Bluetongue virus (BTV) is the 'type' species of the genus Orbivirus within the family Reoviridae. The BTV genome is composed of ten linear segments of double-stranded RNA (dsRNA), each of which codes for one of ten distinct viral proteins. Previous phylogenetic comparisons have evaluated variations in genome segment 3 (Seg-3) nucleotide sequence as way to identify the geographical origin (different topotypes) of BTV isolates. The full-length nucleotide sequence of genome Seg-3 was determined for thirty BTV isolates recovered in the eastern Mediterranean region, the Balkans and other geographic areas (Spain, India, Malaysia and Africa). These data were compared, based on molecular variability, positive-selection-analysis and maximum-likelihood phylogenetic reconstructions (using appropriate substitution models) to 24 previously published sequences, revealing their evolutionary relationships. These analyses indicate that negative selection is a major force in the evolution of BTV, restricting nucleotide variability, reducing the evolutionary rate of Seg-3 and potentially of other regions of the BTV genome. Phylogenetic analysis of the BTV-4 strains isolated over a relatively long time interval (1979-2000), in a single geographic area (Greece), showed a low level of nucleotide diversity, indicating that the virus can circulate almost unchanged for many years. These analyses also show that the recent incursions into south-eastern Europe were caused by BTV strains belonging to two different major-lineages: representing an 'eastern' (BTV-9, -16 and -1) and a 'western' (BTV-4) group/topotype. Epidemiological and phylogenetic analyses indicate that these viruses originated from a geographic area to the east and southeast of Greece (including Cyprus and the Middle East), which appears to represent an important ecological niche for the virus that is likely to represent a continuing source of future BTV incursions into Europe.
Leucocytozoon coracinae sp. nov. is described from the avian family Campephagidae and Hepatozoon apodis sp. nov. from the Apodidae. The distribution of these parasites within their respective families is discussed.
In order to elucidate the taxonomic status of the Fejervarya limnocharis complex relative to Malaysia and Japan populations, morphological observations and molecular phylogenetic analysis were carried out using three populations from Indonesia (type locality), Malaysia, and Japan. In addition, we conducted histological and spermatogenic observations using hybrids among these populations. Principal component and cluster analyses demonstrated that these populations could be clearly separated from one another. Abnormal testes were found in the hybrids between the Japan and Indonesia populations and between the Japan and Malaysia populations, but testes of the controls and hybrids between the Malaysia and Indonesia populations were quite normal. The mean number of univalents per cell was 5.42, 4.58, and 0.20 in hybrids between the Indonesia and Japan populations, Malaysia and Japan populations, and Indonesia and Malaysia populations, respectively. Sequence divergences in 16S rRNA and Cyt b genes were 0-0.4% (xbar=0.2%) and 0.3-1.5% (xbar=1.0%), respectively, between the Malaysia and Indonesia populations, and 2.4-2.6% (xbar=2.5%) and 11.0-12.0% (xbar=11.5%) between the Japan population and F. limnocharis complex, including the Malaysia and Indonesia populations and F. multistriata from China. This study indicated that the Malaysia population and F. multistriata from China should be designated as a subspecies of topotypic F. limnocharis, and that the Japan population should be regarded as a distinct species.
Anopheles sundaicus s.l. is a malaria vector in coastal areas of Southeast Asia. Previous studies showed at least four distinct species within the complex. The present study investigated the phylogeography and the status of A. sundaicus s.l. populations from Cambodia, Thailand, Malaysia and Indonesia with regard to A. sundaicus s.s. from Sarawak, Malaysian Borneo and A. epiroticus in Vietnam and Thailand. Three lineages recovered by analyses of Cyt-b and COI (mtDNA) confirmed the presence of A. sundaicus s.s. in Malaysian Borneo, the distribution of A. epiroticus from southern Vietnam to peninsular Malaysia, and recognised a distinct form in Indonesia that is named A. sundaicus E. The phylogenetic and demographic analyses suggest that the three species were separated during the Early Pleistocene (1.8-0.78 Myr) and experienced bottlenecks followed by a genetic expansion in more recent times. Based on the results and knowledge of the biogeography of the area, we hypothesise that the combination of cyclical island and refugium creation was the cause of lineage isolation and bottleneck events during the Pleistocene.
Two forms of Staurois that are differentiated by body size occur parapatrically in the Crocker Range, Sabah, Borneo. Analyses of a total of 1,499 bp of the mitochondrial cytochrome b, 12S rRNA, and 16S rRNA genes revealed that the two forms could be completely split genetically. The two forms could be also clearly differentiated morphologically, not only by snout-vent length but also by the relative sizes of snout, eye, and finger disk. Comparisons of the two forms with all known species of the genus revealed the large and small forms to be S. tuberilinguis and S. parvus, respectively. The latter species has long been synonymized with the former, but we here consider them to represent different species.
Coptotermes gestroi, the Asian subterranean termite (AST), is an economically important structural and agricultural pest that has become established in many areas of the world. For the first time, phylogeography was used to illuminate the origins of new found C. gestroi in the US Commonwealth of Puerto Rico; Ohio, USA; Florida, USA; and Brisbane, Australia. Phylogenetic relationships of C. gestroi collected in indigenous locations within Malaysia, Thailand, and Singapore as well as from the four areas of introduction were investigated using three genes (16S rRNA, COII, and ITS) under three optimality criteria encompassing phenetic and cladistic assumptions (maximum parsimony, maximum likelihood, and neighbor-joining). All three genes showed consistent support for a close genetic relationship between C. gestroi samples from Singapore and Ohio, whereas termite samples from Australia, Puerto Rico, and Key West, FL were more closely related to those from Malaysia. Shipping records further substantiated that Singapore and Malaysia were the likely origin of the Ohio and Australia C. gestroi, respectively. These data provide support for using phylogeography to understand the dispersal history of exotic termites. Serendipitously, we also gained insights into concerted evolution in an ITS cluster from rhinotermitid species in two genera.
The extent to which response to environmental change is mediated by species-specific ecology is an important aspect of the population histories of tropical taxa. During the Pleistocene glacial cycles and associated sea level fluctuations, the Sunda region in Southeast Asia experienced concurrent changes in landmass area and the ratio of forest to open habitat, providing an ideal setting to test the expectation that habitat associations played an important role in determining species' response to the opportunity for geographic expansion. We used mitochondrial control region sequences and six microsatellite loci to compare the phylogeographic structure and demographic histories of four broadly sympatric species of Old World fruit bats in the genus, Cynopterus. Two forest-associated species and two open-habitat generalists were sampled along a latitudinal transect in Singapore, peninsular Malaysia, and southern Thailand. Contrary to expectations based on habitat associations, the geographic scale of population structure was not concordant across ecologically similar species. We found evidence for long and relatively stable demographic history in one forest and one open-habitat species, and inferred non-coincident demographic expansions in the second forest and open-habitat species. Thus, while these results indicate that Pleistocene climate change did not have a single effect on population structure across species, a correlation between habitat association and response to environmental change was supported in only two of four species. We conclude that interactions between multiple factors, including historical and contemporary environmental change, species-specific ecology and interspecific interactions, have shaped the recent evolutionary histories of Cynopterus fruit bats in Southeast Asia.
Disjunctive distributions across paleotropical regions in the Indian Ocean Basin (IOB) often invoke dispersal/vicariance debates. Exacum (Gentianaceae, tribe Exaceae) species are spread around the IOB, in Africa, Madagascar, Socotra, the Arabian peninsula, Sri Lanka, India, the Himalayas, mainland Southeast Asia including southern China and Malaysia, and northern Australia. The distribution of this genus was suggested to be a typical example of vicariance resulting from the breakup of the Gondwanan supercontinent. The molecular phylogeny of Exacum is in principle congruent with morphological conclusions and shows a pattern that resembles a vicariance scenario with rapid divergence among lineages, but our molecular dating analysis demonstrates that the radiation is too recent to be associated with the Gondwanan continental breakup. We used our dating analysis to test the results of DIVA and found that the program predicted impossible vicariance events. Ancestral area reconstruction suggests that Exacum originated in Madagascar, and divergence dating suggests its origin was not before the Eocene. The Madagascan progenitor, the most recent common ancestor of Exacum, colonized Sri Lanka and southern India via long-distance dispersals. This colonizer underwent an extensive range expansion and spread to Socotra-Arabia, northern India, and mainland Southeast Asia in the northern IOB when it was warm and humid in these regions. This widespread common ancestor retreated subsequently from most parts of these regions and survived in isolation in Socotra-Arabia, southern India-Sri Lanka, and perhaps mainland Southeast Asia, possibly as a consequence of drastic climatic changes, particularly the spreading drought during the Neogene. Secondary diversification from these surviving centers and Madagascar resulted in the extant main lineages of the genus. The vicariance-like pattern shown by the phylogeny appears to have resulted from long-distance dispersals followed by extensive range expansion and subsequent fragmentation. The extant African species E. oldenlandioides is confirmed to be recently dispersed from Madagascar.
Tropical mountains are hot spots of biodiversity and endemism, but the evolutionary origins of their unique biotas are poorly understood. In varying degrees, local and regional extinction, long-distance colonization, and local recruitment may all contribute to the exceptional character of these communities. Also, it is debated whether mountain endemics mostly originate from local lowland taxa, or from lineages that reach the mountain by long-range dispersal from cool localities elsewhere. Here we investigate the evolutionary routes to endemism by sampling an entire tropical mountain biota on the 4,095-metre-high Mount Kinabalu in Sabah, East Malaysia. We discover that most of its unique biodiversity is younger than the mountain itself (6 million years), and comprises a mix of immigrant pre-adapted lineages and descendants from local lowland ancestors, although substantial shifts from lower to higher vegetation zones in this latter group were rare. These insights could improve forecasts of the likelihood of extinction and 'evolutionary rescue' in montane biodiversity hot spots under climate change scenarios.
In many parts of Southeast Asia, the HIV-1 epidemic has been driven by the sharing of needles and equipment among intravenous drug users (IDUs). Over the last few decades, many studies have proven time and again that the diversity of HIV-1 epidemics can often be linked to the route of infection transmission. That said, the diversity and complexity of HIV-1 molecular epidemics in the region have been increasing at an alarming rate, due in part to the high tendency of the viral RNA to recombine. This scenario was exemplified by the discovery of numerous circulating recombinant forms (CRFs), especially in Thailand and Malaysia. In this study, we characterized a novel CRF designated CRF74_01B, which was identified in six epidemiologically unlinked IDUs in Kuala Lumpur, Malaysia. The near-full length genomes were composed of CRF01_AE and subtype B', with eight breakpoints dispersed in the gag-pol and nef regions. Remarkably, this CRF shared four and two recombination hotspots with the previously described CRF33_01B and the less prevalent CRF53_01B, respectively. Genealogy-based Bayesian phylogenetic analysis of CRF74_01B genomic regions showed that it is closely related to both CRF33_01B and CRF53_01B. This observation suggests that CRF74_01B was probably a direct descendent from specific lineages of CRF33_01B, CRF53_01B and subtype B' that could have emerged in the mid-1990s. Additionally, it illustrated the active recombination processes between prevalent HIV-1 subtypes and recombinants in Malaysia. In summary, we report a novel HIV-1 genotype designated CRF74_01B among IDUs in Kuala Lumpur, Malaysia. The characterization of the novel CRF74_01B is of considerable significance towards the understanding of the genetic diversity and population dynamics of HIV-1 circulating in the region.
Brucellosis is an important zoonotic disease that affects both humans and animals. We sequenced the full genome and characterised the genetic diversity of two Brucella melitensis isolates from Malaysia and the Philippines. In addition, we performed a comparative whole-genome single nucleotide polymorphism (SNP) analysis of B. melitensis strains collected from around the world, to investigate the potential origin and the history of the global spread of B. melitensis.
The genus Myotis includes the largest number of species in the family Vespertilionidae (Chiroptera), and its members are distributed throughout most of the world. To re-evaluate the phylogenetic position of East Asian Myotis species with respect to Myotis species worldwide, we analyzed mitochondrial gene sequences of NADH dehydrogenase subunit 1 and cytochrome b from 24 East Asian individuals as well as 42 vespertilionid bats determined previously. The results suggest that: (1) some individuals having the same species name in Europe and Japan do not form a monophyletic clade, indicating that some bat species exhibit morphological convergence, (2) Japanese Myotis mystacinus forms a sister relationship with Myotis brandtii (Palaearctic), and both species are included in the American clade implying that an ancestor of these species originated in North America, and (3) the Black whiskered bat, Myotis pruinosus, is endemic to Japan and forms sister relationships with Myotis yanbarensis and Myotis montivagus collected from Okinawa (Japan) and Selangor (Malaysia), respectively, implying that M. pruinosus originated from the south. The systematics of Japanese and East Asian Myotis bats were revisited by considering their phylogenetic relationships. Our study provides the first extensive phylogenetic hypothesis of the genus Myotis that includes East Asian and Japanese species.
To elucidate the evolution of one of the most species-rich ant-plant symbiotic systems, the association between Crematogaster (Myrmicinae) and Macaranga (Euphorbiaceae) in South-East Asia, we conducted a phylogenetic analysis of the ant partners. For the phylogenetic analysis partial mitochondrial cytochrome oxidase I and II were sequenced and Maximum Parsimony analysis was performed. The analyzed Crematogaster of the subgenus Decacrema fell into three distinct clades which are also characterized by specific morphological and ecological traits (queen morphology, host-plants, and colony structure). Our results supported the validity of our currently used morphospecies concept for Peninsula Malaysia. However, on a wider geographic range (including North and North-East Borneo) some morphospecies turned out to be species complexes with genetically quite distinct taxa. Our phylogenetic analysis and host association studies do not indicate strict cocladogenesis between the subgenus Decacrema and their Macaranga host-plants because multiple ant taxa occur on quite distinct host-plants belonging to different clades within in the genus Macaranga. These results support the view that host-shifting or host-expansion is common in the ants colonizing Macaranga. Additionally, the considerable geographic substructuring found in the phylogenetic trees of the ants suggests that allopatric speciation has also played a role in the diversification and the current distribution of the Decacrema ants.
Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.
Populations of the Asian elephant (Elephas maximus) have been reduced in size and become highly fragmented during the past 3,000 to 4,000 years. Historical records reveal elephant dispersal by humans via trade and war. How have these anthropogenic impacts affected genetic variation and structure of Asian elephant populations? We sequenced mitochondrial DNA (mtDNA) to assay genetic variation and phylogeography across much of the Asian elephant's range. Initially we compare cytochrome b sequences (cyt b) between nine Asian and five African elephants and use the fossil-based age of their separation (approximately 5 million years ago) to obtain a rate of about 0.013 (95% CI = 0.011-0.018) corrected sequence divergence per million years. We also assess variation in part of the mtDNA control region (CR) and adjacent tRNA genes in 57 Asian elephants from seven countries (Sri Lanka, India, Nepal, Myanmar, Thailand, Malaysia, and Indonesia). Asian elephants have typical levels of mtDNA variation, and coalescence analyses suggest their populations were growing in the late Pleistocene. Reconstructed phylogenies reveal two major clades (A and B) differing on average by HKY85/gamma-corrected distances of 0.020 for cyt b and 0.050 for the CR segment (corresponding to a coalescence time based on our cyt b rate of approximately 1.2 million years). Individuals of both major clades exist in all locations but Indonesia and Malaysia. Most elephants from Malaysia and all from Indonesia are in well-supported, basal clades within clade A. thus supporting their status as evolutionarily significant units (ESUs). The proportion of clade A individuals decreases to the north, which could result from retention and subsequent loss of ancient lineages in long-term stable populations or, perhaps more likely, via recent mixing of two expanding populations that were isolated in the mid-Pleistocene. The distribution of clade A individuals appears to have been impacted by human trade in elephants among Myanmar, Sri Lanka, and India, and the subspecies and ESU statuses of Sri Lankan elephants are not supported by molecular data.
The Japanese encephalitis (JE) serocomplex of flaviviruses comprises 10 members, 9 of which: Alfuy (ALF); Koutango (KOU); Kokobera (KOK); Kunjin (KUN); Murray Valley encephalitis (MVE); JE; Stratford (STR); Usutu (USU); and West Nile (WN) have been isolated from Africa, southern Europe, Middle East, Asia, and Australia. The tenth member, St. Louis encephalitis (SLE) virus, is confined to North, Central, and South America. For ALF, KOK, KOU, STR, and USU, no sequence data have as yet been reported, and little molecular phylogeny has been determined for this complex as a whole. Using a rapid, one-step RT-PCR and universal primers, we have amplified and sequenced a 450-600 base pair region of the virus genome encompassing the N terminus of the nonstructural protein NS5 and the 5' end of the 3' noncoding region, for several strains of all of these viruses, except USU and SLE viruses. These data, as well as published sequence data for other flaviviruses, were analyzed with the ClustalW and Phylip computer packages. The resultant phylogenetic data were consistent with some of the current flavivirus serological classification, showing a close relationship between ALF and MVE viruses and between KOK and STR viruses, but suggested that KOK and STR are distantly related to the other viruses and should perhaps be reclassified in their own serocomplex. The data also confirmed the close relationship between KUN and WN viruses and showed that an isolate of KUN virus from Sarawak may represent a "link" between these two virus species. In addition, the primary sequence data revealed a polymorphic region just downstream of the stop codon in the 3' end of the viral genomes.
Biogeographic connections between Australia and other continents are still poorly understood although the plate tectonics of the Indo-Pacific region is now well described. Eupetes macrocerus is an enigmatic taxon distributed in a small area on the Malay Peninsula and on Sumatra and Borneo. It has generally been associated with Ptilorrhoa in New Guinea on the other side of Wallace's Line, but a relationship with the West African Picathartes has also been suggested. Using three nuclear markers, we demonstrate that Eupetes is the sister taxon of the South African genus Chaetops, and their sister taxon in turn being Picathartes, with a divergence in the Eocene. Thus, this clade is distributed in remote corners of Africa and Asia, which makes the biogeographic history of these birds very intriguing. The most parsimonious explanation would be that they represent a relictual basal group in the Passerida clade established after a long-distance dispersal from the Australo-Papuan region to Africa. Many earlier taxonomic arrangements may have been based on assumptions about relationships with similar-looking forms in the same, or adjacent, biogeographic regions, and revisions with molecular data may uncover such cases of neglect of ancient relictual patterns reflecting past connections between the continents.
Tectonic movements, climatic oscillations, and marine transgressions during the Cenozoic have had a dramatic effect on the biota of the tropical rain forest. This study aims to reveal the phylogeography and evolutionary history of a Peninsular Malaysian endemic tropical timber species, Neobalanocarpus heimii (Dipterocarpaceae). A total of 32 natural populations of N. heimii, with 8 samples from each population were investigated. Fifteen haplotypes were identified from five noncoding chloroplast DNA (cpDNA) regions. Overall, two major genealogical cpDNA lineages of N. heimii were elucidated: a widespread southern and a northern region. The species is predicted to have survived in multiple refugia during climatic oscillations: the northwestern region (R1), the northeastern region (R2), and the southern region (R3). These putative glacial refugia exhibited higher levels of genetic diversity, population differentiation, and the presence of unique haplotypes. Recolonization of refugia R1 and R2 could have first expanded into the northern region and migrated both northeastwards and northwestwards. Meanwhile, recolonization of N. heimii throughout the southern region could have commenced from refugia R3 and migrated toward the northeast and northwest, respectively. The populations of Tersang, Pasir Raja, and Rotan Tunggal exhibited remarkably high haplotype diversity, which could have been the contact zones that have received an admixture of gene pools from the northerly and also southerly regions. As a whole, the populations of N. heimii derived from glacial refugia and contact zones should be considered in the conservation strategies in order to safeguard the long-term survival of the species.
The evolutionary history of the colugo, a gliding arboreal mammal distributed throughout Sundaland, was influenced by the location of and connections between forest habitats. By comparing colugo phylogenetic patterns, species ecology, sample distributions, and times of divergence to those of other Sundaic taxa with different life-history traits and dispersal capabilities, we inferred the probable distribution of paleo-forest corridors and their influence on observed biogeographic patterns. We identified a consistent pattern of early diversification between east and west Bornean lineages in colugos, lesser mouse deer, and Sunda pangolins, but not in greater mouse deer. This deep east-west split within Borneo has not been commonly described in mammals. Colugos on West Borneo diverged from colugos in Peninsular Malaysia and Sumatra in the late Pliocene, however most other mammalian populations distributed across these same geographic regions diverged from a common ancestor more recently in the Pleistocene. Low genetic divergence between colugos on large landmasses and their neighboring satellite islands indicated that past forest distributions were recently much larger than present refugial distributions. Our analysis of colugo evolutionary history reconstructs Borneo as the most likely ancestral area of origin for Sunda colugos, and suggests that forests present during the middle Pliocene within the Sunda Shelf were more evergreen and contiguous, while forests were more fragmented, transient, seasonal, or with lower density canopies in the Pleistocene.
Control region polymorphisms in the mitochondrial DNA of 124 unrelated individuals from the Malay population living in or around Kuala Lumpur in Malaysia were investigated and phylogenetic haplogroup lineages were determined. The intergenic COII/tRNALys 9-bp deletion, 3010 and 5178 mutations, and several coding region polymorphisms were examined to discriminate some phylogenetic haplogroups. Sequence comparison of the control regions led to the identification of 117 mitochondrial haplotypes, in which 103 types were observed in only one individual and the other nine types were shared by more than two individuals. Gene diversity was estimated to be 0.997. Phylogenetic haplogroup determination revealed that the gene pool of the modern Malay population in Malaysia consisted mainly of southeast Asian, east Asian, unidentified and unique, and aboriginal southeast-specific haplogroups. These results suggest a multi-original nature for the modern Malay population. The present database may help not only in personal identification but also in determining geographic origin in forensic casework in Malaysian, Southeast Asian and East Asian populations.