Minh Duc Le

Comparing and combining distance-based and character-based approaches for barcoding turtles.

Molecular barcoding can serve as a powerful tool in wildlife forensics and may prove to be a vital aid in conserving organisms that are threatened by illegal wildlife trade, such as turtles (Order Testudines). We produced cytochrome oxidase subunit one (COI) sequences (650 bp) for 174 turtle species and combined these with publicly available sequences for 50 species to produce a data set representative of the breadth of the order. Variability within the barcode region was assessed, and the utility of both distance‐based and character‐based methods for species identification was evaluated. For species in which genetic material from more than one individual was available (n = 69), intraspecific divergences were 1.3% on average, although divergences greater than the customary 2% barcode threshold occurred within 15 species. High intraspecific divergences could indicate species with a high degree of internal genetic structure or possibly even cryptic species, although introgression is also probable in some of these taxa. Divergences between species of the same genus were 6.4% on average; however, 49 species were <2% divergent from congeners. Low levels of interspecific divergence could be caused by recent evolutionary radiations coupled with the low rates of mtDNA evolution previously observed in turtles. Complementing distance‐based barcoding with character‐based methods for identifying diagnostic sets of nucleotides provided better resolution in several cases where distance‐based methods failed to distinguish species. An online identification engine was created to provide character‐based identifications. This study constitutes the first comprehensive barcoding effort for this seriously threatened order.

DNA barcodes for globally threatened marine turtles: a registry approach to documenting biodiversity.

DNA barcoding is a global initiative that provides a standardized and efficient tool to catalogue and inventory biodiversity, with significant conservation applications. Despite progress across taxonomic realms, globally threatened marine turtles remain underrepresented in this effort. To obtain DNA barcodes of marine turtles, we sequenced a segment of the cytochrome c oxidase subunit I (COI) gene from all seven species in the Atlantic and Pacific Ocean basins (815 bp; n = 80). To further investigate intraspecific variation, we sequenced green turtles (Chelonia mydas) from nine additional Atlantic/Mediterranean nesting areas (n = 164) and from the Eastern Pacific (n = 5). We established character‐based DNA barcodes for each species using unique combinations of character states at 76 nucleotide positions. We found that no haplotypes were shared among species and the mean of interspecific variation ranged from 1.68% to 13.0%, and the mean of intraspecific variability was relatively low (0–0.90%). The Eastern Pacific green turtle sequence was identical to an Australian haplotype, suggesting that this marker is not appropriate for identifying these phenotypically distinguishable populations. Analysis of COI revealed a north–south gradient in green turtles of Western Atlantic/Mediterranean nesting areas, supporting a hypothesis of recent dispersal from near equatorial glacial refugia. DNA barcoding of marine turtles is a powerful tool for species identification and wildlife forensics, which also provides complementary data for conservation genetic research.

Molecular phylogenetics of the mud and musk turtle family Kinosternidae.

The turtle family Kinosternidae comprises 25 living species of mud and musk turtles confined to the New World. Previous attempts to reconstruct a phylogenetic history of the group have employed morphological, isozyme, and limited mitochondrial DNA sequence data, but have not been successful in producing a well-resolved phylogeny. With tissues from every recognized species and most subspecies, we sequenced three mitochondrial (cyt b, 12S, 16S) and three nuclear markers (C-mos, RAG1, RAG2). Our analyses revealed the existence of three well-resolved clades within the Kinosterninae (aged >22 mya), only two of which have been named: Sternotherus and Kinosternon. We here describe the third clade as a new genus. The evolutionary relationships among most species were well resolved, although those belonging to the K. scorpioides species group will require more extensive geographic and genetic sampling. Divergence time estimates and ancestral area reconstructions permitted the development of the first rigorous hypothesis of the zoogeographic history of the group, including support for three separate dispersals into South America, at least two of which preceded the closure of the Panamanian portal.

Screening determination of pharmaceutical pollutants in different water matrices using dual-channel capillary electrophoresis coupled with contactless conductivity detection

In this study, the employment of purpose-made dual-channel compact capillary electrophoresis (CE) instrument with capacitively coupled contactless conductivity detection (C(4)D) as a simple and inexpensive solution for screening determination of various pharmaceutical pollutants frequently occurring in surface water and hospital wastewater in Hanoi, Vietnam is reported. Five negatively charged pharmaceutically active compounds, namely ibuprofen, diclofenac, bezafibrate, ketoprofen and mefenamic acid were determined using the first channel whereas three positively charged ones, namely diphenhydramine, metoprolol and atenolol were determined with the second channel of the CE-C(4)D instrument. Two different background electrolytes (BGEs) were used in these two CE channels independently. The best detection limits achieved were in the range of 0.2-0.8mg/L without sample pre-concentration. Enrichment factors up to 200 were obtainable with the inclusion of a solid phase extraction step. Good agreement between results obtained from CE-C(4)D and those with the standard confirmation method (HPLC-DAD) was achieved, with correlation coefficients higher than 0.98.

A phylogeny of softshell turtles (Testudines: Trionychidae) with reference to the taxonomic status of the critically endangered, giant softshell turtle, Rafetus swinhoei

Several important aspects of the evolution of the softshell turtle (family Trionychidae) have not been addressed thoroughly in previous studies, including the pattern and timing of diversification of major clades and species boundaries of the critically endangered Shanghai Softshell Turtle, Rafetus swinhoei. To address these issues, we analyzed data from two mitochondrial loci (cytochrome b and ND4) and one nuclear intron (R35) for all species of trionychid turtles, except Pelochelys signifera, and for all known populations of Rafetus swinhoei in Vietnam and one from China. Phylogenetic analyses using three methods (maximum parsimony, maximum likelihood, and Bayesian inference) produce a well resolved and strongly supported phylogeny. The results of our time-calibration and biogeographic optimization analyses show that trionychid dispersals out of Asia took place between 45 and 49 million years ago in the Eocene. Interestingly, the accelerated rates of diversification and dispersal within the family correspond surprisingly well to global warming periods between the mid Paleocene and the early Oligocene and from the end of the Oligocene to the mid Miocene. Our study also indicates that there is no significant genetic divergence among monophyletic populations of Rafetus swinhoei, and that previous taxonomic revision of this species is unwarranted.

Resolving the phylogenetic history of the short-necked turtles, genera Elseya and Myuchelys (Testudines: Chelidae) from Australia and New Guinea

Phylogenetic relationships and taxonomy of the short-necked turtles of the genera Elseya, Myuchelys, and Emydura in Australia and New Guinea have long been debated as a result of conflicting hypotheses supported by different data sets and phylogenetic analyses. To resolve this contentious issue, we analyzed sequences from two mitochondrial genes (cytochrome b and ND4) and one nuclear intron gene (R35) from all species of the genera Elseya, Myuchelys, Emydura, and their relatives. Phylogenetic analyses using three methods (maximum parsimony, maximum likelihood, and Bayesian inference) produce a single, well resolved, and strongly corroborated hypothesis, which provides support for the three genera, with the exception that the genus Myuchelys is paraphyletic - Myuchelys purvisi is the sister taxon to the remaining Elseya, Myuchelys and Emydura. A new genus is proposed for the species Myuchelys purvisi to address this paraphyletic relationship. Time-calibration analysis suggests that diversification of the group in Australia coincides with periods of aridification in the late Eocene and between the mid-Miocene and early Pliocene. Other speciation events occurred during the faunal exchange between Australia and the island of New Guinea during the late Miocene and early Pliocene. Lineages distributed in New Guinea are likely influenced by the complex geologic history of the island, and include cryptic species diversity.

In-house-made capillary electrophoresis instruments coupled with contactless conductivity detection as a simple and inexpensive solution for water analysis: a case study in Vietnam

A simple and inexpensive method for the determination of various ionic species in different water matrices is discussed in this study. The approach is based on the employment of in-house-made capillary electrophoresis (CE) instruments with capacitively coupled contactless conductivity detection (C(4)D), which can be realized even when only a modest financial budget and limited expertise are available. Advantageous features and considerations of these instruments are detailed following their pilot deployment in Vietnam. Different categories of ionic species, namely major inorganic cations (K(+), Na(+), Ca(2+), Mg(2+), and NH4(+)) and major inorganic anions (Cl(-), NO3(-), NO2(-), SO4(2-), and phosphate), in different water matrices in Vietnam were determined using these in-house fabricated instruments. Inorganic trivalent arsenic (As(iii)), which is the most abundant form of arsenic in reducing groundwater, was determined by CE-C(4)D. The effect of some interfering ions in groundwater on the analytical performance was investigated and is highlighted. The results from in-house-made CE-C(4)D-instruments were cross-checked with those obtained using the standard methods (AAS, AES, UV and IC), with correlation coefficients r(2) ≥ 0.9 and deviations from the referenced results less than 15%.

First Record of the Genus Oreolalax (Anura: Megophryidae) from Vietnam with Description of a New Species

The genus Oreolalax is reported from Vietnam for the first time and a new species is described based on morphological differences, molecular divergence, and phylogenetic placement. Morphologically, the new species is distinguishable from its congeners on the basis of a combination of the following diagnostic characters: size small; tympanum hidden; toes with webbing at base; dorsum with distinct, round, spiny warts; flanks with white, spiny spots; belly and lower surface of limbs smooth, with dark marbling; interorbital region without dark triangular pattern; upper surface of thigh with dark bars; male with black spines present on margin of lower lip, spinal patches on chest small with fine spines, nuptial spines on fingers small, and without vocal sacs. In phylogenetic analyses, the new species is unambiguously nested within the genus Oreolalax.

A new genus and species of xenodermatid snake (Squamata: Caenophidia: Xenodermatidae) from northern Lao People's Democratic Republic.

A male snake collected in Louangphabang Province and a second specimen observed in Houaphan Province, North Laos, share morphological characters with the Asian genus Fimbrios Smith, 1921, including erected edges on the first supra- and infralabial scales, but differ in the following morphological characters: fewer dorsal scale rows (25-27 vs. 30-33), fewer maxillary teeth (27 vs. 30-35), posterior teeth progressively slightly enlarged, and especially the correspondence of two dorsal scale rows per ventral plate throughout the body (i.e. the first dorsal scale row made of a small scale above the fore part of a ventral, followed by a much larger scale above its hind part), a condition known only in Xenodermus Reinhardt, 1836. As the Laotian specimens differ in morphological characters from other genera and species in the family Xenodermatidae, and on the basis of molecular analyses showing a large genetic divergence from the genus Fimbrios (p-distance ≥14.7 %, mitochondrial COI gene), we place these specimens in a new genus, Parafimbrios gen. nov., and describe them as a new species, Parafimbrios lao sp. nov. Besides the characters mentioned above, the new species is diagnosed by a combination of the following ones: small, strongly keeled dorsal scales; rostral and first four supra- and infralabials with raised, erected edges; horizontal tissue ridges above the rostral; loreal single, large, elongate; ventral scales 177-189; subcaudals 55-56, undivided; dorsal colour purplish-grey, neck with a broad, very pale grey collar reaching downwards the pale grey colour of the venter. The morphological characters of the new genus are compared with those of the genera Fimbrios Smith, 1921, Xenodermus Reinhardt, 1836, Stoliczkaia Jerdon, 1870, Achalinus Peters, 1869, and Xylophis Beddome, 1878. A key to the genera Achalinus, Fimbrios and Parafimbrios gen. nov. is provided. Parafimbrios laos spec. nov. is the 111th snake species currently recorded from Laos.

Cyrtodactylus rufford , a new cave-dwelling bent-toed gecko (Squamata: Gekkonidae) from Khammouane Province, central Laos

We describe a new species of the gekkonid genus Cyrtodactylus from Khammouane Province, central Laos based on morphological and molecular data. Morphologically, Cyrtodactylus rufford sp. nov. differs from its congeners by a unique combination of the following characters: medium size, SVL reaching 72.5 mm; dorsal pattern with three or four light transverse bands between limb insertions; one intersupranasal; 14-16 irregular dorsal tubercle rows at midbody, weakly developed in the paravertebral region; 27-29 ventral scale rows between ventrolateral folds; 42-43 precloacal and femoral pores in a continuous row in males, enlarged femoral and precloacal scales present; 4 or 5 postcloacal tubercles on each side; dorsal tubercles present at base of tail; medial subcaudal scales enlarged. Molecular analyses show that the new species is closely related to C. khammouanensis, which was originally described from Khammouane Province.