Intra- and inter-clade cross-reactivity by HIV-1 Gag specific T-cells reveals exclusive and commonly targeted regions: implications for current vaccine trials

The genetic diversity of HIV-1 across the globe is a major challenge for developing an HIV vaccine. To facilitate immunogen design, it is important to characterize clusters of commonly targeted T-cell epitopes across different HIV clades. To address this, we examined 39 HIV-1 clade C infected individuals for IFN-γ Gag-specific T-cell responses using five sets of overlapping peptides, two sets matching clade C vaccine candidates derived from strains from South Africa and China, and three peptide sets corresponding to consensus clades A, B, and D sequences. The magnitude and breadth of T-cell responses against the two clade C peptide sets did not differ, however clade C peptides were preferentially recognized compared to the other peptide sets. A total of 84 peptides were recognized, of which 19 were exclusively from clade C, 8 exclusively from clade B, one peptide each from A and D and 17 were commonly recognized by clade A, B, C and D. The entropy of the exclusively recognized peptides was significantly higher than that of commonly recognized peptides (p = 0.0128) and the median peptide processing scores were significantly higher for the peptide variants recognized versus those not recognized (p = 0.0001). Consistent with these results, the predicted Major Histocompatibility Complex Class I IC₅₀ values were significantly lower for the recognized peptide variants compared to those not recognized in the ELISPOT assay (p<0.0001), suggesting that peptide variation between clades, resulting in lack of cross-clade recognition, has been shaped by host immune selection pressure. Overall, our study shows that clade C infected individuals recognize clade C peptides with greater frequency and higher magnitude than other clades, and that a selection of highly conserved epitope regions within Gag are commonly recognized and give rise to cross-clade reactivities.     

中国沿海日本囊对虾线粒体16S rRNA基因序列变异及遗传分化

对中国东南沿海4个日本囊对虾地理群体16S rRNA基因片段进行了序列测定和分析.在获得的467bp 序列中,共检测到45个变异位点,多态位点比例为9.64%.具有10种单倍型,三亚群体单倍型多样性最高(0.900±0.161).各群体的核苷酸多样性(π)为0.0007～0.0082,其中三亚群体核苷酸多样性最为丰富,远高于湛江、北海和海口群体.群体间遗传分化指数(F_(st))为0.000～0.945.结合已报道的16S rRNA基因序列,构建了NJ分子系统树,中国海域日本囊对虾可分为3支.香港至台湾沿岸所有单倍型聚集为一分支,湛江、海口和北海群体内的所有单倍型聚集成另一分支,两分支分别属于已报道的变种Ⅰ和变种Ⅱ,但三亚群体内的4个单倍型单独聚集成一分支,与前两分支的核苷酸序列差异大(7.95%～8.35%),遗传分化明显,可能为一亲缘关系很远的新种(或变种).研究结果可为日本囊对虾的资源管理和遗传选育提供参考.     

Genetic divergence and phylogenetic independence of Far Eastern species in subfamily Leuciscinae (Pisces: Cyprinidae) inferred from mitochondrial DNA analyses

Classification of freshwater fish in the subfamily Leuciscinae, Cyprinidae is hampered by complexity or lack of morphological diversity. In this study, analyses based on mtDNA sequences were undertaken to clarify phylogenetic relationships among Far Eastern, North American and European species in the Leuciscinae. Evolutionary rate in cytochrome b gene (Cyt-b) and D-loop sequences appear to be almost constant in Leuciscinae. The topology of phylogenetic trees generated by neighbor-joining (NJ) and maximum likelihood (ML) methods based on Cyt-b gene and D-loop sequences was similar. Five major clades, designated clades 1-5, and a minor clade were discriminated. Most of the Far Eastern, North American and European species were included in the major clades. Clade 1, comprised almost entirely of Far Eastern phoxinins, is monophyletic and greatly diverged from the other species of Leuciscinae. From the present phylogenetic relationships and the previous studies, we present the following hypotheses with respect to the evolutionary history of the Far Eastern phoxinins. The Far Eastern species should be classified into Far Eastern-specific genera, although ichthyologists have still insisted that the species should be included in the European genera. The Far Eastern clade 1 consists of two subclades, including genera Pseudaspius-Tribolodon and Far Eastern Phoxinus species. According to our phylogenetic analyses, Pseudaspius leptocephalus and Tribolodon species should be reclassified into the same genus. On the basis of evolutionary rate in Cyt-b gene in Cyprinidae, it is estimated that the Far Eastern lineage diverged approximately 10-14 million years ago (mya) from the common ancestor of Leuciscinae. It is deduced that speciation of the Far Eastern species occurred until approximately 4 mya, in relation to the formation of the Sea of Japan and the Japanese Islands.     

Evolutionary patterns of the mitochondrial genome in the Moorish gecko, Tarentola mauritanica

A previous study on the evolutionary patterns of Tarentola mauritanica demonstrated that low levels of mitochondrial diversity observed in the European populations relative to nuclear markers were consistent with a selective sweep hypothesis. In order to unravel the mitochondrial evolutionary history in this European population and two other lineages of T. mauritanica (Iberian and North African clades), variation within 22 nearly complete mitogenomes was analyzed. Surprisingly, each clade seems to have a distinct evolutionary history; with both the European and Iberian clades presenting a decrease of polymorphism, which in the former is consistent with departure from neutrality of the mtDNA (positive or background selection), but in the latter seems to be the result of a bottleneck after a population expansion. The pattern exhibited by the North African clade seems to be a consequence of adaptation to certain mtDNA variants by positive selection.     

A comprehensive molecular phylogeny of the starlings (Aves: Sturnidae) and mockingbirds (Aves: Mimidae): congruent mtDNA and nuclear trees for a cosmopolitan avian radiation

We generated a comprehensive phylogeny for the avian families Sturnidae (starlings, mynas, Rhabdornis, oxpeckers, and allies) and Mimidae (mockingbirds, thrashers, and allies) to explore patterns of morphological and behavioral diversification. Reconstructions were based on mitochondrial DNA sequences from five coding genes (4108 bp), and nuclear intron sequences from four loci (2974 bp), for most taxa, supplemented with NDII gene sequences (1041 bp) derived from museum skin specimens from additional taxa; together the 117 sampled taxa comprise 78% of the 151 species in these families and include representatives of all currently or recently recognized genera. Phylogenetic analyses consistently identified nine major clades. The basal lineage is comprised of the two Buphagus oxpeckers, which are presently confined to Africa where they are obligately associated with large mammals. Some species in nearly all of the other major clades also feed on or around large vertebrates, and this association may be an ancestral trait that fostered the world-wide dispersal of this group. The remaining taxa divide into sister clades representing the New-World Mimidae and Old-World Sturnidae. The Mimidae are divided into two subclades, a group of Central American and West Indian catbirds and thrashers, and a pan-American clade of mockingbirds and thrashers. The Sturnidae are subdivided into six clades. The Phillipine endemic Rhabdornis are the sister lineage to a larger and substantially more recent radiation of South Asian and Pacific island starlings and mynas. A clade of largely migratory or nomadic Eurasian starlings (within which the basal lineage is the model taxon Sturnus vulgaris) is allied to three groups of largely African species. These reconstructions confirm that Buphagus should not be included in the Sturnidae, and identify many genera that are not monophyletic. They also highlight the substantial diversity among the major Sturnidae subclades in rates of species accumulation, morphological differentiation, and behavioral variation.     

Mitochondrial DNA variation and biogeography of eastern gorillas

Mitochondrial DNA variation in 109 individuals from four populations of wild living gorillas in East Africa was ascertained by sequencing the first hypervariable segment of the control region, or 'd-loop', amplified from noninvasively collected hair and faeces. D-loop haplotypes from eastern gorillas fell into two distinct clades, each with low levels of genetic diversity; most observed haplotypes within each clade differing by only one or two mutations. Both clades show evidence of population bottlenecks in the recent past, perhaps concomitant with the tropical forest reduction and fragmentation brought on by global cooling and drying associated with the last glacial maximum.     

Phylogeography of sable (Martes zibellina L. 1758) in the southeast portion of its range based on mitochondrial DNA variation: highlighting the evolutionary history of the sable

To reveal phylogeographic features of sable (Martes zibellina) in the southeast part of its range, we analyzed variability of the mitochondrial DNA (mtDNA) cytochrome b gene, tRNA (Pro), tRNA (Thr) and control region (D-loop) sequences from 78 specimens in populations of the Russian Far East, northeast China, and Mongolia. Our results revealed the presence of 49 different haplotypes split into two major phylogenetic groups—clades A and B, the latter separated into two clades, B1 and B2. Comparative analysis of D-loop haplotypes in populations originating from the southeast (Russian Far East, China and Mongolia) and the west (northern Urals) portions of sable range indicated that all three mtDNA clades were present in different regional groups. However, highest diversity of clade B1 in northeast China and its nearly complete absence from the Urals suggest that the southeast sable range, being a refuge during Pleistocene glacial periods, can be considered the center of genetic diversification and possibly origin of this species. All divergence estimates fall within the Pleistocene suggesting that Quarternary glaciations played an important role in phylogeographic differentiation of sable.     

Phylogenetic relationships and genetic diversity among members of theFestuca-Lolium complex (Poaceae) based on ITS sequence data

There have been few DNA sequencebased studies of phylogenetic relationships within theFestuca-Lolium complex. Here we infer the phylogeny of 31Festuca-Lolium taxa with a dataset of 116 ITS sequences. The results are consistent with previous studies that resolved two majorFestuca clades: one clade of fine fescues and another clade that containsLolium and associatedFestuca species. This study is unique in suggesting a third, basalFestuca clade, but support for the basal position of this group is low. Extensive sampling permitted investigation of the effects of lineage sorting and reticulate events on the evolution of the complex. Roughly half of the taxa show evidence of lineage sorting or reticulation, and the monophyly ofLolium has likely been obscured by reticulate events. Overall, polyploid species harbor higher levels of ITS sequence diversity than diploids; ITS sequence variants may provide clues to the identity of allopolyploid parents.     

Natural variation, functional divergence, and local adaptation of nucleotide binding site sequences in Rhododendron (Ericaceae)

To further understand natural variation and local adaptation in the evolution of plant defense, we analyzed polymorphism data of nucleotide-binding site (NBS) sequences of Rhododendron at both the species and population levels. Multiple duplication events were found in NBS sequence evolution in Rhododendron genomes, which resulted in six clades: A–F. Our results of several NBS clade pair comparisons showed significant evolutionary rate changes based on differences in substitution rates between NBS-encoding protein clades (type I functional divergence). Pairwise comparisons of NBS clades further revealed that many amino acids displayed radical biochemical property changes causing a shift in amino acid preferences between NBS-encoding protein clades (type II functional divergence). Such divergent evolution of NBSs is likely a consequence of positive selection related to differentiation of recognition signals in response to different pathogens. Primers specific to clades B and C, which differed in the number of radical amino acid changes causing type II functional divergence and levels of nucleotide diversities, were further used to amplify population clades B and C NBS sequences of Rhododendron formosanum populations. Higher levels of net nucleotide divergences (measured by D _a) between R. formosanum populations were found based on NBS sequences of population clade B compared to population clade C, suggesting local adaptation of population clade B NBS sequences. Local adaptation can be further inferred for R. formosanum population clade B NBS sequences because of significant Φ _ST based on variation in nonsynonymous substitutions. Furthermore, local adaptation was also suggested by no significant correlation of population pairwise F _ST between population clades B and C in R. formosanum.     

TESTING FOR ANCIENT ADAPTIVE RADIATIONS IN NEOTROPICAL CICHLID FISHES

Most contemporary studies of adaptive radiation focus on relatively recent and geographically restricted clades. It is less clear whether diversification of ancient clades spanning entire continents is consistent with adaptive radiation. We used novel fossil calibrations to generate a chronogram of Neotropical cichlid fishes and to test whether patterns of lineage and morphological diversification are congruent with hypothesized adaptive radiations in South and Central America. We found that diversification in the Neotropical cichlid clade and the highly diverse tribe Geophagini was consistent with diversity‐dependent, early bursts of divergence followed by decreased rates of lineage accumulation. South American Geophagini underwent early rapid differentiation in body shape, expanding into novel morphological space characterized by elongate‐bodied predators. Divergence in head shape attributes associated with trophic specialization evolved under strong adaptive constraints in all Neotropical cichlid clades. The South American Cichlasomatini followed patterns consistent with constant rates of morphological divergence. Although morphological diversification in South American Heroini was limited, Eocene invasion of Central American habitats was followed by convergent diversification mirroring variation observed in Geophagini. Diversification in Neotropical cichlids was influenced by the early adaptive radiation of Geophagini, which potentially limited differentiation in other cichlid clades.     

Genetic diversity and origin of Chinese cattle revealed by mtDNA D-loop sequence variation

To determine the origin and genetic diversity of Chinese cattle, we analyzed the complete mtDNA D-loop sequences of 84 cattle from 14 breeds/populations from southwest and west China, together with the available cattle sequences in GenBank. Our results showed that the Chinese cattle samples converged into two main groups, which correspond to the two species Bos taurus and Bos indicus. Although a dominant lineage was clearly discerned in both B. taurus and B. indicus mtDNAs, network analysis of the lineages in each of the two species further revealed multiple clades that presented regional difference. The B. taurus samples in China could be grouped into clades T2, T3, and T4, whereas B. indicus harbored two clades I1 and I2. Age estimation of these discerned clades showed a time range of 14,100-44,500 years before present (YBP). The phylogenetic pattern of Chinese cattle was consistent with the recently described cattle matrilineal pool from northeast Asia, but suggested that B. indicus contributed more to the cattle from south and southwest China. The genetic diversity of Chinese cattle varied among the breeds studied.     

中国山羊mtDNA D-loop遗传多样性及其起源研究

采用DNA测序技术分析了中国9个山羊品种（板角山羊、成都麻羊、贵州黑山羊、贵州白山羊、黔北麻羊、马头山羊、陕南白山羊、黄淮山羊和雷州山羊）共计128个个体的mtDNA D-loop全序列。结果表明：山羊mtDNA D-loop全序列长度为1212-1213bp,检测到102个变异位点,约占分析位点总数的8．42％,可变位点中转换占99个,颠换2个,1个转换/颠换共存;界定了92种单倍型,有78种为各品种独享单倍型,另外14种为群体内或群体间共享单倍型。9个山羊品种单倍型多样度为0．9333-1．0000,核苷酸多样度为0．7062％-1．8265％,表明中国山羊品种遗传多样性丰富。根据92种mtDNA单倍型构建了中国山羊的NJ分子系统树,聚类表明,中国山羊mtDNA D-loop序列单倍型分为支系A和支系B两大类。支系A包括75种单倍型,代表95个样本,占总数的74．22％;支系B包括17种单倍型,代表33个样本,占总数的25．78％,说明中国山羊存在支系A和支系B两大母系起源。对中国山羊mtDNA D-loop的支系A和支系B进行核苷酸不配对分布曲线分析和Fu的Fs中性检验,分析表明,支系A的分布曲线呈单峰形,Fs值为-24．6491,P值为0．0000,显著偏离中性,表明山羊支系A曾经历群体扩张;支系B呈近似双峰分布,Fs值为-3．3947,P值为0．0980,中性检验差异不显著,表明山羊支系B没有经历群体扩张,群体大小保持相对稳定。山羊支系B可能起源于中国。     

Single genetic stock of kawakawa Euthynnus affinis (Cantor, 1849) along the Indian coast inferred from sequence analyses of mitochondrial DNA D-loop region

Kawakawa Euthynnus affinis is an epipelagic migratory tuna species, widely distributed in the tropical and subtropical waters of the Indo-Pacific region. Kawakawa constitutes the largest tuna fishery in the Indian waters. In the present study, genetic variation was assessed using sequence analyses of Mitochondrial DNA (mtDNA) D-loop region. A 500 bp segment of D-loop region was sequenced in 400 samples collected from eight localities (Veraval (VE), Ratnagiri (RA), Kochi (KO), Kavaratti (KA), Port-Blair (PB), Tuticorin (TU), Pondicherry (PO), and Vizag (VI)) along the Indian coast. Analysis of molecular variance of mtDNA data revealed no significant genetic differentiation among sites the (Φ _ST ?=?0.0028, P?=?0.20723) indicating a single population along the Indian coast. Phylogenetic analysis revealed no obvious phylogeographic pattern separating the eight samples of kawakawa. However, the genealogical relationships demonstrated that mtDNA D-loop sequences belong to two different clades (clade I and clade II). Clade I is the major clade which consists of more than 98?% specimens from each regional population while clade II has individuals from only three populations (VE, PO, and VI). Results of genetic analyses of the present study support a single stock management of kawakawa along the Indian coast.     

Mitochondrial DNA analysis reveals cryptic large-scale invasion of non-native genotypes of common carp (Cyprinus carpio) in Japan

Wild common carp ( Cyprinus carpio ) are probably suffering from biological invasions of conspecific domesticated strains. However, such invasions may be largely camouflaged by morphological similarities between introduced and native strains. We conducted a large survey of mitochondrial DNA sequences (complete D-loop region) from 11 localities in Japan. From a total of 166 individuals, 28 haplotypes were determined to fit into six divergent clades. One of the six clades included 19 closely related haplotypes with moderate nucleotide differences; however, the remaining five clades each included either a single haplotype or two almost identical haplotypes. Phylogenetic analysis together with the previously published Eurasian haplotypes further demonstrated that the 'monotypic' clades were sisters to various Eurasian lineages, whereas the 19 related haplotypes formed a monophyletic group apart from the whole Eurasian clade. Given their monophyly and genetic diversity, the 19 related haplotypes were thought to originate from the Japanese native strain. Conversely, their phylogenetic affinities to Eurasian lineages and unnaturally low genetic diversities caused the haplotypes of the five monotypic clades to be considered as domesticated strains introduced from Eurasia. These hypotheses were supported by further evidences; i.e. the probable non-native haplotypes were frequently found from Japanese domesticated strains, and the probable native population structure was rescued when the probable non-native haplotypes were excluded from the analyses. This study revealed that almost half or more of the haplotypes in all of the locations studied originated from domesticated strains introduced from Eurasia.     

The Genetic Diversity of mtDNA D-loop and the Origin of Chinese Goats

The complete sequences of mitochondrial DNA D-loop of 128 individuals in nine Chinese goat (Capra hircu) breeds were analyzed by DNA sequencing technology. The results show that the length of mtDNA D-loop in Chinese goats is 1 212-1 213 bp. There are 102 polymorphic sites, accounting for 8.42% of 1 212 bp sequence. Ninety-two mtDNA haplotypes were determined. The haplotype diversity and nucleotide diversity are 0.9333-1 .0000 and 0.7062%-1.8265%, respectively. The results indicate that the genetic diversity of Chinese goats is very abundant. The NJ tree indicates that Chinese goats have two types of maternal origins from lineage A and lineage B. The possibility of lineage B originating from China is also discussed.     

Compartmentalization of HIV-1 within the Female Genital Tract Is Due to Monotypic and Low-Diversity Variants Not Distinct Viral Populations

Background

Compartmentalization of HIV-1 between the genital tract and blood was noted in half of 57 women included in 12 studies primarily using cell-free virus. To further understand differences between genital tract and blood viruses of women with chronic HIV-1 infection cell-free and cell-associated virus populations were sequenced from these tissues, reasoning that integrated viral DNA includes variants archived from earlier in infection, and provides a greater array of genotypes for comparisons.

Methodology/Principal Findings

Multiple sequences from single-genome-amplification of HIV-1 RNA and DNA from the genital tract and blood of each woman were compared in a cross-sectional study. Maximum likelihood phylogenies were evaluated for evidence of compartmentalization using four statistical tests. Genital tract and blood HIV-1 appears compartmentalized in 7/13 women by ≥2 statistical analyses. These subjects'' phylograms were characterized by low diversity genital-specific viral clades interspersed between clades containing both genital and blood sequences. Many of the genital-specific clades contained monotypic HIV-1 sequences. In 2/7 women, HIV-1 populations were significantly compartmentalized across all four statistical tests; both had low diversity genital tract-only clades. Collapsing monotypic variants into a single sequence diminished the prevalence and extent of compartmentalization. Viral sequences did not demonstrate tissue-specific signature amino acid residues, differential immune selection, or co-receptor usage.

Conclusions/Significance

In women with chronic HIV-1 infection multiple identical sequences suggest proliferation of HIV-1-infected cells, and low diversity tissue-specific phylogenetic clades are consistent with bursts of viral replication. These monotypic and tissue-specific viruses provide statistical support for compartmentalization of HIV-1 between the female genital tract and blood. However, the intermingling of these clades with clades comprised of both genital and blood sequences and the absence of tissue-specific genetic features suggests compartmentalization between blood and genital tract may be due to viral replication and proliferation of infected cells, and questions whether HIV-1 in the female genital tract is distinct from blood.     

Molecular diversity of the syndinean genus Euduboscquella based on single-cell PCR analysis

The genus Euduboscquella is one of a few described genera within the syndinean dinoflagellates, an enigmatic lineage with abundant diversity in marine environmental clone libraries based on small subunit (SSU) rRNA. The region composed of the SSU through to the partial large subunit (LSU) rRNA was determined from 40 individual tintinnid ciliate loricae infected with Euduboscquella sampled from eight surface water sites in the Northern Hemisphere, producing seven distinct SSU sequences. The corresponding host SSU rRNA region was also amplified from eight host species. The SSU tree of Euduboscquella and syndinean group I sequences from environmental clones had seven well-supported clades and one poorly supported clade across data sets from 57 to 692 total sequences. The genus Euduboscquella consistently formed a supported monophyletic clade within a single subclade of group I sequences. For most parasites with identical SSU sequences, the more variable internal transcribed spacer (ITS) to LSU rRNA regions were polymorphic at 3 to 10 sites. However, in E. cachoni there was variation between ITS to LSU copies at up to 20 sites within an individual, while in a parasite of Tintinnopsis spp., variation between different individuals ranged up to 19 polymorphic sites. However, applying the compensatory base change model to the ITS2 sequences suggested no compensatory changes within or between individuals with the same SSU sequence, while one to four compensatory changes between individuals with similar but not identical SSU sequences were found. Comparisons between host and parasite phylogenies do not suggest a simple pattern of host or parasite specificity.     

Phylogeography of the fire-bellied toads Bombina: independent Pleistocene histories inferred from mitochondrial genomes

The fire-bellied toads Bombina bombina and Bombina variegata, interbreed in a long, narrow zone maintained by a balance between selection and dispersal. Hybridization takes place between local, genetically differentiated groups. To quantify divergence between these groups and reconstruct their history and demography, we analysed nucleotide variation at the mitochondrial cytochrome b gene (1096 bp) in 364 individuals from 156 sites representing the entire range of both species. Three distinct clades with high sequence divergence (K2P = 8-11%) were distinguished. One clade grouped B. bombina haplotypes; the two other clades grouped B. variegata haplotypes. One B. variegata clade included only Carpathian individuals; the other represented B. variegata from the southwestern parts of its distribution: Southern and Western Europe (Balkano-Western lineage), Apennines, and the Rhodope Mountains. Differentiation between the Carpathian and Balkano-Western lineages, K2P approximately 8%, approached interspecific divergence. Deep divergence among European Bombina lineages suggests their preglacial origin, and implies long and largely independent evolutionary histories of the species. Multiple glacial refugia were identified in the lowlands adjoining the Black Sea, in the Carpathians, in the Balkans, and in the Apennines. The results of the nested clade and demographic analyses suggest drastic reductions of population sizes during the last glacial period, and significant demographic growth related to postglacial colonization. Inferred history, supported by fossil evidence, demonstrates that Bombina ranges underwent repeated contractions and expansions. Geographical concordance between morphology, allozymes, and mtDNA shows that previous episodes of interspecific hybridization have left no detectable mtDNA introgression. Either the admixed populations went extinct, or selection against hybrids hindered mtDNA gene flow in ancient hybrid zones.     

Chloroplast DNA variation and geographical structure of the Aristolochia kaempferi group (Aristolochiaceae)

The present study documents cpDNA variation in the Aristolochia kaempferi group (Aristolochiaceae), which consists of one Chinese and all Japanese and Taiwanese species of the subgenus Siphisia. In a phylogenetic analysis based on the nucleotide sequences of the matK gene, and the atpB-rbcL and trnS-trnG intergenic spacer regions, 38 haplotypes were recognized in the A. kaempferi group and as many as 24 within A. kaempferi. This is the most haplotypes reported for a single species to date. Although six highly significant major clades were identified in the phylogenetic analysis, they were not congruent with previous classifications. This might be attributed to the specific speciation process, such as convergent evolution, incomplete lineage sorting, and/or reticulate evolution. The six major clades had a clear geographical distribution pattern and were significantly associated with geographical distribution of haplotypes in a nested clade analysis and AMOVA. The results allow us to deduce a scenario in which multiple contractions and expansions of the geographical ranges brought about by Quaternary climatic oscillations affected the patterns of genetic diversity. The present geographic patterns of haplotype distribution within the A. kaempferi group can be explained by the last postglacial range expansion from different refugia, and the boundaries may be suture zones.     

Molecular evolution of a family of resistance gene analogs of nucleotide-binding site sequences in <Emphasis Type="Italic">Solanum lycopersicum</Emphasis>

Nucleotide-binding site-leucine-rich repeats (NBS-LRR) gene families are one of the major plant resistance genes. Genomic NBS evolution was studied in many plant species for diverse arrays of NBS gene families. In this study, we focused on one family of NBS sequences in an attempt to understand how closely related NBS sequences evolved in the light of selection in domesticated plant species. A phylogenetic analysis revealed five major clades (A–E) and five subclades (A1–A5) within clade A of cloned NBS sequences. Positive selection was only detected in newly evolved NBS lineages in subclades of clade A. Positively selected codon sites were found among NBS sequences of clade A. A sliding-window analysis revealed that regions with Ka/Ks ratios of >1 were in the inter-motifs when paired clades were compared, but regions with Ka/Ks ratios of >1 were found across NBS sequences when subclades of clade A were compared. Our results based on a family of closely related NBS sequences showed that positive selection was first exerted on specific lineages across all NBS sequences after selective constraints. Subsequently, sequences with mutations in commonly conserved motifs were scrutinized by purifying selection. In the long term, conserved high frequency alleles in commonly conserved motifs and changes in inter-motifs were maintained in the investigated family of NBS sequences. Moreover, codons identified to be under positive selection in the inter-motifs were mainly located in regions involved in functions of ATP binding or hydrolysis.