Dingqi Rao

New insights to the molecular phylogenetics and generic assessment in the Rhacophoridae (Amphibia: Anura) based on five nuclear and three mitochondrial genes, with comments on the evolution of reproduction

The phylogenetic relationships among 12 genera of treefrogs (Family, Rhacophoridae), were investigated based on a large sequence data set, including five nuclear (brain-derived neurotrophic factor, proopiomelanocortin, recombination activating gene 1, tyrosinase, rhodopsin) and three mitochondrial (partial 12S and 16S ribosomal RNA and the complete valine t-RNA) genes. Phylogenetic analysis of the nuclear gene sequences resolved three major clades. The first group included Philautus, Pseudophilautus, Kurixalus, Gracixalus, and Theloderma moloch; Pseudophilautus and Kurixalus were sister taxa. The second group consisted of Nyctixalus and Theloderma. The third group contained Feihyla, Polypedates, Rhacophorus, and Chiromantis vittatus; Polypedates and Feihyla were sister taxa. Analyses of the nuclear and mitochondrial genes supported the following results: (1) Genus Liuixalus formed the sister group of all other rhacophorines. (2) Philautus, Theloderma, and Chiromantis were not resolved as monophyletic genera. Four groups, including Philautus ocellatus and P. hainanus, P. longchuanensis and P. gryllus, P. banaensis, and P. quyeti nested well within the genera Liuixalus, Pseudophilautus, Kurixalus, and Gracixalus, respectively. (3) Theloderma moloch and Chiromantis vittatus did not cluster with other species of Theloderma and Chiromantis, respectively. Foam nesting evolved only once, as did laying eggs in a jelly-like matrix containing some bubbles. Terrestrial direct development evolved twice in the Rhacophoridae.

Is Chytridiomycosis an Emerging Infectious Disease in Asia

The disease chytridiomycosis, caused by the fungus Batrachochytrium dendrobatidis (Bd), has caused dramatic amphibian population declines and extinctions in Australia, Central and North America, and Europe. Bd is associated with >200 species extinctions of amphibians, but not all species that become infected are susceptible to the disease. Specifically, Bd has rapidly emerged in some areas of the world, such as in Australia, USA, and throughout Central and South America, causing population and species collapse. The mechanism behind the rapid global emergence of the disease is poorly understood, in part due to an incomplete picture of the global distribution of Bd. At present, there is a considerable amount of geographic bias in survey effort for Bd, with Asia being the most neglected continent. To date, Bd surveys have been published for few Asian countries, and infected amphibians have been reported only from Indonesia, South Korea, China and Japan. Thus far, there have been no substantiated reports of enigmatic or suspected disease-caused population declines of the kind that has been attributed to Bd in other areas. In order to gain a more detailed picture of the distribution of Bd in Asia, we undertook a widespread, opportunistic survey of over 3,000 amphibians for Bd throughout Asia and adjoining Papua New Guinea. Survey sites spanned 15 countries, approximately 36° latitude, 111° longitude, and over 2000 m in elevation. Bd prevalence was very low throughout our survey area (2.35% overall) and infected animals were not clumped as would be expected in epizootic events. This suggests that Bd is either newly emerging in Asia, endemic at low prevalence, or that some other ecological factor is preventing Bd from fully invading Asian amphibians. The current observed pattern in Asia differs from that in many other parts of the world.

Diversification of rhacophorid frogs provides evidence for accelerated faunal exchange between India and Eurasia during the Oligocene

The accretion of the Indian subcontinent to Eurasia triggered a massive faunal and floral exchange, with Gondwanan taxa entering into Asia and vice versa. The traditional view on the Indian–Asian collision assumes contact of the continental plates during the Early Eocene. Many biogeographic studies rely on this assumption. However, the exact mode and timing of this geological event is still under debate. Here we address, based on an extensive phylogenetic analysis of rhacophorid tree frogs, if there was already a Paleogene biogeographic link between Southeast Asia and India; in which direction faunal exchange occurred between India and Eurasia within the Rhacophoridae; and if the timing of the faunal exchange correlates with one of the recently suggested geological models. Rhacophorid tree frogs showed an early dispersal from India to Asia between 46 and 57 Ma, as reconstructed from the fossil record. During the Middle Eocene, however, faunal exchange ceased, followed by increase of rhacophorid dispersal events between Asia and the Indian subcontinent during the Oligocene that continued until the Middle Miocene. This corroborates recent geological models that argue for a much later final collision between the continental plates. We predict that the Oligocene faunal exchange between the Indian subcontinent and Asia, as shown here for rhacophorid frogs, also applies for other nonvolant organisms with an Indian–Asian distribution, and suggest that previous studies that deal with this faunal interchange should be carefully reinvestigated.

Phylogeny of the Asian spiny frog tribe Paini (Family Dicroglossidae) sensu Dubois

The anuran tribe Paini, family Dicroglossidae, is known in this group only from Asia. The phylogenetic relationships and often the taxonomic recognition of species are controversial. In order to stabilize the classification, we used approximately 2100 bp of nuclear (rhodopsin, tyrosinase) and mitochondrial (12S, 16S rRNA) DNA sequence data to infer the phylogenetic relationships of these frogs. Phylogenetic trees reconstructed using Bayesian inference and maximum parsimony methods supported a monophyletic tribe Paini. Two distinct groups (I,II) were recovered with the mtDNA alone and the total concatenated data (mtDNA+nuDNA). The recognition of two genera, Quasipaa and Nanorana, was supported. Group I, Quasipaa, is widespread east of the Hengduan Mountain Ranges and consists of taxa from relatively low elevations in southern China, Vietnam and Laos. Group II, Nanorana, contains a mix of species occurring from high to low elevation predominantly in the Qinghai-Tibetan Plateau and Hengduan Mountain Ranges. The occurrence of frogs at high elevations appears to be a derived ecological condition. The composition of some major species groups based on morphological characteristics strongly conflicts with the molecular analysis. Some possible cryptic species are indicated by the molecular analyses. The incorporation of genetic data from type localities helped to resolve some of the taxonomic problems, although further combined analyses of morphological data from type specimens are required. The two nuDNA gene segments proved to be very informative for resolving higher phylogenetic relationships and more nuclear data should be explored to be more confident in the relationships.

Re-examination of the phylogeny of Rhacophoridae (Anura) based on mitochondrial and nuclear DNA

The phylogenetic relationships among rhacophorid frogs are under dispute. We use partial sequences of three mitochondrial (12S rRNA, 16S rRNA, and cytochrome b) and three nuclear protein-coding (Rag-1, rhodopsin exon 1, and tyrosinase exon 1) genes from 57 ingroup taxa and eight outgroup taxa to propose a hypothesis for phylogenetic relationships within Rhacophoridae. Our results support recognition of the genus Feihyla, and Chiromantis is the sister taxon to the clade formed by Feihyla, Polypedates and Rhacophorus. We place Aquixalus odontotarsus within Kurixalus, and the remaining species of Aquixalus and Philautus jinxiuensis into the genus Gracixalus. We give Philautus (Kirtixalus) the rank of genus and place Philautus menglaensis within it. The division of species groups among Chinese Rhacophorus needs revision, and a cryptic species is revealed within Rhacophorus nigropunctatus. Rhacophorus pingbianensis is considered a synonym of Rhacophorus omeimontis. The validity of Rhacophorus hui is confirmed by present molecular evidence.

Molecular phylogeography and population structure of a mid-elevation montane frog Leptobrachium ailaonicum in a fragmented habitat of southwest China.

Leptobrachium ailaonicum is a vulnerable anuran restricted to a patchy distribution associated with small mountain streams surrounded by forested slopes at mid-elevations (approximately 2000-2600m) in the subtropical Mount Wuliang and Mount Ailao ranges in southwest China (Yunnan Province) and northern Vietnam. Given high habitat specificity and lack of suitable habitat in lower elevations between these ranges, we hypothesized limited gene flow between populations throughout its range. We used two mitochondrial genes to construct a phylogeographic pattern within this species in order to test our hypothesis. We also examined whether this phylogeographic pattern is a response to past geological events and/or climatic oscillations. A total of 1989 base pairs were obtained from 81 individuals of nine populations yielding 51 unique haplotypes. Both Bayesian and maximum parsimony phylogenetic analyses revealed four deeply divergent and reciprocally monophyletic mtDNA lineages that approximately correspond to four geographical regions separated by deep river valleys. These results suggest a long history of allopatric separation by vicariance. The distinct geographic distributions of four major clades and the estimated divergence time suggest spatial and temporal separations that coincide with climatic and paleogeographic changes following the orogeny and uplift of Mount Ailao during the late Miocene to mid Pliocene in southwest China. At the southern distribution, the presence of two sympatric yet differentiated clades in two areas are interpreted as a result of secondary contact between previously allopatric populations during cooler Pleistocene glacial cycles. Analysis of molecular variance indicates that most of the observed genetic variation occurs among the four regions implying long-term interruption of maternal gene flow, suggesting that L. ailaonicum may represent more than one distinct species and should at least be separated into four management units corresponding to these four geographic lineages for conservation.

Amphibians crossing the Bering Land Bridge: evidence from holarctic treefrogs (Hyla, Hylidae, Anura).

Based on an updated, time-calibrated phylogeny and applying biogeographical model testing and diversification analysis, we re-examined systematics and biogeography of the Holarctic treefrog genus Hyla with a focus on the East Asian species. We analyzed four mitochondrial genes (12S and 16S rRNA, tRNA(Leu), ND1) and one nuclear gene (POMC) for 192 samples representing 30 species of Hyla. Based on our results we suggest that H. ussuriensis is a synonym of H. japonica. Specimens from Sakhalin and Kunashir Islands might represent a cryptic species within H. japonica. We confirm earlier hypotheses that the genus Hyla originated during the Eocene to Early Oligocene and that Eurasian species originated from two independent dispersal events from North America via the Bering Land Bridge. Middle Eocene to Oligocene dispersal gave rise to the most recent common ancestor of the West Palearctic H. arborea-group and the East Palearctic, newly defined, H. chinensis-group. The Northeast Asian H. japonica-group resulted from a second wave of colonization from the Nearctic. A trans-Atlantic dispersal route could be excluded. Dispersal of the H. arborea-group to the western Palearctic coincides with the closure of the Turgai Strait at the end of the Oligocene. Diversification of Hyla decreased at the end of the Middle Miocene, possibly coinciding with the end of the Mid Miocene Climatic Optimum and the advent of cooler and drier climates in the Northern Hemisphere.

Phylogeography and Demographic History of Babina pleuraden (Anura, Ranidae) in Southwestern China

Factors that determine genetic structure of species in southwestern China remain largely unknown. In this study, sequences of two mitochondrial genes (COI and cyt b) were determined to investigate the phylogeography and demography of Babina pleuraden, a pond frog endemic to southwestern China. A total of 262 individuals from 22 populations across the entire range of the species were collected. Our results indicate that B. pleuraden comprises five well-supported mitochondrial lineages roughly corresponding to five geographical areas. The phylogeographic structure of B. pleuraden has been shaped primarily by the unique regional responses of the Yunnan Plateau to the rapid uplift of the Qinghai-Tibetan Plateau occurred c. 2.5 Mya (B phrase of Qingzang Movement) and climatic oscillation during middle Pleistocene (c. 0.64–0.36 Mya), rather than by the paleo-drainage systems. The present wide distribution of the species has resulted from recent population expansion (c. 0.053–0.025 Mya) from multiple refugia prior to the Last Glacial Maximum, corresponding to the scenario of “refugia within refugia”.

Effect of Pleistocene Climatic Oscillations on the Phylogeography and Demography of Red Knobby Newt (Tylototriton shanjing) from Southwestern China

Factors that determine the genetic structure of species in southwestern China remain largely unknown. In this study, phylogeography and demography of Tylototriton shanjing was investigated from a mitochondrial perspective to address the role of the Quaternary ice ages in shaping phylogeographic history and genetic diversity of Yunnan. A total of 146 individuals from 19 populations across the entire range of the species were collected. We detected four maternal phylogenetic lineages corresponding to four population groups, and found that major glaciation events during the Pleistocene have triggered the intra-specific divergence. Coalescent simulations indicated that the populations retreated to different refugia located in southern Yunnan, northwestern Yunnan, the border region of western Yunnan with Myanmar, and middle-western Yunnan, respectively, during previous glacial periods in the Pleistocene, and these four refugia were not retained during the Last Glacial Maximum. Population expansions occurred during the last inter-glaciation, during which ice core and pollen data indicated that the temperature and precipitation gradually increased, and declines of population sizes started after the beginning of the Last Glacial Maximum when the climate became cooler and dryer. The paleo-drainage system had no contribution to the current genetic structure and the rivers were not dispersal barriers for this salamander.

Genealogy and Demographic History of a Widespread Amphibian throughout Indochina

Relatively little is known about spatial patterns of cryptic diversity in tropical species and the processes that generate them. Few studies examine the geographic distribution of genetic lineages in Southeast Asia, an area hypothesized to harbor substantial cryptic diversity. We investigated the evolutionary history of Asian tree frogs of the Polypedates leucomystax complex (n = 172) based on 1800 bp of the mtDNA genes ND1 and cytochrome b and tested hypotheses pertaining to climate, geology, and dispersal patterns. Analyses revealed substantial genetic diversity and lineage divergence throughout the region with evidence for widespread sympatric lineages and a general north versus south clustering. Relaxed molecular clock analysis and tests for demographic expansion identified an initial cladogenesis during the Miocene with subsequent Plio-Pleistocene diversification, with the former corresponding to periods of increased aridity and the onset of monsoonal weather systems. Rates of diversification were relatively constant until the Early Pleistocene when rates increased exponentially. We found equivocal evidence for isolation-by-distance and a potential role of some landscape features as partial barriers to dispersal. Finally, our analyses showed that divergence between insular and mainland populations occurred before Homo sapiens colonized Southeast Asia, suggesting that historical human-mediated dispersal did not drive insular diversification. Our results suggested that demographic expansion in the Late Pleistocene resulted in widespread sympatric lineages in the P. leucomystax complex throughout southern China and Indochina and further clarified the evolutionary history of lineages within P. leucomystax.