Available online at www.sciencedirect.com Molecular Phylogenetics and Evolution 46 (2008) 61–73 www.elsevier.com/locate/ympev Phylogenetic relationships of the mustache toads inferred from mtDNA sequences Ding-Qi Rao a,1, Jeffery A. Wilkinson b,*,1 a Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China b Department of Herpetology, California Academy of Sciences, 875 Howard Street, San Francisco, CA 94103, USA Received 10 February 2007; revised 4 October 2007; accepted 7 October 2007 Available online 16 October 2007 Abstract Species of the genus Vibrissaphora are unique among all anurans in that males exhibit external cornified spines in the maxillary region during the breeding season. They were separated from species of the genus Leptobrachium based on this unique character. We construct a phylogeny using the 16S, ND4, and cytochrome b mitochondrial genes of 42 individuals from eight species of Vibrissaphora and five species of Leptobrachium from mainland China, Southeast Asia, and Hainan Island. Species of both Oreolalax and Scutiger were used as outgroups. The results indicate that: L. huashen and L. chapaense form a clade that is nested within Vibrissaphora, and L. hainanense is the sister taxon to the clade comprising all Vibrissaphora plus L. chapaense and L. huashen; V. boringiae is grouped with a clade consisting of V. leishanensis, V. liui, and V. yaoshanensis; and V. yaoshanensis is a species separate from V. liui. We propose taxonomic changes that reflect these findings. Also based on the resulting phylogenetic trees, we propose that the mustache toads originated in the trans-Hima- layan region of southwest China, and that the evolution of maxillary spines, large body size, and reverse sexual size dimorphism in these frogs was influenced by intrasexual selection due to adopting a resource-defense polygyny mating system. Ó 2007 Elsevier Inc. All rights reserved. Keywords: Biogeography; Character evolution; Intrasexual selection; Leptobrachium; Megophryidae; Molecular phylogeny; Resource-defense polygyny; Trans-Himalayas; Vibrissaphora 1. Introduction nam, V. echinata (Dubios and Ohler, 1998)andV. ngo- clinhensis Orlov (2005). Vibrissaphora jiulongshanensis was The mustache toads of the genus Vibrissaphora (Anura: later considered a synonym of V. liui (Huang et al., 1984; Megophryidae) are restricted to southern China and Viet- Wu and Liu, 1986; Fei et al., 1995), and V. yaoshanensis nam. They are defined by having external cornified spines was demoted to a subspecies of V. liui (Wu and Zhao, in the maxillary region of males during the breeding season 1987; Fei et al., 1995). Vibrissaphora echinata was consid- and reverse sexual size dimorphism in which the males are ered a synonym of V. ailaonica by Ho et al. (1999), but generally larger than the females. Seven species of Vibrissa- its species rank was defended by Ohler et al. (2000) and phora have been described from China; V. ailaonica Yang Grosjean (2001) based on differences in eye color, number et al. (1983), V. boringiae Liu (1945), V. jiulongshanensis of maxillary spines, and larval characters. Wei and Zhao (1981), V. leishanensis Liu and Hu (1973), Species within Vibrissaphora were divided into two spe- V. liui Pope (1947), V. promustache Rao et al. (2006), and cies groups (a V. boringiae species group of V. ailaonica, V. V. yaoshanensis Ye et al. (1978), and two species from Viet- boringiae, and V. echinata, and a V. liui species group of V. leishanensis and V. liui) based on the number and arrange- ment of external spines in the maxillary region but without * Corresponding author. Fax: +1 415 321 8643. E-mail address: [email protected] (J.A. Wilkinson). examining the phylogeny of this genus (Fei et al., 1995; 1 Both authors contributed equally to this work. Dubios and Ohler, 1998; Fei et al., 2005). V. ngoclinhensis 1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.10.005 62 D.-Q. Rao, J.A. Wilkinson / Molecular Phylogenetics and Evolution 46 (2008) 61–73 and V. promustache were not placed within a species group. the nest site (Zheng and Fu, 2007) suggesting that he is V. promustache in particular was considered a distant rela- defending the nest site and not just using it as a calling site. tive of other species of Vibrissaphora based on its posses- Male nest building and defense are uncommon, and are sion of a mixture of morphological characters from usually associated with a resource-defense mating system Vibrissaphora and Leptobrachium. Leptobrachium-like (Sullivan et al., 1995). Halliday and Tejedo (1995) also characters included a small body size, typical anuran sexual noted that in frogs, a relatively larger male body size in size dimorphism with the females larger than males, and relation to the female seems to be associated with presence of vocal sacs, while Vibrissaphora-like characters resource-defense mating systems, whereas males that were presence of maxillary spines, absence of linea mascu- defend only calling sites or fight for possession of females lina, indistinct tympanum, enlarged arms in the male, and are usually considerably smaller than females, even when rough skin (Rao et al., 2006). large males are more likely to mate. The combination of Species of Vibrissaphora are morphologically very simi- large adult body size, reverse sexual size dimorphism, and lar to species of the genus Leptobrachium to the point that weapons has been observed only in species that utilize Vibrissaphora was considered a junior synonym (Dubios, resource-defense polygyny, such as Adelotus brevis (Katsi- 1980) or subgenus of the latter (Tian and Hu, 1985; Dubios karos and Shine, 1997), Hyla rosenborgi (Kluge, 1981), and Ohler, 1998). Dubios (1980) argued that the presence Pyxicephalus adspersus (Cook et al., 2001), P. edulis (Bra- of spines was not sufficient to separate Vibrissaphora from ack and Maguire, 2005), and some species of Limnonectes Leptobrachium. However, Ho et al. (1999) argued that this (Emerson and Inger, 1992; Emerson and Voris, 1992; character distinguishes the two genera and suggested that Orlov, 1997; Tsuji and Matsui, 2002; Tsuji, 2004). Vibrissaphora be retained pending a phylogenetic analysis. Anurans that utilize this type of mating system are typ- Recent molecular phylogenetic studies on megophryid ically prolonged breeders as opposed to explosive breeders frogs have even placed the two genera as sister taxa, with (Sullivan et al., 1995). Mustache toads breed over a period the genera Oreolalax, Scutiger, and Leptolalax occurring of 2 weeks to 2 months depending on the species (Fei, outside a Leptobrachium/Vibrissaphora clade (Zheng 1999). The skin of the breeding male becomes loose (Wu et al., 2004a,b). However, only two species of Vibrissaphora and Yang, 1981; Chen et al., 1984; Gu et al., 1986; Tang, and one to two species of Leptobrachium were sampled in 1990; Ho et al., 1999), which is thought to increase surface these studies; therefore, monophyly of these two genera area facilitating cutaneous respiration (Ho et al., 1999). could not be rigorously tested. Cutaneous respiration would be needed for continual sub- The purpose of the maxillary spines is not known but aerial calling and defense of a nest site during the breeding assumed to be for aggressive behavior (Duelman and season. Multiple egg masses have also been observed Trueb, 1986). This assumption is probably based on the attached to the bottom of the stone within a nest that fact that: (1) maxillary spines are unique to this genus, was defended by a single male of V. ailaonica (Ye et al., and weapons (spines or tusks) and/or reverse sexual size 1993) suggesting that this male mated with the several dimorphism are uncommon, known in only 10% of frog females that oviposited in the nest. Wu and Yang (1981) species (Shine, 1979); and (2) in the few species whose also stated that males of several species engage in multiple males possess weapons and grow larger than females, matings during a breeding season, though they did not male–male combat has been observed (Shine, 1979; Duel- state that they actually observed this behavior. man and Trueb, 1986). Direct male-male interaction, such Based on these observations and comparisons with as fighting, has not been observed in mustache toads, as other anuran species, we hypothesize that the males of behavioral studies have not been conducted. mustache toads engage in a resource-defense polygyny From limited observations, it is known that the maxil- mating system by constructing and then defending nest lary spines develop on males only during the breeding sea- sites in order to increase their attractiveness to females son and drop off afterwards (Wu and Yang, 1981; Chen (Emlen and Oring, 1977). The nest site constructed under et al., 1984; Ho et al., 1999). During the breeding season, an in-stream flat bottom stone with the correct water males move to streams from terrestrial habitat, construct parameters for embryonic development is likely the limit- submerged nest sites under stones by excavating sand cre- ing resource that the male defends. We also hypothesize ating a depression, and then call from within the underwa- that adopting this type of mating system sets the stage ter sites to attract females. Upon entering a nest site, the for sexual selection, specifically intrasexual selection, to females are amplexed by the males and oviposit the eggs act on the evolution of maxillary spines concomitant with as an egg mass, which is then attached to the undersurface larger body sizes in males, producing reverse sexual size of the stone (Wu and Yang, 1981; Chen et al., 1984; Fei dimorphism in these species (Halliday, 1978; Bradbury and Ye, 1984; Gu et al., 1986,1988; Tang, 1990; Dubios and Davies, 1987; Halliday and Tejedo, 1995). and Ohler, 1998; Ho et al., 1999; Zheng and Fu, 2007). The purpose of this study is to reconstruct the phyloge- In V.