sa Kaeo, Pang 100 Lineage 10 L. megastomias Thailand, Sa Kaeo Prov 14.106 102.262 FMNH 266231 Si Da NP

Muang Sa Kaeo, Pang 101 Lineage 10 L. megastomias Thailand, Sa Kaeo Prov 14.106 102.262 FMNH 266232 Si Da NP Thailand, Nakhon Sakaerat Env. Res. 102 †Lineage 10 L. megastomias 14.494 101.871 KU 307774 HM067214 Ratchasima Prov. Station. Thailand, Nakhon Sakaerat Env. Res. 103 †Lineage 10 L. megastomias 14.494 101.871 KU 307775 HM067215 Ratchasima Prov. Station. 104 Lineage 11 Thailand, Loei Prov. Sieo, Mt. Sawan 17.1422 101.706 KU 040185 3.72 105 Lineage 11 Thailand, Loei Prov. Ban Muang Khai 17.4333 101.45 KU 040189 106 Lineage 11 Thailand, Loei Prov. Ban Muang Khai 17.4333 101.45 KU 040190 107 Lineage 11 Thailand, Loei Prov. KU 040192 108 Lineage 11 Thailand, Loei Prov. Ban Muang Khai 17.4333 101.45 KU 040198 109 Lineage 11 Thailand, Loei Prov. Ban Muang Khai 17.4333 101.45 KU 040199 110 ‡Lineage 11 Thailand, Loei Prov. Ban Muang Khai 17.4333 101.45 KU 040200 111 Lineage 11 Thailand, Loei Prov. Phu Luang KUHE 19284 AB526314 Phu Rua Dist., Phu 112 †Lineage 11 Thailand, Loei Prov. 17.334 101.500 FMNH 266212 HM067175 Luang Wildlife Sanctuary Phu Rua Dist., Phu 113 †Lineage 11 Thailand, Loei Prov. 17.259 101.502 FMNH 266218 HM067181 Luang Wildlife Sanctuary 114 Lineage 12 Lao PDR, Bokeo Prov. Ban Tup 20.283 100.7 MNHN1997.3902 115 Lineage 12 Lao PDR, Bokeo Prov. Ban Tup 20.283 100.7 MNHN 1997.3916 116 Lineage 12 Lao PDR, Bokeo Prov. Ban Tup 20.283 100.7 MNHN 1997.4104 117 Lineage 12 Lao PDR, Bokeo Prov. Ban Tup 20.283 100.7 MNHN 1997.4106 118 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.6 119 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.7 120 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.8 121 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.13 122 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.29 123 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.30 124 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.32 125 Lineage 12 Thailand, Chiang Mai Prov. Mueang Dist. 18.8382 98.8964 CUMZ(A)2003.33 126 †Lineage 12 Myanmar, Shan State Kalaw township 20.711 96.487 CAS 221714 HM067036 Taunggyi Dist., Ma 127 Myanmar, Shan State 20.692 96.506 CAS 230949 HM067317 †Lineage 12 Gawe Reserve Vi Xuyen Dist., Cao Bo 128 †Lineage 13 Vietnam, Ha Giang Dist., 22.774 104.867 TAO 699 HM067252 Commune Vi Xuyen Dist., Cao Bo 129 †Lineage 13 Vietnam, Ha Giang Dist., 22.771 104.850 TNE-02 HM067258 Commune

130 Malaysia, Borneo Isl., Kinabalu BOR 22645 AB526323 ‡Lineage 14 Sabah State

131 Lineage 16 Malaysia, Borneo Isl., Tenom Dist., Crocker 5.217 115.95 FMNH238534 Sabah State Range NP

132 Lineage 16 Malaysia, Borneo Isl., Tenom Dist., Crocker 5.217 115.95 FMNH243627 Sabah State Range NP

133 Malaysia, Borneo Isl., Sipitang Dist., 4.9 115.7 FMNH234404 †Lineage 16 Sabah State Mendolong

134 Lineage 19 L. asperatus Indo., Borneo Isl, East Kotawaringin, -1.41667 112.333 FMNH259072 Kalimantan Mentaya Hulu Dist (paratype)

Malaysia, Borneo Isl., Sipitang Dist., 135 4.9 115.7 FMNH234421 †Lineage 20 Sabah State Mendolong

136 Malaysia, Borneo Isl., Sandakan Div. 5.8287 118.061 FMNH76353 †Lineage 21 Sabah State

137 Malaysia, Borneo Isl., Kuching Div., Matang 1.6 110.333 FMNH76385 †Lineage 101 Sarawak State Ranges

138 Malaysia, Borneo Isl., Kuching Div., Matang KUHE 12025 AB526322 ‡Lineage 101 Sarawak State Ranges

139 Malaysia, Borneo Isl., Kapit Div. 1.63333 113.567 FMNH137443 †Sarawak "A" Sarawak State

Malaysia, Borneo Isl., 140 Limbang Div., Pa Main 3.63333 115.517 FMNH128181 †Sarawak "B" Sarawak State

3.73 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited.

Zootaxa 1807: 26–46 (2008) ISSN 1175-5326 (print edition) www.mapress.com/zootaxa/ ZOOTAXA Copyright © 2008 · Magnolia Press ISSN 1175-5334 (online edition)

A new species of big-headed, fanged dicroglossine frog (Genus Limnonectes) from Thailand

DAVID S. McLEOD University of Kansas, Natural History Museum and Biodiversity Research Center, 1345 Jayhawk Boulevard, Lawrence, Kansas 66045-7561, USA. E-mail: [email protected]

Abstract

A new species of the dicroglossine genus Limnonectes from eastern Thailand and its tadpole are described. Analysis of DNA sequence data from 2518 base-pairs of the mitochondrial 12S and 16S gene regions places the species within the complex of frogs currently referred to as Limnonectes kuhlii and demonstrates it to be a separate lineage (>18% sequence divergence from type-material of L. kuhlii from Java). The new species differs from L. kuhlii by having nuptial pads, a greater snout–vent length, and different relative finger lengths than specimens from Java. It has more extensive toe web- bing, a different arrangement of nuptial pads, and a greater snout–vent length than Limnonectes laticeps. The new spe- cies, which lacks vocal slits, also can be distinguished from the morphologically similar Limnonectes namiyei from Japan, which possesses vocal slits.

Key words: dicroglossine, Limnonectes, mitochondrial DNA, morphology, new species, Thailand

Introduction

Among the Asian and Southeast Asian dicroglossine frogs, the fanged frogs of genus Limnonectes comprises a group of species that usually exhibits strong, male-biased sexual dimorphism, unusual secondary sexual characteristics such as greatly enlarged odontoid processes (“fangs”) on the lower jaw, and a great deal of phe- notypic similarity (Emerson, 1994; Emerson et al., 2000). Dubois (1987; Dubois, 1992) proposed two variants of a phenetic classification of the fanged frogs and their relatives. In the latter of these classifications, Dubois recognized three species groups (grunniens, kuhlii, and microdiscus) within the subgenus Limnonectes, which contain 26 species. Emerson and Berrigan (1993) provided morphological evidence to support the monophyly of the fanged frogs, assigning them to the genus Limnonectes, but found no support for Dubois’ phenetic spe- cies groups. Emerson et al. (2000) corroborated the monophyly of Limnonectes with molecular data and rec- ognized five monophyletic species groups within this genus. These results were supported further by Evans et al. (2003) and Frost et al. (2006). In 2003 and 2004, a large-bodied species of Limnonectes, phenotypically similar to Limnonectes kuhlii, was collected from three locations in eastern Thailand. Limnonectes kuhlii Tschudi (1838), as currently recog- nized, seems to be a complex of species, which is found in southern China, in two small areas of northeastern India, and throughout Southeast Asia (including Cambodia, Bryan Stuart, pers. comm.), but which has not been reported from Singapore (IUCN et al., 2006). The relational complexity within the “kuhlii” group and among its relatives is a topic beyond the scope of this paper but is interesting enough to warrant significant attention elsewhere (McLeod, in prep.). The purpose of this paper is to elucidate the diversity within the “kuhlii” complex and recognize the uniqueness of this new species of Limnonectes. To this end, I utilize a phylogenetic hypothesis based on mito-

26 Accepted by M. Vences: 29 April 2008; published: 23 Jun. 2008 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. chondrial DNA fragments of the 12S–16S gene region that places the new species within the “kuhlii” complex and demonstrates its uniqueness when compared to Limnonectes kuhlii from the type locality of Java. Mor- phological evidence is used to support the distinctiveness of this frog that is described herein as a new species. Additional molecular evidence is used to corroborate the relationship between larvae and adults of the new species. A description of the new species and its larvae are provided.

Material and methods

Fieldwork. From 2003–2005, DSM conducted fieldwork at Sakaerat Environmental Research Station (SERS) in Nakhon Ratchasima Province, Thailand (NRCT permit no. 9947). In September 2004, Yodchai Chuaynkern and Bryan L. Stuart conducted fieldwork under the auspices of the Thailand Natural History Museum at Pang Si Da National Park in Sa Kaeo Province, and Phu Luang Wildlife Sanctuary in Loei Province. Post metamorphic specimens were caught in the field by hand, and larvae by dip-net. All specimens were preserved in 10% buffered formalin. Post metamorphic individuals were later transferred to 70% ethanol. Tis- sue samples were taken from frogs by preserving pieces of liver and muscle in either a 20% DMSO-salt satu- rated storage buffer, or in 95% ethanol before the specimen was fixed in formalin. Tissue samples were taken from tadpoles by preserving one or two representatives from a tadpole lot in 20% DMSO-salt saturated stor- age buffer. Specimens were deposited in the Chulalongkorn University Museum of Zoology (CUMZ), the Field Museum of Natural History (FMNH), the Thailand Natural History Museum (THNHM), and the Uni- versity of Kansas Natural History Museum and Biodiversity Research Center (KU). Some of the specimens are cross-cataloged at both FMNH and THNHM (See Appendix 1), in which case vouchers are referenced in the text by their FMNH number. Museum codes correspond to Leviton et al. (1985) and Leviton and Gibbs (1988). Morphology. Measurements were made with digital calipers to the nearest 0.01 mm. Morphometric char- acters of post metamorphic individuals used here are modified from those of Manamendra-Arachchi and Pethiyagoda (2005) and are illustrated in Figure 1. Modifications include the addition of five measurements, which are defined as follows: MH = mandible height—perpendicular distance between dorsal to ventral bor- ders of the mandible measured at the posterior base of the odontoid process; OH = odontoid height—perpen- dicular distance between the ventral border of the mandible and the tip of the odontoid process (Emerson, 1994); RL = rostrum length—distance from the level of the anterior corner of the eye to the anterior-most point of the head (“snout” is most appropriately applied to the portion of the head anterior of the nostrils, but is frequently misused to refer to the region anterior of eye-level); RFL = relative finger length when digits are adpressed—shown, for simplicity, in Arabic numerals in descending order of length (e.g., 3421); RTL = rela- tive toe length when digits are adpressed—shown, for simplicity, in Arabic numerals in descending order of length (e.g., 43521). Abbreviations used are: ED = eye diameter; EN = eye–nostril distance; FEL = thigh (femur) length; FOL = foot length; HL = head length; HW = head width; IN = internarial distance; IO = inter- orbital width; LAL = lower arm length; MN = mandible–nostril distance; PAL = palm length; SVL = snout– vent length; TBL = shank (tibia) length; TD = tympanum diameter; UAW = upper arm length; UEW = upper eyelid width; NM = no measurement taken. Digital webbing formulae follows that of Guayasamin et al. (2006). Digits of the hand are numbered I–IV based on traditional schemes, not homology (Alberch & Gale, 1985). The paratopotypes KU 30774–75 were cleared and double-stained using a modified protocol based on Taylor & Van Dyke (1985). Radiographs and dry skeletal preparations of specimens were also made to exam- ine osteological characters. Sex and life stage were determined by examination of gonads and by inspection of prominent secondary sexual characters (e.g., nuptial pads). Most larval measurements were taken to the near- est 0.1mm using a stereomicroscope equipped with an ocular micrometer; however, tail length, body length, and body width were measured with digital calipers. Larval measurements are modified from those of Lehr et

A NEW SPECIES OF LIMNONECTES FROM THAILAND Zootaxa 1807 © 2008 Magnolia Press · 27 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. al. (2007) and include: BL = body length measured from snout to junction of the posterior body wall with the tail axis (body terminus); TL = tail length measured from body terminus to absolute tip of tail; ToL = total length (sum of BL + TL); BW = body width measured at the widest point right behind the eye; BH = body height at eye level; LED = larval eye diameter; LIO = larval interorbital distance measured between the cen- ters of the dorsal margin of the orbits; LIN = larval internarial distance measured between the centers of the nares (indicated by less pigmentation when closed); LNS = larval naris–snout distance (from center of naris to middle of snout); LEN = larval eye–naris distance (from center of naris to anterior edge of eye); SW = spiracle tube width (at level of opening); TMH = tail muscle height at (1) junction of the body wall with the ventral margin of the tail muscle and (2) at mid-tail length; TH = overall tail height (at mid-length of tail). All illustra- tions were prepared by with the aid of a binocular dissecting microscope and camera lucida. Molecular methods. Tissue samples suitable for molecular analysis were not available for the morpho- logically similar, large-bodied species, Limnonectes namiyei, from Japan. New mitochondrial sequences from Limnonectes sp. nov. and Limnonectes kuhlii (two individuals per species; all specimens from Thailand) were provided by B. J. Evans (Appendix 2). The gene order of the 2518-bp region sequenced (5'–3') is tRNAphe, 12S ribosomal DNA (rDNA), tRNAval, 16S rDNA. Amplification and sequencing protocols follow Evans et al. (2003). Additional partial and complete sequences for the 12S–16S regions for 17 individuals, mostly from Indochina and Asia, were obtained from GenBank (Appendix 2). Sequences were aligned using Se-Al V 2.0a11 Carbon (Rambaut & Charelton, 2002) and then fine-tuned by eye. B. Stuart provided evidence for the genetic corroboration of the relationship between adults and tadpoles of the species described below. Total genomic DNA was extracted from adults and tadpoles using the Pure- Gene protocol for isolation of DNA from animal tissue (Gentra Systems, Inc.). A 694-bp fragment of mito- chondrial DNA that encodes part of the 16S ribosomal RNA gene (16S) was amplified by the polymerase chain reaction (PCR; 94ºC 45s, 60ºC 30 s, 72ºC 1 min) for 35 cycles using the primers L-16SRana (5'- CCTACCGAGCTTAGAGATAGC-3') and H-16SRanaIII (5'-CATGGGGTCTTCTCGTCTTAT-3'). PCR products were electrophoresed in a 1% low melt agarose gel, stained with ethidium bromide, and visualized under ultraviolet light. The band containing the amplified DNA was excised and purified using GELase (Epi- centre Technologies). PCR products were sequenced in both directions using Big Dye version 3 chemistry (Perkin Elmer) and the amplifying primers. Cycle sequencing products were precipitated with ethanol, 3 M sodium acetate, and 125 mM EDTA, and sequenced with a 3730 DNA Analyzer (ABI). Sequences were edited and aligned by eye in Sequencher version 4.1 (Genecodes), and deposited in GenBank (Appendix 2). Phylogenetic Analysis. Based on work by Evans et al. (2003) and others (Che et al., 2007; Emerson et al., 2000; Zhang et al., 2005) Fejervarya limnocharis and Occidozyga laevis were choosen as generic level outgroup taxa. Additionally, Limnonectes gyldenstolpei, L. laticeps, and L. fujianensis were included in the study to help delimit the boundaries of the “kuhlii” complex within the genus Limnonectes. Phylogenetic inference was performed using maximum parsimony (MP), maximum likelihood (ML), and Bayesian (BA) methods. MP heuristic searches were executed in PAUP* V 4.0b10 (Swofford, 2003) using tree bisection- reconnection (TBR) branch swapping and 1000 random taxon replicates. All characters were weighted equally, and gaps were treated as missing data. Nonparametric bootstrap values were obtained with 2000 rep- licates, each with a single random addition replicate, and other settings identical to the original MP heuristic search. For model-based analyses, the best fitting model of evolution was determined using the Akaike Informa- tion Criterion (AIC) in Modeltest V 3.7 (Posada & Crandall, 1988). I conducted ML analyses with Garli V 0.951 (Zwickl, 2006) using 100 bootstrap replicates. Bayesian analyses were conducted using Mr Bayes V 3.1.2 (Ronquist & Huelsenbeck, 2003) under the model selected by Modeltest. Two independent analyses were run with four Metropolis-coupled Markov chains each. All Markov chains were run for 5 million gener- ations, with sampling every 1000 generations. Stationarity of the Markov chains was assessed using Tacer v1.3 (Rambaut and Drummond, 2003). All samples prior to stationarity were discarded as burn-in.

28 · Zootaxa 1807 © 2008 Magnolia Press MCLEOD TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited.

FIGURE 1. Morphological characters measured in this study. (A–B) Dorsal aspect of Limnonectes megastomias (KU 307773, paratopotype; adult female; SVL = 80.1 mm). Abbreviations: IO = interorbital distance; IN = internarial dis- tance; UEW = upper eyelid width; HW = head width; SVL = snout–vent length; FOL = foot length; TBL = Tibia length; FEL = femur length; UAL = upper arm length. (C) Stylized palmar view of hand (CUMZA 2003.134, holotype; adult male; SVL = 121.9 mm). Abbreviations: O = outer metacarpal tubercle; M = middle metacarpal tubercle; T = thenar tubercle; 1 = proximal subarticular tubercle; 2 = distal subarticular tubercle; 3 = postaxial dermal fringe; 4 = preaxial der- mal fringe; 5 = nuptial pad. (D) Lateral view of head (CUMZA 2003.134, holotype; adult male; SVL = 121.9 mm). Abbreviations: RL = rostrum length; EN = eye–nostril distance; ED = eye diameter; TD = tympanum diameter; MN = mandible–nostril distance; HL = Head length; OH = odontoid height; MH = mandible height.

Results

Larvae-adult corroboration. A tadpole (FMNH 266340) and three adults (FMNH 266224, 266229, 266232) share identical haplotypes for a 694-bp fragment of 16S. Phylogeny. Sequences from 23 individuals were sampled. Of these, 391 sites were variable and 567 were parsimony informative. Parsimony searches recovered two equally parsimonious trees of 1940 steps (consis-

A NEW SPECIES OF LIMNONECTES FROM THAILAND Zootaxa 1807 © 2008 Magnolia Press · 29 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. tency index = 0.703, retention index = 0.624). The parsimony trees differed only in the arrangement of L. kuhlii (China 1 and 2) in relation to L. kuhlii (Vietnam 1). The best fitting model of evolution was determined to be the general time reversible model with proportion of invariant sites and discretized gamma distribution to accommodate among site rate heterogeneity (GRT + I + Γ). A Bayesian consensus tree was calculated after a burn-in of 10,000 generations. The ML analysis resulted in a single optimal tree (–ln likelihood = – 11709.728). The ML tree and the 50% majority consensus tree from the Bayesian analysis reveal the same topology (Fig. 2).

FIGURE 2. Phylogenetic hypothesis derived from 12S and 16S mitochondrial data. Occidozyga laevis and Fejervarya limnocharis were used as outgroups, but are not shown. * indicates clades supported by Bayesian posterior probabilities > 90%, and bootstrap values > 70% from ML (100 replicates) and Parsimony analysis (2000 replicates).

Preliminary analysis reveals that Limnonectes kuhlii, as currently recognized, is non-monophyletic, cor- roborating findings of previous authors (Emerson et al., 2000; Evans et al., 2003). It is clear that there is a great amount of diversity within the “kuhlii” complex, and these results suggest that there is even more diver- sity than previously was thought. Phylogenetic results suggest that the new species and Limnonectes kuhlii from Laos and northwestern Thailand are sister taxa (separated by genetic distances of 5% and 9%, respectively). There is considerable genetic distance (17–18%) between the new species from eastern Thailand being described here and samples

30 · Zootaxa 1807 © 2008 Magnolia Press MCLEOD TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. of Limnonectes kuhlii from Java (the type locality for L. kuhlii). For perspective, the difference between the new species and Javan Limnonectes kuhlii is greater than the interspecific genetic distances found between the latter and either L. laticeps (14%), or L. gyldenstolpei (15%). In contrast, the genetic distances between sam- ples known to be from the same population are 0.4% for the northwestern Thailand population of Limnonectes cf. kuhlii, and 0.7% for the new species.

Limnonectes megastomias, new species

Holotype CUMZA 2003.134 deposited at CUMZ, adult male, collected from spring-fed pool at head of intermittent stream in deciduous evergreen forest at Sakaerat Environmental Research Station, Nakhon Rat- chasima Province, Wang Nam Khieo District, Thailand, 14°29.680'N 101°52.257'E, 645 m, on 27 June 2003 at 1900 hr by DSM and Taksin Artchawakom. Paratopotypes. Total of 15 specimens: 11 males, 4 females. CUMZA 2003.135 (female) collected with holotype (Fig. 3B); KU 307760–63, 307766–72 (males), KU 307764–65, 307773 (females), KU 307776 (male, dry skeleton), KU 307774 (male, cleared-and-stained), KU 307777 (female, dry skeleton), KU 307775 (female, cleared-and-stained), collected at same locality as holotype on 15 March 2005 at 1840–2130 hr by DSM, Kyle Hesed and Taksin Artchawakom. Paratypes. Total of 20 specimens: 14 males, 6 females. FMNH 266212, 266219 (females); FMNH 266213–218 (males), collected in Phu Luang Wildlife Sanctuary, Loei Province, Phu Rua District, Thailand, 17°16.812' N 101° 31.125'E, 1460 m, on 2–3 September 2004 by Yodchaiy Chuaynkern, Bryan L. Stuart, Chatchay Chuechat, and Sunchai Makchai. FMNH 266220, 266223, 266229, 266231 (females); 266221–22, 266224–27, 266230, 266232 (males); 266228 (juvenile), collected in Pang Si Da National Park, Sa Kaeo Province, Sa Kaeo District, Thailand, 14°6.345'N 102°15.693'E, 600 m, on 21, 26–27 September 2004 by Yodchaiy Chuaynkern, Bryan L. Stuart, Chatchay Chuechat, and Sunchai Makchai. Other meaterial. FMNH 266340 tadpoles collected in Pang Si Da National Park, Sa Kaeo Province, Sa Kaeo District, Thailand, 14° 6.345'N 102° 15.693'E, 600 m, on 21 September 2004 by Yodchaiy Chuaynkern, Bryan L. Stuart, Chatchay Chuechat, and Sunchai Makchai. KU 307778–307784 tadpoles and eggs (hatched and reared in field lab) collected at type locality on 15 March 2005 at 1430 hr by DSM, Kyle Hesed and Taksin Artchawakom. Diagnosis. Limnonectes megastomias , a large species of Limnonectes allied to the kuhlii species group as characterized by the following combination of characters: (1) adult male SVL 40.0–123.7 mm (mean = 80.2; SD ± 23.66; n = 26), adult female SVL 53.5–86.3 mm (mean = 74.0; SD ± 11.29; n = 10); (2) tympanum obscured by thickened skin; (3) vocal sac and vocal slits absent in males; (4) males have advertisement call; (5) males with nuptial pads on Fingers I and II; (6) males with thick, elongate odontoid processes; females with reduced odontoid processes; (7) males with enlarged heads (HL 41–56% of SVL; 39–45% in females); (8) prominent supratympanic fold from posterior superior corner of eye to angle of jaw; (9) throat, venter, and borders of thigh and leg heavily pigmented (mottled) in males, females with lightly pigmented throat, moder- ate pigmentation on venter and borders of thigh and leg; (10) small, low glandular warts tipped with translu- cent horny spinules on flanks of body, around vent, and on dorsum of shank and foot; dorsal skin moderately rugose except for regions covered with warts, ventral skin smooth; (11) Finger II longer than Finger I when adpressed; (12) no webbing between fingers, toes fully webbed (I0–0+II0–0+III0+–0IV0–0V); (13) larval labial tooth row formula: 2(2)/3(1); (14) narrow gap in ventral oral papillae of larvae; (15) eggs pigmented; eggs deposited in large numbers with no parental care. Comparisons. As noted by Emerson et al. (2000), frogs of the genus Limnonectes are phenotypically similar. Whereas the most distinguishing feature of Limnonectes megastomias is its tremendous maximum

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FIGURE 3. Limnonectes megastomias in life. Photos by DSM. (A) Holotype (CUMZA 2003.134; adult male; SVL = 121.9 mm); (B) Paratopotype (CUMZA 2003.135; adult female; SVL 81.5 mm).

FIGURE 4. Holotype of Limnonectes megastomias in alcohol (CUMZA 2003.134; adult male; SVL = 121.9 mm). (A) Palmar view of hand; (B) plantar view of foot; ventral (C) and dorsal (D) views.

32 · Zootaxa 1807 © 2008 Magnolia Press MCLEOD TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. size (SVL) when compared to other species allied to L. kuhlii, reliance on external morphological characters alone for species determination is inadequate for this group. Based on the results of the molecular phyloge- netic analysis placing the new species within Limnonectes, and because of the genetic distinctiveness of Lim- nonectes megastomias, I have elected to limit morphological comparisons of the new species to those species with which it is (1) most closely related, (2) geographically proximate to, and (3) with those it would most easily be confused: Limnonectes kuhlii (from the type locality, Java, and specimens currently recognized as L. kuhlii from Thailand and Laos), the phenotypically similar Limnonectes laticeps Boulenger (1882), and the large-bodied Limnonectes namiyei Stejneger (1901). Emerson and Berrigan’s (1993) clade consisting of L. kuhlii, L. namiyei, and L. laticeps is united by six synapomorphies, three of which are considered here (an obscured tympanum, short vomerine tooth row, and the presence of nuptial pads), features also shared by L. megastomias. Limnonectes namiyei is endemic to Okinawajima Island in Japan (Maeda & Matsui, 1990), and is consid- ered by Emerson and Berrigan (1993) to be the sister taxon to L. kuhlii. Limnonectes megastomias can be eas- ily distinguished from Limnonectes namiyei by the presence of vocal slits in male L. namiyei (absent in L. megastomias). Limnonectes megastomias is much larger, with a SVL of 123.4 mm, than L. laticeps. Berry (1975), Boulenger (1912), and Chan-ard (2003) reported a maximum SVL = 53 mm for L. laticeps. Limnon- ectes megastomias has more extensive toe webbing than L. laticeps (b–¾ webbed based on Boulenger (1920), I1–2II1–2III1–3-IV3-–1V in examined specimen of L. laticeps; webbing formula = I0–0+II0– 0+III0+–0IV0–0V in L. megastomias), and nuptial pads on Fingers I and II (only on Finger I in L. laticeps). Limnonectes megastomias can be distinguished from the L. kuhlii from the type locality of Java, and other rec- ognized geographic forms of L. kuhlii on the basis of maximum SVL (Table 1). Limnonectes megastomias dif- fers from Javan (type) L. kuhlii in having Finger II longer than Finger I (I > II in Javan L. kuhlii). Based on specimens examined, it seems that males of L. kuhlii from Java lack nuptial pads (see discussion), whereas males of L. megastomias have well-developed nuptial pads. Description of holotype. Adult male (Figs. 3a and 4). Habitus robust with greatly enlarged head (HL = 56% of SVL); head longer than wide (HW = 92% of HL). Rostrum rounded in dorsal view, projecting beyond lower jaw, obtuse (sloping) in profile; nostril dorsolaterally oriented, closer to tip of snout than eye; internarial distance 74% of interorbital distance; canthus rounded; lores concave; upper lip distinctly swollen and flared, extending to post-rictal tubercle; eye diameter 15% head length; width of upper eyelid 63% of interorbital dis- tance; pupil diamond shaped. Supratympanic fold moderate, extending from eye to angle of jaw (insertion of arm); tympanum not visible. Vomerine teeth on oblique ridges, separated from each other by width of one ridge. Choanae oval, perpendicular to the longitudinal axis of the body. Odontoid processes robust, more than twice the depth of the mandible at base of processes. Symphysial knob at mandibular symphysis. Tongue oval, deeply notched posteriorly. Tips of all four fingers rounded, not expanded into discs but with rounded distal pad; relative lengths of fingers: 3421; no webbing between fingers; distinct, movable fringe of skin on pre- and postaxial sides of Fingers II and III, indistinct fringes of skin on Finger IV; digits indicated by roman numeral (tubercle count in parentheses): IV (1), III (2) II (1), I (1); proximal subarticular tubercles prominent, round, elevated, bifid on Fingers II and III (Fig. 4A); distal subarticular tubercles low, flat and indistinct, bifid on Finger III; thenar metacarpal tubercle large, oval, not elevated; inner metacarpal tubercle oval, smaller than thenar tubercle, not contacting outer or thenar tubercles; outer metacarpal tubercle smaller than inner tubercle, oval, elevated; prominent nuptial pads composed of minute spines on medial surface of Finger I and dorsome- dial surface of Finger II above preaxial fringe. Tips of toes rounded, not expanded into discs, toe pads ele- vated; relative lengths of toes: 43521; toes webbed to middle of terminal phalanx (webbing formula = I0– 0+II0–0+III0+–0IV0–0V); distinct, movable flap of skin on postaxial side of Toe V from middle of terminal phalanx to proximal end of metatarsus; distinct, movable flap of skin on preaxial side of Toe I from middle of terminal phalanx to level of inner metatarsal tubercle, continuing as weak fold on distal one third of tarsus; subarticular tubercles prominent, elevated, round; digits indicated by roman numeral (tubercle count in paren- theses): V (2), IV (3) III (2), II (1), I (1); inner metatarsal tubercle oval, elongate with elevated post axial bor- der. Skin on top of head, dorsal surface of limbs, and dorsum moderately rugose; skin on sides, dorsum of lower arm, around vent, dorsum of shank and foot distinctly rugose, covered with small, low glandular warts with pearl tips; ventral skin smooth; weak, transverse fold between posterior margins of eyes.

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Measurements. Morphometric data for the holotype (male, CUMZA 2003.134) are: SVL = 121.9; ED = 10.2; EN = 10.7; RL = 19.7; FEL = 58.6; FOL = 68.2; HL = 68.1; HW = 62.7; IN = 8.9; IO = 12.0; LAL = 23.2; MN = 57.6; PAL = 27.3; TBL = 48.3; UEW = 7.6; OH = 9.6; MH = 4.5.

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Color of holotype in life. Based on color images shown in Figure 3A. Dorsum olive-brown with dark brown blotch over shoulders and indistinct, dark transverse bars on upper surface of hind limbs; side of head and lateral surfaces of body yellowish brown; chin brownish white; belly white with brown vermiform mark- ings; dark brown bar between eyes bordered by thin yellowish-brown bars; lower half of iris gold, upper half brown, separated by dark brown horizontal band; nuptial pad white. Color of holotype in perservative. Similar to color in life. Areas colored yellowish brown in life appear gray brown in preservative; white coloration on venter appears gray. Description of Tadpole. The following description is based on an individual at Gosner (1960) Stage 40 (FMNH 266340). The tadpole is illustrated in Figures 5A and B. Body oval, slightly depressed, with mid- body height being about 24% of body length; tail slightly higher than body, dorsal fin arising slightly behind origin of caudal musculature, maximum height just posterior to mid-length, tapering gradually until near tip where it narrows abruptly to narrow, rounded tip; tail about 2 times body length. Maximum body width 59% of body length; body height 24% of body length. Eyes large (approximately 12% of body length; 50% of body height at level of eyes); positioned dorsolaterally, oriented laterally; interorbital distance 41% of body width. Nares between tip of snout and eyes, rim not raised; internarial distance 61% of interorbital distance. Spiracle closer to eye than to end of body, midway up side, end of tube free of body wall. Oral disk ventral, near anterior end of body; posterior labium with single, staggered row of short, thick papillae, with narrow median interruption; papillae of anterior labium in single row, confined to corners; labial tooth formula 2(2)/3(1), outer posterior row much shorter than other rows; jaw sheaths marginally black, ser- rate, anterior jaw sheath with weak median convexity. Color of Tadpole in life. (Fig. 5C) Body brownish gold with faint dark brown mottling on dorsum, with- out pigment on venter; iris gold with median dark brown horizontal bar; caudal muscle and dorsal fin brown- ish gold with faint dark brown mottling and distinct spots over entire length; ventral fin without brown pigmentation in proximal half, mottled and spotted in distal half.

FIGURE 5. Larvae of Limnonectes megastomias (FMNH 266340; paratype; Gosner (1960) stage 40). Oral (A) and lat- eral (B) views. (C) Larvae (paratopotype) in life. Photo by DSM.

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Color of Tadpole in preservative. Similar to color in life. Areas colored brownish gold in life appear yel- lowish white in preservative. Measurement of Tadpole. Morphometric data for the Stage 40 individual (FMNH 266340) are: BL = 14.4; BW = 8.5; BH = 3.5; ToL = 42.4; LED = 1.8; LIO = 3.5; LIN = 2.2; LNS = 1.8; LEN = 1.4; SW = 0.8; TMH1 = 3.5; TMH2 = 3.3; TH = 7.0; TL = 28.0 Variation. Measurements of paratypes and congeners are summarized in Table 1. Most striking is the enlargement of the head in males of Limnonectes megastomias. The progressive, disproportionate, enlarge- ment of the head in males is well documented in Limnonectes kuhlii (Hiroshi & Matsui, 2002; Inger, 1966; Pope, 1931), and is associated with male-biased sexual dimorphism and male-male combat behaviors (Hiroshi & Matsui, 2002). Though no male-male combat behavior was observed in L. megastomias, specimens were collected at SERS that had missing digits and limbs, fresh bite marks, and scars indicative of either predation attempts or conspecific combat. Additionally, in L. megastomias, when head length is subtracted from SVL, adult males have smaller bodies (53% SVL) than adult females (58% SVL), a trend also seen in populations of L. kuhlii (Pope, 1931). Curiously, there is a great deal of variation in the SVL of males with nuptial pads (40– 123.7 mm), which also may be related to sexual selection and competitive male-male interactions. Hiroshi & Matsui (2002) reported that larger males are more successful in male-male combat, and suggested that males defending suitable oviposition sites are more likely to have access to females, and as a result, mate more fre- quently. At SERS, very large males (i.e., SVL > 90 mm) were encountered less frequently, were more wary and likely to seek cover in mud and debris when disturbed, and thus, were more difficult to collect than smaller males (i.e., SVL < 70 mm). Most specimens are uniformly brown in dorsal coloration and lack dark-colored bars on the forearm and hind limbs. A few specimens from each population have a lighter dorsal coloration and have distinct bars on the forearms and hind limbs. The holotype, and some (23 of 36) of the syntypes have bifid subarticular tuber- cles (Fig. 4). This character occasionally varies from side-to-side in the same animal. In the SERS population, all specimens examined (16 individuals) have bifid subarticular tubercles, most frequently the proximal subar- ticular tubercles on Fingers II and III, but occasionally distal tubercles on the same fingers. In the Pang Si Da population, eight of thirteen individuals have bifid subarticular tubercles, most frequently the distal tubercle on Finger III (6 of 8). None of the individuals from the Pu Luang population examined has bifid subarticular tubercles. Additional variation is seen in the degree to which the tympanic annulus was visible. In 12 of 37 specimens of L. megastomias, the tympanic annulus was sufficiently visible (though always partially occluded by the supra tympanic fold) to be measured, though it is unclear whether this is an artifact of preservation, or a variable character within this species (see discussion below). Morphological variation observed in a series of 14 tadpoles is summarized in Table 3. The largest total length of 44.0 mm was observed in a Stage-41 tadpole, the smallest (28.0 mm) in a Stage-28 tadpole. During development, the relative difference between internarial and interorbital distances increases (LIN/LIO = 70% at Stage 28 and 46–51% at Stage 41). Though there is variation in individual measurements, none seems to be noteworthy. Etymology. The specific name megastomias is a combination of the Greek words mega, meaning "large" and stomias, which is the masculine noun for "large" or "hard-mouthed" animal. The most striking character- istics of the males of this species are their exceptionally large mouths and powerful jaws. It would not be inap- propriate to describe this frog as an enormous mouth with a body attached to it. The specific name is used as a noun in apposition. Distribution and ecology. Limnonectes megastomias is known from only three localities in eastern Thai- land (Fig. 6). This aquatic frog inhabits streams in dry evergreen forest with year-round water supply and is rarely found away from water. Pang Si Da National Park , located on the southwestern margin of the Khorat Basin, was sampled at elevations between 90 and 600 m along roads, around buildings, in disturbed bamboo and evergreen forests, and in hill evergreen forest. Phu Luang Wildlife Sanctuary, located on the northwestern margin of the Khorat Basin, was sampled at elevation between 850 and 1460 m in hill evergreen, bamboo mixed with evergreen, and rhododendron heath forests. The last forest type was encountered at 1460 m on the

A NEW SPECIES OF LIMNONECTES FROM THAILAND Zootaxa 1807 © 2008 Magnolia Press · 39 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. summit of Kok Nok Kraba, where weather conditions were cool and foggy. At SERS, located on the southwest margin of the Khorat Basin, this frog is known from only one closed- canopy stream. For most of the year, this spring-fed stream is reduced to standing pools connected by a con- tinuous trickle of water. At the time of our visit, the pools were 10–15cm deep, except the largest pool at the head of the stream, was slightly less than 1m at the deepest point. Stream substrate is sand-silt overlaid with an accumulation of leaf detritus, under which these frogs are quick to seek cover when disturbed. Males were observed calling from 1400–2200 hrs. During two visits to the SERS site (2003 and 2005), only one other anuran (Limnonectes gyldenstolpei) was found to co-occur with L. megastomias. Clutches of newly deposited eggs were found in = 10 mm water at the SERS site on 15 March 2005. There was no evidence of parental egg care. Individual jelly capsules of eggs were 12–15 mm in diameter, with brown-pigmented ova 2.3–2.8 mm in diameter. Embryos maintained in captivity began hatching within 2 days of collection and had com- pleted hatching after 7 days. Larvae had reached Gosner (1960) Stage 39 by 12 July when all remaining spec- imens were preserved. Limnonectes megastomias seems to be a sit-and-wait predator and is known to consume insects, frogs (L. gyldenstolpei), and birds.

FIGURE 6. Known distribution of Limnonectes megastomias in Thailand. From north to south: Phu Luang Wildlife Sanctuary, Loei Province; Sakaerat Environmental Research Station, Nakhon Ratchasima Province (star = type locality); Pang Si Da National Park, Sa Kaeo Province.

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TABLE 3. Measurements of tadpole series (FMNH 266340). See text for character definitions. All larvae were staged based on Gosner (1960). * indicates the stage 40 tadpole illustrated in Figure 5.

Stage283030313234343640*4040404141 BL 10.2 10.3 11.1 11.0 11.2 12.1 11.0 13.8 14.4 13.7 13.1 13.6 13.7 12.9 TL 17.8 20.8 20.4 20.7 22.0 20.0 18.8 23.2 28.0 23.5 26.8 25.9 30.3 30.8 ToL 28.0 31.1 31.5 31.8 33.3 32.1 29.8 37.0 42.4 37.2 40.0 39.5 44.0 43.7 BW4.75.25.35.35.45.95.27.68.58.17.47.47.57.0 BH3.52.62.63.53.52.63.63.63.53.82.84.03.84.0 LED1.31.31.41.51.51.51.51.81.81.81.51.81.81.7 LIO2.52.32.72.72.72.82.63.03.53.53.53.53.53.6 LIN1.81.81.91.91.91.91.92.32.22.12.02.01.81.7 LNS1.51.01.81.51.71.81.31.81.82.01.42.01.51.5 LEN1.11.11.21.11.21.31.11.31.41.51.51.21.31.3 SW0.70.70.70.70.70.50.70.80.80.80.51.00.71.0 TMH12.82.82.82.83.03.03.03.53.53.53.33.53.33.3 TMH22.02.52.52.52.42.52.72.93.33.02.83.42.02.5 TH4.98.05.35.25.55.85.57.37.08.06.06.88.05.6 BH/BL0.340.250.240.310.310.210.320.260.240.270.210.300.280.31 BL/ToL 0.36 0.33 0.35 0.35 0.34 0.38 0.37 0.37 0.34 0.37 0.33 0.34 0.31 0.29 BW/BL0.460.510.480.480.480.490.470.550.590.590.560.550.550.54 LED/BL0.120.120.130.130.130.120.140.130.120.130.110.130.130.13 LED/BH0.360.480.530.420.410.560.420.490.500.470.530.440.460.41 LIO/BW 0.53 0.43 0.50 0.50 0.49 0.47 0.50 0.39 0.41 0.43 0.47 0.47 0.46 0.51 LIN/LIO0.700.780.720.700.720.690.730.760.610.600.570.570.510.46 TL/ToL0.640.670.650.650.660.620.630.630.660.630.670.660.690.71 TL/BL 1.75 2.02 1.83 1.88 1.96 1.65 1.71 1.67 1.95 1.72 2.04 1.90 2.22 2.39 TH/ToL 0.17 0.26 0.17 0.16 0.17 0.18 0.18 0.20 0.17 0.22 0.15 0.17 0.18 0.13 TH2/TH 0.41 0.31 0.48 0.48 0.44 0.43 0.49 0.40 0.46 0.38 0.46 0.50 0.25 0.45

Discussion

The genetic distinctiveness of Limnonectes megastomias and the morphological differences that separate it from its congeners are certainly sufficient to warrant the recognition of this frog as a new species. There is, however, a frustrating amount of external morphological similarity between the new species and L. kuhlii, enough to warrant a brief discussion of the “kuhlii complex” and some of the characters shared between L. kuhlii and L. megastomias.

Historically, the identification of Limnonectes kuhlii was credited to Dumeril and Bibron (1841) who pro- vided a description of a single specimen from Java. Tschudi (1838), however, had previously (albeit briefly) described the same taxon under the same name, also from Java. Several names have been synonomized with L. kuhlii, including Rana conspicillata Günther (1872) from Sarawak (Borneo), Nyctibatrachus sinensis Peters (1882) from Guangzhou Province, China, Rana khasiana Boulenger (1882) from the Khasi Hills, India, and Rana paradoxa Mocquard (1890) from Sabah (Borneo) (Frost, 2007). Boulenger (1920) indicated that though he observed great intraspecific variation in Limnonectes kuhlii he was unable to find characters that defined “geographic races. ” Inger (1966) also noted variation among the populations of L. kuhii from Borneo, but found insufficient evidence using external morphology to warrant the

A NEW SPECIES OF LIMNONECTES FROM THAILAND Zootaxa 1807 © 2008 Magnolia Press · 41 TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. recognition of separate species. While examining specimens of the “kuhlii” complex for this study, I have observed a great deal of similarity between L. megastomias and L. kuhlii, enough so that it would be difficult to identify individuals of the same approximate size to the level of species in the field. Population-level variation in morphological characters (in particular, the presence and location of nuptial pads, and the degree to which the tympanum is obscured) is potentially confounding when one is trying to diagnose Limnonectes kuhlii and separate it from Limnonectes megastomias. All males of L. megastomias examined in this study (26 individuals from three populations; SVL 40–123 mm) all have nuptial pads on Fin- gers I and II. Inger (1966) noted that specimens of L. kuhlii from China and Thailand also possessed nuptial pads on Fingers I and II, whereas specimens from Sabah and Sarawak presented nuptial pads only on Finger I. These observations are corroborated by specimens I examined, with the exception of a single male from Thai- land (Chiang Mai Province), which had nuptial pads only on Finger I. Additionally, specimens examined from Laos also have nuptial pads on Fingers II and III. Three adult males from Java examined in this study (sex determined by enlarged head and odontoids, and gonadal evidence): a poorly preserved and bleached lecto- type (MNHN 4469), a paralectotype (RMNH 4297), and a recently preserved specimen. It is clear that the RMNH paralectotype and recent specimens lacks nuptial pads. It appears that the MNHN lectotype also lacks nuptial pads, but the poor state of preservation makes it difficult to say this with certainty. Examination of additional specimens hopefully will resolve this uncertainty. Emerson & Berrigan (1993) described L. kuhlii, L. laticeps, and L. namiyei as all having concealed, but present, tympana. In general, the same character also diagnoses Limnonectes megastomias, yet in 12 of 37 specimens examined, the tympanic annulus was partially obscured, but visible enough to permit a measure- ment of the tympanic diameter. Similarly, Boulenger (1920), considered the tympanum of L. kuhlii hidden or slightly distinct, but provided measurements of the tympanic diameter. Inger (1966) noted that in Bornean populations of L. kuhlii, the tympanic annulus usually was not visible under the skin. Although Tschudi (1838) does not mention the tympanum of L. kuhlii, Duméril and Bibron (1841) discuss its size in relation to the upper eyelid, presumably visible and measurable without dissection of the single Javan specimen from which they based their description. In Javan specimens of L. kuhlii examined here, 12 of 13 have obscured tympana, but still visible enough to allow for measurement. The tympanic annulus is not visible in the poorly preserved lectotype. The specimens of L. kuhlii that I examined from Thailand and Laos also varied in the degree to which the tympanum was obscured (obscure, but visible in 7 of 14, and 4 of 5 specimens, respec- tively), sometimes even varying from side to side in the same animal. It is possible that the state of preserva- tion and hydration of a specimen affects the degree to which the tympanic annulus is visible through the skin. Because most systematic works are based on fluid-preserved specimens, this may influence how this character appears to the investigator. It is reasonable to consider that as a specimen dries, the skin covering of the head covering the tympanic structures tightens and presents an “obscure, but visible” tympanic annulus. The varia- tion in this character among populations of L. kuhlii and its allies warrants closer examination (both in pre- served and live specimens). The presence of bifid subarticular tubercles (proximal or distal, or both) on Fingers II or III (or both) was noted only in some specimens of Limnonectes megastomias. This same character was also observed in four individuals of L. kuhlii from Chiang Mai (Thailand), one specimen of L. kuhlii from Laos, and in none of the specimens from Java. In the Chiang Mai population, only Finger III has bifid tubercles (proximal in all cases, distal in one). The specimen from Laos has a bifid distal subarticular tubercle on Finger III. It is interesting that in the SERS population of L. megastomias bifid subarticular tubercles are present in all individuals exam- ined, but in other populations it seems to be highly variable. The apparent small size and isolation of the SERS population to only one stream may affect the frequency of inheritance of this trait. In Thailand, Limnonectes kuhlii is distributed along the mountainous western edge of the Kingdom from north to south, including the central portion of the Thai-Malay Peninsula (Chan-ard, 2003). The type locality of L. megastomias and Pang Si Da National Park are more than 400 km distant from the western edge of the

42 · Zootaxa 1807 © 2008 Magnolia Press MCLEOD TERM OF USE This pdf is provided by Magnolia Press for private/research use. Commercial sale or deposition in a public library or website site is prohibited. known distribution of L. kuhlii in Thailand. There are records, however, of L. kuhlii (a small-bodied form; SVL < 90 mm) from Loei Province where specimens of L. megastomias also were collected (Phu Luang Wildlife Sanctuary). The amount of genetic divergence between samples of L. kuhlii from Chiang Mai Prov- ince (Thailand) and L. kuhlii from Java (type locality) (14–16%) would suggest that what is currently recog- nized, as “kuhlii” in Thailand is a distinct species. Furthermore, based on the genetic distance between L. megastomias and Chiang Mai populations of L. kuhlii (±9%), it is plausible that there are multiple species of “kuhlii”-like frogs in Thailand and that some of those populations may occur sympatrically with L. megasto- mias. Because of the wide distribution of L. kuhlii and the broad range of morphological variation, it is also possible that populations currently recognized, as L. kuhlii will be subsumed by L. megastomias. A broad and robust sampling of specimens from across the known distribution of L. kuhlii is currently underway to investi- gate the molecular structure of this species at the population level (McLeod, in prep). There remains a great deal of work necessary to resolve fully the identity of the frogs in the kuhlii-com- plex. Phylogenetic resolution of this enigmatic group, and other groups like it, will not be possible without the detailed study of internal and external morphology coupled with a robust molecular analysis (McLeod, in prep).

Acknowledgments

Funding for this project was provided by grants from the United States Geological Survey, The David L. Boren graduate fellowship, The University of Kansas Department of Ecology and Evolutionary biology, and the Natural History Museum and Biodiversity Research Center. Thanks to Mr. Taksin Artchawakom and his excellent staff at SERS who facilitated the adventures that led to the discovery of this frog; Kyle Hesed, an outstanding field assistant; Dr. Kumthorn Thirakhupt, Anchalee Aowphol and the other members of Chula- longkorn University’s “Thai Turtle Lab” for their partnership and support; Heather and Jadelin McLeod made this work possible and took care of larvae in the field; R. Inger and H. Voris provided invaluable input and support that facilitated the development of this paper. A. Ohler, J. van Egmond, J. Vindum, T. LaDuc, J. McGuire, M. Kearney, H.Voris, A. Resetar, and C. Austin facilitated loans of preserved specimens from their respective institutions. Sequencing was done by B. Stuart at The Field Museum’s Pritzker Laboratory for Molecular Systematics and Evolution operated with support from the Pritzker Foundation. Critical reviews and constructive comments on early drafts of this manuscript were provided by R. Brown, J. M. Guyasamin, E. Lehr, R. Inger, C. Siler, and L. Trueb.

References

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Appendix 1: Specimens examined

Comparative material was examined from the holdings of the California Academy of Sciences (CAS), the Chula- longkorn University Museum of Zoology (CUMZ), the Field Museum of Natural History (FMNH), the Louisiana State University Museum of Natural Science (LSUMNS), the Museum National d’Histoire Naturalle (MNHN), the Museum Zoologicum Bogoriense (MZB), Rijksmuseum van Natuurlijke Historie (RMNH), the Texas Natural His- tory Collection of the Texas Memorial Museum (TNHC), the Thailand Natural History Museum (THNHM),the Uni- versity of California Museum of Vertebrate Zoology (MVZ), and the University of Kansas Natural History Museum and Biodiversity Research Center (KU).

Limnonectes laticeps: MALAYSIA: SARAWAK: Bintulu District: KU 155687. Limnonectes kuhlii: INDONESIA: Java Is.: MNHN 4469 (lectotype), RZB 4297 (two specimens: paralectotypes); JAWA BARAT PROVINCE: Java Is.: Kecamatan Kadudampit: TNHC 59826, 59829, LSU 81895; BSI-FS 0026–0032, 0067– 69, 0081–92 (uncataloged specimens, property of MZB); LAO PEOPLE’S REPUBLIC: BOKEO PROVINCE: MNHN 1997.3902, 1997.3904, 1997.3916, 1997.4104, 1997.4106; MALAYSIA: SARAWAK: Bintulu District: KU 155685– 86; Kapit District: KU 155681–84; THAILAND: Chaing Mai Province: CUMZA 2003.4–8, 2003.13, 2003.30, 2003.32–33; LOEI PROVINCE: KU 40185, 40189–90, 40192, 40198–200.

Limnonectes megastomias: THAILAND: LOEI PROVINCE: Phu Rua District: KU 30776073; FMNH 266212 (THMNH 05003), FMNH 266213 (THMNH 05006), FMNH 266214 (THMNH 05004), FMNH 266215 (THMNH 05002), FMNH 266216–17 (THMNH 05007–08), FMNH 266218 (THMNH 05001); NAKHON RATCHASIMA PROVINCE: Wang Nam Khieo District: CUMZA 2003.134–135, KU 307760–83; SA KAEO Province: Sa Kaeo District: FMNH 266220–223 (= THMNH 05379–83), FMNH 266224 (= THMNH 05342), FMNH 266225–32 (= THMNH 05384– 90).

Limnonectes namiyei: JAPAN: NAGO: Okinawa Shima Loo Choo Isls: CAS 22614, 22622, 22809, 22810–18.

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Appendix 2. Samples and sequences used in this study.

Species Country of Origin Specimen voucher numbers Accession number(s) Occidozyga laevis Philippines VUB0967 (DLSUD 002) DQ347024 Fejervarya limnocharis China SCUM04H001 DQ458239 Limnonectes megastomias Thailand CUMZA 2003.134 L. megastomias Thailand CUMZA 2003.135 L. megastomias Thailand FMNH 266224 L. megastomias Thailand FMNH 266229 L. megastomias Thailand FMNH 266232 L. megastomias Thailand FMNH 266340 L. laticeps Malaysia SBE 071 AF183125, AF183126 L. fujianensis China YNUHU20026017 DQ458234 L. fujianensis China KIZYP028 DQ118473, DQ118518 L. gyldenstolpei Thailand PWRC 002 AF183123, AF183124 L. kuhlii China ZNAC 21014 AY703868, AY703855 L. kuhlii China ZNAC 21020 AY703869, AY703856 L. kuhlii China KIZ-RD05DT1 DQ458245 L. kuhlii Vietnam ROM 19384 AF206464, AF206083, AF206128 L. kuhlii Vietnam AMNH A161202 DQ283370 L. kuhlii Vietnam VUB 0930 DQ346995 L. kuhlii Malaysia (Borneo) FMNH 230302 AF183136, AF183135 L. kuhlii Indonesia (Borneo) AMNH 167141 AY313686 L. kuhlii Malaysia (Brunei) FMNH 248357 AF183134, AF183133 L. kuhlii Laos MNHN 1997.3904 AF215415, AF215209 L. kuhlii Thailand CUMZA 2003.5 L. kuhlii Thailand CUMZA 2003.8 L. kuhlii Taiwan FMNH 257133 AF183132, AF183131 L. kuhlii Indonesia (Java) MZB Amphib6501 AY313687 L. kuhlii Indonesia (Java) Deposited in MZB AF183138, AF183137

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