A preliminary genetic distinctness of four Coilia fishes (Clupeiformes: Engraulidae) inferred from mitochondrial DNA sequences

In order to understand the genetic distinctness between four Coilia fishes,i.e., C. ectenes, C. e. taihuensis, C. mystus, and C. grayii, the combined cytochrome b (cytb), 12S rRNA and 16S rRNA gene sequences were obtained and analysed. The results are as follows: 1) neighbour-joining (NJ) and maximum parsimony (MP) trees were built with 1000 bootstrap replicates. They supported that Coilia fishes consisted of two clades: C. grayii form a monophyletic group, and the rest samples form the other monophyletic group; 2) the topological differences between the NJ tree and the MP tree are checked by Templeton test, indicating that their differences are insignificant (P > 0.05); and 3) Kimura-2-parameter distances between the samples of C. ectenes, C. e. taihuensis and C. mystus are all no bigger than 1.6%, and most of them are between 0.0% and 0.9%. C. ectenes, C. e. taihuensis and C. mystus are closely related and that their differences may be under subspecies.     

Molecular phylogeny of Clupeiformes (Actinopterygii) inferred from nuclear and mitochondrial DNA sequences

The taxonomy of clupeiforms has been extensively studied, yet phylogenetic relationships among component taxa remain controversial or unresolved. Here we test current and new hypotheses of relationships among clupeiforms using mitochondrial rRNA genes (12S and 16S) and nuclear RAG1 and RAG2 sequences (total of 4749bp) for 37 clupeiform taxa representing all five extant families and all subfamilies of Clupeiformes, except Pristigasterinae, plus seven outgroups. Our results, based on maximum parsimony, maximum likelihood, and Bayesian analyses of these data, show that some traditional hypotheses are supported. These include the monophyly of the families Engraulidae, consisting of two monophyletic subfamilies, Engraulinae (Engraulis and Anchoa) and Coilinae (Coilia and Setipinna), and Pristigasteridae (here represented only by Ilisha and Pellona). The basal position of Denticeps among clupeiforms is consistent with the molecular data when base compositional biases are accounted for. However, the monophyly of Clupeidae was not supported. Some clupeids were more closely related to taxa assigned to Pristigasteridae and Chirocentridae (Chirocentrus). These results suggest that a major revision in the classification of clupeiform fishes may be necessary, but should await a more complete taxonomic sampling and additional data.     

Mitochondrial DNA diversity of Coilia mystus (Clupeiformes: Engraulidae) in three Chinese estuaries

We used sequences of mitochondrial cytb and 16SrRNA gene segments in order to clarify the genetic diversity and population structure in three Chinese estuary populations of Coilia mystus: 21 individuals from ChangJiang River (Yangtze River) estuary, 22 from MinJiang River estuary, and 22 from ZhuJiang River (Pearl River) estuary (65 individuals total). We obtained 607 base pairs of consensus cytb sequence. Thirty four distinct haplotypes were detected among the 65 cytb sequences. The indexes of nucleotide diversity (π) in these three populations were ChangJiang 0.533%, MinJiang 1.135%, and ZhuJiang 0.268%. MinJiang is the largest of the three populations. Genetic distances within the populations were between 0.3 and 1.2%, and 0.8 to 10.8% among populations. The largest genetic distance was 10.8% between the ChangJiang and ZhuJiang populations, and the smallest was 0.8% between MinJiang and ZhuJiang populations. Analysis of molecular variance (AMOVA) analysis revealed that variation among populations accounts for 90.25% of total variation, suggesting that this is the main source of total variance. We obtained 470 base pairs of consensus sequence of 16SrRNA. We detected 19 distinct haplotypes among the 65 sequences. The indexes of nucleotide diversity (π) in these three populations were ChangJiang 0.108%, MinJiang 0.843%, and ZhuJiang 0.097%. MinJiang is also the largest among these three populations. Genetic distances were between 0.1 and 0.9% within populations and 0.5 to 1.9% between populations. The largest genetic distance was the 1.9% between the ChangJiang and MinJiang populations, and the smallest was 0.5% between the MinJiang and the ZhuJiang populations. AMOVA analysis disclosed that variation among populations accounts for 74.61% of total variation, suggesting that this is the main source of total variation. The results of this study suggest that the three Coilia mystus populations, especially the most isolated Changjiang population, have developed significant genetic structure.     

Genetic structure and differentiation of four lake populations of Coilia ectenes (Clupeiformes: Engraulidae) based on mtDNA control region sequences

To investigate genetic structure and differentiation of four lake populations of Coilia ectenes, a total of eighty-one individuals from four largest freshwater lakes (Dongting [DT], Poyang [PY], Chaohu [CH], Taihu [TH]) were analyzed based on mtDNA control region sequences. Seventy-five haplotypes were detected, and four to seven 38 bp tandem repeats were found. The indexes of nucleotide diversity (π) in these four populations were DT 0.78%, PY 1.09%, CH 0.81%, and TH 0.62%. Genetic distances within populations were between 0.63 and 1.14%, and from 0.76 to 1.60% among populations. A Neighbor–Joining (NJ) tree consisted of three major clades. Clade 1 consisted of 88.89, 26.09, and 4.55% individuals of DT, PY, and CH. Analysis of molecular variance (AMOVA) showed that most variances occurred within populations, suggesting that this is the main source of total variance. Our results suggest that the four lake populations of C. ectenes have not developed significant genetic structure.     

Phylogenetic relationships of elopomorph fishes inferred from mitochondrial ribosomal DNA sequences

The superorder Elopomorpha, a grouping which includes all teleost fishes that possess a specialized leptocephalous larva [true eels (Anguilliformes), gulpers and bobtail snipe eels (Saccopharyngiformes), bonefishes, spiny eels, and halosaurs (Albuliformes, including Notacanthiformes), ladyfishes and tarpons (Elopiformes, including Megalopiformes)] comprises >800 species for which phylogenetic relationships are poorly understood. In the present study, we analyzed mitochondrial DNA sequences in segments of the 12S and 16S rRNA genes in 33 elopomorph taxa encompassing all of the previously proposed orders, and 9 of the 15 currently recognized families of the Anguilliformes, as well as outgroup representatives from the superorders Osteoglossomorpha (nine species) and Clupeomorpha (three species), to develop phylogenetic hypotheses based on distance and parsimony methods. Both methods failed to support the monophyly of the Elopomorpha, casting doubt on the validity of the leptocephalus as an elopomorph synapomorphy. The orders Elopiformes, Albuliformes, and Anguilliformes, however, were resolved as monophyletic assemblages. Parsimony analysis supported the separation of the Anguilliformes into two groups (primitive and advanced) based on the presence of divided versus fused frontal bones. In addition, the molecular data indicated a close affinity of the anguilliform Thalassenchelys coheni (incertae sedis), known only from the leptocephalus, with the family Serrivomeridae. The implications of these data as regards the evolution of the elopomorph assemblage are discussed.     

Phylogenetic relationships of neopterygian fishes, inferred from mitochondrial DNA sequences.

To investigate the relationships among the three main groups of extant neopterygian fishes--Amiidae, Lepisosteidae, and Teleostei--we sequenced fragments of three mitochondrial genes from 12 different actinopterygian fishes and translated the nucleotide sequences into amino acid sequences. When all three regions are considered together, Amiidae clusters with Lepisosteidae in the most parsimonious cladograms, but other clades, such as Neopterygii and Teleostei, that are well supported by morphological evidence fail to emerge as monophyletic. When the cytochrome b sequences are analyzed together with previously published sequences for other taxa, the majority-rule consensus tree is consistent with the monophyly of Teleostei and Neopterygii and marginally supports the Amiidae + Lepisosteidae clade. In either analysis, when Neopterygii and Teleostei are constrained to monophyly, all the most-parsimonious cladograms support the Amiidae + Lepisosteidae topology. Where molecules and morphology disagree, provisional morphology-based constraints on the analysis of molecular data offer a practical means of integrating the two types of data.     

Classification and phylogenetic relationships of African tilapiine fishes inferred from mitochondrial DNA sequences

African cichlid fishes are composed of two major lineages, the haplochromines and the tilapiines. Whereas the phylogenetic relationships of the haplochromines have been studied extensively, primarily because of their spectacular adaptive radiations in the Great Lakes of East Africa, little is known about the relationships among the tilapiine species, despite the fact that they have become an important component of African, indeed world, aquaculture. To remedy this situation, molecular phylogenetic analysis of tilapiine fishes was undertaken. A segment of mitochondrial DNA encompassing the terminal part of the tRNA(Pro) gene and the most variable part of the control region was amplified by the polymerase chain reaction with DNA samples isolated from 42 tilapiine species, and the amplification products were subjected to heteroduplex analysis and sequencing. Phylogenetic trees based on 68 sequences revealed the existence of 11 sequence groups and 11 single-sequence branches. The groups, designated Ti1 through Ti11, were distinguished by specific combinations of diagnostic substitutions, formation of monophyletic clusters, and separation by genetic distances in excess of 0.04. Although the relationships among the groups could not be resolved, the sequences separated Oreochromis and Sarotherodon from Tilapia, as defined by Trewavas. The Oreochromis sequences clustered with the Sarotherodon sequences and thus supported the hypothesis that the mouthbrooding behavior of the tilapiine fishes evolved only once from the substrate-spawning behavior. Since on phylogenetic trees the O. alcalicus (sub)species were always separated from O. amphimelas by other Oreochromis species, it was concluded that the adaptation to life in water with a high salt concentration and high pH values evolved independently at least twice in the tilapiine fishes. The tilapiines diverged from the haplochromines more than 8 million years ago; most of the intragroup divergences among the tilapiines took place an estimated 1.1 to 6 million years ago.     

Phylogenetic analysis of hampala fishes (subfamily Cyprininae) in Malaysia inferred from partial mitochondrial cytochrome B DNA sequences

This study examined 396 base pairs of the mitochondrial cytochrome b gene from 110 individuals belonging to the genus Hampala, a group of freshwater cyprinids that inhabit Southeast Asia. The samples were taken from various locations throughout Sarawak, Sabah, and peninsular Malaysia. The nucleotide sequences were subjected to phylogenetic analyses by using the neighbor-joining, maximum parsimony, and maximum likelihood methods. All three methods revealed the reciprocally monophyletic relationship of Hampala macrolepidota to the other Hampala forms, thus strongly supporting its status as a distinct species. Phylogenetic analysis also discovered the existence of two H. bimaculata lineages endemic to Borneo: (1) a newly identified species from the southern and central part of Sarawak assigned as H. bimaculata Type A and (2) the previously described H. bimaculata from northern Sarawak and the west coast of Sabah assigned as H. bimaculata Type B. However, the status of H. sabana and an intermediate form were not elucidated. The results suggest that the intermediate form from the Tawau population is actually a subpopulation of H. sabana, while the highly divergent intermediate form from Kalabakan could represent a cryptic species. The sharing of H. macrolepidota haplotypes in the southern peninsular Malaysia and southern and central Sarawak samples (Hm1 and Hm2) reflected the recent disconnection of the two regions, during the late Pleistocene. Overall, the partial sequence of the mitochondrial cytochrome b gene was useful for resolving the phylogenetic relationships among Hampala fishes in Malaysia.     

Molecular phylogeny of the stromateoid fishes (Teleostei: Perciformes) inferred from mitochondrial DNA sequences and compared with morphology-based hypotheses

The phylogenetic relationships among 21 species of stromateoid fishes, representing five families and 13 genera, were reconstructed using 3263bp of mitochondrial DNA sequences, including the posterior half of the 16S rRNA and entire COI and Cytb genes. The resultant molecular phylogenies were compared with previous phylogenetic hypotheses inferred from morphological characters. Molecular phylogenetic trees were constructed using the maximum parsimony, maximum likelihood, and Bayesian methods. All three methods resulted in well-resolved trees with most nodes being supported by moderate to high support values. In contrast to previous morphological analyses, which resulted in non-monophyly of Centrolophidae, all three methods utilized for the present molecular analyses supported the monophyly of Centrolophidae, as well as the reciprocal monophyly of the other stromateoid families, previous morphological hypotheses being rejected by the Templeton and Shimodaira-Hasegawa tests. In addition, the three methods indicated a sister-group relationship between Ariommatidae and Nomeidae. The position of Tetragonuridae was, however, incongruent between the MP method and the ML and Bayesian methods, being placed in the most basal position of Stromateoidei in the former, but occupying a sister relationship to Stromateidae in the latter. Comparison of the molecular phylogenies to previous morphological hypotheses suggested that evolutionary changes in morphological characters have not occurred equally among the stromateoid lineages, the evolution of the centrolophids not having been accompanied by appreciable morphological changes, whereas other stromateoids have undergone considerable morphological changes during their evolutionary history. The molecular phylogenies also shed some light on the evolutionary pattern of the pharyngeal sac, two of the four types of sac corresponding to two main lineages of Stromateoidei. Some taxonomic implications were also discussed.     

Molecular phylogeny of Sinocyclocheilus (Cypriniformes: Cyprinidae) inferred from mitochondrial DNA sequences

More than 10 species within the freshwater fish genus Sinoncyclocheilus adapt to caves and show different degrees of degeneration of eyes and pigmentation. Therefore, this genus can be useful for studying evolutionary developmental mechanisms, role of natural selection and adaptation in cave animals. To better understand these processes, it is indispensable to have background knowledge about phylogenetic relationships of surface and cave species within this genus. To investigate phylogenetic relationships among species within this genus, we determined nucleotide sequences of complete mitochondrial cytochrome b gene (1140 bp) and partial ND4 gene (1032 bp) of 31 recognized ingroup species and one outgroup species Barbodes laticeps. Phylogenetic trees were reconstructed using maximum parsimony, Bayesian, and maximum likelihood analyses. Our phylogenetic results showed that all species except for two surface species S. jii and S. macrolepis clustered as five major monophyletic clades (I, II, III, IV, and V) with strong supports. S. jii was the most basal species in all analyses, but the position of S. macrolepis was not resolved. The cave species were polyphyletic and occurred in these five major clades. Our results indicate that adaptation to cave environments has occurred multiple times during the evolutionary history of Sinocyclocheilus. The branching orders among the clades I, II, III, and IV were not resolved, and this might be due to early rapid radiation in Sinocyclocheilus. All species distributed in Yunnan except for S. rhinocerous and S. hyalinus formed a strongly supported monophyletic group (clade V), probably reflecting their common origins. This result suggested that the diversification of Sinocyclocheilus in Yunnan may correlate with the uplifting of Yunnan Plateau.     

Angiosperm phylogeny inferred from sequences of four mitochondrial genes

An angiosperm phylogeny was reconstructed in a maximum likelihood analysis of sequences of four mitochondrial genes, atpl, matR, had5, and rps3, from 380 species that represent 376 genera and 296 families of seed plants. It is largely congruent with the phylogeny of angiosperms reconstructed from chloroplast genes atpB, matK, and rbcL, and nuclear 18S rDNA. The basalmost lineage consists of Amborella and Nymphaeales (including Hydatellaceae). Austrobaileyales follow this clade and are sister to the mesangiosperms, which include Chloranthaceae, Ceratophyllum, magnoliids, monocots, and eudicots. With the exception of Chloranthaceae being sister to Ceratophyllum, relationships among these five lineages are not well supported. In eudicots, Ranunculales, Sabiales, Proteales, Trochodendrales, Buxales, Gunnerales, Saxifragales, Vitales, Berberidopsidales, and Dilleniales form a basal grade of lines that diverged before the diversification of rosids and asterids. Within rosids, the COM (Celastrales-Oxalidales-Malpighiales) clade is sister to malvids (or rosid Ⅱ), instead of to the nitrogen-fixing clade as found in all previous large-scale molecular analyses of angiosperms. Santalales and Caryophyllales are members of an expanded asterid clade. This study shows that the mitochondrial genes are informative markers for resolving relationships among genera, families, or higher rank taxa across angiosperms. The low substitution rates and low homoplasy levels of the mitochondrial genes relative to the chloroplast genes, as found in this study, make them particularly useful for reconstructing ancient phylogenetic relationships. A mitochondrial gene-based angiosperm phylogeny provides an independent and essential reference for comparison with hypotheses of angiosperm phylogeny based on chloroplast genes, nuclear genes, and non-molecular data to reconstruct the underlying organismal phylogeny.     

Phylogenetic relationships of glyptosternoid fishes (Siluriformes: Sisoridae) inferred from mitochondrial cytochrome b gene sequences

To explore phylogenetic relationships among glyptosternoid fishes, we determined nucleotide sequences of the complete mitochondrial cytochrome b gene region (1138 base pair). Thirteen species of glyptosternoid fishes and six species of non-glyptosternoids represent 10 sisorid genera were examined. Molecular phylogenetic trees were constructed using the maximum parsimony, minimum evolution, maximum likelihood, and Bayesian methods. Bayesian and maximum likelihood analyses support the monophyly of glyptosternoids, but our hypothesis of internal relationships differs from previous hypothesis. Results indicated that glyptosternoid is a monophyletic group and genera Glyptosternum and Exostoma are two basal species having a primitive position among it. Genera Euchiloglanis and Pareuchiloglanis form a sister-group. Then they form a sister-group with Pseudexostoma plus Oreoglanis. Our result also found that Pareuchiloglanis anteanalis might be considered as the synonyms of Parechiloglanis sinensis, and genus Euchiloglanis might have only one valid species, Euchiloglanis davidi.     

Phylogeography of white-spotted Charr (Salvelinus leucomaenis) inferred from mitochondrial DNA sequences

The white-spotted charr (Salvelinus leucomaenis) is a coldwater-adapted fish distributed in far-eastern Asia. To assess phylogeographic patterns of this species over most of its range in the Japanese archipelago and Sakhalin Island, Russia, we examined nucleotide sequences of the mitochondrial DNA (mtDNA) cytochrome b region (557 bp) in 141 individuals from 50 populations. A total of 33 (5.5%) nucleotide positions were polymorphic and defined 29 haplotypes. Phylogenetic analysis assigned the observed haplotypes to four main clades, which were characterized by the idiosyncrasies and discontinuity of geographic distributions. The nested clade analyses revealed that the geographical distribution patterns of some haplotypes and clades were explained by historical event such as past fragmentation. Although substantial genetic differentiation was found among the four main clades, their geographic distributions overlapped extensively in several regions. Since white-spotted charr can potentially use both freshwater and marine environments, coexistence among different lineages can be attributed to secondary contact through range expansion by migratory individuals during multiple glacial periods after interglacial isolation. Finally, our data demonstrate that the current subspecies designation does not reflect the phylogeography of this species based on mtDNA analysis. Hierarchical analysis (AMOVA) also showed that genetic variation was far more pronounced within subspecies than among subspecies (i.e., among discrete regions). These results suggest that each population, rather than each subspecies, must be treated as an evolutionarily significant unit.     

Molecular systematics of pikas (genus Ochotona) inferred from mitochondrial DNA sequences

The phylogenetic relationships among worldwide species of genus Ochotona were investigated by sequencing mitochondrial cytochrome b and ND4 genes. Parsimony and neighbor-joining analyses of the sequence data yielded congruent results that strongly indicated three major clusters: the shrub-steppe group, the northern group, and the mountain group. The subgeneric classification of Ochotona species needs to be revised because each of the two subgenera in the present classification contains species from the mountain group. To solve this taxonomic problem so that each taxon is monophyletic, i.e. , represents a natural clade, Ochotona could be divided into three subgenera, one for the shrub-steppe species, a second for the northern species, and a third for the mountain species. The inferred tree suggests that the differentiation of this genus in the Palearctic Region was closely related to the gradual uplifting of the Tibet (Qinghai-Xizang) Plateau, as hypothesized previously, and that vicariance might have played a major role in the differentiation of this genus on the Plateau. On the other hand, the North American species, O. princeps, is most likely a dispersal event, which might have happened during the Pliocene through the opening of the Bering Strait. The phylogenetic relationships within the shrub-steppe group are worth noting in that instead of a monophyletic shrub-dwelling group, shrub dwellers and steppe dwellers are intermingled with each other. Moreover, the sequence divergence within the sister taxa of one steppe dweller and one shrub dweller is very low. These findings support the hypothesis that pikas have entered the steppe environment several times and that morphological similarities within steppe dwellers were due to convergent evolution.     

Phylogeny of east asian bufonids inferred from mitochondrial DNA sequences (Anura: amphibia)

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei, B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B. bankorensis from Taiwan is evaluated.     

Phylogenetic relationships among Bactrocera species (Diptera: Tephritidae) inferred from mitochondrial DNA sequences

Several members of the dipteran family Tephritdae are serious pests because females lay eggs in ripening fruit. The genus Bactrocera is one of the largest within the family with over 500 described species arranged in 28 subgenera. The phylogenetic relationships among the various species and subgenera, and the monophyly of specific groups have not been examined using a rigorous phylogenetic analysis. Therefore, phylogenetic relationships among 24 Bactrocera species belonging to 9 subgenera were inferred from DNA sequence of portions of the mitochondrial 16S rRNA, cytochrome oxidase II, tRNA(Lys), and tRNA(Asp) genes. Two morphological characters that traditionally have been used to define the four groups within the subgenus Bactrocera were evaluated in a phylogenetic context by mapping the character states onto the parsimony tree. In addition, the evolutionary trend in male-lure response was evaluated in a phylogenetic context. Maximum parsimony analyses suggested the following relationships: (1) the genus Bactrocera is monophyletic, (2) the subgenus B. (Zeugodacus) is paraphyletic, (3) the subgenus B. (Daculus) is a sister group to subgenus B. (Bactrocera), and (4) the subgenus B. (Bactrocera) is monophyletic. The mapping analyses suggested that the morphological characters exhibit a simple evolutionary transition from one character state to another. Male-lure response was identified as being a labile behavior that has been lost on multiple occasions. Cue-lure response was plesiomorphic to methyl-eugenol response, and the latter has evolved independently within the Bactrocera and Zeugodacus groups of subgenera. The implications of our results for devising a coherent, consolidated classification for Bactrocera is discussed.     

Isolation and characterization of microsatellite loci in the fish Coilia mystus (Clupeiformes: Engraulidae) using PCR-based isolation of microsatellite arrays

Coilia mystus is the most important harvested fish species in China; it inhabits quite different water environments during the different life history stages. Populations of C. mystus have dropped sharply due to overharvesting and water pollution. We developed eight microsatellite loci in C. mystus for conservation genetics studies. These new markers were tested in 20 individuals from the Min River in ChangLe. The number of alleles ranged from 3 to 8, the expected heterozygosity from 0.621 to 0.853 and the observed heterozygosity from 0.473-0.800. Only two loci deviated significantly from Hardy-Weinberg expectations due to heterozygote deficiency. These primers may provide a tool for understanding demography and population structure of this economically important and threatened species.     

Evolutionary relationships among the true vipers (Reptilia: Viperidae) inferred from mitochondrial DNA sequences

Nucleotide sequences of mitochondrial cytochrome b and 16S rRNA genes, totaling 946 bp, were used to reconstruct a molecular phylogeny of 42 species of the subfamily Viperinae representing 12 of the 13 recognized genera. Maximum-parsimony and maximum-likelihood were used as methods for phylogeny reconstruction with and without a posteriori weighting. When representatives of the Causinae were taken as outgroup, five major monophyletic groups were consistently identified: Bitis, Cerastes, Echis, the Atherini (Atheris s.l.), and the Eurasian viperines. Proatheris was affiliated with Atheris, and Adenorhinos clustered within Atheris. The African Bitis consisted of at least three monophyletic groups: (i) the B. gabonica group, (ii) the B. caudalis group, and (iii) the B. cornuta group. B. worthingtoni and B. arietans are not included in any of these lineages. Eurasian viperines could be unambiguously devided into four monophyletic groups: (i) Pseudocerastes and Eristicophis, (ii) European vipers (Vipera s.str.), (iii) Middle East Macrovipera plus Montivipera (Vipera xanthina group), and (iv) North African Macrovipera plus Vipera palaestinae and Daboia russelii. These evolutionary lineages are consistent with historical biogeographical patterns. According to our analyses, the viperines originated in the Oligocene in Africa and successively underwent a first radiation leading to the five basal groups. The radiation might have been driven by the possession of an effective venom apparatus and a foraging startegy (sit-wait-strike) superior in most African biomes and might have been adaptive. The next diversifications led to the Proatheris-Atheris furcation, the basal Bitis splitting, and the emergence of the basal lineages within the Eurasian stock. Thereafter, lineages within Echis, Atheris, and Cerastes evolved. The emergence of three groups within Vipera s.l. might have been forced by the existence of three land masses during the early Miocene in the area of the Paratethys and the Mediterranean Seas. Taxonomic consequences of these findings are discussed.     

A phylogeny of Chinese species in the genus Phrynocephalus (Agamidae) inferred from mitochondrial DNA sequences

We investigated the phylogenetic relationships among most Chinese species of lizards in the genus Phrynocephalus (118 individuals collected from 56 populations of 14 well-defined species and several unidentified specimens) using four mitochondrial gene fragments (12S rRNA, 16S rRNA, cytochrome b, and ND4-tRNA(LEU)). The partition-homogeneity tests indicated that the combined dataset was homogeneous, and maximum-parsimony (MP), neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian (BI) analyses were performed on this combined dataset (49 haplotypes including outgroups for 2058bp in total). The maximum-parsimony analysis resulted in 24 equally parsimonious trees, and their strict consensus tree shows that there are two major clades representing the Chinese Phrynocephalus species: the viviparous group (Clade A) and the oviparous group (Clade B). The trees derived from Bayesian, ML, and NJ analyses were topologically identical to the MP analysis except for the position of P. mystaceus. All analyses left the nodes for the oviparous group, the most basal clade within the oviparous group, and P. mystaceus unresolved. The phylogenies further suggest that the monophyly of the viviparous species may have resulted from vicariance, while recent dispersal may have been important in generating the pattern of variation among the oviparous species.     

Phylogeography of field voles (Microtus agrestis) in Eurasia inferred from mitochondrial DNA sequences

In a distribution-wide phylogeographic survey of the field vole (Microtus agrestis), 75 specimens from 56 localities across Eurasia were examined for DNA sequence variation along the whole 1140 base pair (bp) mitochondrial (mt) cytochrome b gene. The species is subdivided into three main mtDNA phylogeographic groups - western, eastern and southern - with largely allopatric distributions. The western phylogeographical group is found in west and central Europe and spread most probably from a glacial refugium in the Carpathians. The eastern group covers a large range from Lithuania to central Asia, and probably originated from a southeast European source (e.g. the southern Urals or the Caucasus). The southern group occupies an area from Portugal to Hungary, with division into two distinct mtDNA sublineages that presumably derive from separate glacial refugia in the Iberian Peninsula. Molecular clock estimates suggest that the western and eastern field vole populations separated during the last glaciation, whereas the southern population dates back 0.5-0.9 Myr. High levels of mtDNA variation indicate relatively large population sizes and subdivisions within phylogeographic groups during the last glaciation. We report a possible new suture zone in east Europe.