A taxonomic revision of the Ingerophrynus parvus species complex (Amphibia, Anura, Bufonidae) with the description of a new species from Thailand

The hitherto monotypic opilionid genus Fangensis Rambla, 1994 is re-evaluated from new material collected in northern, western and southern Thailand. The original description of F. leclerci Rambla (type material not traceable) was based on specimens belonging to at least three distinct species. Fangensis leclerci is thus redefined and the binomen restricted to specimens from a cave in northern Thailand only. Three new species are described, F. spelaeus, sp. nov. and F. cavernarus, sp. nov. from two caves in western Thailand and F. insulanus, sp. nov. from forests on two islands in southern Thailand. Taxonomic characters are discussed and relationships among Fangensis species and between Fangensis and other cyphophthalmids are evaluated using morphological data and ~4 Kb of molecular data from three loci, two nuclear ribosomal genes and a mitochondrial coding gene. Overall, data indicate monophyly of Stylocellidae and morphological and ribosomal data suggest monophyly of Fangensis, which is in turn divided into two clades, one composed of the three species from northern and western Thailand and the other represented by the species from southern Thailand. Observations on mating in F. leclerci are provided.

Utilizing the COI barcoding approach, cryptic diversity has previously been detected within the morphologically recognized Simulium (Gomphostilbia) trangense Jitklang, Kuvangkadilok, Baimai, Takaoka & Adler, 2008 and S. (G.) sheilae Takaoka & Davies, 1995, of the S. (G.) ceylonicum species-group. Here, an unknown black fly species belonging to the S. ceylonicum species-group from southern Thailand was discovered and described as a new species, S. (G.) sipoense sp. nov. In addition, S. (G.) trangense is herein fully redescribed based on specimens collected from its type locality. Based on an integrative taxonomic approach combining morphological and molecular data, the validity of the newly described S. sipoense sp. nov. and the redescribed S. trangense is confirmed. Comparative morphological characteristics and phylogenetic analysis, inferred from COI sequences, suggest that the new species is conspecific with the species redescribed as S. trangense, using specimens collected from Malaysia, and is morphologically and phylogenetically closely related to S. sheilae, particularly to the specimens from Indonesia. The redescribed S. trangense is genetically highly similar or even identical to the species that was apparently misidentified as S. sheilae from southern and western Thailand, and is morphologically very similar to the new species, from which it is clearly distinguished by the relative length of the female claw tooth, shape of the male ventral plate, and color of the larval body. A detailed information on the morphological characteristics separates the new species, and the redescribed S. trangense from all other known species of the same species-group in Thailand and neighboring countries is provided. Further studies are warranted to clarify the taxonomic status of several cryptic species recognized within the morphologically defined S. trangense and S. sheilae.

The rock-dwelling gecko genus Cnemaspis is one of the most species-diverse genera of gekkonid in Thailand. Earlier studies relied on morphological data to identify species, but cryptic morphology often obscured species diversity in Cnemaspis. In this study, an integrative taxonomic approach based on morphological characters and sequences of the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene were used to clarify current taxonomy of the Cnemaspis siamensis complex and delimit a new species from Lan Saka District, Nakhon Si Thammarat Province, southern Thailand. Cnemaspis lineatubercularissp. nov. is distinguished from other congeneric species by the combination of morphological characters: (1) maximum snout-vent length (SVL) of 40.6 mm (mean 38.8 ± SD 1.4, N = 12) in adult males and maximum SVL of 41.8 mm (mean 39.5 ± SD 1.9, N = 7) in adult females; (2) 8–9 supralabial and infralabial scales; (3) gular, pectoral, abdominal, and subcaudal scales keeled; (4) rostral, interorbitals, supercilium, palmar scales, and ventral scales of brachia smooth; (5) 5–6 small, subconical spine-like tubercles present on flanks; (6) 19–21 paravertebral tubercles linearly arranged; (7) 27–29 subdigital lamellae under the fourth toe; (8) 4–7 pore-bearing precloacal scales, pores rounded arranged in chevron shape and separated only in males; (9) one postcloacal tubercles each side in males; (10) ventrolateral caudal tubercles present anteriorly; (11) caudal tubercles restricted to a single paravertebral row on each side; (12) single median row of subcaudal scales keeled and lacking enlarged median row; and (13) gular region, abdomen, limbs and subcaudal region yellowish only in males. Genetically, the uncorrected pairwise divergences between the new species and their congeners in the C. siamensis group were between 15.53–28.09%. The new species is currently known only from granitic rocky streams at Wang Mai Pak Waterfall in the Nakhon Si Thammarat mountain range. Its discovery suggests that additional unrecognized species of Cnemaspis may still occur in unexplored areas of southern Thailand.

he Chinese tiger frog Hoplobatrachus rugulosus is widely distributed in southern China, Malaysia, Myanmar, Thailand, and Vietnam. It is listed in Appendix II of CITES as the only Class II nationally-protected frog in China. The bred tiger frog known as the Thailand tiger frog, is also identified as H. rugulosus. Our analysis of the Cyt b gene showed high genetic divergence (13.8%) between wild and bred samples of tiger frog. Unexpected genetic divergence of the complete mt genome (14.0%) was also observed between wild and bred samples of tiger frog. Yet, the nuclear genes (NCX1, Rag1, Rhod, Tyr) showed little divergence between them. Despite this and their very similar morphology, the features of the mitochondrial genome including genetic divergence of other genes, different three-dimensional structures of ND5 proteins, and gene rearrangements indicate that H. rugulosus may be a cryptic species complex. Using Bayesian inference, maximum likelihood, and maximum parsimony analyses, Hoplobatrachus was resolved as a sister clade to Euphlyctis, and H. rugulosus (BT) as a sister clade to H. rugulosus (WT). We suggest that we should prevent Thailand tiger frogs (bred type) from escaping into wild environments lest they produce hybrids with Chinese tiger frogs (wild type).

Myanmar and Thailand often experience severe droughts and floods that cause irreparable damage to the socio-economy condition of both countries. In this study, the Southeastern Asian Summer Monsoon variation is found to be the main element of interannual precipitation variation of the region, more than the El Niño/Southern Oscillation (ENSO). The ENSO influence is evident only during the boreal spring season. Although the monsoon is the major factor, the existing Indian Monsoon Index (IMI) and Western North Pacific Monsoon Index (WNPMI) do not correlate well with the precipitation variation in the study regions of Southern Myanmar and Thailand. Therefore, a new set of indices is developed based on the regional monsoon variations and presented here for the first time. Precipitation variations in Southern Myanmar and Thailand differ as well as the elements affecting the precipitation variations in different seasons. So, separate indices are proposed for each season for Southern Myanmar and Thailand. Four new monsoon indices based on wind anomalies are formulated and are named as the Indochina Monsoon Indices. These new indices correlate better with the precipitation variations of the study region as compared to the existing IMI and WNPMI.

Morphological variation of the skull was examined in the northern treeshrew (Tupaia belangeri) from various localities across Southeast Asia. Through a multivariate analysis, the treeshrews from South Vietnam exhibited distinct morphological characteristics compared to other populations from Thailand and Laos, and Malaysia. The plots of the specimens of North Vietnam are not randomly mixed with Thailand plots segregation in the scatteregrams of canonical discriminant analysis. Since the skulls of the population from North Vietnam were morphologically similar to those form central Laos and northern and northeastern Thailand, the zoogeographical barrier effect of Mekong River was not clearly confirmed. The population of the Kanchanaburi in western Thailand is clearly smaller in size compared to the other populations. The southern border of the distribution of this species is determined by the Isthmus of Kra or Kangar-Pattani Line. In the northern treeshrew, which is distributed from southern China to Bangladesh and southern Thailand, we have detected osteometrical geographical variation driven by geography. These results indicate that the skull morphology in the Tupaia glis-belangeri complex distinctively differs in South Vietnam, western Thailand, and southern Thailand. The zoogeographical barrier and factor separating these districts are expected to clarify in the future.

Morphological characters were studied in 90 accessions of Capsicum frutescens in Southeast and East Asia, and cluster analysis was carried out to qualitatively and quantitatively assess the characters. Four major clusters appeared both in the qualitative characters (XA, XB, XC and XD) and quantitative characters (YA, YA, YC and YD), and 90 accessions were classified into 9 types based on cluster combinations. Four major types (XAYB, XAYC, XBYB and XBYD) including 61 accessions were distributed evenly in Southeast Asia. Five minor types, on the other hand, were distributed unevenly: XAYA in the Ryukyu Islands, Indonesia, northern Thailand; XBYA in Indonesia, southern Thailand, Vietnam; XCYD in northern and southern Thailand; XCYD in Vietnam and Laos; XDYD in southern Thailand. Morphological characters were more diversified in Thailand. This is partly because C. frutescens is used more frequently in Thailand than in other regions. All 17 accessions from the Ryukyu Islands, on the contrary, showed exactly the same qualitative characters and high similarity in quantitative characters. These results agree with the results of biochemical analyses. Accessions from the Ryukyu Islands were both morphologically and biochemically very close to each other, indicating that the Ryukyu Islands is the end of the dispersal route of C. frutescens in Southeast and East Asia.

The population systematics of the cobras of the genus Naja in southern Thailand, Malaysia and Indonesia are investigated, using multivariate analysis of a large number of morphological characters. These populations are found to constitute three distinct groups: a northern form, which occurs in Thailand and northern Peninsular Malaysia; an equatorial form, which occurs in southern Thailand, Peninsular Malaysia, Sumatra and Borneo; and a southern form, which occurs on Java and the Lesser Sunda Islands. The first two forms are sympatric in northern Peninsular Malaysia and southern Thailand, and therefore constitute separate species. This is of importance for the treatment of snakebite in the region. The distribution of the three forms can be related both to present ecological conditions and to Pleistocene geological and climatic events. The reliability of the results is demonstrated by the relationship between character number and congruence of patterns of geographic variation, investigated by random resampling. The pattern of geographic variation within two of the three main forms is investigated and related to current ecological conditions and Pleistocene events.

Simple SummaryNew species of frogs continue to be discovered at a rapid rate in Southeast Asia, often as a result of reexamining populations of geographically widespread species using new molecular and bioacoustic tools. Here, we show that members of the fanged frog genus Limnonectes from Ko Pha-ngan, Ko Samui, and Ko Lanta Yai Islands in southern Thailand can be distinguished from the morphologically similar species Limnonectes doriae in molecular, advertisement call, morphometric, and qualitative morphological characters. On the basis of these multiple lines of evidence, we describe the insular populations in southern Thailand as a new species, Limnonectes pseudodoriae sp. nov. The new species occurs near small streams in low to mid-elevation forests and breeds in terrestrial nests consisting of moist, clay depressions in which the eggs and larvae develop.A new species in the dicroglossid frog genus Limnonectes is described from Ko Pha-ngan, Ko Samui, and Ko Lanta Yai Islands in southern Thailand. Males of Limnonectes pseudodoriae sp. nov. lack a caruncle on top of the head and very closely resemble L. doriae (Boulenger, 1887) from Myanmar and western and southern Thailand. However, the new species is distinguished from L. doriae and its congeners using an integrative taxonomic approach of morphology, mitochondrial DNA, and bioacoustics. Limnonectes pseudodoriae sp. nov. differs from L. doriae and its congeners by having a unique combination of morphological characters, including body size; skin texture of the interorbital region, dorsum, and shank; toe webbing; relative size of the inner metatarsal tubercle; and coloration of the tympanum, venter, and ova. The advertisement call of the new species is also readily differentiated from that of L. doriae in temporal parameters. Limnonectes pseudodoriae sp. nov. is highly divergent in mitochondrial DNA from L. doriae and its congeners, but its phylogenetic position within the genus is not resolved. The natural history of the new species is presented, and the geographic range of L. doriae in Thailand is clarified.

Summary Gurney's Pitta Pitta gurneyi was not seen in the wild for over 30 years before its rediscovery in a small area in southern Thailand in 1986. In 2003, it was found over a much larger area in southern Myanmar (Burma) after an even longer absence of records. This paper reports the results of recent research on both populations. In Myanmar, birds were found in a high proportion of visited sites, including sites up to 40 km further north than any previous records in the country. Occupied forest sites had a higher density of seedlings, saplings, bamboo and rattan than unoccupied forest sites, suggesting a preference for regenerating forest. There was no evidence of a decline in the species's likelihood of occurrence up to at least the highest visited altitude of 230 m. Maximum entropy models suggested a potential range size of between 3,200 and 5,800 km2 and the predicted range extended just over the border into Thailand, suggesting that birds might persist there. Population estimates for southern Myanmar ranged from 9,300 to 35,000 territories, with a mid-point estimate of 20,000 territories, based upon range sizes modelled from different parameters and a range of territory densities estimated from Thailand. The population in southern Thailand was estimated at between 15 and 20 territories in 2003, 2005 and 2007, and there was a reduction in the historic rate of forest loss in the core range over the same period, suggesting that long-term declines have been at least temporarily contained by recent conservation intervention. However, nesting success in Thailand was very low, due to heavy nest predation by cat snakes Boiga. Nests were usually built close to waterlogged areas and damp gullies, and nestlings were fed almost exclusively on earthworms. Occupied sites in Thailand had a higher density of seedlings, saplings, spiny palms and rattans, but sparser ground cover and a lower density of large trees and bamboo, than forests in Myanmar, indicating their secondary nature. Occupied and unoccupied sites in Myanmar were structurally more similar to each other than either was to occupied sites in Thailand. The results suggest that the species might inhabit a wider range of altitudes, slopes and forest types than previously thought, and so might persist in previously unsurveyed areas and might survive or even benefit from a degree of forest disturbance. However, the species's conservation-dependent status in Thailand and accelerating forest clearance in Myanmar suggest that forest protection measures are urgently needed to secure its long-term future.

Resolving an unnoticed diversity within the Schistura robertsi species complex (Teleostei: Nemacheilidae) using molecules and morphology

BackgroundBiting midges of the genus Culicoides play an important role in the transmission of pathogenic arboviruses and parasites. Thailand has documented more than 100 species of Culicoides; however, several cryptic species complexes remain to be clarified. Recent studies in areas with leishmaniasis indicate that several species of Culicoides might be potential vectors of Leishmania in the subgenus Mundinia, but evidence supporting the hypothesis is still lacking. Therefore, the diversity of Culicoides biting midges and their potential role as vectors of leishmaniasis in southern Thailand remains uncertain.MethodsFemale Culicoides biting midges were collected using Centers for Disease Control and Prevention (CDC) ultraviolet (UV) light traps from four locations within leishmaniasis-affected areas in three provinces of southern Thailand, including Nakhon Si Thammarat, Krabi, and Surat Thani. Culicoides species were identified based on the morphology of wing spot patterns and subsequently confirmed by cytochrome c oxidase subunit I (COI) Sanger sequencing. A potential cryptic species was classified using an integrative taxonomic approach associated with DNA barcoding identification by Barcode of Life Database (BOLD) and Basic Local Alignment Search Tool (BLAST) searches. Furthermore, three different methods of species delimitation, namely ASAP [Assemble Species by Automatic Partitioning], TCS [Templeton, Crandall, and Sing], and PTP [Poisson Tree Processes], were employed to verify the sequences into the molecular operational taxonomic unit (MOTU). Detection of Leishmania and other trypanosomatid parasites was performed by polymerase chain reaction (PCR) based on the ITS1 region and small subunit SSU ribosomal RNA (rRNA) gene, followed by Sanger sequencing and haplotype diversity analysis. The identification of host blood sources was carried out using host-specific multiplex PCR.ResultsA total of 716 unfed midges and 159 blood-fed specimens were morphologically identified into 25 species belonging to five subgenera (Avaritia, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and four species groups (Clavipalpis, Ornatus, Shermani, and Shortti). Two unidentified specimens were classified into two subgenera (Trithecoides and Avaritia). The DNA barcoding identification exhibited an 82.20% success rate. Species delimitation analyses demonstrated the presence of cryptic species complexes, categorized into six species: Culicoides actoni, C. orientalis, C. huffi, C. palpifer, C. clavipalpis, and C. jacobsoni. Furthermore, 6.42% of the Culicoides biting midges tested positive for Leishmania DNA in three sampling sites in Nakhon Si Thammarat and Surat Thani provinces (with no positive results in Krabi province). Furthermore, the sympatric infection of Leishmania martiniquensis and Leishmania orientalis was identified in several Culicoides species in Ron Phibun and Phunphin districts in Nakhon Si Thammarat and Surat Thani, respectively. In contrast, L. orientalis was detected in Sichon district, Nakhon Si Thammarat province. A genetic diversity analysis revealed high haplotype diversity and relatively low nucleotide diversity in both parasite populations. Additionally, Crithidia sp. and Crithidia brevicula were detected in Culicoides peregrinus and Culicoides subgenus Trithecoides. The analysis of the host blood meal from Ron Phibun also demonstrated that Culicoides had fed on cows, dogs, and chickens, and mixed blood preferences for humans and cows or chickens and cows were detected.ConclusionsThe findings of the present study demonstrate the presence of mixed blood hosts and co-circulation of L. martiniquensis and L. orientalis in Culicoides in areas of leishmaniasis, as well as cryptic species of Culicoides biting midges, through an integrative taxonomic approach. These findings support the hypothesis that Culicoides biting midges may serve as potential vectors in southern Thailand, and vector diversity is a contributing factor to the risk of zoonotic transmission.Graphical

Cyrtodactylusmonilatus sp. nov. is described from Si Sawat District, Kanchanaburi Province, in western Thailand. The new species superficially resembles C.zebraicus Taylor, 1962 from southern Thailand. However, differences between the new species from C.zebraicus and other congeners were supported by an integrative taxonomic analysis of molecular and morphological data. Phylogenetic analyses based on the mitochondrial NADH dehydrogenase subunit 2 (ND2) gene showed that the new species is a member of the C.oldhami group and closely related to Cyrtodactylus sp. MT468911 from Thong Pha Phum National Park, Thong Pha Phum District, Kanchanaburi Province. Uncorrected pairwise genetic divergences (p-distances) between the new species and its congeners, including C.zebraicus, ranged from 7.7-17.7%. Cyrtodactylusmonilatus sp. nov. can also be distinguished from all members of the C.oldhami group by having a unique combination of morphological characters, including a snout to vent length of 53.7-63.3 mm in adult males and 58.6-75.8 mm in adult females; 22-34 paravertebral tubercles; 34-42 ventral scales; 30-39 enlarged contiguous femoroprecloacal scales; femoral pores and precloacal pores absent in both sexes; four or five rows of postprecloacal scales; enlarged median subcaudal scales absent; weak ventrolateral folds present; 4-7 rows of paired, paravertebral, dark-brown blotches edged in yellow or yellowish white; and two rows of small, diffuse, yellow or yellowish white spots on flanks. The new species occurs in a narrow range of forest at mid to low elevations associated with karst landscapes in the Tenasserim mountain range.

Background: Snakes possess adaptive characteristics of morphology that may result in incorrect reconstruction of phylogeny. Molecular approaches have become the major source of new information for advancing our understanding of evolutionary, genetic relationships, and species identification. Objective: We studied DNA sequences of Ophiophagus hannah in different parts of Thailand and compared them with those of O. hannah from other countries. Materials and Methods: We studied the mitochondrial NADH dehydrogenase subunit 2 (ND2) and control region (CR) sequences of 12 individuals O. hannah from different localities across Thailand. Phylogenetic analysis of DNA sequences were compared to the published sequences of O. hannah deposited in NCBI GenBank database from other countries. Results: O. hannah could be categorized into 2 Clades, 5 haplotypes, and 4 localities based on 43 different nucleotide positions from the 736 bp of ND2 and 673 bp of CR. Clade A was haplotype A from southern Thailand. Clade B consisted of haplotypes B, C, D, and E. Haplotype B and C came from northern Thailand. Haplotype D came from western Thailand, while haplotype E was O. hannah from central Thailand. The DNA sequences of Clade B were similar to the sequences of O. hannah in Myanmar and southern China that are already deposited in NCBI GenBank database. Conclusion: We found a different genotype of O. hannah from southern Thailand and suggest that this may be a new species of O. hannah.

BackgroundBactrocera dorsalis s.s. (Hendel) and B. papayae Drew & Hancock, are invasive pests belonging to the B. dorsalis complex. Their species status, based on morphology, is sometimes arguable. Consequently, the existence of cryptic species and/or population isolation may decrease the effectiveness of the sterile insect technique (SIT) due to an unknown degree of sexual isolation between released sterile flies and wild counterparts. To evaluate the genetic relationship and current demography in wild populations for guiding the application of area-wide integrated pest management using SIT, seven microsatellite-derived markers from B. dorsalis s.s. and another five from B. papayae were used for surveying intra- and inter-specific variation, population structure, and recent migration among sympatric and allopatric populations of the two morphological forms across Southern Thailand and West Malaysia.ResultsBasic genetic variations were not significantly different among forms, populations, and geographical areas (P > 0.05). Nonetheless, two sets of microsatellite markers showed significantly different levels of polymorphisms. Genetic differentiation between intra- and inter-specific differences was significant, but low. Seventeen populations revealed three hypothetical genetic clusters (K = 3) regardless of forms and geographical areas. The genetic structure of sympatric populations slightly changed during the different years of collection. Recent gene flow (m ≥ 0.10) was frequently detected whether samples were sympatric or allopatric. Ninety-five of 379 individuals distributed across the given area were designated as recent migrants or of admixed ancestry. As a consequence of substantial migration, no significant correlation between genetic and geographic distances was detected (R2 = 0.056, P = 0.650).ConclusionsAccording to the 12 microsatellite variations, weak population structure and recent gene flow suggest that there is no status for cryptic species between B. dorsalis s.s. and B. papayae forms in Southern Thailand and West Malaysia. Both forms can be treated as a single target pest for the SIT program in an area-wide sense. Additionally, the result of species identification based on molecular data and morphological character are not congruent. The use of independent, multiple approaches in the characterization of the target population may ensure the effectiveness and feasibility of SIT-based control in the target area.