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Mainland Southeast Asia, also known as Indochina, is recognized as a global biodiversity hotspot for freshwater mussels, hosting a significant number of species and exhibiting high levels of endemism. Recently, the monotypic genus Songkhlanaia was described from the Songkhla Lake Basin in southern Thailand. In this study, two additional lineages are revealed, S.moreleti comb. nov. and S.songkhramensis sp. nov. , from the Mekong Basin through an integrative taxonomic approach combining morphological characters and molecular phylogenetic analyses. The multi-locus phylogenetic inference supported the monophyly of the genus and further elucidated the sister relationship between S.moreleti and the new species, and with S.tamodienica positioned as a basal lineage. Pairwise uncorrected COI p-distances among these three species also supported the species validity and ranged from 4.2% to 8.24%. Notably, S.songkhramensis sp. nov. and S.moreleti exhibit similarities in shell morphology; however, the new species can be differentiated by more robust pseudocardinal teeth. Both species are distinguishable from S.tamodienica by their approximately twice larger size, more inflated shells, and more prominent, roughened, irregular growth lines on the shell surface. Furthermore, based on the current data, these three species are recognized as endemic and are restricted to disjunct biogeographic areas in Indochina: S.tamodienica in the Songkhla Lake Basin in southern Thailand, S.moreleti in the Tonle Sap and Lower Mekong basins, and S.songkhramensis sp. nov. in the Songkhram Basin and its nearby tributaries of the Middle Mekong Basin., Competing Interests: The authors have declared that no competing interests exist., (Ekgachai Jeratthitikul, Chirasak Sutcharit, Pongpun Prasankok.)

Stingless beekeeping, also known as meliponiculture, has gained increasing popularity in many tropical and subtropical countries for its use in commercial pollination and high-value honey and propolis production. However, this rising interest in stingless beekeeping has led to significant geographical displacements of bee colonies by beekeepers, occasionally surpassing their native ranges. Consequently, this affects local bee populations by disrupting gene flow across unnaturally large geographic scales. For Heterotrigona itama, one of the most common stingless bee species in Southeast Asian countries, including Thailand, there is concern that large-scale artificial propagation by beekeepers utilizing a limited number of bee colonies will lead to inbreeding. This practice leads to increased inbreeding within managed populations and introgression into wild populations. These concerns highlight the need for careful management practices in stingless beekeeping to mitigate potential adverse effects. To assess the genetic structure of H. itama in Thailand, 70 colonies were sampled, and partially sequenced cytochrome c oxidase subunit 1 (COI) gene, large ribosomal subunit rRNA gene (16S rRNA), and 28S large ribosomal subunit rDNA gene (28S rRNA) were analyzed. Our results showed slightly lower nuclear genetic variability, but higher mitochondrial genetic variability, which can be attributed to gene flow, colony transport, and nest division. We suggest that increasing the number of colonies maintained through nest division does not negatively affect genetic variability, as it is maintained by small-scale male dispersal and human-mediated nest transport. However, caution should be exercised when transporting nests from distant localities, considering the high genetic differentiation observed between samples from Narathiwat and those from Krabi and Nakhon Si Thammarat provinces, which might indicate local adaptation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Wongsa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

The taxonomy of the pill millipede genus Rhopalomeris Verhoeff, 1906, which is restricted to Indochina and currently comprises six described species, is refined and updated. An integrative taxonomic approach was employed that combines morphological examination with DNA barcoding using the cytochrome c oxidase subunit I (COI) gene for species identification and delineation. The first objective was to confirm the identity of Rhopalomeriscarnifex (Pocock, 1889), a charismatic species known as the "candy pill millipede" due to its vivid coloration, based on specimens collected near the type locality in Myanmar. The second objective was to describe a new species, Rhopalomerisnigroflava Likhitrakarn, sp. nov. , discovered in Linno Gu, Kayin State, Myanmar. This new species is distinguished by its small body size (5.1-9.7 mm long) and yellow body with contrasting brown to blackish markings on certain terga. In addition, the position of the telopod syncoxital lobe relative to the lateral syncoxite horns separates it from other Rhopalomeris species. The interspecific divergence between R.nigroflava Likhitrakarn, sp. nov. and other congeners ranges from 10.85% to 16.13%, based on uncorrected COI p-distances, while the intraspecific divergence was 0%-7.44%. A distribution map of and a revised identification key to all known species of Rhopalomeris are also provided., Competing Interests: The authors have declared that no competing interests exist., (Natdanai Likhitrakarn, Sergei I. Golovatch, Ruttapon Srisonchai, Parin Jirapatrasilp, Pichsinee Sapparojpattana, Ekgachai Jeratthitikul, Somsak Panha, Chirasak Sutcharit.)

The razor clam genus Novaculina is a secondary marine-derived freshwater taxa within the otherwise exclusively marine family Pharidae. Novaculina currently comprises four valid species that are distributed allopatrically across several drainages in Asia. We employed an integrated approach, combining morphology and molecular phylogenetic analyses to elucidate the taxonomic placement of members within this genus. The multi-locus phylogenetic trees based on cytochrome c oxidase subunit I (COI ), 16S rRNA and 28S rRNA gene sequences demonstrate that Novaculina is polyphyletic. Specimens identified as N. siamensis form a distinct clade that is not sister group to other currently recognised congeners. Furthermore, morphological examination reveals distinct characteristics in 'N. siamensis ', namely a fused, fringed siphon, in contrast to the separated, smooth siphons observed in other species. Based on these findings, we propose the establishment of a new genus, Cenonovaculina gen. nov., to accommodate 'N. siamensis '. The new genus is distinguished from other genera in having a short shell, deep pallial sinus, elongate, oval to bean-shaped anterior adductor scar and long fused siphons surrounded by conical tentacles. ZooBank: urn:lsid:zoobank.org:pub:6E16FC43-5BBA-4791-A805-1C84859877A3.

Macrobrachiumlanchesteri (De Man, 1911), a translucent freshwater prawn has a wide distribution range throughout mainland Southeast Asia. A high morphological variation and genetic divergence between different geographical M.lanchesteri populations in Thailand have peculiarly extended the uncertainty of species boundaries and blended confusingly with several Macrobrachium species. To clarify these circumstances, broad sample examinations of the morphological variation, including topotype specimens, and phylogenetic reconstruction based on the concatenated mitochondrial dataset (16s rRNA and COI genes) were performed. Broad morphological examination of M.lanchesteri has shown congruency with phylogenetic analyses by revealing prominent lineages of M.lanchesteri sensu stricto and two new sibling lineages with interspecific variation between 6.48-8.76% for COI and 3.06-4.23% for 16S. Descriptions of two new species, named herein as M.panhai Chaowvieng & Siriwut, sp. nov. and M.rostrolevatus Chaowvieng & Siriwut, sp. nov. are provided. Morphological investigation of rostral form suggested plasticity in M.rostrolevatus populations showing the morphological trait associated with their habitat preferences. Furthermore, phylogenetic positions of the three taxa affirmed the hidden diversity of Thai freshwater Macrobrachium fauna correlated with the river network in the Mekong and Chao Phraya basins, Thailand. The genetic data and distribution records obtained in this study may also assist future river conservation plans as well as the sustainable management of freshwater prawn diversity., Competing Interests: The authors have declared that no competing interests exist., (Apisara Chaowvieng, Chirasak Sutcharit, Ratmanee Chanabun, Ruttapon Srisonchai, Ekgachai Jeratthitikul, Warut Siriwut.)

The pill millipede genus Hyperglomeris Silvestri, 1917 is reported from Laos for the first time. Two new species, namely H.bicaudata Likhitrakarn, sp. nov. and H.inkhavilayi Likhitrakarn, sp. nov. , from Houaphanh and Khammouane provinces, northern Laos, are described and illustrated based on morphological characters and molecular analyses. Sequences of COI gene were used as DNA barcoding markers, and successfully supported the accurate identification of other Glomeridae species. Interspecific divergence of the COI uncorrected p-distance between these new species and other Hyperglomeris species ranged from 7.84-13.07%, while the intraspecific divergence was 0.45% in H.inkhavilayi sp. nov. and 5.3% in H.bicaudata sp. nov. The updated status of Hyperglomeris , a map of its distribution, and identification keys for all species are given., (Natdanai Likhitrakarn, Ruttapon Srisonchai, Warut Siriwut, Parin Jirapatrasilp, Ekgachai Jeratthitikul, Somsak Panha, Chirasak Sutcharit.)

The snorkel snail genus Rhiostoma Benson, 1860 is comprised of terrestrial cyclophorid snails with wide-ranging species diversity and radiation in Southeast Asia. The typical characters of the genus are a depressed shell, a detached and descending portion of the last whorl with a distinctive peristomal breathing device attached, and a calcareous cup-shaped operculum. Herein, we have revised the systematics of extant species based on shell morphology combined with COI barcoding. From these thirty recognised species, twelve are described as new to science: R. ? amarapuraense sp. nov. , R.anceyi sp. nov. , R.breviocollar sp. nov. , R.ebenozostera sp. nov. , R.cheliopegma sp. nov. , R.furfurosum sp. nov. , R.gnomus , sp. nov. , R.lannaense sp. nov. , R.laoense sp. nov. , R.platymorpha sp. nov. , R.rhothonotaphrosa sp. nov. , and R.tigrina sp. nov. All conchological characters are provided via illustrations of type specimens and living snails, and descriptions of the shells and radulae. Phylogenetic analysis based on partial COI gene sequences strongly supports the designated morphospecies and a monophyletic Rhiostoma , confirming that all pterocyclinid snails with a calcareous, cup-shaped operculum belong to the same clade. A high intra-specific divergence was observed in R.jalorensis and R.housei populations from locations in close proximity, suggesting a lower dispersal and higher level of isolation. The low inter-specific divergence found in R.hainesi , R.samuiense , R.asiphon , and R.rhothonotaphrosa sp. nov. supports their recent diversification and local adaptation, and is congruent with their marked morphological differences. Finally, nine formerly Rhiostoma -placed species were reclassified into either the genus Cyclotus or the genus Opisthoporus ., (Piyoros Tongkerd, Sakboworn Tumpeesuwan, Khamla Inkhavilay, Pongpun Prasankok, Ekgachai Jeratthitikul, Somsak Panha, Chirasak Sutcharit.)

Leeches in the genus Hirudinaria Whitman, 1886, also known as buffalo leeches, are blood-sucking ectoparasites of vertebrates. Although they are widely distributed in Asia and had been highly abundant in the past, studies on diversity and taxonomy of this genus are still scarce. There is probably a large amount of cryptic diversity yet to be discovered, particularly from mainland Southeast Asia. In this study, we used morphology and DNA barcoding with a COI gene fragment to explored the diversity of Hirudinaria leeches in the southern region of Thailand, where a unique geographic feature could have led to the diversification of freshwater biota. Molecular phylogenetic analyses and species delimitation approaches (ABGD, bPTP, GMYC, and BOLD) revealed the presence of four putative species of Hirudinaria leeches from southern Thailand, including H. bpling , H. thailandica , and two morphologically cryptic lineages of H. manillensis . Compared to other leech genera, genetic distances of Hirudinaria leeches were relatively low (0.11-0.65% within species; 3.72-14.36% between species) and barcoding gaps were very narrow (1.54-2.88%). The species diversity, distribution pattern, and a phenomenon of low genetic divergence of Hirudinaria leeches in southern Thailand could be explained by an ancient seaway, paleo-drainage, and anthropogenic activities.

The body of knowledge regarding the classification and evolution of freshwater mussels in the family Unionidae (Bivalvia) in Indochina has recently increased. However, the taxonomic revision of all extant taxa in the region is still ongoing. In this study, the genus Pilsbryoconcha was revised based on an integrative analysis of shell morphology, biogeography, and molecular data. Multi-locus phylogeny indicated the availability of eight species within the genus. Four previously recognized species are P. exilis (Lea, 1838), P. schomburgki (Martens, 1860) stat. rev., P. linguaeformis (Morelet, 1875), and P. carinifera (Conrad, 1837), while four other species are described herein as P. acuta sp. nov., P. mekongiana sp. nov., P. kittitati sp. nov., and P. hoikaab sp. nov. In addition, the neotype of P. carinifera is also designated to clarify its long taxonomic ambiguity. Divergent time estimation and historical biogeography analysis revealed that Pilsbryoconcha originated in the area now called the Khorat Plateau around the middle of the Eocene (mean age = 43.12 Mya), before its range was expanded across Indochina through a series of complex geomorphological changes of river systems, which also led to diversification of the genus., (© 2022. The Author(s).)

Natural hybridization has played various roles in the evolutionary history of primates. Its consequences range from genetic introgression between taxa, formation of hybrid zones, and formation of new lineages. Hylobates lar, the white-handed gibbon, and Hylobates pileatus, the pileated gibbon, are largely allopatric species in Southeast Asia with a narrow contact zone in Khao Yai National Park, Thailand, which contains both parental types and hybrids. Hybrid individuals in the zone are recognizable by their intermediate pelage and vocal patterns, but have not been analyzed genetically. We analyzed mitochondrial and microsatellite DNA of 52 individuals to estimate the relative genetic contributions of the parental species to each individual, and the amount of introgression into the parental species. We obtained fecal samples from 33 H. lar, 15 H. pileatus and four phenotypically intermediate individuals in the contact zone. Both mitochondrial and microsatellite markers confirmed distinct differences between these taxa. Both H. lar and H. pileatus contributed to the maternal lineages of the hybrids based on mitochondrial analysis; hybrids were viable and present in socially normal reproductive pairs. The microsatellite analysis identified ten admixed individuals, four F1 hybrids, which corresponded to phenotypic hybrids, and six H. lar-like backcrosses. All 15 H. pileatus samples were identified as originating from genetically H. pileatus individuals with no H. lar admixture; hence, backcrossing is biased toward H. lar. A relatively low number of phenotypic hybrids and backcrossed individuals along with a high number of parental types indicates a bimodal hybrid zone, which suggests relatively strong bias in mate selection between the species., (© 2021. Japan Monkey Centre.)

The diversity of Indochinese prawns in genus Macrobrachium is enormous due to the habitat diversification and broad tributary networks of two river basins: the Chao Phraya and the Mekong. Despite long-standing interest in SE-Asian decapod diversity, the subregional Macrobrachium fauna is still not yet comprehensively clarified in terms of taxonomic identification or genetic diversification. In this study, integrative taxonomic approaches including morphological examination, DNA barcoding, and molecular species delimitation were used to emphasize the broad scale systematics of Macrobrachium prawns in Indochina. Twenty-seven nominal species were successfully re-verified by traditional and molecular taxonomy. Barcode gap analysis supported broad overlapping of species boundaries. Taxonomic ambiguity of several deposited samples in the public database is related to inter- and intraspecific genetic divergence as indicated by BOLD discordance. Diagnostic nucleotide positions were found in six Macrobrachium species. Eighteen additional putative lineages are herein assigned using the consensus of species delimitation methods. Genetic divergence indicates the possible existence of cryptic species in four morphologically complex and wide-ranging species: M. lanchesteri, M. niphanae, M. sintangense, and some members of the M. pilimanus group. The geographical distribution of some species supports the connections and barriers attributed to paleo-historical events of SE-Asian rivers and land masses. Results of this study show explicitly the importance of freshwater ecosystems in Indochinese subregions, especially for the Mekong River Basin due to its high genetic diversity and species composition found throughout its tributaries., Competing Interests: The authors have declared that no competing interests exist.

Spiny-backed orb-weaving spiders of the subfamily Gasteracanthinae are broadly distributed in the Old World. Despite their use as a model species in biology, evolution, and behavior because of their extraordinary characteristics, the systematics of this group of spiders are still poorly understood. This study elucidates the systematics of Gasteracanthinae in Thailand based on morphological and molecular-based analyses. In total, seven species from three genera, namely Gasteracantha , Macracantha , and Thelacantha , were recorded in Thailand. Shape of abdominal spines, pattern of sigilla, and female genitalia are significant characters for species identification. In contrast, coloration shows highly intraspecific variation in most species within Gasteracanthinae. A phylogenetic tree based on partial sequences of COI, 16S, and H3 genes recovered Gasteracanthinae as a monophyletic group and supports the existence of three clades. Gasteracantha hasselti is placed as a sister taxon to Macracantha arcuata . Hence, we propose to transfer G. hasselti to Macracantha. Moreover, molecular species delimitation analyses (ABGD, bPTP, and GMYC) using 675 bp of COI gene support all nominal species, with evidence of possible additional cryptic species., (Kongkit Macharoenboon, Warut Siriwut, Ekgachai Jeratthitikul.)

Specific status and species boundaries of several freshwater prawns in the Macrobrachium pilimanus species group remain ambiguous, despite the taxonomic re-description of type materials and additional specimens collected to expand the boundaries of some species. In this study, the " pilimanus " species group of Macrobrachium sensu Johnson (1958) was studied using specimens collected from montane streams of Thailand. Molecular phylogenetic analyses based on sequences of three molecular markers (COI, 16S and 18S rRNA) were performed. The phylogenetic results agreed with morphological identifications, and indicated the presence of at least nine putative taxa. Of these, six morphospecies were recognised as M. malayanum , M. forcipatum , M. dienbienphuense , M. hirsutimanus , M. eriocheirum , and M. sirindhorn . Furthermore, three morphologically and genetically distinct linages were detected, and are described herein as M. naiyanetri Siriwut sp. nov. , M. palmopilosum Siriwut sp. nov. and M. puberimanus Siriwut sp. nov. The taxonomic comparison indicated wide morphological variation in several species and suggested additional diagnostic characters that are suitable for use in species diagnoses, such as the shape and orientation of fingers, the rostrum form, and the presence or absence of velvet pubescence hairs and tuberculated spinulation on each telopodite of the second pereiopods. The " pilimanus " species group was portrayed as non-monophyletic in both ML and BI analyses. The genetic structure of different geographical populations in Thailand was detected in some widespread species. The species delimitation based on the four delimitation methods (BIN, ABGD, PTP and GMYC) suggested high genetic diversity of the " pilimanus " species group and placed the candidate members much higher than in previous designations based on traditional morphology. This finding suggests that further investigation of morphological and genetic diversity of Southeast Asian freshwater prawns in the genus Macrobrachium is still required to provide a comprehensive species list to guide efforts in conservation and resource management., Competing Interests: The authors declare there are no competing interests., (©2020 Siriwut et al.)

The micropolydesmoid millipede family Haplodesmidae is here recorded from Cambodia for the first time through the discovery of the first, new species of the genus Eutrichodesmus Silvestri, 1910: E. cambodiensis sp. nov. This new species is described from two limestone habitats in Kampot Province, based on abundant material. It is easily distinguished from all related congeners by the following combination of characters: body greyish-brown; limbus roundly lobulate; solenomere partially divided from acropodite by a digitiform lobe, but without hairpad. Brief remarks on the previously-proposed " pecularis -group" are provided and a second group, the " demangei -group", is established and discussed on the basis of morphological evidence, updating the number of recognised species groups of Eutrichodesmus to two. Detailed morphological illustrations, photographs and a distribution map, as well as remarks on its habitat and mating behaviour of the new species are presented. Furthermore, the current distributions of all 55 presently-known species of Eutrichodesmus are provided and a key to all 23 species that occur in mainland Southeast Asia is given., (Ruttapon Srisonchai, Natdanai Likhitrakarn, Chirasak Sutcharit, Ekgachai Jeratthitikul, Warut Siriwut, Phanara Thrach, Samol Chhuoy, Peng Bun Ngor, Somsak Panha.)

The Tonle Sap Lake in Cambodia is a crucial freshwater biodiversity hotspot and supports one of the world's largest inland fisheries. Within the Tonle Sap basin, freshwater molluscs provide vital ecosystem services and are among the fauna targetted for commercial harvesting. Despite their importance, freshwater molluscs of the Tonle Sap basin remain poorly studied. The historical literature was reviewed and at least 153 species of freshwater molluscs have been previously recorded from throughout Cambodia, including 33 from the Tonle Sap basin. Surveys of the Tonle Sap Lake and surrounding watershed were also conducted and found 31 species, 15 bivalves (five families) and 16 gastropods (eight families), in the Tonle Sap basin, including three new records for Cambodia ( Scaphula minuta , Novaculina siamensis , Wattebledia siamensis ), the presence of globally invasive Pomacea maculata and potential pest species like Limnoperna fortunei . This study represents the most comprehensive documentation of freshwater molluscs of the Tonle Sap basin, and voucher specimens deposited at the Inland Fisheries Research and Development Institute, Cambodia, represent the first known reference collection of freshwater molluscs in the country. In order to combat the combined anthropogenic pressures, including invasive species, climate change and dams along the Mekong River, a multi-pronged approach is urgently required to study the biodiversity, ecology, ecosystem functioning of freshwater molluscs and other aquatic fauna in the Tonle Sap basin., (Ting Hui Ng, Ekgachai Jeratthitikul, Chirasak Sutcharit, Samol Chhuoy, Kakada Pin, Arthit Pholyotha, Warut Siriwut, Ruttapon Srisonchai, Zeb S. Hogan, Peng Bun Ngor.)

Prior to this study, few collections and records were made of the land snails in Cambodia and the historical taxa had never been reviewed. Herein a report on the land snail diversity based on specimens collected recently from karstic and non-karstic areas in southern Cambodia is provided. This checklist presents 36 species of land snails (two Neritimorpha, six Caenogastropoda, and 28 Heterobranchia). Illustrations and brief taxonomic notes/remarks are provided for every species. We also described Georrisa carinata Sutcharit & Jirapatrasilp, sp. nov. based on some distinct shell morphological characters. Since the first descriptions during the colonial period in the nineteenth century, some land snail species (e.g., Trichochloritis norodomiana , Durgella russeola , Anceyoconcha siamensis obesula comb. nov. , Anceyoconcha chaudoensis comb. nov ., and Succinea tenuis ) have not been reported subsequently. This probably reflects a lack of knowledge concerning land snail biodiversity in this country. To our knowledge, this is the first comprehensive survey of land snails in southern Cambodia. A need for more field research and systematic revision of the land snails in this interesting region is also highlighted and demonstrated., (Chirasak Sutcharit, Phanara Thach, Samol Chhuoy, Peng Bun Ngor, Ekgachai Jeratthitikul, Warut Siriwut, Ruttapon Srisonchai, Ting Hui Ng, Arthit Pholyotha, Parin Jirapatrasilp, Somsak Panha.)

Hirudinaria manillensis (Lesson, 1842), commonly known as the buffalo leech, shows a polymorphism of two ventral colorations. The green color morph has a plain green ventral surface and the red color morph has a brick-red ventral surface with two black submarginal stripes. Based on molecular and morphological evidence in the present study, these two color morphs were revealed as two different species. The red color morph fits well with the description of H. manillensis , while the green color morph showed some distinctions, and therefore is described herein as Hirudinaria thailandica Jeratthitikul & Panha, sp. nov. The new species can be distinguished from its congeners by the dark greenish or dark olive ventral surface and a round atrium with ventral insertion of ejaculatory ducts in the male reproductive organ. A phylogenetic tree based on concatenated data of COI and 28S genes supported the new species and further indicated it as a sister species to H. bpling Phillips, 2012., (Ekgachai Jeratthitikul, Putita Jiranuntskul, Takafumi Nakano, Chirasak Sutcharit, Somsak Panha.)

A new, giant species of Trachyjulus from a cave in southern Thailand is described, illustrated, and compared to morphologically closely related taxa. This new species, T. magnus sp. nov. , is much larger than all other congeners and looks especially similar to the grossly sympatric T. unciger Golovatch, Geoffroy, Mauriès & VandenSpiegel, 2012, which is widespread in southern Thailand. Phylogenetic trees, both rooted and unrooted, based on a concatenated dataset of the COI and 28S genes of nine species of Cambalopsidae ( Trachyjulus , Glyphiulus , and Plusioglyphiulus ), strongly support the monophyly of Trachyjulus and a clear-cut divergence between T. magnus sp. nov. and T. unciger in revealing very high average p -distances of the COI gene (20.80-23.62%)., (Natdanai Likhitrakarn, Sergei I. Golovatch, Ekgachai Jeratthitikul, Ruttapon Srisonchai, Chirasak Sutcharit, Somsak Panha.)

The Asian rice gall midge (RGM) Orseolia oryzae (Wood Mason) (Diptera: Cecidomyiidae) is a major pest of rice, leading to yield losses in Thailand and many Asian countries. Despite an increasing number of reported midge outbreaks and the presence of many susceptible rice varieties, only a few studies have focused on the genetic variation of the midges. Therefore, we analyzed the phylogeography among Thai RGM populations covering north, northeast and central Thailand. Two mitochondrial DNA genes, cytochrome C oxidase I (COI) and 12S, and a non-coding repeat region (RR) situated just before COI were amplified. Overall, the haplotype diversity for COI and 12S genes of the Thai population was high, but the nucleotide diversity was quite low. Altogether, the phylogenetic tree and pairwise F st values indicated that Thai RGM populations recently expanded and were homogeneously distributed throughout the country, except for some populations in the north, which most likely became recently isolated from the main population. Two non-coding repeat motifs, that were recently observed in the mitogenome of RGM in India, were absent in Thai populations and replaced by an 89 bp non-coding sequence. Tandem nucleotide repeats of the sequence TA were also observed. The repeat copy number varied from 2 to 11 and was not correlated with geographical repartition of the midge. Finally, COI barcoding divergence between Indian and Thai populations was high (6.3% in average), giving insights into the potential existence of an RGM species complex in Asia.

In total, 435 specimens of the Southeast Asian freshwater leech species within the Hirudinidae family were collected from 17 locations of various types of aquatic habitats in northeastern Thailand. They were all morphologically placed within the genus Hirudinaria Whitman, 1886 and there were three distinct species: the common Hirudinariamanillensis, 78.2% of all collected specimens and at all 17 locations, Hirudinariajavanica at 20.3% of collected samples and from five locations and a rarer unidentified morphospecies (Hirudinaria sp.) with six samples from only two locations. The karyotypes of these three species were examined across their range in this study area for 38, 11 and 6 adult specimens of Hirudinariamanillensis, Hirudinariajavanica and Hirudinaria sp., respectively. This revealed different chromosome numbers among all three species, with Hirudinariajavanica having n = 13, 2n = 26, Hirudinariamanillensis lacked one small chromosome pair with n = 12, 2n = 24, and the unknown Hirudinaria sp. differed from any known Hirudinaria karyotypes in exhibiting a higher chromosome number (n = 14, 2n = 28) and a gradual change in size from large to small chromosomes. This suggests that the unknown Hirudinaria sp. is a new biological species. However, phylogenetic analysis based upon a 658 bp fragment of the cytochrome oxidase subunit I gene placed this unknown morphospecies within the Hirudinariamanillensis clade, perhaps then suggesting a recent sympatric speciation, although this requires further confirmation. Regardless, the chromosomes of all three species were asymmetric, most with telocentric elements. A distinct bi-armed chromosome marker was present on the first chromosome pair in Hirudinariajavanica, whilst it was on pairs 1, 2, 3 and 5 in Hirudinariamanillensis, and on pairs 3 and 5 for the unknown Hirudinaria sp.

A high degree of intraspecific variation, both genetic and in shell morphology, of the operculate land snail Cyclophorus fulguratus (Pfeiffer, 1854) suggests that its classification as a single species warrants reconsideration. We sequenced two nuclear (18S and 28S) and two mitochondrial (16S and COI) genes of 46 C. fulguratus specimens and used them to estimate the phylogeny and to determine the validity of species boundaries. Molecular phylogenetic analyses revealed the presence of three lineages corresponding to three geographically disjunctive populations of C. fulguratus in Thailand. Likelihood tests of topologies significantly supported the non-monophyly of the C. fulguratus-complex and Bayesian species delimitation analysis significantly supported the potential representation as distinct species of these three lineages. Discriminant function analysis based on geometric-morphometrics of shell shape allowed for significant distinction of these three candidate species, although they revealed a considerable degree of overlap of shell shape reflecting their crypsis morphologically. The diagnostic characters are provided by color pattern, pattern of protoconch and pattern of jaw. In conclusion, the results support that the C. fulguratus s.l., as currently recognized, consists of three distinct species in Thailand: C. fulguratus s.s., C. rangunensis and C. abditus sp.nov., which are described herein.

The widespread lycaenid butterfly Tongeia fischeri is distributed from eastern Europe to northeastern Asia and represented by three geographically isolated populations in Japan. In order to clarify the phylogeographic history of the species, we used sequences of three mitochondrial (COI, Cyt b and ND5) and two nuclear (Rpl5 and Ldh) genes of 207 individuals collected from 55 sites throughout Japan and five sites on the Asian continent. Phylogenetic trees and the median-joining network revealed six evolutionary mitochondrial haplotype clades, which corresponded to the geographic distribution of the species. Common ancestors of Japanese T. fischeri might have come to Japan during the mid-Pleistocene by multiple dispersals of continental populations, probably via a land bridge or narrow channel between western Japan and the Korean Peninsula. The geographical patterns of variation of mitochondrial and nuclear markers are discordant in northeastern Kyushu, possibly as a result of introgressive hybridization during the ancient contact between the Kyushu and Shikoku populations in the last glacial maximum. The phylogeographic pattern of T. fischeri in Japan are probably related to the geological history, Pleistocene climatic oscillations and distribution of the host plant., (Copyright © 2012 Elsevier Inc. All rights reserved.)