Two New Species of Ansonia from Thailand (Anura: Bufonidae).

The taxonomic status of two populations of stream toads of the genus Ansonia from Thailand, previously suspected to be specifically distinct on the basis of molecular data, are assessed using morphological and additional genetic data. In mtDNA phylogeny, each of the two Thai lineages, one from Pilok Subdistrict, Kanchanaburi, and another from Phuket Island, are separated from other congenerics from the Thai-Malay Peninsula by large genetic distances, comparable with those observed between other species in the genus. Although each of the two lineages is superficially similar to other species morphologically, they are distinguishable in several morphological traits and are considered to represent valid, independently evolving species. We therefore describe them as A. pilokensis sp. nov. and A. phuketensis sp. nov.

A new tree frog of the genus Gracixalus from Thailand (Amphibia: Rhacophoridae).

We report a new tree frog of the genus Gracixalus from western Thailand and describe it as a new species Gracixalus seesom based on results of morphological and molecular analyses. The new species is a small-sized Gracixalus (male snout-vent length ca. 22 mm) and is morphologically similar to G. gracilipes, but is easily distinguished from it by its dorsal tan color in life, absence of white spot on lower lip, and black markings on its foot webbing. The new species also clearly differs from all the other members of the genus by the combination of small body size, triangular snout, and light yellowish brown dorsum without distinct tuberculations. Problems of phylogeny and taxonomy of the genus Gracixalus are briefly discussed.

Two new species of Tylototriton from Thailand (Amphibia: Urodela: Salamandridae).

Three morphological groups are found in a salamandrid newt Tylototriton shanjing from Thailand. We describe two of them as new species, one from northern and the other from northeastern Thailand, based on molecular and morphological data, however we could not make a taxonomic decision on the remaining one group because of the lack of voucher specimens and sufficient genetic data. The northern species differs morphologically from all known congeners by having the combination of orange to reddish brown markings, narrow and sharply protruding dorsolateral bony ridges on head, weakly segmented vertebral ridge, and long and high tail. The northeastern species is characterized by having the combination of yellow, orange, or reddish brown markings, wide and moderately protruding dorsolateral bony ridges on head, smooth vertebral ridge, black limbs, and black tail except for edges. Validity of taxonomic subdivision of the genus Tylototriton is discussed.

Phylogenetic relationships among Hoplobatrachus rugulosus in Thailand as inferred from mitochondrial DNA sequences of the cytochrome-b gene (Amphibia, Anura, Dicroglossidae).

A fragment (564 bp) of the mitochondrial cytochrome-b (Cyt-b) gene was studied for 73 individual rice field frogs (Hoplobatrachus rugulosus) from 18 geographical locations (populations) within Thailand. Sequence analysis revealed the presence of 12 haplotypes, with five haplotypes being represented in two or more populations, and the other seven being population-distinct haplotypes. Phylogenetic analysis by maximum parsimony, maximum likelihood, and neighbor joining analyses all placed the 12 haplotypes into two distinct and well-separated clades with high bootstrap support, reflecting the high sequence divergences between the clades (25.3-32.3%). The mountain ranges and the Isthmus of Kra are likely to have played important roles in hindering gene flow among H. rugulosus populations in Thailand. From the sequence divergence values, the two clades of H. rugulosus can be classified into two distinct species, and therefore, the strains of H. rugulosus bred in farm stocks should be restricted to a population of one clade so as to avoid cross breeding between the two clades.

Hepatic metallothionein and Glutathione-S-Transferase responses in two populations of rice frogs, Fejervarya limnocharis, naturally exposed to different environmental cadmium levels.

Glutathione-S-Transferase (GST) and metallothionein are important biomarker endpoints in studying the effect of Cd exposure. The purpose of this research was to study the correlation between hepatic GST and metallothionein with hepatic Cd in wild Fejervarya limnocharis exposed to environmental Cd. Results showed that frogs from contaminated sites had significantly higher hepatic metallothionein (3.58 mg/kg wet weight) and GST activity (0.259 µmol/min/mg total protein) than those from the reference site (2.36 mg/kg wet weight and 0.157 µmol/min/mg total protein respectively). There was a significantly positive correlation between hepatic Cd and GST activity (r = 0.802, p = 0.009) but not between hepatic Cd and metallothionein (r = 0.548, p = 0.139). The results concluded that while frogs from the contaminated site had higher GST and metallothionein, only GST showed significant positive correlation with hepatic Cd levels, indicating that hepatic GST activity may be used as a biomarker endpoint.

Systematic relationships of Oriental tiny frogs of the family Microhylidae (Amphibia, Anura) as revealed by mtDNA genealogy.

We estimated the genealogical relationships and assessed systematic relationships among 45 out of 89 named species and four unnamed taxa from 11 of 14 genera of the Oriental microhylids from 1767 bp sequences of the mitochondrial DNA genes 12S rRNA and 16S rRNA using maximum parsimony, maximum likelihood, and Bayesian inference methods. Monophyly was rejected for the subfamily Microhylinae, and our data reveal four well-supported clades whose relationships to each other are unresolved: (A) Microhyla, Calluella, and Glyphoglossus, (B) Chaperina, (C) Kaloula, Phrynella, and Metaphrynella, and (D) Micryletta. They were genetically as divergent from each other as from another Oriental subfamily Kalophryninae, and could be recognized as distinct subfamilies. Within Clade A, our data reveal three well-supported subclades whose relationships to each other are unresolved: (AI) Microhyla-I, (AII) Calluella and Glyphoglossus, and (AIII) Microhyla-II. Of the two enigmatic Malaysian genera, whose subfamilial placement has been undetermined, Phrynella was found to be the sister species of Metaphrynella in Clade C, whereas Gastrophrynoides was grouped in the Papua-Australian subfamily Asterophryinae. Currently recognized subgenera and species groups within Microhyla based on morphology were not supported phylogenetically, and require thorough reassessments.

A new species of the genus Tylototriton (Urodela, Salamandridae) from western Thailand.

We describe a new species of the newt genus Tylototriton from Umphang Wildlife Sanctuary, Tak Province, western Thailand based on molecular and morphological evidence and named here as Tylototritonumphangensis sp. nov. The new species is assigned to the subgenus Tylototriton and differs from other species in having dark-brown to blackish-brown body and limbs, truncate snout, prominent antero-medial ends of the expansion of the dentary bones, laterally protruding quadrate regions, indistinct and small rib nodules, a well-segmented vertebral ridge, and rough dorsolateral bony ridges, which are steeper anterior, and curved medially at the posterior ends. The molecular data show that Tylototritonumphangensis sp. nov. differs from T.uyenoi sensu stricto by a 5% genetic sequence divergence of the mitochondrial NADH dehydrogenase subunit 2 region gene. The new species and T.uyenoi are both endemic to Thailand, distributed along the Northwest Thai (Dawna) Uplands of Indochina. To clarify the species boundary between Tylototritonumphangensis sp. nov. and T.uyenoi, additional field research is needed in adjacent areas. Tylototritonumphangensis sp. nov. is restricted to evergreen hill forests in Umphang Wildlife Sanctuary. We suggest that the new species should be classified as Endangered (EN) in the IUCN Red List.

Phylogenetic relationships of megophryid frogs of the genus Leptobrachium (Amphibia, Anura) as revealed by mtDNA gene sequences.

By investigating genealogical relationships, we estimated the phylogenetic history and biogeography in the megophryid genus Leptobrachium (sensu lato, including Vibrissaphora) from southern China, Indochina, Thailand and the Sundaland. The genealogical relationships among the 30 named and unnamed taxa were estimated using 2009 bp of sequences from the mitochondrial DNA genes 12S rRNA, tRNA(val), and 16S rRNA using maximum parsimony, maximum likelihood, and Bayesian inference methods. The genus Leptobrachium was a well-supported monophyletic group that contained two major clades. One clade had three subclades primarily from disjunct regions including Borneo, Peninsular Malaysia and Java, and Thailand. The Bornean subclade included one species each from the Philippines and Sumatra. The other major clade consisted of two subclades, one from Indochina and the other from southern China (Vibrissaphora). Divergence times estimated an old evolutionary history of each subclade, one that could not be explained by the geohistory of Southeast Asian major landmasses.

Phylogenetic relationships of Ansonia from Southeast Asia inferred from mitochondrial DNA sequences: systematic and biogeographic implications (Anura: Bufonidae).

We investigated the phylogenetic relationships and estimated the history of species diversification and biogeography in the bufonid genus Ansonia from Southeast Asia, a unique organism with tadpoles adapted to life in strong currents chiefly in montane regions and also in lowland rainforests. We estimated phylogenetic relationships among 32 named and unnamed taxa using 2461bp sequences of the mitochondrial 12S rRNA, tRNA(val), and 16S rRNA genes with equally-weighted parsimony, maximum likelihood, and Bayesian methods of inference. Monophyletic clades of Southeast Asian members of the genus Ansonia are well-supported, allowing for the interpretation of general biogeographic conclusions. The genus is divided into two major clades. One of these contains two reciprocally monophyletic subclades, one from the Malay Peninsula and Thailand and the other from Borneo. The other major clade primarily consists of Bornean taxa but also includes a monophyletic group of two Philippine species and a single peninsular Malaysian species. We estimated absolute divergence times using Bayesian methods with external calibration points to reconstruct the relative timing of faunal exchange between the major landmasses of Southeast Asia.

Cadmium accumulation in two populations of rice frogs (Fejervarya limnocharis) naturally exposed to different environmental cadmium levels.

Contaminant accumulation analysis is important in the study of sentinels. This research determined cadmium accumulation and bioconcentration factors of whole organism, liver, kidney, ovary and testis of Fejervarya limnocharis exposed to different environmental cadmium levels. Frogs from contaminated sites had significantly higher hepatic (1.939 mg/kg), renal (7.253 mg/kg) and testicular (1.462 mg/kg) cadmium than those from the reference sites (0.205, 0.783 and 0.379 mg/kg, respectively). Cadmium accumulation was the highest during the late dry and early rainy seasons. If this species is used as a sentinel for cadmium accumulation, the utilization of its whole organism, liver, kidney and testis is appropriate.

Genetic divergences and phylogenetic relationships among the Fejervarya limnocharis complex in Thailand and neighboring countries revealed by mitochondrial and nuclear genes.

To clarify the genetic divergence in the F. limnocharis complex from Thailand and neighboring countries and to elucidate the phylogenetic problems of this taxon, we analyzed partial sequences of the mitochondrial 12S and 16S rRNA genes and the nuclear CXCR4, NCX1, RAG-1, and tyrosinase genes. The F. limnocharis complex from Thailand had three distinct haplotypes for 12S and 16S rRNA genes. Nucleotide similarities and the phylogenetic relationships indicated that the haplotype 1 group corresponded to the real "F. limnocharis", the haplotype 2 group was F. orissaensis or closely related to it, and the haplotype 3 group was possibly an undescribed species. Mitochondrial gene data also showed two major clades of the genus Fejervarya, the Southeastern and South Asian groups. Although F. orissaensis is so far known only from Orissa in India, the haplotype 2 group was observed in Thailand. This distribution pattern and the phylogeny suggested that the origin of F. orissaensis and the haplotype 2 group might lie in Southeast Asia. There was also evidence suggesting that the haplotype 3 group originated in the South Asian area and has spread to northern Thailand. The nuclear gene data did not support the monophyly of the haplotypes recognized by mitochondrial genes. This incongruence between the mitochondrial and nuclear data seems to be caused by ancestral polymorphic sites contained in nuclear genes. Although neither the mitochondrial nor the nuclear data clarified intergeneric relationships, the nuclear data rejected the monophyly of the genus Fejervarya.

Genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia (Java) and other Asian countries.

In order to elucidate the genetic relationships and reproductive-isolation mechanisms among the Fejervarya limnocharis complex from Indonesia and other Asian countries, allozyme analyses and crossing experiments were carried out using 208 individuals from 21 localities in eight Asian countries. The allozyme analyses revealed that 17 enzymes examined were controlled by genes at 27 loci, and that 7.9 phenotypes were produced by 5.2 alleles on average. The two species recognized in F. limnocharis sensu lato from Southeast Asia (i.e., F. limnocharis sensu stricto and F. iskandari) were found to occur sympatrically at three localities (Bogor, Cianjur and Malingping), all on Java, Indonesia. Fejervaya iskandari was dominant at each of these localities and showed substantial geographic genetic variation. Laboratory-produced hybrids between F. limnocharis and F. iskandari from Java became underdeveloped and died at the tadpole stage, suggesting that these species are completely isolated by hybrid inviability. Hybrids between topotypic F. limnocharis and the Malaysian and Japanese conspecific populations developed normally to metamorphosis. Likewise, hybrids between topotypic F. iskandari and the Thailand and Bangladesh conspecific populations also showed normal viability throughout larval development. The present allozyme analyses and crossing experiments strongly suggested the presence of two distinct forms, the large type and the small type, in the F. limnocharis complex from Asia, and further subdivision of the large type into the F. limnocharis assemblage and the F. iskandari assemblage. The small type was found in samples from India, Thailand, Bangladesh and Sri Lanka, and included at least three different species. The sample from Pilok, Thailand, was considered to represent an undescribed species.

Evolutionary relationships and reproductive isolating mechanisms in the rice frog (Fejervarya limnocharis) species complex from Sri Lanka, Thailand, Taiwan and Japan, inferred from mtDNA gene sequences, allozymes, and crossing experiments.

The rice frog (Fejervarya limnocharis) species complex is widely distributed, from India to Japan, and most prevalently in Southeast Asia. Conspicuous morphological variation has been reported for this species complex throughout its distribution range. In the present study, we used mtDNA gene sequence and allozyme analyses to infer evolutionary affinities within this species complex using eight populations (Sri Lanka; Bangkok and Ranong in Thailand; Taiwan; and Hiroshima, Okinawa, Ishigaki and Iriomote in Japan). We also conducted crossing experiments among four populations from Japan, Thailand, and Sri Lanka in order to find out more about the reproductive isolating mechanisms that might exist among the East, Southeast, and South Asian populations of this species complex. The crossing experiments revealed that the Sri Lanka population is reproductively isolated from the Hiroshima, Bangkok, and Ranong populations by complete hybrid inviability, and that the Bangkok population may be reproductively isolated from the Hiroshima population by partial hybrid inviability. Thus, it is not unreasonable to regard the Sri Lanka population as a species separated from F. limnocharis. The mtDNA and allozyme data showed that the Ranong population is most closely related to the Bangkok population in nuclear genome, but more similar to the Okinawa and Taiwan populations in mtDNA genome. The present, preliminary survey may raise questions about the species status of these particular populations and also about the nature of the biological species concept.

A new Ansonia from the Isthmus of Kra, Thailand (Amphibia, Anura, Bufonidae).

A new species of torrent-dwelling bufonid frog of the genus Ansonia is described from the Isthmus of Kra, Thailand. Ansonia kraensis is morphologically similar to Malaysian A. malayana, but differs from it in ventral coloration and larval morphology. Occurrence of A. kraensis in this region suggests a heterogeneous nature of the anuran fauna between northern and southern regions of the Malay Peninsula.

Taxonomic relationships within the pan-oriental narrow-mouth toad Microhyla ornata as revealed by mtDNA analysis (Amphibia, Anura, Microhylidae).

A molecular phylogenetic survey was conducted using mtDNA sequences of 12S and 16S rRNA, and cyt-b genes to examine taxonomic relationships among populations of the Pan-Oriental microhylid, Microhyla ornata, from India, Bangladesh, Thailand, Laos, China, Taiwan, and the Ryukyu Archipelago of Japan. Two discrete clades are recognized within this species, one consisting of populations from India and Bangladesh, and the other encompassing the remaining populations. In the latter clade, populations from the Ryukyu Archipelago are clearly split from the rest (populations from Taiwan and the continent) with considerable degrees of genetic differentiations. Each of the three lineages is judged to represent a good species, and the name Microhyla ornata is restricted to the South Asian populations. For the populations from Taiwan and a wide region from China to Southeast Asia, the name Microhyla fissipes should be applied, whereas the Ryukyu populations are most appropriately referred to as Microhyla okinavensis, although further substantial genetic differentiations are recognized among some island group populations within this last species.

Reproductive Mode of Fejervarya limnocharis (Anura: Ranidae) Caught from Mae Sot, Thailand Based on Its Gonadosomatic Indices.

Amphibians show a variety of reproductive modes and strategies. The cyclicity and continuity of reproduction can often be predicted from the annual gonadosomatic index trends in a species specific manner. This research aims to document the female and male gonadosomatic index profiles and to ascertain the reproductive cyclicity or continuity of Fejervarya limnocharis (Anura: Ranidae) caught in Mae Sot, Tak Province, Thailand. Frogs were collected monthly between November 2007 and October 2009 in rice fields and their surrounding areas in the study site. For each frog, total weight and ovarian/testicular weight were measured to obtain the female and male gonadosomatic index. The number of female individuals with mature eggs (postvitellogenic eggs) was also counted. The results showed that female frogs had two main surges of increased GSI in March and September of 2008. Alternatively, male frogs showed a more gradual increase and decrease in gonadosomatic index, thus the index remaining high throughout the year. This study concluded that while F. limnocharis in this area is essentially a continuous breeder, it is more optimized for a cyclic reproduction mode with two breeding cycles during the rainy season.

Phylogenetic relationships of Oriental torrent frogs in the genus Amolops and its allies (Amphibia, Anura, Ranidae).

We investigated the phylogenetic relationships among 20 species of Oriental torrent frogs in the genus Amolops and its allies from China and Southeast Asia based on 1346-bp sequences of the mitochondrial 12S and 16S rRNA genes. Oriental species of the tribe Ranini form a monophyletic group containing 11 clades (Rana temporaria + Pseudoamolops, R. chalconota, four clades of Amolops, Meristogenys, three clades of Huia species, and Staurois) for which the phylogenetic relationships are unresolved. The genus Amolops consists of southern Chinese, southwestern Chinese, Thai, and Vietnamese-Malaysian lineages, but their relationships are also unresolved. The separation of southern and southwestern lineages within China conforms to previous morphological and karyological results. Species of Huia do not form a monophyletic group, whereas those of Meristogenys are monophyletic. Because P. sauteri is a sister species of R. temporaria, distinct generic status of Pseudoamolops is unwarranted.

Gonadotropin and estrogen responses in freshwater turtle (Chrysemys picta) from Cape Cod, Massachusetts.

As a result of chemical waste disposal on the Massachusetts Military Reservation, a Superfund site on Cape Cod, MA, contaminated groundwater plumes have formed. These plumes are of concern due to the widespread use of groundwater wells as a drinking water source by the local population. Prior observations on a sentinel species Chrysemys picta field-trapped from ponds on Cape Cod suggested deficits in reproductive processes including lower levels of vitellogenin, estradiol-17beta, oviduct weights, and oocyte numbers in females and lower testicular weight and sperm count in males. Possible loci in the hypothalamic-pituitary-gonadal-liver axis at which xenobiotics may act were determined in turtles trapped from Moody Pond (a test site) and Washburn Pond (a reference site). Specifically, gonadotropin and estrogen responses were assessed using plasma steroids and vitellogenin as markers. Basal vitellogenin levels were significantly lower in Moody Pond females; however, vitellogenin responses to estradiol-17beta were the same in both groups, indicating a normal hepatic response to estrogen. In contrast, estradiol-17beta secretion was not stimulated by gonadotropin in Moody Pond females, compared to Washburn animals. Basal plasma testosterone and the response to gonadotropin in males were similar, although steroid levels in Moody Pond animals were slower to return to baseline after gonadotropin injection. The results suggest that a low-level mixture of xenobiotic contaminants may interfere with the steroid metabolic pathways in turtles exposed to the test site, but not the reference site, environment.