Nikolai L. Orlov

Cryptic Species of a Cascade Frog from Southeast Asia: Taxonomic Revisions and Descriptions of Six New Species

Abstract The Southeast Asian cascade frog, Rana livida (Blyth, 1856), has long been suspected to be a complex of species. Several different forms are described from across its vast range. The loss of type material and disparate sampling efforts are challenges. Is variability in this species due to geographic variation or to the presence of multiple species? We use concordant evidence from morphology, morphometrics, cellular DNA content, and allozyme electrophoresis to investigate diversity in R. livida from Vietnam. Three distinct species are recognized on the basis of morphology, as are four other suspect groups (morphotypes). Discriminant function analyses of morphometric data detect patterns of morphological variation among all seven groups. Pairwise comparison of cellular DNA content using t-tests shows significant differences among sympatric morphotypes, suggesting they represent distinct species. This hypothesis is supported by an analysis of 14 allozymic loci, in which fixed allelic differences are found among specimens in sympatry and allopatry. Examination of available type material of four junior synonyms of R. livida results in their recognition as species. One of these species, R. chloronota, is a wide-ranging species erroneously referred to as R. livida. Seven species occur in Vietnam. We describe six new cryptic species belonging to the Rana chloronota complex, redescribe R. chloronota, R. livida, R. sinica, and R. graminea, and give comments on R. leporipes. Three of these new species (R. bacboensis, new species, R. hmongorum, new species, and R. daorum, new species) occur in montane forests in northern Vietnam, and two (R. banaorum, new species and R. morafkai, new species) are known only from the Tay Nguyen Plateau of Vietnams Central Highlands. One species, R. megatympanum, new species, occurs in portions of both northern Vietnam and the Central Highlands. An identification key for the Rana chloronota complex from Vietnam is provided. The finding of six cryptic species within a small portion of the geographic region of R. chloronota suggests that many more cascade ranids await discovery. This documentation has serious implications for conservation; each of the new species occurs in sympatry with at least one other member of the complex. Consequently, far more species are being affected by habitat loss than was previously thought.

The Phylogenetic Relationships of the Chinese and Vietnamese Waterfall frogs of the genus Amolops

Ranid frogs of the genus Amolops occur in Southeast Asia and are typically found near waterfalls. Their phylogenetic relationships have not been resolved. We include 2,213 aligned nucleotide sites of the 12S, 16S and tRNA(val) gene regions of the mitochondrial DNA genome from 43 individuals of Chinese and Vietnamese Amotops, Huia, Hylarana, Meristogenys, Odorrana and Rana. The outgroup species were from the genera Chaparana, Limnonectes, Nanorana, and Paa. The data were analyzed within the framework of a refutationist philosophy using maximum parsimony. Four clades of waterfall frogs were resolved. Meristogenys was not resolved as the sister group to either Huia nor Amolops. The hypothesis Of evolutionary relationships placed Amolops chapaensis and Huia nasica in the genus Odorrana.

Molecular phylogeny and biogeography of caecilians from Southeast Asia (Amphibia, Gymnophiona, Ichthyophiidae), with special reference to high cryptic species diversity in Sundaland

We investigated the phylogenetic relationships and estimated the history of species diversification and character evolution in two ichthyophiid genera: Caudacaecilia and Ichthyophis. We estimated the phylogenetic relationships of 67 samples from 33 localities in Southeast Asia from 3840-bp sequences of the mitochondrial 12S rRNA, 16S rRNA, and cyt b genes using Bayesian inference, maximum likelihood, and maximum parsimony methods. The Southeast Asian samples formed a well-supported clade differentiated from a South Asian sample. The Southeast Asian clade was divided into two subclades, one containing samples from South China, Indochina, Malay Peninsula, and Java. The other consisted of samples from Borneo and the Philippines. Neither Caudacaecilia nor Ichthyophis was monophyletic, nor did samples with or without light stripes lateral to the body form clades. We found several distinct sympatric lineages and undescribed species, especially from Sundaland.

Two new species of Leptolalax (Anura: Megophryidae) from northern Vietnam

Two new species of Leptolalax are described from two mountain ranges in northern Vietnam (Song Gam and Tam Dao) that are less than 150 km apart. Currently, only two species of Leptolalax, L. pelodytoides and L. bourreti, are described from Vietnam. The two new species are distinguished from other Leptolalax by a combination of characters including skin texture, ventral color pattern, presence or absence of spots on the flanks, and lateral fringes on the toes. Furthermore, the new species can be distinguished from the widely distributed and sympatric L. pelodytoides by their large size, indistinct color pattern, and an absence of granules under the chin. In addition, one of the new species has a higher cellular DNA content than sympatric L. pelodytoides.

A New Species of Chirixalus from Vietnam (Anura: Rhacophoridae)

Abstract A new rhacophorid species is described on the basis of two specimens collected from Vu Quang Nature Reserve, Ha Tinh Province, central Vietnam. The species has inner and outer fingers that are not opposable, but in order to avoid taxonomic confusion, it is tentatively assigned to the genus Chirixalus. It is a large Chirixalus, having robust body with warty, grayish dorsum and immaculate ventrum, and lacking large pollex, white granules around anus and on limbs, and dark markings on sides of body. It is most similar to C. eiffingeri and C. idiootocus in external morphology, and much different from the other congeners. Generic definition of the genera Chirixalus and Kurixalus is discussed.

Flow cytometry in biodiversity surveys: methods, utility, and constraints

Flow cytometry of blood is a powerful tool for rapidly sorting individual specimens on the basis of cellular DNA content. During biodiversity surveys, the method enabled the early identification of both cryptic sympatric and allopatric species of Vietnamese ranid frogs. This method may be extremely valuable in sorting individuals from other taxa and geographic regions, especially when cellular DNA content is known to vary among closely related taxa, and in tropical situations where crypsis is a relatively common phenomenon. Protocols for preparation of freezing solution, field procedures, preparation of reference standards, and flow cytometric analysis are provided. The best method for field preservation of blood is freezing in liquid nitrogen; field fixation of blood in ethanol was less efficient and resulted in drastically increased coefficients of variation. Once samples have been transferred to freezer storage, they should not be returned to a lower storage temperature in liquid nitrogen.

Mitochondrial phylogeny shows multiple independent ecological transitions and northern dispersion despite of Pleistocene glaciations in meadow and steppe vipers (Vipera ursinii and Vipera renardi)

The phylogeny and historical demography of small Eurasian vipers of the Vipera ursinii and V. renardi complexes were studied using mitochondrial DNA sequences analysed with Bayesian inference, Maximum Likelihood and Maximum Parsimony approaches, and mismatch distributions. Diversification in the group resulted from an initial dispersion in the later Pliocene - Pleistocene in two directions: north-westwards via the Balkans (V. ursinii complex) and north-eastwards from Asia Minor via the Caucasus (V. renardi complex). An independent, comparatively recent transition occurred from montane habitats to lowland grasslands in different mitochondrial lineages during the Late Pleistocene, when representatives of the both complexes had reached lowland steppes to the north. Effective population size showed clear signs of rapid growth in eastern V. renardi, triggered by colonization of vast lowland steppes, but in western V. ursinii complex grew during the Last Glaciation and experienced stabilization in Holocene. Expansion and population growth in lowland lineages of V. renardi was not strongly affected by Pleistocene climatic oscillations, when cold, dry conditions could have favoured species living in open grasslands. The high diversity of closely related haplotypes in the Caucasus and Tien-Shan could have resulted from repetitive expansion-constriction-isolation events in montane regions during Pleistocene climate fluctuations. The mitochondrial phylogeny pattern conflicts with the current taxonomy.

Three New Indochinese Species of Cascade Frogs (Amphibia: Ranidae) Allied to Rana archotaphus

Abstract Three new frog species allied to Rana archotaphus are described from Laos and Vietnam. One new species from the Hoang Lien Mountains of northwestern Vietnam is characterized by having females with snout-vent length 66–68 mm, no visible pineal body, the first finger shorter than second, width of third finger disc about equal to tympanum diameter, and tympanum of males relatively larger than females. The second new species from northern Laos is characterized by having males with snout-vent length 38–44 mm, no visible pineal body, first finger shorter than second, width of third finger disc equal to the tympanum diameter, venter with very light or no spotting, and venter translucent. The third new species from the Northern Truong Son (Annamite Highlands) of Laos and the Kon Tum Plateau of Vietnam is characterized by having females with snout-vent length 56–57 mm, a visible pineal body, tympanum of males relatively equal to females, the first finger shorter than second, and males with width of third finger disc about half the tympanum diameter. Descriptions are expanded of several species that resemble the two new species: Rana archotaphus, R. daorum, R. iriodes, and Amolops chunganensis.

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.

A New Species of Goniurosaurus (Squamata: Eublepharidae) from Hainan Island, China

Abstract A new species of Goniurosaurus described from Hainan Island is differentiated from all other species by having a series of enlarged, flat, imbricate scales on the mediolateral portions of the wrist; 37–46 preanal pores as opposed to 18–32 for all other species that have preanal pores; thin as opposed to wide juvenile body bands; faint, whitish colored bands in the caudal interspaces; and caudal bands being incomplete ventrally in adults. A phylogenetic analysis reveals that the new species is the basal member of the Goniurosaurus luii group. It differs from the other members of this group, Goniurosaurus luii and Goniurosaurus araneus, in being less than 107 mm in maximum snout vent length, having a more robust body stature, lacking elongate curved penultimate phalanges, having short robust digits, and having thin dark borders on the body bands. Three species of Goniurosaurus occur on Hainan Island.