A Phylogeny of the High-Elevation Tibetan Megophryid Frogs and Evidence for the Multiple Origins of Reversed Sexual Size Dimorphism J
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Journal of Zoology. Print ISSN 0952-8369 A phylogeny of the high-elevation Tibetan megophryid frogs and evidence for the multiple origins of reversed sexual size dimorphism J. Fu, C. J. Weadickà &K.Bi Department of Integrative Biology, University of Guelph, Guelph, ON, Canada Keywords Abstract phylogeny; sexual dimorphism; DNA sequences; megophryids; Scutiger; A molecular phylogeny of the high-elevation Tibetan megophryid frogs was Oreolalax; Vibrissaphora. reconstructed using mitochondrial and nuclear gene sequences. Both parsimony and Bayesian analysis produced similar tree topologies, which identified the genus Correspondence Leptobrachium as a sister group to Vibrissaphora, and the genera Oreolalax and Jinzhong Fu, Department of Integrative Scutiger as monophyletic groups. Within the latter two genera, several species Biology, University of Guelph, Guelph, ON groups were also clearly recognized. At least 13 megophryids display reversed N1G 2W1, Canada. Tel: 519 824 4120 ext. sexual size dimorphism (RSSD), where males are equal to or larger than females. 52715; Fax: 519 767 1656 According to our phylogenetic hypothesis, RSSD may have independently evolved Email: [email protected] at least five times or as many as seven times in this group. The multiple origins of RSSD within this group provide an excellent opportunity for studying the ÃCurrent address: Department of Ecology & relationship between body size sexual dimorphism and other life-history traits Evolutionary Biology, University of Toronto, and for determining the costs and benefits of RSSD. 25 Harbord St, Toronto, ON M5S 3G5, Canada. Received 8 December 2006; accepted 8 May 2007 doi:10.1111/j.1469-7998.2007.00330.x Introduction between the sexes in breeding populations could explain most of the dimorphism. As such, the evolutionary factors Females are larger than males in most animal species, with operating on body size in anurans remain largely controver- avian and mammalian species being the main exceptions. sial, and a study of the origin of RSSD will undoubtedly The widely accepted explanation for large female size is improve our understanding of how natural and sexual fecundity advantage, as egg production increases with body selection operate in this large and important group. RSSD size (Andersson, 1994). Most anurans follow this rule, but in is particularly concentrated among the high-elevation Tibetan c. 10% of species, males tend to be of an equal or of a larger megophryid frog species of the genera Oreolalax, Scutiger and size than females (Shine, 1979). For instance, male Vibrissa- Vibrissaphora; in at least 13 of them, males are of an equal or phora boringiae may attain a body length of 69.8–89.0 mm, a larger size than females (Liu & Hu, 1961; Yang, 1991; Ye, whereas females fall within the range of 58.6–76.0 mm (Liu Fei & Hu, 1993; Fei & Ye, 2000, Supplementary Material & Hu, 1961). This is known as reversed sexual size dimorph- Appendix S1). The repeated presence of RSSD in this group ism (RSSD). Several mechanisms have been proposed to offers us an excellent opportunity to examine the adaptive explain the large male size in anurans: male-to-male physical values and tradeoffs that have shaped the degree of sexual size combat (Shine, 1979), different selection gradients for size dimorphism over time, from both the male’s and female’s between males and females (Arak, 1988), female choice perspective. (Morris & Yoon, 1989) and several others (see Andersson, Hypotheses on the evolutionary origin(s) of RSSD can 1994 for a review). Monnet & Cherry (2002) pointed out only be tested within a solid phylogenetic framework. For that the previous studies suffered from either methodologi- instance, a shared single origin of RSSD would suggest that cal problems (e.g. non-independent data points) or unrealis- the multiple occurrences of RSSD simply reflect phyloge- tic assumptions, and rejected the sexual selection-based netic inertia, and are unrelated to variation in present-day explanations. Instead, they suggested that the age difference selection pressures. Conversely, independent origins of Journal of Zoology 273 (2007) 315–325 c 2007 The Authors. Journal compilation c 2007 The Zoological Society of London 315 A phylogeny of Tibetan megophryid frogs J. Fu, C. J. Weadick and K. Bi RSSD might reflect convergence due to similar selection RSSD. Three species from two other genera, Leptolalax and pressures, a possibility that could be investigated by con- Megophrys, were selected as outgroups for phylogenetic sidering how RSSD and environmental conditions are dis- analysis. The genus Leptolalax has been considered to be tributed across the phylogeny. Phylogenetic hypotheses closely related to the ingroup genera by many taxonomists provide the foundation necessary for making informed (e.g. Dubois, 1980; Fei et al., 2005), while the genus evolutionary comparisons that account for shared history Megophrys is relatively distantly related to the ingroup. All in addition to contemporary selective pressures (Harvey & voucher specimens are deposited in the herpetological collec- Pagel, 1991). tions of the Chengdu Institute of Biology (Chengdu), the Several phylogenetic works on the high-elevation Tibetan Institute of Zoology (Beijing) and the Royal Ontario Mu- megophryids have been published. For example, Fu, seum at Toronto (Table 1). Lathrop & Murphy (1997) and Fu & Murphy (1997) analysed some morphological data of the genera Scutiger Laboratory protocols and Oreolalax, respectively. A few recent studies have focused on inter-generic relationships within the Mego- DNA sequence data were used for phylogenetic reconstruc- phryidae; Delorme & Dubois (2001) presented a preliminary tion. Two fragments were targeted for sequencing. One is phylogeny of 15 megophryid frogs based on 54 external from the mitochondrial genome, and includes the second morphological characters, and Zheng (2004) studied the half of the 12S gene, most of the 16S gene, plus the tRNAÀval relationships among eight genera of the family Megophryi- gene between them. This fragment is one of the most dae using partial 16S and cytochrome b gene sequence data. conservative parts of the mitochondrial genome and has Frost et al. (2006) also included eight megophryids in their been used extensively in the reconstruction of species-level amphibian tree of life. Unfortunately, these previous studies phylogenies in various vertebrate groups, especially in am- had either limited number of taxa or limited number of phibians (e.g. Vences et al., 2004). The other fragment is part informative characters, as well as some analytical short- of the recombination activating gene 1 (RAG-1), a single- comings, as Frost et al. (2006) pointed out. As such, the copy protein-coding nuclear gene that has also been widely phylogenetic relationships of megophryid frogs in general used in amphibian phylogenetics (e.g. Mauro et al., 2004). and among species with RSSD in particular remain largely DNA was extracted from muscle tissues preserved in 95% unknown. The lack of a solid phylogeny for this group has ethanol or liquid nitrogen using the Promega Wizard geno- constrained the pursuit of several research avenues. mic DNA extraction protocols. A standard polymerase We recently initiated a project aiming at understanding chain reaction (PCR) was used to amplify the mitochondrial the evolution of sexual dimorphism and sexual selection in 12S–16S gene fragment. PCR products were purified using these high-elevation megophryid frogs. As the first step of Qiaquick protocols (Qiagen, Germantown, MA, USA), and this long-term study, we used a combination of nuclear and were directly sequenced with BigDye-labelled terminator mitochondrial DNA (mtDNA) sequence data to reconstruct sequencing kits on an ABI 377 automatic sequencer (Applied a molecular phylogeny of the genera Oreolalax, Scutiger and Biosystems, Foster City, CA, USA). The primers, 12SA-50, Vibrissaphora. Furthermore, by mapping size dimorphism 16Sbr-30 (Palumbi, 1996), 1602L (50-GTA TAC CGG AAG on to the reconstructed tree, we tested the single versus GTG TAC TTG GAA CAG-30, this study) and 2571H (50- multiple origins hypotheses of RSSD among these mego- TAC CTT CGC ACG GTC AGA ATA CCG C-30,this phryids. study), were used to amplify and sequence the 12S–16S fragment. Most sequencing was conducted in both directions Material and methods with a 70–80% overlap. In a few cases, strings of eight or more consecutive guanines or cytosines disrupted sequencing and, as a result, sequences from only one direction were Species sampling obtained. For the RAG-1 gene, five of the samples were A total of 34 specimens representing 23 species of the genera amplified, purified and directly sequenced using the same Oreolalax, Scutiger and Vibrissaphora were collected during protocols as the mitochondrial genes. The primers, Amp- 1999–2006. Zheng (2004) suggested that the three genera RAG1 F (50-AGC TGC AGY CAR TAC CAY AAR ATG might form a monophyletic group. All eight species with TA-30, Mauro et al., 2004) and RAG1-R (50-GCA AAG reported RSSD in the genera Oreolalax and Scutiger TTT CCG TTC ATT CTC AT-30, this study), were used for (O. jingdongensis, O. nanjiangensis, O. omeimontis, O. popei, both amplification and sequencing. For the rest of the O. rhodostigmatus, S. glandulatus, S. mammatus and samples, the targeted fragments were first amplified and then S. muliensis) were included in our analysis. Five of the six cloned into the pCR2.1/TOPO vector (Invitrogen, Carlsbad, species of the genus Vibrissaphora (V. ailaonica, V. boringiae, CA, USA). The standard M13 forward and reverse primers V. leishanensis, V. liui and V. ngoclinhensis) have larger were then used to sequence the inserted fragment. males, but we were only able to obtain two species in this analysis. One species from the genus Leptobrachium, which Analysis was often considered to be closely related to Vibrissaphora (but see Zheng, 2004), was also included in the analysis. Sequence alignment was conducted with the computer None of the Leptobrachium species are known to display programs ClustalX (version 1.8; Thompson et al., 1997) 316 Journal of Zoology 273 (2007) 315–325 c 2007 The Authors.