Human mitochondrial DNAs (mtDNAs) from 153 independent samples encompassing seven Asian populations were surveyed for sequence variation using the polymerase chain reaction (PCR), restriction endonuclease analysis and oligonucleotide hybridization. All Asian populations were found to share two ancient AluI/DdeI polymorphisms at nps 10394 and 10397 and to be genetically similar indicating that they share a common ancestry. The greatest mtDNA diversity and the highest frequency of mtDNAs with HpaI/HincII morph 1 were observed in the Vietnamese suggesting a Southern Mongoloid origin of Asians. Remnants of the founding populations of Papua New Guinea (PNG) were found in Malaysia, and a marked frequency cline for the COII/tRNA(Lys) intergenic deletion was observed along coastal Asia. Phylogenetic analysis indicates that both insertion and deletion mutations in the COII/tRNA(Lys) region have occurred more than once.
We report the fact that D. albomicans invaded into Shanghai suddenly in the autumn of 1991. Using 9 restriction enzymes, we analyse the RFLPs of mitochondrial DNA of 29 isofemale lines belonging to 4 populations of Shanghai, Jiading, Qinpu and Nanhui. We find that all 29 haplotypes are different from each other. Comparing with the populations of Canton, Kunming, Sanhutan (Taiwan), Sumoto (Japan), and Kuala Lumper (Malaysia), we come to the conclusion that D. albomicans caught in Shanghai and areas nearby is from a few of places in the south of China-mainland. This conclusion agrees with the viewpoint that this species is on the speciation stage of migration towards north. We also discuss the mtDNA polymorphism within the species.
Laboratories intending to adopt cycle sequencing of PCR products in their routine analysis often face a confusing range of methods and kits. Through the study of mitochondrial cytochrome b, we have shown that clean and highly reproducible sequences could be obtained by using a combination of existing simple and economical methods in the preparation of DNA templates, PCR, purification of PCR products and sequencing. Our protocol is useful in itself or as a standard in typing other PCR-amplified DNA at the population level.
Mitochondrial DNA variation was surveyed in nine populations of the pigtail macaque (Macaca nemestrina), covering all three recognized subspecies in Southeast Asia. To do this, a 2,300 base pair fragment spanning the mitochondrial NAD 3 and NAD 4 genes and flanking tRNA subunits leucine and glycine was targeted for amplification and digested with a battery of 16 restriction endonucleases. Out of a total of 107 individuals, 32 unique haplotypes could be distinguished. Parsimony and neighbor-joining analyses grouped the haplotypes into five strongly supported assemblages representing China/Thailand, Malaysia, Sumatra, Borneo, and Siberut. These results indicate that the mainland and island mtDNA haplotypes are strictly and uniquely limited to the geographic ranges of the recognized morphological subspecies. Cladistic and neighbor-joining analyses indicate that inferred phylogenies of mtDNA haplotypes are congruent with subspecies designations. Furthermore, in support of morphological studies, results indicate that the Mentawai macaque is most likely not a distinct species but a subspecies of M. nemestrina.
The sequences of the mitochondrial ND4 gene (1339 bp) and the ND4L gene (290 bp) were determined for all the 14 extant taxa of the Drosophila nasuta subgroup. The average A + T content of ND4 genes is 76.5% and that of ND4L genes is 83.5%. A total of 114 variable sites were scored. The ND4 gene sequence divergence ranged from 0 to 5.4% within the subgroup. The substitution rate of the ND4 gene is about 1.25% per million years. The base substitution of the genes is strongly transition biased. Neighbor-joining and parsimony were used to construct a phylogeny based on the resultant sequence data set. According to these trees, five distinct mtDNA clades can be identified. D. niveifrons represents the most diverged lineage. D. sulfurigaster bilimbata and D. kepulauana form two independent lineages. The other two clades are the kohkoa complex and the albomicans complex. The kohkoa complex consists of D. sulfurigaster sulfurigaster, D. pulaua, D. kohkoa, and Taxon-F. The albomicans complex can be divided into two groups: D. nasuta, D. sulfurigaster neonasuta, D. sulfurigaster albostrigata, and D. albomicans from Chiangmai form one group; and D. pallidifrons, Taxon-I, Taxon-J, and D. albomicans from China form the other group. High genetic differentiation was found among D. albomicans populations. Based on our phylogenetic results, we hypothesize that D. niveifrons diverged first from the D. nasuta subgroup in Papua New Guinea about 3.5 Mya. The ancestral population spread to the north and when it reached Borneo, it diversified sequentially into the kohkoa complex, D. s. bilimbata, and D. kepulauana. About 1 Mya, another radiation occurred when the ancestral populations reached the Indo-China Peninsula, forming the albomicans complex. Discrepancy between morphological groupings and phylogenetic results suggests that the male morphological traits may not be orthologous.
Varroa jacobsoni was first described as a natural ectoparasitic mite of the Eastern honeybee (Apis cerana) throughout Asia. It later switched host to the Western honeybee (A. mellifera) and has now become a serious pest of that bee worldwide. The studies reported here on genotypic, phenotypic and reproductive variation among V. jacobsoni infesting A. cerana throughout Asia demonstrate that V. jacobsoni is a complex of at least two different species. In a new classification V. jacobsoni is here redefined as encompassing nine haplotypes (mites with distinct mtDNA CO-I gene sequences) that infest A. cerana in the Malaysia Indonesia region. Included is a Java haplotype, specimens of which were used to first describe V. jacobsoni at the beginning of this century. A new name, V. destructor n. sp., is given to six haplotypes that infest A. cerana on mainland Asia. Adult females of V. destructor are significantly larger and less spherical in shape than females of V. jacobsoni and they are also reproductively isolated from females of V. jacobsoni. The taxonomic positions of a further three unique haplotypes that infest A. cerana in the Philippines is uncertain and requires further study. Other studies reported here also show that only two of the 18 different haplotypes concealed within the complex of mites infesting A. cerana have become pests of A. mellifera worldwide. Both belong to V. destructor, and they are not V. jacobsoni. The most common is a Korea haplotype, so-called because it was also found parasitizing A. cerana in South Korea. It was identified on A. mellifera in Europe, the Middle East, Africa, Asia, and the Americas. Less common is a Japan/Thailand haplotype, so-called because it was also found parasitizing A. cerana in Japan and Thailand. It was identified on A. mellifera in Japan, Thailand and the Americas. Our results imply that the findings of past research on V. jacobsoni are applicable mostly to V. destructor. Our results will also influence quarantine protocols for bee mites, and may present new strategies for mite control.
The lung fluke, Paragonimus westermani (Kerbert, 1878), is widely distributed in Asia, and exhibits much variation in its biological properties. Previous phylogenetic studies using DNA sequences have demonstrated that samples from north-east Asia form a tight group distinct from samples from south Asia (Philippines, Thailand, Malaysia). Among countries from the latter region, considerable molecular diversity was observed. This was investigated further using additional DNA sequences (partial mitochondrial cytochrome c oxidase subunit 1 (COI) and the second internal transcribed spacer of the nuclear ribosomal gene repeat (ITS2)) from additional samples of P. westermani. Phylogenies inferred from these again found three or four groups within P. westermani, depending on the method of analysis. Populations of P. westermani from north-east Asia use snail hosts of the family Pleuroceridae and differ in other biological properties from populations in south Asia (that use snail hosts of the family Thiaridae). It is considered that the populations we sampled can be divided into two species, one in north-east Asia and the other in south Asia.
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.
Complete sequences were obtained for the coding portions of the mitochondrial (mt) genomes of Schistosoma mansoni (NMRI strain, Puerto Rico; 14 415 bp), S. japonicum (Anhui strain, China; 14 085 bp) and S. mekongi (Khong Island, Laos; 14 072 bp). Each comprises 36 genes: 12 protein-encoding genes (cox1-3, nad1-6, nad4L, atp6 and cob); two ribosomal RNAs, rrnL (large subunit rRNA or 16S) and rrnS (small subunit rRNA or 12S); as well as 22 transfer RNA (tRNA) genes. The atp8 gene is absent. A large segment (9.6 kb) of the coding region (comprising 14 tRNAs, eight complete and two incomplete protein-encoding genes) for S. malayensis (Baling, Malaysian Peninsula) was also obtained. Each genome also possesses a long non-coding region that is divided into two parts (a small and a large non-coding region, the latter not fully sequenced in any species) by one or more tRNAs. The protein-encoding genes are similar in size, composition and codon usage in all species except for cox1 in S. mansoni (609 aa) and cox2 in S. mekongi (219 aa), both of which are longer than homologues in other species. An unexpected finding in all the Schistosoma species was the presence of a leucine zipper motif in the nad4L gene. The gene order in S. mansoni is strikingly different from that seen in the S. japonicum group and other flatworms. There is a high level of identity (87-94% at both the nucleotide and amino acid levels) for all protein-encoding genes of S. mekongi and S. malayensis. The identity between genes of these two species and those of S. japonicum is less (56-83% for amino acids and 73-79% for nucleotides). The identity between the genes of S. mansoni and the Asian schistosomes is far less (33-66% for amino acids and 54-68% for nucleotides), an observation consistent with the known phylogenetic distance between S. mansoni and the other species.
Vivipary with precocious seedlings in mangrove plants was thought to be a hindrance to long-range dispersal. To examine the extent of seedling dispersal across oceans, we investigated the phylogeny and genetic structure among East Asiatic populations of Kandelia candel based on organelle DNAs. In total, three, 28 and seven haplotypes of the chloroplast DNA (cpDNA) atpB-rbcL spacer, cpDNA trnL-trnF spacer, and mitochondrial DNA (mtDNA) internal transcribed spacer (ITS) were identified, respectively, from 202 individuals. Three data sets suggested consistent phylogenies recovering two differentiated lineages corresponding to geographical regions, i.e. northern South-China-Sea + East-China-Sea region and southern South-China-Sea region (Sarawak). Phylogenetically, the Sarawak population was closely related to the Ranong population of western Peninsula Malaysia instead of other South-China-Sea populations, indicating its possible origin from the Indian Ocean Rim. No geographical subdivision was detected within the northern geographical region. An analysis of molecular variance (AMOVA) revealed low levels of genetic differentiation between and within mainland and island populations (phiCT = 0.015, phiSC = 0.037), indicating conspicuous long-distance seedling dispersal across oceans. Significant linkage disequilibrium excluded the possibility of recurrent homoplasious mutations as the major force causing phylogenetic discrepancy between mtDNA and the trnL-trnF spacer within the northern region. Instead, relative ages of alleles contributed to non-random chlorotype-mitotype associations and tree inconsistency. Widespread distribution and random associations (chi2 = 0.822, P = 0.189) of eight hypothetical ancestral cytotypes indicated the panmixis of populations of the northern geographical region as a whole. In contrast, rare and recently evolved alleles were restricted to marginal populations, revealing some preferential directional migration.
A morphological and molecular analysis was undertaken with the objective of identifying markers for geographical populations of Old World screwworm flies, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). The morphological analysis involved 192 adult flies from 14 countries, and the molecular analysis involved 45 larvae or adults from 14 populations in 11 countries. Principal components and cluster analysis of 10 morphological characters indicated that flies from Papua New Guinea (PNG) were a distinct group and most similar to flies from nearby Asian islands (Java, Sabah). There was poor resolution of other geographical regions, but some support for clustering of flies from Africa or India. Cladistic analysis of mitochondrial DNA sequences gave strong support for recognizing two races of Old World screwworm, one from sub-Saharan Africa and the other from the Gulf region and Asia. This latter race could be further divided into two lineages, i.e. one from mainland Asia (from Iraq to the Malay Peninsula) and the other from two islands of PNG.
Recent electrophoretic data have indicated that Schistosoma japonicum in mainland China may be a species complex, with the existence of a cryptic species being predicted from the analysis of schistosome populations from Sichuan province. To investigate the Sichuan form of S. japonicum, 4.9 kbp of mitochondrial DNA from each of three samples of the parasite from China (two from Sichuan and one from Hunan) and one from Sorsogon in the Philippines were amplified, sequenced and characterized. The sequence data were compared with those from the related South-east Asian species of S. mekongi (Khong Island, Laos) and S. mlayensis (Baling, Malaysia) and that from S. japonicm from Anhui (China). At both the nucleotide and amino-acid levels, the variation among the five S. japonicum samples was limited (< 1%). This was consistent with the conclusions drawn from previous molecular studies, in which minimal variation among S. japonicum populations was also detected. In contrast, S. mekongi and S. malayensis, species recognized as separate but closely related, differ from each other by about 10%, and each differs by 25%-26% from S. japonicum. Phylogenetic trees provided a graphic representation of these differences, showing all S. japonicum sequences to be very tightly clustered and distant from S. mekongi and S. malayensis, the last two being clearly distinct from each other. The results thus indicate no significant intra-specific genetic variation among S. japonicum samples collected from different geographical areas and do not support the idea of a distinct form in Sichuan.
In a previous study of Southeast Asian genetic variation, we characterized mitochondrial DNAs (mtDNAs) from six populations through high-resolution restriction fragment length polymorphism (RFLP) analysis. Our analysis revealed that these Southeast Asian populations were genetically similar to each other, suggesting they had a common origin. However, other patterns of population associations also emerged. Haplotypes from a major founding haplogroup in Papua New Guinea were present in Malaysia; the Vietnamese and Malaysian aborigines (Orang Asli) had high frequencies of haplogroup F, which was also seen in most other Southeast Asian populations; and haplogroup B, defined by the Region V 9-base-pair deletion, was present throughout the region. In addition, the Malaysian and Sabah (Borneo) aborigine populations exhibited a number of unique mtDNA clusters that were not observed in other populations. Unfortunately, it has been difficult to compare these patterns of genetic diversity with those shown in subsequent studies of mtDNA variation in Southeast Asian populations because the latter have typically sequenced the first hypervariable segment (HVS-I) of the control region (CR) sequencing rather than used RFLP haplotyping to characterize the mtDNAs present in them. For this reason, we sequenced the HVS-I of Southeast Asian mtDNAs that had previously been subjected to RFLP analysis, and compared the resulting data with published information from other Southeast Asian and Oceanic groups. Our findings reveal broad patterns of mtDNA haplogroup distribution in Southeast Asia that may reflect different population expansion events in this region over the past 50,000-5,000 years.
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.
Houseflies (Musca domestica L., Diptera: Muscidae) are cosmopolitan, colonizing, and eusynanthropic. Their distribution in the Malaysian archipelago provides an opportunity to study successive waves of colonization and extinction during the Pleistocene and Recent epochs. We scored single-strand conformation polymorphisms (SSCPs) at 16S2 and COII mitochondrial loci in 47 housefly samples from the Australian, Austro-Malayan, Indo-Malayan, Manchurian and Indo-Chinese subregions of Wallace's zoogeographical classification. We discuss the results in light of the Pleistocene vs. post-Pleistocene dispersal and faunal exchange in the Asia-Pacific area. Fourteen haplotypes were detected, of which 10 were confined to a single subregion. No haplotype was ubiquitous and only one was found in four subregions. Population diversity, HS, was greatest in the Indo-Malayan (0.36) and heterogeneous among subregions. The mean subregional diversity was 0.21 +/- 0.03, representing the probability that two randomly chosen flies, from any subregion, had different haplotypes. The hierarchical partition of diversity indicated restricted maternal gene flow among subregions (GRT = 0.60, Nm approximately 0.32). These results suggest long-standing genetic isolation of houseflies in the Malaysian archipelago and support the hypothesis that they dispersed widely during the Pleistocene. Haplotypes common among mainland populations but shared with island groups in low frequencies (<1%) indicate surprisingly little recent gene flow.
We investigate the evolution of host association in a cryptic complex of mutualistic Crematogaster (Decacrema) ants that inhabits and defends Macaranga trees in Southeast Asia. Previous phylogenetic studies based on limited samplings of Decacrema present conflicting reconstructions of the evolutionary history of the association, inferring both cospeciation and the predominance of host shifts. We use cytochrome oxidase I (COI) to reconstruct phylogenetic relationships in a comprehensive sampling of the Decacrema inhabitants of Macaranga. Using a published Macaranga phylogeny, we test whether the ants and plants have cospeciated. The COI phylogeny reveals 10 well-supported lineages and an absence of cospeciation. Host shifts, however, have been constrained by stem traits that are themselves correlated with Macaranga phylogeny. Earlier lineages of Decacrema exclusively inhabit waxy stems, a basal state in the Pachystemon clade within Macaranga, whereas younger species of Pachystemon, characterized by nonwaxy stems, are inhabited only by younger lineages of Decacrema. Despite the absence of cospeciation, the correlated succession of stem texture in both phylogenies suggests that Decacrema and Pachystemon have diversified in association, or codiversified. Subsequent to the colonization of the Pachystemon clade, Decacrema expanded onto a second clade within Macaranga, inducing the development of myrmecophytism in the Pruinosae group. Confinement to the aseasonal wet climate zone of western Malesia suggests myrmecophytic Macaranga are no older than the wet forest community in Southeast Asia, estimated to be about 20 million years old (early Miocene). Our calculation of COI divergence rates from several published arthropod studies that relied on tenable calibrations indicates a generally conserved rate of approximately 1.5% per million years. Applying this rate to a rate-smoothed Bayesian chronogram of the ants, the Decacrema from Macaranga are inferred to be at least 12 million years old (mid-Miocene). However, using the extremes of rate variation in COI produces an age as recent as 6 million years. Our inferred timeline based on 1.5% per million years concurs with independent biogeographical events in the region reconstructed from palynological data, thus suggesting that the evolutionary histories of Decacrema and their Pachystemon hosts have been contemporaneous since the mid-Miocene. The evolution of myrmecophytism enabled Macaranga to radiate into enemy-free space, while the ants' diversification has been shaped by stem traits, host specialization, and geographic factors. We discuss the possibility that the ancient and exclusive association between Decacrema and Macaranga was facilitated by an impoverished diversity of myrmecophytes and phytoecious (obligately plant inhabiting) ants in the region.
Anopheles sundaicus s.l. is a principal malaria vector taxon on islands and along the coastal areas of Southeast Asia. It has a wide geographical distribution and exhibits a high level of ecological and behavioral variability. Study of this taxon is crucial for understanding its biology and implementing effectise vector control measures. We compared populations of An. sundaicus from Vietnam, Thailand, and Malaysian Borneo by using two mitochondrial DNA markers: cytochrome oxidase I and cytochrome b. Genetic divergence, geographic separation, and cladistic analysis of relationships revealed the presence of two cryptic species: Anopheles sundaicus s.s. on Malaysian Borneo and An. sundaicus species A in coastal areas of Thailand and Vietnam. A polymerase chain reaction (PCR) assay was developed to easily identify these two species throughout their geographic distributions. The assay was based on sequence characterized amplified region derived from random amplified polymorphic DNA. This PCR identification method needs to be validated and adapted for the recognition of other possible species in the Sundaicus Complex.
We analyse molecular and phenotypic evolution in a group of taxonomically problematic Indomalayan pitvipers, the Trimeresurus sumatranus group. Mitochondrial DNA sequencing provides a well-resolved phylogeny, with each species representing a distinct lineage. Multivariate morphological analysis reveals a high level of phenotypic differentiation, which is congruent between the sexes but does not reflect phylogenetic history. An adaptive explanation for the observed pattern of differentiation is supported by independent contrasts analysis, which shows significant correlations between current ecology and the characters that most account for the variation between taxa, including those that are presently used to identify the species. Reduced precipitation and altitude, and increased temperature, are correlated with higher numbers of scales on the head, body and tail. It is hypothesized that scale number plays an important role in heat and water exchange by influencing the area of exposed of interstitial skin, and that colour pattern variation reflects selection pressures involving camouflage and thermoregulation. Ecological convergence in traits used for classification is found to have important implications for species identification where taxa are distributed over varying environments.
Taxonomic relationships within the Old World fruit bat genus, Cynopterus, have been equivocal for the better part of a century. While nomenclature has been revised multiple times on the basis of phenotypic characters, evolutionary relationships among taxa representing the entire geographic range of the genus have not been determined. We used mitochondrial DNA sequence data to infer phylogenetic relationships among the three most broadly distributed members of the genus: C. brachyotis, C. horsfieldi, and C. sphinx, and to assess whether C. brachyotis represents a single widespread species, or a complex of distinct lineages. Results clearly indicate that C. brachyotis is a complex of lineages. C. sphinx and C. horsfieldi haplotypes formed monophyletic groups nested within the C. brachyotis species complex. We identified six divergent mitochondrial lineages that are currently referred to C. brachyotis. Lineages from India, Myanmar, Sulawesi, and the Philippines are geographically well-defined, while in Malaysia two lineages, designated Sunda and Forest, are broadly sympatric and may be ecologically distinct. Demographic analyses of the Sunda and Forest lineages suggest strikingly different population histories, including a recent and rapid range expansion in the Sunda lineage, possibly associated with changes in sea levels during the Pleistocene. The resolution of the taxonomic issues raised in this study awaits combined analysis of morphometric characters and molecular data. However, since both the Indian and Malaysian Forest C. brachyotis lineages are apparently ecologically restricted to increasingly fragmented forest habitat, we suggest that reevaluation of the conservation status of populations in these regions should be an immediate goal.
A recent dispersal of modern humans out of Africa is now widely accepted, but the routes taken across Eurasia are still disputed. We show that mitochondrial DNA variation in isolated "relict" populations in southeast Asia supports the view that there was only a single dispersal from Africa, most likely via a southern coastal route, through India and onward into southeast Asia and Australasia. There was an early offshoot, leading ultimately to the settlement of the Near East and Europe, but the main dispersal from India to Australia approximately 65,000 years ago was rapid, most likely taking only a few thousand years.