Detection and identification of Legionella pneumophila by PCR-restriction fragment length polymorphism analysis of the RNA polymerase gene (rpoB)

The partial RNA polymerase beta-subunit coding gene (rpoB) sequences of 38 Legionella species (59 reference strains) were used to select both Legionella genus-specific and Legionella pneumophila species-specific primers to amplify the 347-bp and 217-bp DNAs, respectively. Enzyme restriction sites for PCR-restriction fragment length polymorphism (PCR-RFLP) analysis were also generated by a computer program. Thirty-eight Legionella species were well differentiated by the identification scheme for Legionella genus-specific PCR-RFLP using HaeIII, AluI, CfoI, PstI, and MaeII. The most common and important pathogenic species, L. pneumophila, was differentiated into two subspecies (L. pneumophila subsp. pneumophila and L. pneumophila subsp. fraseri) by both Legionella genus-specific PCR-RFLP and L. pneumophila species-specific PCR-RFLP using BamHI. Eighty-two Korean culture isolates could also be easily identified by both PCR-RFLP methods as 68 strains of L. pneumophila subsp. pneumophila, 11 strains of L. pneumophila subsp. fraseri, and three novel strains that were separately confirmed by 16S rDNA and rpoB sequence analysis. These results suggest that the rpoB PCR-RFLP for Legionella is a simple and convenient method, not only for specific detection, but also for the rapid identification of Legionella species.     

Molecular evolution of the dotA gene in Legionella pneumophila

The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15). It was found that dotA has a complex mosaic structure. The whole dotA gene of Legionella pneumophila subsp. pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp. fraseri. A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences of dotA, which suggests that multiple hits, resulting in synonymous substitutions, have occurred. Gene phylogenies inferred from three different segments (the 5'-end region, the central, large periplasmic domain, and the 3'-end region) showed impressively dissimilar topologies. This was concordant with the sequence polymorphisms, indicating that each region has experienced an independent evolutionary history, and was evident even within the same domain of each strain. For example, the PP2 domain was found to have a heterogeneous structure, which led us hypothesize that the dotA gene of L. pneumophila may have originated from two or more different sources. Comparisons of synonymous and nonsynonymous substitutions demonstrated that the PP2 domain has been under strong selective pressure with respect to amino acid change. Split decomposition analysis also supported the intragenic recombination of dotA. Multiple recombinational exchange within the dotA gene, encoding an integral cytoplasmic membrane protein that is secreted, probably provided increased fitness in certain environmental niches, such as within a particular biofilm community or species of amoebae.     

Molecular evolution of Legionella pneumophila dotA gene,the contribution of natural environmental strains

Given the role of DotA protein in establishing successful infections and the diversity of host cells interacting with Legionella pneumophila in nature, it is possible that this gene product is a target for adaptive evolution. We investigated the influence of L. pneumophila isolates from natural environments with the molecular evolution of this crucial virulence‐related gene. The population genetic structure of L. pneumophila was inferred from the partial sequences of rpoB and dotA of 303 worldwide strains. The topology of the two inferred trees was not congruent and in the inferred dotA tree the vast majority of the natural environmental isolates were clustered in a discrete group. The Ka/Ks ratio demonstrated that this group, contrary to all others, has been under strong diversifying selection. The alignment of all DotA sequences allowed the identification of several alleles and the amino acid variations were not randomly distributed. Moreover, from these results we can conclude that dotA from L. pneumophila clinical and man‐made environmental strains belong to a sub‐set of all genotypes existing in nature. A split graph analysis showed evidence of a network‐like organization and several intergenic recombination events were detected within L. pneumophila strains resulting in mosaic genes in which different gene segments exhibited different evolutionary histories. We have determined that the allelic diversity of dotA is predominantly found in L. pneumophila isolates from natural environments, suggesting that niche‐specific selection pressures have been operating on this gene. Indeed, the high level of dotA allelic diversity may reflect fitness variation in the persistence of those strains in distinct environmental niches and/or tropism to various protozoan hosts.     

基于线粒体控制区的嘉陵裸裂尻鱼种群遗传结构分析

嘉陵裸裂尻鱼为青藏高原特有鱼类,近年来随自然地理气候的变迁和人类活动的影响,种群数量急剧减少。为了解嘉陵裸裂尻鱼的遗传背景以便更好的保护其遗传资源,本研究采用线粒体控制区部分序列变异,分析了嘉陵裸裂尻鱼6个地理种群的遗传结构和分布动态。在147尾个体中共发现17个变异位点,定义了14种单倍型,群体总的单倍型多样性较高为0.810,核苷酸多样性低为0.00698。AMOVA分析显示,44.29%的分子差异源于群体间,55.71%的分子差异源于群体内,群体间遗传分化极显著。Fst值统计检验表明,除宕昌群体和舟曲群体之间差异不显著外,其余两两群体之间Fst值统计检验均显著。基因流估计显示各群体间的基因流水平较高,遗传交流较频繁。Mantel test检验表明,嘉陵裸裂尻鱼种群之间遗传分化程度与地理距离存在显著相关。系统树和单倍型网络进化图显示,6个地理群体的单倍型按照嘉陵江水系和渭河水系形成两个大的类群。错配分布和中性检验表明嘉陵裸裂尻鱼群体在近期历史上群体大小保持稳定,未出现显著的种群扩张。根据本文所揭示的嘉陵裸裂尻鱼种群遗传结构特征,建议将分布在嘉陵江水系的嘉陵裸裂尻鱼作为一个整体进行保护,嘉陵裸裂尻鱼渭河种群属高度分化的单倍型类群,且遗传多样性极低,需对该种群进行优先保护。     

Population genetic structure of Chilo suppressalis (Walker) (Lepidoptera: Crambidae): strong subdivision in China inferred from microsatellite markers and mtDNA gene sequences

Chilo suppressalis (Walker) displays significant geographical differences in ecological preference that may be congruent with patterns of molecular variation. To test this, we collected and analysed 381 individuals of this species from cultivated rice at 18 localities in China during the rice-growing season of 2005–2006. We used four microsatellite DNA markers and four mitochondrial DNA gene fragments. We found that this species is highly differentiated, coupled with an estimated population expansion date of at least 60 000  bp . Phylogenetic analyses, Bayesian clustering, and phylogeographical analyses of statistical parsimony haplotype network consistently divided the populations into three clades: a central China (CC) clade, a northern plus northeastern China (NN) clade and a southwestern China (SW) clade. Analysis of molecular variance indicated a high level of geographical differentiation at different hierarchical levels [ F _ST for microsatellite markers, COI, COII, 16S and ND1 is 0.06004 ( P  < 0.0001), 0.27607 ( P  < 0.0001), 0.22949 ( P  < 0.0001), 0.19485 ( P  < 0.0001) and 0.29285 ( P  < 0.0001), respectively]. Isolation by distance appeared among the samples from within China ( r  = 0.404, P  = 0.0002); N_em values estimated using a coalescent-based method were small (< 2 migrants per generation), suggesting that the observed levels of differentiation are a result of migration–drift equilibrium. Our results imply that the genetic differentiation of this borer, which is approximately in accordance with its observed number of generations per year in different Chinese geographical regions, is probably attributed to climatic and/or geological events (e.g. the last glacial maximum) and subsequently strengthened by the domestication of rice.     

Population structure and recombination in environmental isolates of Legionella pneumophila

Legionella pneumophila is a water-borne bacteria responsible for most cases of legionellosis, an emerging disease with an increasing incidence in industrialized countries. Although early analysis based on multilocus enzyme electrophoresis (MLEE) described the population structure of this species as clonal, more recent reports have suggested that recombination also contributes to shaping variation across its genome. We report here the results of analysing the nucleotide sequences of 19 loci in 31 environmental samples of L. pneumophila from a small Spanish region (near Alcoi, province of Alicante) where legionellosis has become almost endemic. We analysed the six loci currently incorporated to the sequence-based typing scheme developed by European Working Group for Legionella Infections (EWGLI) for L. pneumophila and 13 intergenic regions, for which we developed primers anchored in flanking, conserved genes. Our results show that recombination among natural isolates of this species is a common phenomenon, as 20 of the 31 isolates contained at least one locus in which recombination was revealed by at least three different methods. The mapping of the recombination events on the maximum likelihood tree of the concatenate sequence of the 19 loci indicated that at least nine independent recombination events might explain the observed distribution of recombinant loci among isolates. In consequence, we have shown that recombination in L. pneumophila is much more frequent than previously considered and that it does not seem to be restricted to already described pathogenicity islands or other genome constituents which provide it with a high plasticity.     

Higher-level phylogeny of Foraminifera inferred from the RNA polymerase II (RPB1) gene

Macroevolutionary relations among main lineages of Foraminifera have traditionally been inferred from the small subunit ribosomal genes (SSU rDNA). However, important discrepancies in the rates of SSU rDNA evolution between major lineages led to difficulties in accurate interpretation of SSU-based phylogenetic reconstructions. Recently, actin and beta-tubulin sequences have been used as alternative markers of foraminiferal phylogeny and their analyses globally confirm results obtained with SSU rDNA. In order to test new protein markers, we sequenced a fragment of the largest subunit of the RNA polymerase II (RPB1), a nuclear encoded single copy gene, for 8 foraminiferal species representing major orders of Foraminifera. Analyses of our data robustly confirm previous SSU rDNA and actin phylogenies and show (i) the paraphyly and ancestral position of monothalamid Foraminifera; (ii) the independent origin of miliolids; (iii) the monophyly of rotaliids, including buliminids and globigerinids; and (iv) the polyphyly of planktonic families Globigerinidae and Candeinidae. Additionally, the RPB1 phylogeny suggests Allogromiidae as the most ancestral foraminiferal lineage. In the light of our study, RPB1 appears as a valuable phylogenetic marker, particularly useful for groups of protists showing extreme variations of evolutionary rates in ribosomal genes.     

Molecular phylogeny of songbirds (Passeriformes) inferred from mitochondrial 16S ribosomal RNA gene sequences

Phylogenetic relationships among the families of passerine birds have been the subject of many debates. These relationships have been investigated by using a number of different character sets, including morphology, proteins, DNA-DNA hybridization, and mitochondrial DNA gene sequences. Our objective was to examine the phylogenetic relationships of a set of passerine songbirds (Oscines) and to test the taxonomic relationships proposed by. We sequenced 1403 aligned bases encompassing the mitochondrial transfer-RNA-Valine and 16S ribosomal RNA genes in 27 species from 14 families (including a Suboscine outgroup). Our results differ in significant ways from the superfamily designations of Sibley and Ahlquist by questioning the monophyly of the Sylvioidea and by placing the Regulidae in the Corvoidea.     

Phylogenetic analysis of the Metastrongyloidea (Nematoda: Strongylida) inferred from ribosomal RNA gene sequences

Phylogenetic relationships among nematodes of the strongylid superfamily Metastrongyloidea were analyzed using partial sequences from the large-subunit ribosomal RNA (LSU rRNA) and small-subunit ribosomal RNA (SSU rRNA) genes. Regions of nuclear ribosomal DNA (rDNA) were amplified by polymerase chain reaction, directly sequenced, aligned, and phylogenies inferred using maximum parsimony. Phylogenetic hypotheses inferred from the SSU rRNA gene supported the monophyly of representative taxa from each of the 7 currently accepted metastrongyloid families. Metastrongyloid taxa formed the sister group to representative trichostrongyloid sequences based on SSU data. Sequences from either the SSU or LSU RNA regions alone provided poor resolution for relationships within the Metastrongyloidea. However, a combined analysis using sequences from all rDNA regions yielded 3 equally parsimonious trees that represented the abursate Filaroididae as polyphyletic, Parafilaroides decorus as the sister species to the monophyletic Pseudaliidae, and a sister group relationship between Oslerus osleri and Metastrongylus salmi. Relationships among 3 members of the Crenosomatidae, and 1 representative of the Skrjabingylidae (Skrjabingylus chitwoodorum) were not resolved by these combined data. However, members of both these groups were consistently resolved as the sister group to the other metastrongyloid families. These relationships are inconsistent with traditional classifications of the Metastrongyloidea and existing hypotheses for their evolution.     

Quantitative detection of Legionella pneumophila in water samples by immunomagnetic purification and real-time PCR amplification of the dotA gene

A new real-time PCR assay was developed and validated in combination with an immunomagnetic separation system for the quantitative determination of Legionella pneumophila in water samples. Primers that amplify simultaneously an 80-bp fragment of the dotA gene from L. pneumophila and a recombinant fragment including a specific sequence of the gyrB gene from Aeromonas hydrophila, added as an internal positive control, were used. The specificity, limit of detection, limit of quantification, repetitivity, reproducibility, and accuracy of the method were calculated, and the values obtained confirmed the applicability of the method for the quantitative detection of L. pneumophila. Moreover, the efficiency of immunomagnetic separation in the recovery of L. pneumophila from different kinds of water was evaluated. The recovery rates decreased as the water contamination increased (ranging from 59.9% for distilled water to 36% for cooling tower water), and the reproducibility also decreased in parallel to water complexity. The feasibility of the method was evaluated by cell culture and real-time PCR analysis of 60 samples in parallel. All the samples found to be positive by cell culture were also positive by real-time PCR, while only eight samples were found to be positive only by PCR. Finally, the correlation of both methods showed that the number of cells calculated by PCR was 20-fold higher than the culture values. In conclusion, the real-time PCR method combined with immunomagnetic separation provides a sensitive, specific, and accurate method for the rapid quantification of L. pneumophila in water samples. However, the recovery efficiency of immunomagnetic separation should be considered in complex samples.     

Sequence Polymorphism of dotA and mip Alleles Mediating Invasion and Intracellular Replication of Legionella pneumophila

Legionella pneumophila inhabit a variety of natural and man-made aquatic environments, where they live primarily as intracellular parasites of protozoans. Given the proper exposure, however, they can cause opportunistic pneumonic infections in humans. The products of two L. pneumophila genes, dotA and mip, are part of the mechanism mediating the initial invasion of eukaryotic cells, and subsequent intracellular survival and multiplication. In this study, DNA polymorphism of the dotA and mip genes was assessed for 17 clinical and environmental isolates by nucleotide sequencing to determine the level of sequence variation, rates of molecular evolution, and history of gene divergence. The mip gene is highly conserved, whereas dotA is extremely variable, with an average level of nucleotide diversity four times greater than that of mip. Gene trees for each locus support a division of the L. pneumophila isolates into two clonal lineages. There are several disagreements between the gene trees suggesting that although L. pneumophila has a clonal population structure, genetic exchange has contributed to genotypic variation among strains in nature. Received: 12 July 2001 / Accepted: 20 August 2001     

Population history and gene dispersal inferred from spatial genetic structure of a Central African timber tree,Distemonanthus benthamianus (Caesalpinioideae)

African rainforests have undergone major distribution range shifts during the Quaternary, but few studies have investigated their impact on the genetic diversity of plant species and we lack knowledge on the extent of gene flow to predict how plant species can cope with such environmental changes. Analysis of the spatial genetic structure (SGS) of a species is an effective method to determine major directions of the demographic history of its populations and to estimate the extent of gene dispersal. This study characterises the SGS of an African tropical timber tree species, Distemonanthus benthamianus, at various spatial scales in Cameroon and Gabon. Displaying a large continuous distribution in the Lower Guinea domain, this is a model species to detect signs of past population fragmentation and recolonization, and to estimate the extent of gene dispersal. Ten microsatellite loci were used to genotype 295 adult trees sampled from eight populations. Three clearly differentiated gene pools were resolved at this regional scale and could be linked to the biogeographical history of the region, rather than to physical barriers to gene flow. A comparison with the distribution of gene pools observed for two other tree species living in the same region invalidates the basic assumption that all species share the same Quaternary refuges and recolonization pathways. In four populations, significant and similar patterns of SGS were detected. Indirect estimates of gene dispersal distances (sigma) obtained for three populations ranged from 400 to 1200 m, whereas neighbourhood size estimates ranged from 50 to 110.     

Phylogenetic relationships of Sparassis inferred from nuclear and mitochondrial ribosomal DNA and RNA polymerase sequences

Sparassis species show extensive morphological variation, especially when materials from eastern Asia and Australia are compared with collections from North America and Europe. We have been studying the taxonomy of Sparassis from eastern Asia, North America, Australia and Europe, using both morphological and molecular data. DNA was extracted from 32 recent collections of Sparassis from Australia, Canada, China, Finland, France, Germany, Japan, Switzerland, Thailand, the United Kingdom and the United States. The report of a Sparassis taxon from Australia is the first report of this genus from the Southern Hemisphere. Sequences of nuclear and mitochondrial rDNA and the gene encoding RNA polymerase subunit II (RPB2) were used to examine relationships both within the genus Sparassis and between Sparassis species and other members of the polyporoid clade. Equally weighted parsimony analyses and Bayesian analyses were performed using independent datasets and combined datasets of sequences from different regions. Our results suggest that: (i) Polyporoid fungi producing a brown rot may form a clade; (ii) as suggested in a previous study, Sparassis and Phaeolus form a monophyletic group, which is united by the production of a brown rot, the presence of a bipolar mating system and the frequent habit of growing as a root and butt rot on living trees; (iii) at least seven lineages are within Sparassis, represented by S. spathulata, S. brevipes, S. crispa, S. radicata and three taxa that have not been described, which can be distinguished on the basis of fruiting body structure, presence or absence of clamp connections, presence or absence of cystidia and spore size.     

Population history of Manihot esculenta (Euphorbiaceae) inferred from nuclear DNA sequences

The nature of gene flow in plants -- including the propensity for interspecific introgression -- makes them interesting candidates for phylogeographical analysis. Plant phylogeography studies have been limited, however, by the availability of suitable intraspecific variation. In this study, DNA sequence variation from a nuclear gene [Glyceraldehyde 3-phosphate dehydrogenase; (G3pdh)] was used to examine the population history of Manihot esculenta ssp. flabellifolia and a potentially hybridizing species, M. pruinosa. These species occur in the rainforest-savanna ecotone adjoining the Amazon basin, a region believed to have undergone major habitat shifts since the Pleistocene. Geographical distributions of the G3pdh haplotypes indicate genetic isolation-by-distance across the range of M. esculenta ssp. flabellifolia. However, there is greater genetic similarity between northeastern and western populations than would be expected given the present species distribution. A nested clade analysis suggests that northeastern and western populations were connected by gene flow until relatively recently, when they became fragmented. This inferred fragmentation event is consistent with post-Pleistocene habitat shifts proposed for the Amazon basin. At the interspecific level, haplotype sharing with M. pruinosa may reflect either recent interspecific introgression or incomplete lineage sorting between these closely related species.     

Population genetic structure of Korean pen shell (Atrina pectinata) in Korea inferred from microsatellite marker analysis

The Korean pen shell Atrina pectinata is a commercially valuable species in Korea. The commercial catch of this fish has decreased continuously since 1990. However, its genetic characteristics have never been studied. In order to explore the population genetic structure of this species, 125 pen shells sampled from three major habitats along the western coast of Korea were genotyped at 21 microsatellite loci. Relatively high levels of genetic variability (mean number of allelic richness (A_R) = 14.74; mean hetrerozygosity (He) = 0.849) was found among localities. However, despite the potential of the Korean pen shell to exhibit genetic structure (adults are sedentary), none of the genetic tests applied in this study detected significant genetic differentiation among the samples. The lack of genetic differentiation among samples may be due to high levels of larval dispersal, through passive drift with ocean currents. Alternatively, populations may have diverged too recently for significant genetic differentiation to have become evident. Furthermore, small sample sizes and the limited number of samples may have hampered the detection of genetic structure. The results of this study therefore suggested a lack of genetic structure among the populations of pen shell of Korean waters and should be managed as a single unit. This information on the genetic characteristics of Korean pen shell populations has important implications for the sustainable exploitation of the fishing resources and the preservation of biodiversity.     

Population structure of the yellow-footed rock-wallaby Petrogale xanthopus (Gray, 1854) inferred from mtDNA sequences and microsatellite loci

The yellow-footed rock-wallaby Petrogale xanthopus is considered to be potentially vulnerable to extinction. This wallaby inhabits naturally disjunct rocky outcrops which could restrict dispersal between populations, but the extent to which that occurs is unknown. Genetic differences between populations were assessed using mitochondrial DNA (control region) sequencing and analysis of variation at four microsatellite loci among three geographically close sites in south-west Queensland (P. x. celeris) and, for mtDNA only, samples from South Australia (P. x. xanthopus) as well. Populations from South Australia and Queensland had phylogenetically distinct mtDNA, supporting the present classification of these two groups as evolutionarily distinct entities. Within Queensland, populations separated by 70 km of unsuitable habitat differed significantly for mtDNA and at microsatellite loci. Populations separated by 10 km of apparently suitable habitat had statistically homogeneous mtDNA, but a significant difference in allele frequency at one microsatellite locus. Tests for Hardy-Weinberg equilibrium and micro-geographical variation at microsatellite loci did not detect any substructuring between two wallaby aggregations within a colony encircling a single rock outcrop. Although the present study was limited by small sample sizes at two of the three Queensland locations examined, the genetic results suggest that dispersal between colonies is limited, consistent with an ecological study of dispersal at one of the sites. Considering both the genetic and ecological data, we suggest that management of yellow-footed rock-wallabies should treat each colony as an independent unit and that conservation of the Queensland and South Australian populations as separate entities is warranted.     

Population structure and colonization of Bactrocera dorsalis (Diptera: Tephritidae) in China,inferred from mtDNA COI sequences

The oriental fruit fly, Bactrocera dorsalis, is a serious pest of fruits and vegetables in South‐east Asia, and, because of quarantine restrictions, impedes international trade and economic development in the region. Revealing genetic variation in oriental fruit fly populations will provide a better understanding of the colonization process and facilitate the quarantine and management of this species. The genetic structure in 15 populations of oriental fruit fly from southern China, Laos and Myanmar in South‐east Asia was examined with a 640‐bp sequence of the mitochondrial cytochrome oxidase subunit I (COI) gene. The highest levels of genetic diversity were found in Laos and Myanmar. Low to medium levels of genetic differentiation (F_ST ≤ 0.134) were observed among populations. Pooled populations from mainland China differed from those in Laos and Myanmar (F_ST = 0.024). Genetic structure across the region did not follow the isolation‐by‐distance model. The high genetic diversity observed in Laos and Myanmar supports the South‐east Asian origin of B. dorsalis. High genetic diversity and significant differentiation between some populations within mainland China indicate B. dorsalis populations have been established in the region for an extended period of time. High levels of genetic diversity observed among the five populations from Hainan Island and similarity between the Island and Chinese mainland populations indicate that B. dorsalis was introduced to Hainan from the mainland and has been on the island for many years. High genetic diversity in the recently established population in Shanghai (Pudong) suggests multiple introductions or a larger number of founders.     

Population structure and phylogeography of Solanum pimpinellifolium inferred from a nuclear gene

Phylogeographical studies are emerging as a powerful tool for understanding the population structure and evolution of wild relatives of crop species. Because of their value as genetic resources, there is great interest in exploring the distribution of variation in wild relatives of cultivated plants. In this study, we use sequence variation from the nuclear gene, fruit vacuolar invertase (Vac), to investigate the population history of Solanum pimpinellifolium. Solanum pimpinellifolium is a close relative of the cultivated tomato and has repeatedly served as a source of valuable traits for crop improvement. We sequenced the second intron of the Vac gene in 129 individuals, representing 16 populations from the northern half of Peru. Patterns of haplotype sharing among populations indicate that there is isolation by distance. However, there is no congruence between the geographical distribution of haplotypes and their genealogical relationships. Levels of outcrossing decrease towards the southernmost populations, as previously observed in an allozyme study. The geographical pattern of Vac variation supports a centre of origin in northern Peru for S. pimpinellifolium and a gradual colonization along the Pacific coast. This implies that inbreeding populations are derived from outcrossing ones and that variation present at the Vac locus predates the spread of S. pimpinellifolium. The expansion of cities and human agricultural activity in the habitat of S. pimpinellifolium currently pose a threat to the species.     

Population genetic structure and phylogeography of the oak gall wasp Andricus chodjaii (Hymenoptera: Cynipidae) in Turkey as inferred from mitochondrial and nuclear DNA sequences

Sequence data of mitochondrial cytochrome b gene and nuclear ITS2 region were used to assess genetic diversity, intraspecific phylogeography and population genetic structure of the oak gall wasp Andricus chodjaii from Turkey. We examined 293 individuals from 21 localities which generated 57 cyt b haplotypes and 8 ITS2 alleles. The average genetic diversity was 0.575 for cyt b and 0.202 ITS2, and the average nucleotide diversity 0.015 for cyt b gene and 0.001 for the ITS2 region. Phylogenetic analyses of cyt b haplotypes produced mostly similar topologies with geographically significant groupings. The ITS2 data provided less resolution without robust and apparent geographic structure. Population demographic analysis indicated that some eastern populations expanded, however, some others underwent either expansion or decline resulting in genetically structured populations. Molecular clock applied to the mtDNA data indicated that ingroup haplotypes diversified from the outgroup haplotypes around Early Pliocene. Further diversification events throughout Pleistocene resulted in major clade formations. It appears that geographic formations and glacial and interglacial cycles of Pleistocene were crucial for shaping the phylogeographic structure of A. chodjaii in Turkey.