anatomy, histology, and systematic implications of the head ornamentation in the males of four...

Anatomy, histology, and systematic implications of thehead ornamentation in the males of four species ofLimnonectes (Anura: Dicroglossidae)

MARKUS LAMBERTZ1*, TIMO HARTMANN2, SHANNON WALSH3, PETER GEISSLER2

and DAVID S. MCLEOD3

1Institut für Zoologie, Rheinische Friedrich-Wilhelms-Universität Bonn, Poppelsdorfer Schloss, 53115Bonn, Germany2Sektion Herpetologie, Zoologisches Forschungsmuseum Alexander Koenig, Adenauerallee 160, 53113Bonn, Germany3University of Kansas Biodiversity Institute, 1345 Jayhawk Blvd., Lawrence, Kansas, USA

Received 10 March 2014; revised 24 April 2014; accepted for publication 6 May 2014

The males of four species of the Asian frog genus Limnonectes [Limnonectes dabanus (Smith, 1922a), Limnonectesgyldenstolpei (Andersson, 1916), Limnonectes macrognathus (Boulenger, 1917), and Limnonectes plicatellus (Stoliczka,1873)] exhibit remarkable ornamentation in the form of a swollen, or cap-like, structure (caruncle) on the top oftheir heads. These caruncles vary in their appearance among species, and neither their function nor their actualsystematic value is known. We compared their anatomy via dissections, morphometrics, radiography, and histol-ogy, and analysed the available mitochondrial DNA sequences as well as new data to place these species withinthe context of a larger phylogenetic hypothesis for Limnonectes. Despite the externally different morphology, theunderlying histological structure is virtually identical. Beneath skin that is densely packed with mucous glandslies a pad of connective tissue overlaying the parietal bone. The actual function of the caruncle, however, remainsenigmatic. In addition to the presence of the caruncle, independent evidence from osteological characters and mo-lecular data support the monophyly of a clade comprising of L. dabanus, L. gyldenstolpei, L. macrognathus, andL. plicatellus. The caruncles are therefore interpreted as a robust autapomorphy for this clade, and suggest thatthe subgenus Elachyglossa should be restricted to the four species in question.

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014doi: 10.1111/zoj.12171

ADDITIONAL KEYWORDS: Elachyglossa – functional morphology – integrative taxonomy – L. dabanus –L. gyldenstolpei – L. macrognathus – L. plicatellus – phylogeny – Ranoidea – Southeast Asia.

INTRODUCTION

The Asian frog genus Limnonectes Fitzinger, 1843(Ranoidea: Dicroglossidae, following Pyron & Wiens,2011) comprises 61+ extant species (Frost, 2014). Inrecent years, molecular phylogenetic analyses of thisgroup have revealed a remarkable level of diversity(e.g. Emerson, Inger & Iskandar, 2000; Evans et al.,2003; Zhang et al., 2005; McLeod, 2008, 2010; Matsui

et al., 2010a; McLeod et al., 2011). Much of this diver-sity is embodied in brown-coloured, stream-dwellingfrogs that are morphologically conservative and un-impressive in appearance. Collectively referred to asthe ‘fanged frogs’, because of the presence of odontoidprocesses in both sexes, the genus is characterized byan unusual suite of secondary sexual characteristics.Males of the genus typically exhibit enlarged odontoids,hypertrophied heads, and subsequently male-biased sizedimorphism. Among the more unusual members ofLimnonectes are four species in which adult males have*Corresponding author. E-mail: [email protected]

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Zoological Journal of the Linnean Society, 2014. With 7 figures

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well-developed head ornamentation: Limnonectesdabanus (Smith, 1922a), Limnonectes gyldenstolpei(Andersson, 1916), Limnonectes macrognathus(Boulenger, 1917), and Limnonectes plicatellus (Stoliczka,1873). These species have an Indo-Chinese distribu-tion and are considered species of least concern or datadeficient by the International Union for Conserva-tion of Nature (IUCN, 2013). Limnonectes gyldenstolpeiand L. plicatellus were originally described based onfemale specimens (see Ohler & Dubois, 1999), whichlack the unusual head ornamentation. Boulenger (1917)was the first to mention a structure on the head inL. macrognathus, and Smith (1922a, b) documented thisin the males of all four species and used the superfi-cial morphological similarity as evidence for their re-latedness. Smith (1922a) also provided a very briefdescription of the structure in L. macrognathus andL. gyldenstolpei (as Rana pileata), which was charac-terized as being mainly composed of dense fibrous tissue.These structures have been referred to variably as ‘caps’(Chan-ard, 2003), ‘flaps’ (Smith, 1922a), and as a ‘fleshybutton’ (Emerson & Berrigan, 1993). In order to es-tablish consistency for the terminology of these struc-tures we recommend the term caruncle, as it is usedin ornithology to describe flap-like integumentary struc-tures (Campbell & Lack, 1985).

Despite nearly a century of acknowledging theseunusual head ornaments, no study has undertaken adetailed examination of the histological structure orpossible function of the caruncles in these species. Inour experience the presence of such an unusual andconspicuous structure leads the observer to wonder atits possible function, with the most obvious questionbeing: can the frog independently move this struc-ture? Several plausible explanations for the presenceof the caruncle can be imagined, including sexual se-lection or defensive posturing. Moreover, the system-atic value of this structure has not been fully explored,nor has any study included all of the ornamentedspecies. Results of recent molecular phylogeneticstudies are inadequate for addressing the relation-ships among these taxa because they have includedonly L. gyldenstolpei (e.g. Evans et al., 2003; McLeod,2010; McLeod, Kelly & Barley, 2012; Suwannapoomet al., 2012), or L. dabanus and L. gyldenstolpei (Inger& Stuart, 2010; Pyron & Wiens, 2011). Emerson &Berrigan (1993) included L. gyldenstolpei (as R. pileata)and L. plicatellus (as Rana plicatella) in theirmorphology-based phylogenetic analysis, and indeedconsidered the presence of the caruncle as asynapomorphy for these two taxa.

The goals of this study are: (i) to describe the com-parative anatomy and address possible functions of thedifferent caruncles; and (ii) to present a phylogenetichypothesis based on morphological and molecular datafrom all four of these species of Limnonectes.

MATERIAL AND METHODSSPECIMENS EXAMINED

The adult male specimens examined in this study aredeposited at the Field Museum of Natural History(FMNH, Chicago, IL, USA), the University of KansasBiodiversity Institute (KU, Lawrence, KS, USA), andthe Zoologisches Forschungsmuseum Alexander Koenig(ZFMK, Bonn, Germany). Reproductive status and sexwere determined by gonadal examination and/or thepresence of secondary sexual characters (nuptial pads,odontoid processes, caruncles). Specimens were fixedin 10% formalin and/or were preserved in 70% ethanol(EtOH). Details on the specimens are summarized inTable 1. Specimens were identified based on morpho-logical characters such as the presence and nature ofthe caruncle, extent of digital webbing of the foot, andrelative size of the tympanum (Smith, 1922a,b; Taylor,1962).

MACROSCOPIC ANATOMY

Measurements were made with digital calipers to thenearest 0.1 mm, as follows: CW, maximum width ofcaruncle; EC, distance from the posterior margin ofthe eye to the posterior margin of the caruncle; HL,distance from tip of snout to mouth angle; HW, headwidth at mouth angle; SC, distance from tip of the snoutto the posterior margin of the caruncle; SVL, snout–vent length. For L. gyldenstolpei, a Pearson correla-tion with Bonferroni-corrected significance levels(P = 0.05/9) for SC, CW, and EC, all with SVL, HL,and HW, respectively, was performed using CRAN R(R Core Team, 2013).

The cranial portion of one male L. gyldenstolpei(ZFMK 95609) was mediosagitally sectioned with a razorblade and the right half was excised from the speci-men posterior to the level of the tympanum. The re-maining internal view of the head was photographedin situ. To achieve a better resolution of the overalltopographical anatomy of the head, a thin slicewas removed with a razor blade parasagitally fromthe specimen afterwards and decalcified in 2.5%ethyleneaminotetraacetic acid (EDTA) for 3 days andembedded in paraffin according to standard proto-cols. The cut surface of this block was deparaffinizedwith xylene, the tissue rehydrated through graded seriesof EtOH, and then stained with Mayer’s haematoxylin,counterstained with eosin, and finally photographedfor a comparison with the in situ image.

A Canon EOS 5D Mark-II DSLR coupled with a CanonMacro Lens EF 100 mm 1:2.8 L IS was used to photo-graph surface anatomy in adult male specimensof L. dabanus (ZFMK 95611), L. gyldenstolpei(ZFMK 95610), L. macrognathus (FMNH 270104),and L. plicatellus (FMNH 186577), as well as the

2 M. LAMBERTZ ET AL.

© 2014 The Linnean Society of London, Zoological Journal of the Linnean Society, 2014

mediosagitally cut head of the aforementionedL. gyldenstolpei.

Radiographs of the complete body in dorsal view, aswell as details of the head in dorsal and lateral viewof L. dabanus (ZFMK 88991), L. gyldenstolpei (ZFMK90254), L. macrognathus (FMNH 270104), andL. plicatellus (FMNH 186577), were made using an LX60digital X-ray (Faxitron X-ray LLC; Lincolnshire, IL,USA).

HISTOLOGY

Three male specimens of L. gyldenstolpei (KU 336134,336135, ZFMK 95610), and one male each of L. dabanus(ZFMK 95611), L. macrognathus (FMNH 270104), andL. plicatellus (FMNH 186577) were used for histologi-cal studies. The caruncle, concomitant tissues, andunderlying bone were excised by making a rectangu-lar incision around this structure. The resulting blockof soft tissue and bone was decalcified in 2.5% EDTAfor at least 3 days. The left halves of caruncles fromL. dabanus and L. gyldenstolpei (ZFMK 95610, 95611;mediosagitally sectioned with a razor blade) were placedin 30% sucrose overnight and then transferred toShandon M-1 Embedding Matrix (Thermo Fisher Sci-entific Germany Ltd. & Co KG, Bonn, Germany) andsectioned at 25–50 μm on a CM1850 cryostat (LeicaMicrosystems Nussloch GmbH, Nussloch, Germany).

After obtaining several good parasagittal sections fromthe medial portion of the L. gyldenstolpei carunclesample, the tissue block again was covered with cryo-medium and rotated by 90° to facilitate transverse sec-tions through the lateral margin of the caruncle.

Right halves of the two species mentioned above andthe entire caruncle from L. macrognathus andL. plicatellus were dehydrated in graded solutions ofEtOH and embedded in 2-hydroxyethyl methacrylate(Technovit 7100; Heraeus Kulzer GmbH, Wehrheim,Germany). Parasagittal sections of 2–3 μm in thick-ness were cut at 20-μm intervals through the entirecaruncle specimen with an HM 350 rotary microtome(Microm International GmbH, Walldorf, Germany). Allsections were stained with toluidine blue (0.1%) andmounted in Roti Histokitt II (Carl Roth GmbH + Co.KG, Karlsruhe, Germany) on glass slides. The carunclesof two other L. gyldenstolpei specimens (KU 336134,336135) were embedded in paraffin according to stand-ard procedures and serially sectioned at 10 μm usinga TBS CUT 4060 rotary microtome (Fisher Scientific,Waltham, MA, USA). Paraffin-mounted sections weresubsequently stained using two protocols. For prelimi-nary examination of tissue structure, we usedDelanfield’s haematoxylin, counterstained with eosin,cleared in xylene, and mounted in Canada balsam onglass slides. Alcian Blue was used to visualize thegylcosaminoglycans in the tissue (glands, cartilage, and

Table 1. List of specimens and morphometric characters examined. All measurements are given in mm and specimensare ordered according to increasing snout–vent length (SVL) within their species

Species SVL HL HW SC EC CW Voucher

Limnonectes dabanus 53.4 24.5 26.2 19.7 8.1 5 ZFMK 88990Limnonectes dabanus 53.9 24.7 27.5 24.6 – 8 ZFMK 95611*Limnonectes dabanus 56.4 28.2 30.3 24.1 10.6 7.2 ZFMK 88992Limnonectes dabanus 66.8 33 32.7 25.9 12.3 9.2 ZFMK 88991Limnonectes gyldenstolpei 41 17.7 18.3 10.5 3.3 4.4 ZFMK 89292Limnonectes gyldenstolpei 43.7 20.9 21.4 15.4 3.6 8.7 ZFMK 89287Limnonectes gyldenstolpei 50.4 21.9 23.1 14.1 2.3 5 ZFMK 89290Limnonectes gyldenstolpei 51 24.6 23.6 15.7 2.9 6.5 ZFMK 90258Limnonectes gyldenstolpei 51.1 24 24 15.4 4 7.5 ZFMK 90262Limnonectes gyldenstolpei 53.8 24.6 25.7 18.7 5.3 8.2 ZFMK 90253Limnonectes gyldenstolpei 54 27.3 26.7 21.3 – 9 ZFMK 95610*Limnonectes gyldenstolpei 54.5 25.9 26.4 18.4 5.3 10.9 ZFMK 89293Limnonectes gyldenstolpei 57.7 27.4 27.9 18.7 5.2 9.6 ZFMK 90259Limnonectes gyldenstolpei 58.1 26.7 26.8 16.3 3.1 6.4 ZFMK 89294Limnonectes gyldenstolpei 58.6 28.9 27.3 19.0 3.5 7.4 KU 336135*Limnonectes gyldenstolpei 59.3 30 30.8 26.8 5.1 11.9 ZFMK 95609Limnonectes gyldenstolpei 61.2 30.5 29.7 20.7 5.3 8.4 KU 336134*Limnonectes gyldenstolpei 62.6 32 31.1 22.3 6.4 11.5 ZFMK 90254Limnonectes macrognathus 44.7 17 19.5 15 3 7.4 FMNH 270104*Limnonectes plicatellus 33.1 12.6 15 11.8 2.6 2.6 FMNH 186577*

*Specimens used for histology.

HEAD ORNAMENTATION OF LIMNONECTES SPP. 3


lamina calcarea) using the following protocol: 100%Xylene (3x, 2 min); 100% EtOH (3x, 2 min); 70% EtOH(1x, 2 min); deionized (DI) H2O (1x, 2 min); 3% aceticacid (3x, 2 min); Alcian Blue (1x, 30 min); 3% aceticacid (rinse); DI H2O (1x, 10 min using running water);Nuclear Fast Red (1x, 5 min); DI H2O (1x, 1 min usingrunning water); 100% isopropyl alcohol (3x, 2 min); 100%xylene (3x, 2 min); mounted in Canada balsam on glassslides. All sections were analysed with a Leica DMREor a Zeiss Axioskop 2 Plus compound microscope.

MOLECULAR SYSTEMATICS

To take advantage of readily available comparative ma-terial from previous work we used the approximately1400-bp region of 16S mitochondrial DNA (mtDNA)data (Emerson et al., 2000; Evans et al., 2003; Jiang& Zhou, 2005; Zhang et al., 2005, 2009; Frost et al.,2006; Che et al., 2007; Inger & Stuart, 2010; McLeod,2010; Matsui et al., 2010a,b). Clade nomenclature followsthat of McLeod (2010) and McLeod et al. (2011). Severalnew molecular sequences, including one sample ofL. macrognathus (FMNH 270104) and three samplesof L. plicatellus (LSUHC 4001, 6710, 6582) were addedto the data matrix following DNA extraction and se-quencing protocols detailed in McLeod (2010). Taxa in-cluded in this study were selected based on previouslyreported relationships with the caruncle-bearing species,the availability of data, and an effort to broadly rep-resent the relationships of the focal taxa to othermembers of the genus Limnonectes. The Limnonecteskuhlii (Tschudi, 1838) complex was sampled from ex-tensively because of previous uncertainties regardingthe relationship between L. gyldenstolpei andLimnonectes kuhlii (from Java) and other members ofthis complex (D.S.M., unpubl. data). Occidozyga laevis(Günther, 1858), Hoplobatrachus rugulosus (Wiegmann,1834), and Fejervara limnocharis (Gravenhorst, 1829)were used to root the tree.

A data matrix comprising 82 taxa (Appendix) wasaligned using MUSCLE (Edgar, 2004) and then ad-justed by eye in SE-AL CARBON 2.0a11 (Rambaut,2002). Maximum-likelihood (ML) analyses were per-formed using RAxML-HPC BLACKBOX 7.3.2 on theCIPRES Science Gateway (Stamatakis, 2006; Miller,Pfeiffer & Schwartz, 2010) via 1000 non-parametricrapid bootstrap replicates. A thorough ML search withbootstrap scores was mapped onto the best-scoring MLtree. Bayesian analyses (BA) were conducted usingMrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) onXSEDE accessed through the CIPRES Science Gateway(Miller et al., 2010). Four independent analyses wererun with four Metropolis-coupled Markov chains each.All Markov chains were run for 10 million genera-tions, sampling every 1000 generations. To assess con-vergence between chains, we verified that the average

standard deviation of split frequencies approached zero,the potential scale reduction factor approached 1, andthat the log likelihood scores had reached stationarity.The output files were examined in TRACER 1.5(Rambaut et al., 2013) to determine the number of gen-erations to exclude as burn-in and as a final check forconvergence [ensuring that all parameters and statis-tics had reached stationarity and sufficient (> 100)effective sample sizes].

RESULTSMACROSCOPIC ANATOMY

The caruncle in L. macrognathus is the smallest andmost inconspicuous among the four species examined(Fig. 1). In gross appearance it is a small, low-profile,domed structure without a free posterior edge. It extendsslightly (7% of SVL) beyond the posterior margin ofthe eyes, obtains a thickness of less than 1 mm, andis relatively wide (38% of HW). Limnonectes gyldenstolpeipresents a U-shaped, flap-like caruncle that is thicker,more robust, and has a free posterior edge. The largerseries that was available for this species revealed acertain level of variation between specimens. The carun-cle reaches 5–10% of SVL beyond the eye and 22–41% of head width. Our results reveal a slight tendencytowards a larger caruncle with increased SVL, HL, andHW, respectively. Statistically significant correlationsexist between SC and SVL (N = 12, r = 0.784, P = 0.003),HL and CW (N = 12, r = 0.785, P = 0.002), HL and SC(N = 12, r = 0.881, P < 0.001), HW and CW (N = 12,r = 0.804, P = 0.002), as well as HW and SC (N = 12,r = 0.906, P < 0.001). There are, however, both largespecimens with a small caruncle and relatively smallspecimens with a rather large caruncle. No statisti-cally significant correlations were detected betweenCW and SVL (N = 12, r = 0.661, P = 0.019), EC and SVL(N = 11, r = 0.622, P = 0.041), HL and EC (N = 11,r = 0.697, P = 0.017), and HW and EC (N = 11, r = 0.714,P = 0.014). In L. plicatellus, the caruncle takes the formof a horn, as also evidenced by its common name ‘Rhi-noceros frog’ (Chan-ard, 2003). The horn-like struc-ture extends slightly behind the eyes (8% SVL), andis relatively narrow (17% HW) at its base. The morerobust and relatively high-profile, domed, caruncle inL. dabanus extends well beyond the posterior marginof the eyes (15–18% of SVL), and is wider at its base(19–28% of HW). Morphometric values for all speci-mens examined are summarized in Table 1.

The in situ section of the entire head inL. gyldenstolpei reveals the caruncle as a dense padon top of the parietal bone. In the image of the stainedcut surface, the mass making up the caruncle can beseparated from the surrounding tissues and appearsas a relatively dense mass of connective tissue on top



of the parietal bone (Fig. 2). The body of this carun-cle extends dorsally from the cranium and then swingscaudad, resulting in the flap-like appearance. The skinseems to follow this swing and appears to be firmlyattached to the underlying connective tissue only atthe dorsalmost and posterior margins.

The radiographs (Fig. 3) show that the translu-cence of the caruncle tissue in all four species is similar

to that of the remaining soft tissue of the body. Thereis no indication of an osseous or similar solid centralsupporting element of the actual caruncle; however,there appears to be a dense, apparently osseous bulgingof the parietal bone at the anterior margin of the carun-cle, which is most prominent in L. dabanus (Fig. 3,arrowhead). The caruncle always arises from the dorsalside of the parietal bone and may extend further caudad,

Figure 1. Appearance of caruncles in dorsal (top row), lateral (middle row), and frontal (bottom row) views of the fourspecies of Limnonectes. Only the dorsal images are to scale. Scale bar: 1 cm.

Figure 2. Head of a male Limnonectes gyldenstolpei, mediosagittally split. The actual specimen is shown on the left,whereas the right-hand image shows the H&E stained surface of the corresponding view. Note the flap-like caudad curveof the posterior margin of the caruncle (ca). Abbreviations: br, brain; ca, caruncle; to, tongue. Scale bar: 1 cm.



also covering the occipital region. The remaining cranialbones show no notable abnormalities. It is, however,worth noting that the pterygoid bones in all four speciesexamined exhibit a concave lateral curvature of theiranterior ramus (Fig. 3, arrows).

HISTOLOGY

Histologically, the skin covering the caruncles of allfour species examined was found to be typical of am-phibian skin (Fig. 4). The skin itself can be subdivid-ed into an epidermis with a thin stratum corneum anda moderate stratum germinativum, as well as a dermiscomposed of a stratum spongiosum and a stratumcompactum. Both dermal layers are clearly separat-ed from each other by a distinct lamina calcarea(Fig. 5A). All observed glands are mucous producingand occur in high density over the entire caruncle, in-cluding the lateral margins. These glands are embed-ded within the stratum spongiosum of the dermis(Fig. 5A,B). Irregular osseous projections arising fromthe parietal bone are present at the anterior base ofthe caruncle (Fig. 5C,D). Beneath the dermal part of

the integument lies a pad of fibrous connective tissuethat varies in thickness according to the species in ques-tion (Figs 4, 5E). The flap-like bending of the carun-cle (in species exhibiting it) occurs within the pad ofconnective tissue and leaves a sharply marked cleav-age between adjacent ‘layers’ (Fig. 5F). The whole padof connective tissue conducts a network of variably sizedcapillaries that vascularize the skin (Fig. 5A,B,E–G).Occasionally fat cells were found scattered within thispad of connective tissue, generating an adipose-likeappearance (Fig. 5G).

MOLECULAR SYSTEMATICS

Results of the molecular analyses provide evidencefor a monophyletic clade of caruncle-bearingLimnonectes. Both ML and BA analyses recovered thesame relationships among the species of interest, al-though support for nodes deeper in the tree differedbetween analyses. Results of the BA are presented inFigure 6.

In both analyses, the caruncule-bearing Limnonectes(L. gyldenstolpei, L. dabanus, L. plicatellus, and

Figure 3. Radiographs of the four species of Limnonectes examined in the present study in dorsal (top row), ventral(middle row), and lateral (bottom row) views. Note the translucent caruncle and the underlying osseous bulging of theparietal bone, most pronounced in Limnonectes dabanus (arrowhead). The arrows indicate the concave lateral curvatureof the anterior ramus of the pterygoid bone. Only the dorsal images are to scale. Scale bar: 1 cm.



L. macrognathus) form a monophyletic clade that is wellsupported in the BA (Bayesian posterior probabil-ity = 1.0), but that is only marginally supported in theML analysis (63% bootstrap support). Within this cladethere is full support (both analyses) for sister rela-tionships between L. gyldenstolpei and L. dabanus, andbetween L. plicatellus and L. macrognathus. Thecaruncule-bearing Limnonectes are sister to the cladecontaining Limnonectes microdiscus (Boettger, 1892),Limnonectes kadarsani Iskandar, Boeadi & Sancoyo,1996, and Limnonectes laticeps (Boulenger, 1882).Results of the BA present these two clades as recip-rocally monophyletic, but in the ML analysis, the cladecontaining L. microdiscus and L. kadarsani is sisterto L. laticeps, which is sister to the caruncularLimnonectes. This hypothesis, however, is only weaklysupported in the ML analysis, with < 65% nodal boot-strap support.

Clade A comprises Limnonectes kuhlii (Tschudi, 1838)and Limnonectes sisikdagu McLeod et al., 2011. Clades Band C contain Indo-Chinese members of the L. kuhliicomplex, and clade D comprises all Bornean L. kuhliicomplex taxa. Clade E2–4 contains non-kuhlii complexspecies of Limnonectes from the study of Evans et al.(2003).

DISCUSSIONANATOMY AND HISTOLOGY OF THE CARUNCLES

Our results on the outer appearance of the carunclesin L. dabanus, L. gyldenstolpei, L. macrognathus, andL. plicatellus are largely congruent with the earlierdescriptions and illustrations given by Smith (1922a,b) and Taylor (1962). The caruncles in all taxa exam-ined extend from the parietal bone, and each showsa species-specific and therefore diagnostic shape.

Using L. gyldenstolpei as the exemplar, we providehere the first morphometric evaluation of caruncle char-acteristics for one of the four species. Our data indi-cate that the size of the caruncle correlates with thesize of the specimen. Larger specimens, in general, alsoappear to have larger caruncles, but there are alsoseveral exceptions from this rule. Whether these gen-eralized relationships are also shared by the other threespecies remains to be studied. Nevertheless, our ob-servations suggest that although the general extentof the caruncles is intraspecifically variable, it servesas a diagnostic character of interspecific variation(Table 1).

Additionally, we provide here the first microscopicinvestigation of caruncle morphology for any of these

Figure 4. Comparative overview of the histological structure of the caruncles in the four species of Limnonectes: A, Limnonectesdabanus; B, Limnonectes gyldenstolpei; C, Limnonectes macrognathus; D, Limnonectes plicatellus. Note that in spite ofthe fundamentally differing external morphology, the histology is virtually identical: a size-variable pad of connectivetissue (ct) lies between the skin (sk) and the parietal bone (pb). Scale bars: 2 mm.



four species of Limnonectes. Despite the species-specific differences in external morphology, especiallywith regard to extent and shape, the internal histo-logical structure is virtually identical in all four speciesstudied. The caruncle is always composed of a pad ofmainly fibrous connective tissue that is covered by skintypical of other anurans.

POSSIBLE FUNCTIONAL IMPLICATIONS OF THE

CARUNCLES

Despite the array of possible functions that we couldimagine for the caruncle in these four species, the resultsof our study do not provide any direct evidence forits purpose. In contrast to the superficially similar

Figure 5. Details of the histological structure of the caruncle. A, dorsal portion of the caruncle parasagitally sectionedand showing the typically layered skin, with underlying connective tissue (ct). Abbreviations: ct, connective tissue;lc, lamina calcarea; sc, stratum corneum; sco, stratum compactum; sg, stratum germinativum; ss, stratum spongiosum.B, transverse section through the lateral margin showing the overall homogeneity of the caruncle. C, D, irregularosseous projections (op) of the parietal bone at the anterior margin of the caruncle. E, detail of dense connectivetissue. F, cleavage within the connective tissue of a flap-like caruncle. G, detail of the connective tissue showingcapillaries (arrowheads) of various dimensions and fat cells (fc). Limnonectes macrognathus is shown in (A), (D), and(E); L. gyldenstolpei is shown in (B), (F), and (G); L. dabanus is shown in (C). Scale bars: A, D, E, G, 100 μm; C, 200 μm;B, F, 500 μm.



swellings on the head of certain South American speciesof Melanophryniscus Gallardo, 1961 (Anura, Bufonidae)(see Naya, Langone & de Sá, 2004), there are no in-dications that the caruncles in Limnonectes act asa macrogland or anything similar. Only mucousglands typical of amphibian skin were observed in ourspecimens.

Similarly, the direct value of the caruncle as a sec-ondary sexual character, functioning as an optic cue,seems doubtful. Given the current understanding ofoptic signalling in anurans (Hödl & Amézquita, 2001),it seems that the caruncle is simply too small (ex-tending only a few millimetres above the dorsal surfaceof the head) to be an effective visual cue, although thishypothesis needs further investigation in order toaddress it adequately. Furthermore, muscular controlof the structure would be expected if the caruncle isused in visual signalling between conspecifics. Exam-

ples of such signalling behaviours are known fromseveral anuran taxa (e.g. Orlov et al., 2012), but usuallyinvolve active movements of larger or more conspicu-ous structures, such as the extremities.

Vocalization in frogs is produced during expiratoryair movements from the lungs to the buccal cavity orvocal sacs (Gans, 1973). Ultimately, this results in anoscillation of the entire body, which is transduced tothe surrounding air and can then be perceived as soundby other animals. There is no indication that thecaruncles can actively be recruited as an inflatableacoustic device that is directly involved in the attrac-tion of female conspecifics or the deterrence of malerivals. Although small cavities were observed, particu-larly in species with flap-like caruncles, no central reso-nating cavity could be detected. Additionally, the absenceof connections to the external environment rendersinflation of the existing cavities impossible. These

Figure 6. Simplified phylogram demonstrating the relationships among the caruncle-bearing Limnonectes, within thecontext of the genus Limnonectes and the Limnonectes kuhlii complex, based on a Bayesian analysis of 16S mtDNA se-quences. Numbers above branches are Bayesian posterior probabilities. Fejervara limnocharis (Gravenhorst, 1829),Hoplobatrachus rugulosus (Wiegmann, 1834), and Occidozyga laevis (Günther, 1858) were used to root the tree (not shown).



cavities only appear to result from the lack of dermalconnections presented by certain caruncles in pre-served specimens. Caruncles are individually vari-able masses located at the centre of oscillation duringvocalization, namely the head, and it can be hypoth-esized, purely on basic physical principles, that theyhave a modulatory influence on the frequency of theemitted sound. Behavioural and bioacoustic data areneeded to test this hypothesis and demonstrate the in-fluence of the caruncle on sound production.

Another potential role for this structure could be foundin combat behaviour. Although in general only very littleis known about the ecology of Limnonetces spp., thereare a few studies that report male–male combat in thistaxon (Orlov, 1997; Tsuji & Matsui, 2002; Tsuji, 2004).The overall male-biased sexual size dimorphism indeedmakes male–male combat behaviour a likely trait forthe entire genus (Shine, 1979; Kupfer, 2007). Whereasa general function of the caruncles as a helmet-likestructure seems unlikely (there are no signs of a thick-ened epidermis, for example), the high density of ex-clusively mucous glands could serve a ‘lubricating’function during combat. More research on the basicecology of the species in question is needed to test thishypothesis, however.

The observed relationships between the size of thecaruncle and SVL, HL, and HW in L. gyldenstolpei,in conjunction with the remarkable outliers (Table 1),may be interpreted as an indication of individual fitness-related expression of this structure. The presence ofadipose tissue in the body of the caruncle suggests thatcaruncle size may reflect the nutritional condition ofa specimen, and in turn be related to individual fitness.

SYSTEMATIC VALUE AND IMPLICATIONS

OF THE CARUNCLES

On the basis of morphological similarities (vomerineteeth, skin, toe webbing, and coloration), Smith (1922a,b) argued that L. macrognathus, L. gyldenstolpei (aspileatus), Limnonectes kochangae (Smith, 1922a),L. dabanus (as macrognathus dabana), L. plicatellus,and Limnonectes doriae (Boulenger, 1887) comprisedan evolutionary clade. Interestingly, although Smith(1922a, b) discusses the significance and uniquenessof the caruncle in four of these species, he seems tohave given greater weight to other aspects of mor-phology than to the presence or absence of the carun-cle, and consequently he includes two species(L. kochangae and L. doriae) in the group that lack thisstructure.

Based on molecular evidence, Inger & Stuart(2010) present a well-supported hypothesis in whichtwo caruncle-bearing species (L. dabanus andL. gyldenstolpei) are each other’s closest relative, andsister to a clade comprising L. kohchangae, L. doriae,

Limnonectes limborgi (Sclater, 1892), and Limnonecteshascheanus (Stoliczka, 1870), all of which lack the carun-cle. Other recent studies have proposed a clade com-prising L. microdiscus, L. kadarsani, L. laticeps, andL. gyldenstolpei, in which a sister relationship betweenL. laticeps and L. gyldenstolpei is hypothesized (Emersonet al., 2000; Evans et al., 2003; Inger & Stuart, 2010;McLeod, 2010; McLeod et al., 2011; Pyron & Wiens,2011). Results of our molecular analyses corroboratethese phylogenetic hypotheses and suggest that a cladecomprising L. laticeps, L. microdiscus, and L. kadarsaniis sister to the clade comprising the four species ofcaruncle-bearing Limnonectes: L. gyldenstolpei,L. dabanus, L. macrognathus, and L. plicatellus. Withinthis latter clade, two well-supported pairs of sistertaxa exist: L. gyldenstolpei + L. dabanus and L.macrognathus + L. plicatellus.

In addition to molecular evidence and the presenceof the caruncle, a third and independent osteologicalsynapomorphy presented here corroborates the hy-pothesis of monophyly for the four caruncle-bearingspecies. We found that the anterior ramus of the ptery-goid bone exhibits a concave curvature in L. dabanus,L. gyldenstolpei, L. macrognathus, and L. plicatellus,but is convex in other congenerics (Emerson & Berrigan,1993). Because the pterygoid bone does not have a directconnection to the caruncle it seems reasonable that thesetwo characters are not influenced by each other andshould therefore be regarded as functionally and evo-lutionarily uncoupled.

A limitation of this study is the absence of com-parative material in analyses (both molecular and mor-phological) for L. doriae and L. kohchangae. In addition,the use of a single mitochondrial gene for phylogeneticanalyses has recognized drawbacks, as evidenced bythe poor resolution and weak support at deeper nodesin the Limnonectes tree. Nevertheless, our results usingonly 16S mtDNA achieve consistent and comparableresults that corroborate other studies that have em-ployed multiple genes. To fully test the phylogenetichypothesis of Smith (1922a, b), molecular and mor-phological (especially osteological) data from L. doriaeand L. kohchangae, and additionally from L. limborgiand L. hascheanus (taxa proposed to be most closelyrelated to L. doriae by Inger & Stuart, 2010), are re-quired. We propose the following hypothesis (Fig. 7)to be tested by future studies that incorporate a morerobust molecular data set and a careful examinationof osteological characters, particularly with regard tothe pterygoid and other cranial elements: the clade com-prising L. kohchangae, L. doriae, L. limborgi, andL. hascheanus, which lack the caruncle, are expectedto have a convex curve of the anterior ramus of thepterygoid, and will be sister to a clade comprisingL. gyldenstolpei, L. dabanus, L. macrognathus, andL. plicatellus. Furthermore, on the basis of existing



molecular evidence, we propose that these two cladeswill be most closely related to a clade containingL. laticeps, L. microdiscus, and L. kadarsani.

CONCLUSION

Given our current knowledge of the genus Limnonectes,only the males of four species possess unusual andconspicuous head ornamentation. We interpretthe caruncle as an autapomorphy for a monophyleticclade comprising L. dabanus, L. gyldenstolpei,L. macrognathus, and L. plicatellus. Monophyly is furthersupported by a concave curvature of the anterior ramusof the pterygoid bone and congruence of 16Smitochondrial DNA data.

Based on the results of this study, we suggest thatthe subgenus Elachyglossa Andersson, 1916 (follow-ing Ohler & Dubois, 1999) should be restricted to thecaruncle-bearing Limnonectes. In an effort to avoid con-founding the nomenclatural and systematic under-standing of Limnonectes we do not recommend elevatingElachyglossa to the generic level (as intended byAndersson, 1916) at this time. We do, however, rec-ommend a revision of the entire genus employing multi-ple lines of evidence. Behavioural and bioacoustic datawould not only facilitate our understanding of the re-lationships among the Limnonectes, but may help toelucidate the functional aspects and biological rel-evance of the remarkable and enigmatic caruncle.

ACKNOWLEDGEMENTS

We are grateful to Stefan Hertwig (NHMB), Chan KinOnn (KU), and Bryan L. Stuart (NCSM) for provid-ing sequence data for L. macrognathus and L. plicatellus.We thank Alan Resetar (FMNH) for the loan of speci-mens under his care and Michael H. Hofmann (Uni-versity of Bonn) for granting M.L. the infrastructureand support for dissections and histology. Kirsten Jensen(KU) provided equipment, supplies, and histologicaltraining to S.W. for this project. Steven F. Perry (Uni-versity of Bonn) and Matthew Vickaryous (Univer-sity of Guelph) are thanked for valuable discussionson the observed histology, as is Wolfgang Böhme (ZFMK)for discussions on the biological significance of thecaruncles. T.H. warmly thanks Catherine Wood (NewYork) for provision of fixatives. P.G. and T.H. are grate-ful to Thomas Ziegler (Zoologischer Garten Köln),Nguyen Quang Truong (Hanoi), and Markus Handschuh(ACCB) for their support during fieldwork. We grate-fully acknowledge Le Xuan Canh and Ta Huy Thinh(both Hanoi) for their support and loan of specimens.Collaborative work on this project was funded in partby a Deutscher Akademischer Austauschdienst (DAAD)fellowship to D.S.M. Fieldwork in Cambodia andVietnam was partly funded through grants from theAlexander Koenig Gesellschaft (AKG), the AngkorCentre for Conservation of Biodiversity (ACCB), theWilhelm-Peters-Fond of the Deutsche Gesellschaftfür Herpetologie und Terrarienkunde (DGHT), and the

Figure 7. Phylogeny depicting hypothesized relationships between the caruncle-bearing Limnonectes and their allies.Greyscale bars indicate taxa considered in this study and in previous studies to comprise monophyletic groups.



Zoological Society for Conservation of Species and Popu-lations (ZGAP) to T.H. and P.G. Financial support ofthis project and its publication was provided by TheUniversity of Kansas Biodiversity Institute and De-partment of Ecology and Evolutionary Biology and theZoologisches Forschungsmuseum Alexander Koenig.

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.246

105.

683

AM

NH

1063

81H

M06

7268

McL

eod

(201

0)L

.ban

nae

nsi

sV

ietn

am,

Qu

ang

Bin

hP

rov.

Min

hH

oaD

ist.

17.6

8710

5.75

0A

MN

H10

6382

HM

0672

69M

cLeo

d(2

010)

L.b

ann

aen

sis

ZN

AC

2102

0N

C01

2837

Zh

ang

etal

.(2

009)

L.d

aban

us

Cam

bodi

aM

ondo

lkir

iP

rov.

,P

ich

rada

Dis

t.12

.533

107.

533

FM

NH

2619

37G

U93

4329

Inge

ran

dS

tuar

t(2

010)

L.d

aban

us

Vie

tnam

Yok

Don

RO

M22

081

AF

2064

96C

hen

etal

.(2

005)

L.f

ern

eri

Ph

il.,

Min

dan

aoIs

l.,D

avao

del

Nor

teP

rov.

Mu

n.

Mon

kayo

,M

t.P

asia

n7.

971

126.

297

CM

NH

5572

L.f

ern

eri

Ph

il.,

Min

dan

aoIs

l.,D

avao

del

Nor

teP

rov.

Mu

n.

Mon

kayo

,M

t.P

asia

n7.

971

126.

297

CM

NH

5573

L.f

ragi

lis

Ch

ina,

Hai

nan

Isl.

Mt.

Lim

u19

.135

109.

773

SC

UM

H00

8D

Q45

8235

Ch

eet

al.

(200

7)L

.fu

jian

ensi

sC

hin

a,A

nh

ui

Pro

v.N

C00

7440

L.f

uji

anen

sis

Taiw

anR

OC

Nan

toC

o.23

.923

120.

890

NM

NS

T16

650

HM

0672

36M

cLeo

d(2

010)

L.f

uji

anen

sis

Taiw

anR

OC

Taoy

uan

Co.

24.7

8412

1.28

1N

MN

ST

1660

2H

M06

7231

McL

eod

(201

0)L

.fu

jian

ensi

sV

ietn

amZ

ISP

TA

O93

6T

his

stu

dyL

.fu

jian

ensi

sV

ietn

amZ

ISP

TA

O93

9T

his

stu

dy



L.g

yld

enst

olpe

iT

hai

lan

d,L

oei

Ph

ulu

ang

Wil

dlif

eR

esea

rch

Cen

ter

PW

RC

002

AF

1831

23,

AF

1831

24E

mer

son

etal

.(2

000)

L.g

yld

enst

olpe

iT

hai

lan

d,S

aK

aeo

Pro

v.S

aK

aeo

Dis

t.,

Pan

gS

iD

aN

atio

nal

Par

k13

.983

102.

200

FM

NH

2662

03G

U93

4331

Inge

ran

dS

tuar

t(2

010)

L.i

san

ensi

sT

hai

lan

d,L

oei

Pro

v.P

hu

Ru

aD

ist.

,P

hu

Lu

ang

Wil

dlif

eS

anct

uar

y17

.334

101.

500

FM

NH

2662

12H

M06

7175

McL

eod

(201

0)

L.i

san

ensi

sT

hai

lan

d,L

oei

Pro

v.P

hu

Lu

ang

KU

HE

1928

4A

B52

6314

Mat

sui

etal

.(2

010a

)L

.jar

uji

ni

Th

aila

nd,

Ph

etch

abu

riP

rov.

Kae

ng

Kra

chan

KU

HE

2010

1A

B55

8942

Mat

sui

etal

.(2

010b

)L

.kad

arsa

ni

Indo

.,L

ombo

kIs

l.L

SU

MZ

8172

2A

Y31

3693

Eva

ns

etal

.(2

003)

L.k

uh

lii

Indo

nes

ia,

Java

Isl.

Pu

rwer

ojo

GM

Uu

nn

um

bere

dA

B52

6316

Mat

sui

etal

.(2

010a

)L

.lat

icep

sM

alay

sia

Sel

ango

rD

ist.

,G

omba

kF

ield

Stu

dyC

ente

rS

BE

071′

depo

site

dat

the

Un

iver

sity

ofM

alay

sia

AF

1831

25,

AF

1831

26E

van

set

al.

(200

3)

L.l

atic

eps

Mal

aysi

aS

elan

gor

Dis

t.,

Gom

bak

Fie

ldS

tudy

Cen

ter

AB

5306

23

L.l

eyte

nsi

sP

hil

.,M

inda

nao

Isl.,

Dav

aode

lN

orte

Pro

v.M

un

.M

alag

os,

Dav

aoC

ity

RM

B37

88JX

9113

22O

aks

etal

.(2

013)

L.m

acro

gnat

hu

sT

hai

lan

dN

akh

onS

iT

ham

arat

FM

NH

2701

04K

J720

984

Th

isst

udy

L.m

ales

ian

us

Mal

aysi

a,B

orn

eoIs

l.,S

arw

awk

Pro

v.G

un

un

gB

uda

,n

ear

Mu

luN

.P.

4.49

411

9.76

7A

Y31

3692

Eva

ns

etal

.(2

003)

L.m

egas

tom

ias

Th

aila

nd,

Sa

Kae

oP

rov.

Mu

ang

Sa

Kae

o,P

ang

Si

Da

NP

14.1

0610

2.25

6F

MN

H26

6220

HM

0671

83M

cLeo

d(2

010)

L.m

egas

tom

ias

Th

aila

nd,

Sa

Kae

oP

rov.

Mu

ang

Sa

Kae

o,P

ang

Si

Da

NP

14.1

0610

2.25

6F

MN

H26

6221

HM

0671

84M

cLeo

d(2

010)

L.m

egas

tom

ias

Th

aila

nd,

Nak

hon

Rat

chas

ima

Pro

v.S

akae

rat

En

v.R

es.

Sta

tion

.14

.494

101.

871

KU

3077

60H

M06

7201

McL

eod

(201

0)L

.meg

asto

mia

sT

hai

lan

d,N

akh

onR

atch

asim

aP

rov.

Sak

aera

tE

nv.

Res

.S

tati

on.

14.4

9410

1.87

1K

U30

7761

HM

0672

02M

cLeo

d(2

010)

L.m

icro

dis

cus

Indo

.,Ja

vaIs

l.S

uka

bum

iL

SU

MZ

8173

9A

Y31

3688

Eva

ns

etal

.(2

003)

L.m

icro

tym

pan

um

Indo

.,S

ula

wes

iIs

l.,S

ula

wes

iS

elat

anP

rov.

Bar

ruo

4.49

411

9.76

7A

MN

H16

7146

AY

3137

43E

van

set

al.

(200

3)

L.m

icro

tym

pan

um

Indo

.,S

ula

wes

iIs

l.,S

ula

wes

iS

elat

anP

rov.

Bar

ruo

4.49

411

9.76

7A

MN

H16

7145

Eva

ns

etal

.(2

003)

L.m

odes

tus

Indo

nes

ia,

Su

law

esi,

Su

law

esi

Uta

raP

rov.

Gor

onta

loT

NH

C59

710

AY

3137

49E

van

set

al.

(200

3)

L.n

amiy

eiJa

pan

,O

kin

awaj

ima

Isla

nd,

Oki

naw

aK

UH

EL

0809

191

AB

5263

09M

atsu

iet

al.

(201

0a)

L.n

amiy

eiJa

pan

,O

kin

awaj

ima

Isla

nd,

Oki

naw

aK

UH

EL

0809

192

AB

5263

10M

atsu

iet

al.

(201

0a)

L.p

arvu

sP

hil

.,M

inda

nao

Isl.,

Dav

aode

lN

orte

Pro

v.M

un

.C

alin

an,

Bar

anga

yM

alag

os7.

186

125.

416

PN

M74

47A

Y31

3694

Eva

ns

etal

.(2

003)

L.p

lica

tell

us

Mal

aysi

aS

elan

gor

Dis

t.,

Kep

ong,

FR

IML

SU

HC

4001

KJ7

2098

3T

his

stu

dyL

.pli

cate

llu

sM

alay

sia

Pu

lau

Pin

ang,

Em

pan

gan

Air

Hit

amL

SU

HC

6710

KJ7

2098

1T

his

stu

dy

L.p

lica

tell

us

Mal

aysi

aS

elan

gor

Dis

t.,

Gom

bak

Sw

amp

LS

UH

C65

82K

J720

982

Th

isst

udy

L.s

isik

dag

uIn

don

esia

,S

um

atra

Bat

uL

ayan

g−3

.464

102.

316

RM

BR

515

HM

0672

45M

cLeo

d(2

010)

L.s

isik

dag

uIn

don

esia

,S

um

atra

Bat

uL

ayan

g−3

.464

102.

320

RM

BR

393

HM

0672

44M

cLeo

d(2

010)

L.s

isik

dag

uIn

don

esia

,S

um

atra

Sar

asah

Bu

nta

−0.0

9410

0.67

1F

MN

H26

6612

JF83

6881

McL

eod

etal

.(2

011)

L.s

isik

dag

uIn

don

esia

,S

um

atra

Sar

asah

Bu

nta

−0.0

9410

0.67

1F

MN

H26

6617

JF83

6880

McL

eod

etal

.(2

011)



AP

PE

ND

IXC

onti

nu

ed

Spe

cies

Gen

eral

loca

lity

Spe

cifi

clo

cali

ty

GP

Sco

ordi

nat

es

Mu

seu

mvo

uch

erG

enB

ank

acce

ssio

nn

o.F

irst

publ

icat

ion

N/S

E

L.s

isik

dag

uIn

don

esia

,S

um

atra

Bat

ang

Har

au−0

.068

100.

655

FM

NH

2666

10JF

8368

73M

cLeo

det

al.

(201

1)L

.sis

ikd

agu

Indo

nes

ia,

Su

mat

raS

aras

ahB

un

ta−0

.094

100.

671

FM

NH

2666

15JF

8368

74M

cLeo

det

al.

(201

1)L

.sp.

Mal

aysi

a,B

orn

eoIs

l.,S

arw

awk

Pro

v.M

atan

gK

UH

E12

025

AB

5263

22M

atsu

iet

al.

(201

0a)

L.t

aylo

riL

aoP

DR

,P

hon

gsal

yP

rov.

Ph

ongs

aly

Dis

t.,

Ph

ouD

endi

nN

at.

Bio

div.

Con

serv

.Are

a22

.094

102.

213

FM

NH

2585

17H

M06

7156

McL

eod

(201

0)

L.t

aylo

riL

aoP

DR

,P

hon

gsal

yP

rov.

Ph

ongs

aly

Dis

t.,

Ph

ouD

endi

nN

at.

Bio

div.

Con

serv

.Are

a22

.094

102.

213

FM

NH

2585

18H

M06

7157

McL

eod

(201

0)

L.t

aylo

riT

hai

lan

d,C

han

gM

aiD

oiIn

than

onK

UH

E19

101

AB

5589

29M

atsu

iet

al.

(201

0b)

L.t

aylo

riT

hai

lan

d,C

han

gM

aiD

oiIn

than

onK

UH

E19

046

AB

5589

28M

atsu

iet

al.

(201

0b)

Lin

eage

13V

ietn

am,

Ha

Gia

ng

Dis

t.,

Vi

Xu

yen

Dis

t.,

Cao

Bo

Com

mu

ne

22.7

7110

4.85

0Z

ISP

TN

E-0

2H

M06

7258

McL

eod

(201

0)

Lin

eage

13V

ietn

am,

Ha

Gia

ng

Dis

t.,

Vi

Xu

yen

Dis

t.,

Cao

Bo

Com

mu

ne

22.7

7410

4.86

7Z

ISP

TA

O69

7H

M06

7250

McL

eod

(201

0)

Lin

eage

13V

ietn

am,

Ha

Gia

ng

Dis

t.,

Vi

Xu

yen

Dis

t.,

Cao

Bo

Com

mu

ne

22.7

7410

4.86

7Z

ISP

TA

O69

9H

M06

7252

McL

eod

(201

0)

Lin

eage

13V

ietn

am,

Ha

Gia

ng

Pro

v.V

iX

uye

nD

ist.

22.7

6110

4.88

2A

MN

H10

6355

HM

0672

67M

cLeo

d(2

010)

Lin

eage

14M

alay

sia,

Bor

neo

Isl.,

Sab

ahS

tate

Kin

abal

uN

P6.

035

116.

547

FM

NH

2571

55H

M06

7144

McL

eod

(201

0)L

inea

ge14

Mal

aysi

a,B

orn

eoIs

l.,S

abah

Sta

teK

inab

alu

NP

6.03

511

6.54

7F

MN

H25

7154

HM

0671

43M

cLeo

d(2

010)

Lin

eage

14M

alay

sia,

Bor

neo

Isl.,

Sab

ahS

tate

Kin

abal

uN

P6.

035

116.

547

FM

NH

2343

75H

M06

7116

McL

eod

(201

0)L

inea

ge14

Mal

aysi

a,B

orn

eoIs

l.,S

abah

Sta

teC

rock

erR

ange

NP

5.04

611

6.07

1S

P20

839

Lin

eage

14M

alay

sia,

Bor

neo

Isl.,

Sab

ahS

tate

Cro

cker

Ran

geN

P5.

470

116.

054

SP

2094

4L

inea

ge20

Mal

aysi

a,B

orn

eoIs

l.,S

araw

akS

tate

Mir

iD

ist.

,L

ambi

rH

ills

NP

4.19

811

4.06

3L

SU

HC

4089

HM

0672

26M

cLeo

d(2

010)

Lin

eage

22K

alim

anta

n,

Indo

nes

iaK

uta

iN

.P

0.53

211

7.46

5A

MN

H16

7141

AY

3136

86E

van

set

al.

(200

3)L

inea

ge4

Cam

bodi

a,S

tun

gTr

eng

Pro

vS

iem

Pan

gD

ist.

Vir

ach

eyN

P14

.268

106.

629

FM

NH

2627

26H

M06

7170

McL

eod

(201

0)L

inea

ge4

Cam

bodi

a,S

tun

gTr

eng

Pro

v.S

iem

Pan

gD

ist.

Vir

ach

eyN

P14

.268

106.

629

FM

NH

2627

30H

M06

7174

McL

eod

(201

0)L

inea

ge5

Lao

PD

R,

Xe

Kon

gP

rov.

Kal

eum

Dis

t.,

Xe

Sap

Nat

.B

iodi

v.C

onse

rv.A

rea

16.0

0910

6.91

7F

MN

H25

8505

HM

0671

46M

cLeo

d(2

010)

Lin

eage

5L

aoP

DR

,X

eK

ong

Pro

v.K

aleu

mD

ist.

,X

eS

apN

at.

Bio

div.

Con

serv

.Are

a16

.009

106.

925

FM

NH

2585

09H

M06

7150

McL

eod

(201

0)

Lin

eage

6M

alay

sia,

Per

akTe

men

gor

For

est

Res

erve

5.56

910

1.65

5L

SU

HC

7034

HM

0672

30M

cLeo

d(2

010)

Lin

eage

6M

alay

sia,

Pah

ang

Bat

ang

Kal

i,G

enti

ng

Hig

hla

nd

3.42

310

1.78

6F

RIM

1141

HM

0672

00M

cLeo

d(2

010)

Lin

eage

6M

alay

sia,

Pah

ang

Su

nga

iL

embi

ng

Log

gin

gC

amp

3.08

710

3.05

0L

SU

HC

5008

HM

0672

29M

cLeo

d(2

010)

Lin

eage

6M

alay

sia,

Pah

ang

Su

nga

iL

embi

ng

Log

gin

gC

amp

3.08

710

3.05

0L

SU

HC

4922

HM

0672

28M

cLeo

d(2

010)

Lin

eage

9M

yan

mar

,S

agai

ng

Div

.Ala

un

gdaw

Kat

hap

aN

.P.

22.3

0094

.414

CA

S20

5260

HM

0672

85M

cLeo

d(2

010)

Lin

eage

9M

yan

mar

,S

agai

ng

Div

.Ala

un

gdaw

Kat

hap

aN

.P.

22.3

0094

.414

CA

S20

5263

HM

0672

88M

cLeo

d(2

010)



anatomy, histology, and systematic implications of the head ornamentation in the males of four...

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