Fine-scale phylogeographical analysis of Mediterranean Anacamptis palustris (Orchidaceae) populations based on chloroplast minisatellite and microsatellite variation

The phylogeographical history of the rare marsh orchid Anacamptis palustris (Orchidaceae) was reconstructed using highly polymorphic chloroplast minisatellite and microsatellite loci. Allelic variation at chloroplast microsatellite loci was due to length variation in poly(A/T) repeats and was informative on a regional scale, but was not sufficient to unravel relationships among populations on a local geographical scale. The minisatellite locus, however, was found to be highly variable. Nine distinct repeat types were found and variation in repeat number occurred in five repeat types. The distribution of chloroplast haplotypes, combining microsatellite and minisatellite repeat type variation, provided a clear phylogeographical picture on a large geographical scale, whereas length variation in one highly polymorphic minisatellite repeat type provided fine-scale phylogeographical information. Mediterranean populations could be divided into four main lineages, a western European lineage, a northern and central Italian lineage, a well-isolated southern Italian (Apulian) lineage, and an eastern European lineage. Variation at the most variable minisatellite repeat type N revealed 19 alleles and allowed the study of seed-mediated gene flow and an estimation of the ratio of pollen to seed flow among neighbouring populations.     

Chloroplast DNA inheritance in the orchid Anacamptis palustris using single-seed polymerase chain reaction

The modality of chloroplast inheritance in orchids has been investigated only in a few species due to the difficulties associated with the analysis of large progeny numbers from experimental crosses. To test chloroplast DNA inheritance in the orchid Anacamptis palustris, we took advantage of the presence of a highly variable minisatellite repeat located in the tRNA(LEU) intron in the chloroplast genome. Seed progeny obtained from experimental crosses between parental individuals carrying different chloroplast DNA (cpDNA) minisatellite repeat numbers were analyzed using a single-seed polymerase chain reaction (PCR) protocol. All examined seeds displayed the maternal cpDNA haplotypes, indicating that cpDNA inheritance is strictly maternal in this Mediterranean orchid species. No evidence for paternal leakage was found. This finding concurs with results obtained from PCR amplifications of pollen massulae that exclude the presence of chloroplast DNA in the pollen tetrads.     

Hypervariable plastid locus variation and intron evolution in the Anacamptis palustris lineage.

Data on the organization of a hypervariable chloroplast locus in the Anacamptis palustris (Orchidaceae) lineage are provided and used to infer the pattern of molecular evolution in this group. A large survey of sequence variation in A. palustris and allied taxa reveals several repeat types differing in number and organization that occur in the same plastid region. The resulting repeat type network suggests that at least seven different minisatellite loci evolved near each other in the tRNALEU intron and indicates the presence of at least three main phyletic lines in the A. palustris lineage.     

Variation at a chloroplast minisatellite locus reveals the signature of habitat fragmentation and genetic bottlenecks in the rare orchid Anacamptis palustris (Orchidaceae)

Geoclimatic changes during the Oligocene and more recent anthropogenic influences have shaped the current distribution and population structure of Mediterranean plant species. Anacamptis palustris (Orchidaceae) is a typical member of coastal wetlands, which have become increasingly fragmented and isolated. As a consequence, this orchid has become rare in the recent past. Length variation at a chloroplast minisatellite locus was used to estimate genetic variation within and between the largest extant populations of A. palustris. Genetic diversity was positively correlated with population size. Estimation of observed and expected gene diversity and analyses of haplotype number and haplotype frequency distributions provided evidence for population bottlenecks in the history of small populations. Comparison with an earlier study suggests that nuclear allozyme diversity was most likely lost during the Oligocene and could not recover subsequently due to low mutation rates, whereas genetic variation was restored at the highly variable chloroplast minisatellite locus. Population bottlenecks indicated by cpDNA variation occurred most likely as a consequence of more recent anthropogenic changes. The comparison of molecular markers with different levels of polymorphism provided valuable insights into the processes shaping genetic diversity and population structure in this rare orchid.     

Minisatellite and microsatellite length variation at a complex bovine VNTR locus

Length variation at the BTGL1 variable number of tandem repeat (VNTR ) locus, which includes both minisatellite and microsatellite motifs, was examined in a wide sample of cattle. A total of 22 alleles were uncovered. The distribution of allele size variation implicated mechanisms involving both minisatellite and microsatellite sequences. This was confirmed by direct sequencing of two alleles. Differences between the two alleles involved repeat number variation of microsatellite motifs and a complex event involving the minisatellite motif, but point mutations were not observed.     

Minisatellite allele diversification: the origin of rare alleles at the HRAS1 locus.

Three genetic markers within the promoter-exon 1 region of the HRAS1 locus have been employed to investigate lineage relationships among alleles of the highly polymorphic variable tandem repeat (VTR) immediately downstream of the HRAS1 gene. These markers were in absolute linkage disequilibrium with the HRAS1 VTR, allowing the assignment of unique upstream haplotypes to each of the four common VTR alleles. Analysis of 17 rare alleles revealed a stratification of allele fragment size and upstream haplotype in which each rare VTR allele possessed the markers characteristic of the common allele nearest in size. Therefore, hyperallelism emanated from the four common alleles in a defined fashion, the size of a rare allele specifying its origin. As discussed below, this result implies that unequal crossing-over between homologues is unlikely to be the predominant mechanism for generating new VTR alleles at this minisatellite locus.     

Slipped-strand mispairing at noncontiguous repeats in Poecilia reticulata: a model for minisatellite birth

The standard slipped-strand mispairing (SSM) model for the formation of variable number tandem repeats (VNTRs) proposes that a few tandem repeats, produced by chance mutations, provide the "raw material" for VNTR expansion. However, this model is unlikely to explain the formation of VNTRs with long motifs (e.g., minisatellites), because the likelihood of a tandem repeat forming by chance decreases rapidly as the length of the repeat motif increases. Phylogenetic reconstruction of the birth of a mitochondrial (mt) DNA minisatellite in guppies suggests that VNTRs with long motifs can form as a consequence of SSM at noncontiguous repeats. VNTRs formed in this manner have motifs longer than the noncontiguous repeat originally formed by chance and are flanked by one unit of the original, noncontiguous repeat. SSM at noncontiguous repeats can therefore explain the birth of VNTRs with long motifs and the "imperfect" or "short direct" repeats frequently observed adjacent to both mtDNA and nuclear VNTRs.     

Evolution of killer cell Ig-like receptor (KIR) genes: definition of an orangutan KIR haplotype reveals expansion of lineage III KIR associated with the emergence of MHC-C

Orangutan (Pongo pygmaeus) MHC-C appears less evolved than human HLA-C: Popy-C is not fixed and its alleles encode only one (C1) of the two motifs for killer cell Ig-like receptor (KIR) ligands. To assess the structure and complexity of the orangutan KIR locus, the complete nucleotide sequence of an orangutan KIR haplotype was determined. The PopyKIR locus is flanked by LILR and FCAR and consists of seven genes and pseudogenes, two novel and five corresponding to known cDNA. Distinguishing all KIRs in this rapidly evolving KIR locus from the KIR3DX1 gene is an LTR33A/MLT1D element in intron 3. These two forms of KIR represent lineages that originated by duplication of a common ancestor. The conserved, framework regions of primate KIR loci comprise the 5' part of a lineage V KIR, the 3' part of a pseudogene, the complete 2DL4 gene, and the 3' part of a lineage II KIR. Although previously defined PopyKIR2DL4 alleles contain premature termination codons, the sequenced haplotype's PopyKIR2DL4 allele encodes a full-length protein. A model for KIR evolution is proposed. Distinguishing the orangutan KIR haplotype from the proposed common ancestor of primate KIR haplotypes is an increased number to give three lineage III KIR genes in the centromeric part of the locus, the site for most human lineage III genes encoding HLA-C specific KIR. Thus, expansion of lineage III KIR is associated with emergence of MHC-C.     

Ancient diversity of splicing motifs and protein surfaces in the wild emmer wheat (Triticum dicoccoides) LR10 coiled coil (CC) and leucine-rich repeat (LRR) domains

In this study, we explore the diversity and its distribution along the wheat leaf rust resistance protein LR10 three-dimensional structure. Lr10 is a leaf rust resistance gene encoding a coiled coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) class of protein. Lr10 was cloned and sequenced from 58 accessions representing diverse habitats of wild emmer wheat in Israel. Nucleotide diversity was very high relative to other wild emmer wheat genes (π= 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposition of the diversity on the CC three-dimensional structure showed that some of the variable and positively selected residues were solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but showed a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat was longer than the other LRRs, and three-dimensional modelling suggested that an extensive α helix structure was formed in this region. The two haplotypes also differed in splicing regulation motifs. In genotypes with one haplotype, an intron was alternatively spliced in this region, whereas, in genotypes with the other haplotype, this intron did not splice at all. The two haplotypes are proposed to be ancient and maintained by balancing selection.     

Sequence rearrangement in the AT-rich minisatellite of the novel rice transposable element Basho.

In the process of characterizing a rice wx deletion mutant, an AT-rich minisatellite sequence that consisted of units of approximately 80 bp was detected about 2.3 kb downstream of the wx gene. This AT-rich minisatellite was a multiple-copy element (1 x 10(3) to 2 x 10(3) copies per haploid genome) and interspersed in the rice genome. By BLAST homology search it was indicated that not only the tandem repeat but also both flanking sequences were conserved among copies. According to the characteristics of the termini (5'-CHH ... CTAG-3') and a target site preference for T, this AT-rich minisatellite accompanying the flanking sequences was classified into a novel transposon, Basho. The results of direct amplification of Basho showed that relatively large variation in size existed in the Basho family. We estimate the variation to be generated by not only alteration of the number of units in the minisatellite but also by duplications of larger blocks including the conserved flanking sequences caused by single-strand mispairing (SSM) at noncontiguous repeats. Because the AT-rich minisatellite contained in Basho possessed several motifs of the matrix attachment region (MAR) in its repeat unit, the functional role as MAR in the rice genome was discussed.     

A unique A. palustris lineage across the Otranto strait: botanical evidence for a past land-bridge?

Phylogeography allows to reconstruct the history of genealogic lineages at the intra- and interspecific level. Previous studies on the distribution of minisatellite and microsatellite plastid haplotypes in populations of the Mediterranean marsh orchid Anacamptis palustris revealed that populations from the Salento peninsula (Apulia region, west coast of Otranto strait) formed a distinct and isolated lineage. To further explore the peculiarity of this biogeographic region, in the present study we examined both fresh samples and herbarium specimens of the rare orchid Anacamptis palustris from populations located on the eastern coast of the Otranto strait (i.e. on the Greek-Albanian side). All the investigated samples exhibited a unique combination of chloroplast microsatellites, which was previously found exclusively in populations from the Salento peninsula. Samples from Corfu, Igoumenitsa and Durazzo showed a novel minisatellite repeat type, while those of Zakynthos possessed the widespread ancestral repeat type. Interestingly, the novel repeat type and those previously found in Salento populations clustered together on a well-differentiated evolutionary branch in a minisatellite repeat type network of the A. palustris lineages. This finding indicates a close relationship between the populations from both sides of the Otranto strait and supports the geological evidence for the occurrence of an ancient land-bridge connection between the southern most part of Italy and Greece during the Messinian desiccation and/or the ``Lago-Mare' period.     

Incongruence in the diploid B-genome species complex of Glycine (Leguminosae) revisited: histone H3-D alleles versus chloroplast haplotypes.

Variation at the single-copy nuclear locus histone H3-D was surveyed in the diploid B-genome group of Glycine subgenus Glycine (Leguminosae: Papilionoideae), which comprises three named Australian species and a number of distinct but as yet not formally recognized taxa. A total of 23 alleles was identified in the 44 accessions surveyed. Only one individual was clearly heterozygous, which is not surprising given the largely autogamous breeding system of subgenus Glycine. Alleles differed by as many as 19 nucleotide substitutions, nearly all in the three introns; length variation was minimal. Phylogenetic analysis identified two shortest allele trees with very little homoplasy, suggesting that recombination has been rare. Both topological and data set incongruence were statistically significant between histone H3-D allele trees and trees inferred from chloroplast DNA haplotypes previously described from these same accessions. Whereas the distribution of H3-D alleles agrees well with morphologically based taxonomic groupings, chloroplast DNA haplotype polymorphisms transgress species boundaries, suggesting that the chloroplast genome is not tracking taxic relationships. Divergences among chloroplast DNA haplotypes involved in such transgressive patterns appear to be more recent than speciation events, suggesting hybridization rather than lineage sorting.     

Unique haplotypes of co-segregating major histocompatibility class II <Emphasis Type="Italic">A</Emphasis> and class II <Emphasis Type="Italic">B</Emphasis> alleles in Atlantic salmon (<Emphasis Type="Italic">Salmo salar</Emphasis>) give rise to diverse class II genotypes

Sequence-based typing of a breeding population (G1) consisting of 84 Atlantic salmon individuals revealed the presence of 7 Sasa-DAA and 7 Sasa-DAB expressed alleles. Subsequent typing of 1,182 individuals belonging to 33 families showed that Sasa-DAA and Sasa-DAB segregate as haplotypes. In total seven unique haplotypes were established, with frequencies in the population studied ranging from 0.01 to 0.49. Each haplotype is characterized by a unique minisatellite marker size embedded in the 3' untranslated region of the Sasa-DAA gene. These data corroborate the fact that Atlantic salmon express a single class II locus, consisting of tightly linked class II A and class B genes. The seven haplotypes give rise to 15 genotypes with frequencies varying between 0.01 and 0.23; 21 class II homozygous individuals were present in the G1 population. We also studied the frequency distribution in another breeding population (G4, n=374) using the minisatellite marker. Only one new marker size was present, suggesting the presence of one new class II haplotype. The marker frequency distribution in the G4 population differed markedly from the G1 population. The genomic organizations of two Sasa-DAA and Sasa-DAB alleles were determined, and supported the notion that these alleles belong to the same locus. In contrast to other studies of salmonid class II sequences, phylogenetic analyses of brown trout and Atlantic class II A and class II B sequences provided support for trans-species polymorphism.     

Incongruent plastid and nuclear DNA phylogenies reveal ancient intergeneric hybridization in Pilosella hawkweeds (Hieracium, Cichorieae, Asteraceae)

Phylogenetic relationships for Hieracium subgen. Pilosella were inferred from chloroplast (trnT-trnL, matK) and nuclear (ITS) sequence data. Chloroplast markers revealed the existence of two divergent haplotype groups within the subgenus that did not correspond to presumed relationships. Furthermore, chloroplast haplotypes of the genera Hispidella and Andryala nested each within one of these groups. In contrast, ITS data were generally in accord with morphology and other evidence and were therefore assumed to reflect the true phylogeny. They revealed a sister relationship between Pilosella and Hispidella and a joint clade of Hieracium subgenera Hieracium and Chionoracium (Stenotheca) while genus Andryala represented a third major lineage of the final ingroup cluster. Detailed analysis of trnT-trnL character state evolution along the ITS tree suggested two intergeneric hybridization events between ancestral lineages that resulted in cytoplasmic transfer (from Hieracium/Chionoracium to Pilosella, and from the introgressed Pilosella lineage to Andryala). These chloroplast capture events, the first of which involved a now extinct haplotype, are the most likely explanation for the observed incongruencies between plastid and nuclear DNA markers.     

Chloroplast microsatellites and mitochondrial nad1 intron 2 sequences indicate congruent phylogenetic relationships among Swiss stone pine (Pinus cembra), Siberian stone pine (Pinus sibirica), and Siberian dwarf pine (Pinus pumila)

We studied the phylogenetic relationships among the three stone pine species, Pinus cembra, P. sibirica, and P. pumila, using chloroplast microsatellites and mitochondrial nad1 intron 2 sequences. The three chloroplast microsatellite loci combined into a total of 18 haplotypes. Fourteen haplotypes were detected in 15 populations of P. cembra and one population of P. sibirica, five of which were shared between the two species, and the two populations of P. pumila comprised four species-specific haplotypes. Mitochondrial intron sequences confirmed this grouping of species. Sequences of P. cembra and P. sibirica were identical, but P. pumila differed by several nucleotide substitutions and insertions/deletions. A repeat region found in the former two species showed no intraspecific variation. These results indicate a relatively recent evolutionary separation of P. cembra and P. sibirica, despite their currently disjunct distributions. The species-specific chloroplast and mitochondrial markers of P. sibirica and P. pumila should help to trace the hybridization in their overlapping distribution area and to identify fossil remains with respect to the still unresolved postglacial re-colonization history of these two species.     

Molecular variation and speciation in Antitrichia curtipendula s.l. (Leucodontaceae, Bryophyta)

Molecular variation in Antitrichia curtipendula (Hedw.) Brid. s.l. was studied based on the nuclear internal transcribed spacer (ITS) and the chloroplast markers trn L- trn F and rpl 16, and analysed by neighbour joining (for ITS; recombination present), maximum parsimony (chloroplast markers) and TCS (haplotype network). Old World plus E North American populations belong to a different lineage than those of W North America. These are molecularly well differentiated and are treated as A. curtipendula and A. gigantea (Sull. & Lesq.) Kindb. Two distinct groups of Old World haplotypes are separated by one 'missing' haplotype and are interpreted as cryptic species. Tropical African populations share one ITS deletion and form a lineage within one of the cryptic species. Molecular variation within A. gigantea , within each of the two cryptic Old World plus E North American (except tropical African) haplotype groups, and among tropical African populations are of similar magnitude, suggesting that analogous mechanisms and similar time spans explain the found variation. Events related to Pleistocene climatic oscillations are suggested as having caused this differentiation within each group, whereas the African lineage was probably split off before this. Identical tRNA-Gly sequences were found in 33 specimens; new primers were designed for rpl 16 and ITS 1+2.  © 2008 The Linnean Society of London, Botanical Journal of the Linnean Society , 2008, 156 , 341–354.     

Development and characterization of chloroplast microsatellite markers in Macaranga (Euphorbiaceae).

As part of our study on the phylogeography of the ant-plant genus Macaranga, we have screened for polymorphic regions in the chloroplast genome. Initially, ten universal PCR primer pairs targeted at chloroplast microsatellite loci were applied to a small set of specimens, covering various taxonomic levels from intrafamilial to intraspecific. Eight primer pairs produced PCR fragments that behaved as single and discrete bands on agarose gels. The five most promising candidate pairs were further analysed with an extended set of DNA templates, and PCR products were separated on sequencing gels. The number of size variants per locus varied from two to eight, combining into 17 haplotypes among 29 Macaranga accessions from 10 species. Comparative sequencing demonstrated that microsatellites were responsible for the observed size variation at three of five loci, whereas variation at the other loci was caused by larger insertions and (or) deletions (indels). In addition to poly(A) and poly(T) repeats, which are typically found in chloroplast DNA, we also identified a variable (CT)n repeat, with n = 4 to n = 8. Sequencing revealed three examples of size homoplasy, one of which was caused by a single base substitution that raised the actual number of haplotypes to 18. Relationships between haplotypes were assessed by phenetic analyses of size variants and by constructing a parsimony network based on sequence variation. For both types of analysis, the distribution of haplotypes correlated with geographically circumscribed regions rather than with taxonomic boundaries.     

New insights into the intraspecific cytoplasmic DNA diversity,maternal lineages classification and conservation issues of Tunisian pearl millet landraces

Tunisian pearl millet (Pennisetum glaucum L.) landraces are still growing in contrasting agro-ecological environments and are considered potentially useful for national and international breeders. Despite its genetic potential, the cropping areas of this species are still limited and scattered which increases the risk of genetic erosion. The chloroplast DNA polymorphism and maternal lineages classification of forty nine pearl millet landraces representing seven populations covering the main distribution area of this crop in Tunisia were undertaken based on informative cpSSR molecular markers. A total of 21 alleles combining to 9 haplotypes were detected with a mean value of 3.5 alleles per locus and a haplotype genetic diversity (Hd) of 0.82. The number of chloroplast haplotypes per population ranged from 1 to 4 with an average of 1.28. The haplotypes median-joining network and UPGMA analyses revealed two probable ancestral maternal lineages with a differential pearl millet seed-exchange rate between the investigated areas. Northern and Central populations presented unique genetic backgrounds while historical farmers’ practices in the South-East area resulted in the isolation of their own local landraces. The genetic evidences strongly support at least two introduction origins of pearl millet in Tunisia, one in the North and the other in the South followed by distinct local dispersal histories. Complementary in-situ and ex-situ conservation strategies taking into account the conservation of the maternal lineage cytoplasmic diversity are required. The investigated chloroplast SSRs provide useful molecular markers which could be used in further genetic studies and breeding surveys of pearl millet genetic resources.     

基于叶绿体DNA变异的湖北梨属种质系统进化及遗传多样性分析

利用trnL intron、trnL-trnF、trnS-psbC和accD-psa I等4个叶绿体DNA片段对来自湖北省的88份梨属种质资源进行系统进化和遗传多样性分析。结果表明,4个cpDNA片段共检测到变异位点11个,其中单一突变位点6个,插入/缺失(Indel)位点5个。acc D-psa I多态性最高,其变异位点数、核苷酸多态性和单倍型多样性均为最高。供试梨种质的核苷酸多样性和单倍型多样性分别为0.00112和0.769;Tajima's D检验值在P0.10水平上均不显著,表明所检测的4个区域以及合并后的片段均遵循中性进化模型;4个序列合并共检测到叶绿体单倍型10个,其中兴山梨种质中检测到的单倍型最多,荆门其次;Hap2和Hap5是2个主要单倍型,分别占总样本数的31.82%和30.68%;中介邻接网络图显示东方梨和西洋梨独立进化,而较为原始的稀有单倍型Hap8和Hap9均位于荆门,暗示该地区可能为砂梨的起源中心或多样性中心之一。