Extensive intraspecific polymorphism detected by SSCP at the nuclear C-mos gene in the endemic Iberian lizard Lacerta schreiberi

C-mos is a highly conserved intronless gene that has proved useful in the analysis of ancient phylogenetic relationships within vertebrates. We selected the Iberian endemic Schreiber's green lizard (Lacerta schreiberi) that persisted in allopatric refugia since the late Pliocene to investigate the utility of the C-mos nuclear gene for intraspecific phylogeographic studies. Our combination of DNA sequencing with the high resolving power of single-strand conformational polymorphism (SSCP) effectively discriminated four common alleles showing strong population structuring (F(ST) = 0.46). In addition, reconstruction of allele phylogenetic relationships further improved our understanding of C-mos spatial patterns of variation and allowed a comparison with previously described mitochondrial DNA data. Finally, limited sequencing of an extended C-mos fragment in six additional Lacerta species showed extensive polymorphism, to our knowledge representing a rare example of variation in a highly conserved nuclear gene.     

The evolutionary history of a mammal species with a highly fragmented range: the phylogeography of the European snow vole

The European snow vole Chionomys nivalis has a patchy distribution restricted to rocky habitats across southern Europe and the Near and Middle East. We carried out a phylogeographic study to provide a biogeographic scenario, based on molecular data, outlining the major processes that determined the current distribution of the species. The samples include 26 snow voles from 14 different populations across the entire species range from Spain to Anatolia and Israel. Nearly complete sequences (1037 bp) of the mitochondrial gene for cytochrome b were sequenced. Relationships among haplotypes were inferred with neighbour-joining, maximum likelihood, maximum parsimony analyses and minimum spanning network. An analysis of mismatch distribution was used to cast light on past demographic expansion. We found 22 different haplotypes that fall into six distinct lineages, all but one is supported by high bootstrap values with all methods. Four lineages are allopatric (Tatra Mts., Iberia, Balkans and Middle East) while divergent haplotypes from two lineages show sympatry in the Alps and the Apennines. The basal relationships of these lineages could not be established by any tree. The mean pairwise genetic distance between lineages ranges from 2.4 to 4.2%. The shape of the mismatch distribution indicated a past expansion event dating back to between 158 000 and 84 000 years ago. These data can be interpreted with the existence of southern glacial refugia (Iberia, Balkans, Middle East and Italy) and one additional northern glacial refugium. The lack of phylogenetic resolution among lineages and the shape of mismatch distribution are indicative of a simultaneous and rapid splitting due to a relatively fast initial expansion of populations. Moreover, the analysis supports the hypothesis of the European origin of C. nivalis and its subsequent eastward dispersion during the Middle Pleistocene.     

High levels of population subdivision in a morphologically conserved Mediterranean toad (Alytes cisternasii) result from recent,multiple refugia: evidence from mtDNA,microsatellites and nuclear genealogies

Pleistocene glaciations often resulted in differentiation of taxa in southern European peninsulas, producing the high levels of endemism characteristic of these regions (e.g. the Iberian Peninsula). Despite their small ranges, endemic species often exhibit high levels of intraspecific differentiation as a result of a complex evolutionary history dominated by successive cycles of fragmentation, expansion and subsequent admixture of populations. Most evidence so far has come from the study of species with an Atlantic distribution in northwestern Iberia, and taxa restricted to Mediterranean‐type habitats remain poorly studied. The Iberian Midwife toad (Alytes cisternasii) is a morphologically conserved species endemic to southwestern and central Iberia and a typical inhabitant of Mediterranean habitats. Applying highly variable genetic markers from both mitochondrial and nuclear genomes to samples collected across the species’ range, we found evidence of high population subdivision within A. cisternasii. Mitochondrial haplotypes and microsatellites show geographically concordant patterns of genetic diversity, suggesting population fragmentation into several refugia during Pleistocene glaciations followed by subsequent events of geographical and demographic expansions with secondary contact. In addition, the absence of variation at the nuclear β‐fibint7 and Ppp3caint4 gene fragments suggests that populations of A. cisternasii have been recurrently affected by episodes of extinction and recolonization, and that documented patterns of population subdivision are the outcome of recent and multiple refugia. We discuss the evolutionary history of the species with particular interest in the increasing relevance of Mediterranean refugia for the survival of genetically differentiated populations during the Pleistocene glaciations as revealed by studies in co‐distributed taxa.     

Maintenance of genetic differentiation across a transition zone in the sea: discordance between nuclear and cytoplasmic markers

To investigate the origin and maintenance of the genetic discontinuity between Atlantic and Mediterranean populations of the common sea bass (Dicentrarchus labrax) we analysed the genetic variation at a fragment of mitochondrial cytochrome b sequence for 18 population samples. The result were also compared with new or previously published microsatellite data. Seven mitochondrial haplotypes and an average nucleotidic divergence of 0.02 between Atlantic and Mediterranean populations that matches a Pleistocene allopatric isolation were found. The frequency variation at the cytochrome b locus was many times greater between Atlantic and Mediterranean populations (theta(C) = 0.67) than at microsatellite loci (theta(N)= 0.02). The examination of the different evolutionary forces at play suggests that a sex-biased hybrid breakdown is a likely explanation for part of the observed discrepancy between mitochondrial and nuclear loci. In addition, an analysis is made of the correlation between microsatellite loci points towards the possible existence of a hybrid zone in samples from the Alboran Sea.     

European Y-chromosomal lineages in Polynesians: a contrast to the population structure revealed by mtDNA.

We have used Y-chromosomal polymorphisms to trace paternal lineages in Polynesians by use of samples previously typed for mtDNA variants. A genealogical approach utilizing hierarchical analysis of eight rare-event biallelic polymorphisms, seven microsatellite loci, and internal structural analysis of the hypervariable minisatellite, MSY1, has been used to define three major paternal-lineage clusters in Polynesians. Two of these clusters, both defined by novel MSY1 modular structures and representing 55% of the Polynesians studied, are also found in coastal Papua New Guinea. Reduced Polynesian diversity, relative to that in Melanesians, is illustrated by the presence of several examples of identical MSY1 codes and microsatellite haplotypes within these lineage clusters in Polynesians. The complete lack of Y chromosomes having the M4 base substitution in Polynesians, despite their prevalence (64%) in Melanesians, may also be a result of the multiple bottleneck events during the colonization of this region of the world. The origin of the M4 mutation has been dated by use of two independent methods based on microsatellite-haplotype and minisatellite-code diversity. Because of the wide confidence limits on the mutation rates of these loci, the M4 mutation cannot be conclusively dated relative to the colonization of Polynesia, 3,000 years ago. The other major lineage cluster found in Polynesians, defined by a base substitution at the 92R7 locus, represents 27% of the Polynesians studied and, most probably, originates in Europe. This is the first Y-chromosomal evidence of major European admixture with indigenous Polynesian populations and contrasts sharply with the picture given by mtDNA evidence.     

The phylogeographical history of the Iberian steppe plant Ferula loscosii (Apiaceae): a test of the abundant-centre hypothesis

The geology and climate of the western Mediterranean area were strongly modified during the Late Tertiary and the Quaternary. These geological and climatic events are thought to have induced changes in the population histories of plants in the Iberian Peninsula. However, fine-scale genetic spatial architecture across western Mediterranean steppe plant refugia has rarely been investigated. A population genetic analysis of amplified fragment length polymorphism variation was conducted on present-day, relict populations of Ferula loscosii (Apiaceae). This species exhibits high individual/population numbers in the middle Ebro river valley and, according to the hypothesis of an abundant-centre distribution, these northern populations might represent a long-standing/ancestral distribution centre. However, our results suggest that the decimated southern and central Iberian populations are more variable and structured than the northeastern ones, representing the likely vestiges of an ancestral distribution centre of the species. Phylogeographical analysis suggests that F. loscosii likely originated in southern Spain and then migrated towards the central and northeastern ranges, further supporting a Late Miocene southern-bound Mediterranean migratory way for its oriental steppe ancestors. In addition, different glacial-induced conditions affected the southern and northern steppe Iberian refugia during the Quaternary. The contrasting genetic homogeneity of the Ebro valley range populations compared to the southern Iberian ones possibly reflects more severe bottlenecks and subsequent genetic drift experienced by populations of the northern Iberia refugium during the Pleistocene, followed by successful postglacial expansion from only a few founder plants.     

Genealogy of the nuclear beta-fibrinogen locus in a highly structured lizard species: comparison with mtDNA and evidence for intragenic recombination in the hybrid zone

The study of nuclear genealogies in natural populations of nonmodel organisms is expected to provide novel insights into the evolutionary history of populations, especially when developed in the framework of well-established mtDNA phylogeographical scenarios. In the Iberian Peninsula, the endemic Schreiber's green lizard Lacerta schreiberi exhibits two highly divergent and allopatric mtDNA lineages that started to split during the late Pliocene. In this work, we performed a fine-scale analysis of the putative mtDNA contact zone together with a global analysis of the patterns of variation observed at the nuclear beta-fibrinogen intron 7 (beta-fibint7). Using a combination of DNA sequencing with single-strand conformational polymorphism (SSCP) analysis, we show that the observed genealogy at the beta-fibint7 locus reveals extensive admixture between two formerly isolated lizard populations while the two mtDNA lineages remain essentially allopatric. In addition, a private beta-fibint7 haplotype detected in the single population where both mtDNA lineages were found in sympatry is probably the result of intragenic recombination between the two more common and divergent beta-fibint7 haplotypes. Our results suggest that the progressive incorporation of nuclear genealogies in investigating the ancient demography and admixture dynamics of divergent genomes will be necessary to obtain a more comprehensive picture of the evolutionary history of organisms.     

Phylogeography of a mountain lizard species: an ancient fragmentation process mediated by riverine barriers in the Liolaemus monticola complex (Sauria: Liolaemidae)

Liolaemus monticola is a mountain lizard species, with a widespread distribution from central Chile that displays several highly polymorphic chromosomal races. Our study determined the phylogeographic structuring and relationships among three chromosomal races of L. monticola in Chile. Mitochondrial DNA (mtDNA) sequences of the cytochrome b gene were examined using the following phylogenetic methods: maximum parsimony, maximum likelihood, Bayesian inference and nested clade phylogeographic analyses (NCPAs). These methods revealed two major monophyletic clades (north and south) in the L. monticola species, with non-overlapping geographical locations separated by the Maipo and Yeso rivers (except one hybrid, from a zone of secondary contact). The NCPA showed that a past fragmentation process likely resulted in the separation of the two clades. The southern clade includes all samples of the 'Southern, 2 n  = 34' race; the northern clade is comprised of all remaining derived chromosomal races: the 'Northern, 2 n  = 38–40 and the Multiple Fission, 2 n  = 42–44' races. Our results support the hypothesis of a geographical and genetic split resulting from allopatric processes caused by riparian barriers acting over a long time period. The inferred biogeographical scenario shows that populations have moved from the south to the north using the Andean mountains as the primary corridor for dispersal.     

Molecular evidence for a species complex in the patagonian lizard Liolaemus bibronii and phylogeography of the closely related Liolaemus gracilis (Squamata: Liolaemini)

The lizard genus Liolaemus is endemic to temperate South America and includes 190 species. Liolaemus bibronii has a large geographic distribution and inhabits a great diversity of habitats, including the Monte, Steppe, and high Andean grassland environments. Liolaemus gracilis has a similar body size and shape to L. bibronii; the two are parapatrically distributed, and L. gracilis is also widely distributed. Here we use the mtDNA cytb sequence data of these two species to investigate lizard phylogeographic patterns in southern South America. L. bibronii is paraphyletic with respect to L. gracilis, Liolaemus ramirezae, Liolaemus robertmertensi and Liolaemus saxatilis; it is composed of many genetically different allopatric haploclades, some of which are reciprocally monophyletic. We also found evidence for introgression between L. bibronii and L. gracilis in the same area that introgression was hypothesized in the Liolaemus darwinii complex. We discuss the distribution of the major haploclades with inferences of their population histories, the concordance of these clades' distributions and histories with other lizard complexes studied with the same markers and methods, and taxonomic implications of these results.     

Multi-locus DNA sequence data reveal a history of deep cryptic vicariance and habitat-driven convergence in the desert night lizard Xantusia vigilis species complex (Squamata: Xantusiidae)

The lizard genus Xantusia of southwestern North America has received recent attention in relation to delimiting species. Using more than 500 lizards from 156 localities, we further test hypothesized species boundaries and clarify phylogeographical patterns, particularly in regions of potential secondary contact. We sequenced the entire mitochondrial cytochrome b gene for every lizard in the study, plus a second mitochondrial DNA (mtDNA) region and two nuclear introns for subsets of the total sample. Phylogenetic analyses of the mtDNA recover a well-resolved, novel hypothesis for species in the Xantusia vigilis complex. The nuclear DNA (nDNA) data provide independent support for the recognition of X. arizonae, X. bezyi and X. wigginsi. Differences between the respective mtDNA and nDNA topologies result from either the effects of lineage sorting or ancient introgression. Nuclear data confirm the inference that some populations of X. vigilis in northwestern Arizona converged on rock-crevice-dwelling morphology and are not X. arizonae with an introgressed X. vigilis mtDNA genome. The historical independence of ancient cryptic lineages of Xantusia in southern California is also corroborated, though limited introgression is detected. Our proposed biogeographical scenario indicates that diversification of this group was driven by vicariance beginning in the late Miocene. Additionally, Pleistocene climatical changes influenced Xantusia distribution, and the now inhospitable Colorado Desert previously supported night lizard presence. The current taxonomy of the group likely underestimates species diversity within the group, and our results collectively show that while convergence on the rock-crevice-dwelling morphology is one hallmark of Xantusia evolution, morphological stasis is paradoxically another.     

Genetic diversity and host plant preferences revealed by simple sequence repeat and mitochondrial markers in a population of the arbuscular mycorrhizal fungus Glomus intraradices

Arbuscular mycorrhizal fungi (AMF) are important symbionts of plants that improve plant nutrient acquisition and promote plant diversity. Although within-species genetic differences among AMF have been shown to differentially affect plant growth, very little is actually known about the degree of genetic diversity in AMF populations. This is largely because of difficulties in isolation and cultivation of the fungi in a clean system allowing reliable genotyping to be performed. A population of the arbuscular mycorrhizal fungus Glomus intraradices growing in an in vitro cultivation system was studied using newly developed simple sequence repeat (SSR), nuclear gene intron and mitochondrial ribosomal gene intron markers. The markers revealed a strong differentiation at the nuclear and mitochondrial level among isolates. Genotypes were nonrandomly distributed among four plots showing genetic subdivisions in the field. Meanwhile, identical genotypes were found in geographically distant locations. AMF genotypes showed significant preferences to different host plant species (Glycine max, Helianthus annuus and Allium porrum) used before the fungal in vitro culture establishment. Host plants in a field could provide a heterogeneous environment favouring certain genotypes. Such preferences may partly explain within-population patterns of genetic diversity.     

The complex history of the genus Ilex L. (Aquifoliaceae): evidence from the comparison of plastid and nuclear DNA sequences and from fossil data

 A plastid phylogeny of the genus Ilex based on three different loci (the atpB-rbcL spacer, trnL-trnF and rbcL) is compared with its nuclear phylogeny based on two different loci (the ribosomal ITS and the 5S RNA spacer). These two sets of molecular data are then compared to geographical and temporal data from the fossil record. The plastid phylogeny is strongly correlated with the geographic distribution of extant species. However, the nuclear phylogeny is strongly incongruent with the plastid phylogeny, suggesting frequent interlineage hybridizations. Moreover, the comparison of the ribosomal ITS tree and the 5S RNA spacer tree indicates also possible lineage sorting. Particularly interesting is the finding of two different Ilex lineages in the plastid American clade showing different biogeographic patterns in South America. One of them has a simple North American/South American biogeographical relationship. The other has complex biogeographical relationships, some species showing direct Asian/South American biogeographical relationships. During its history, the genus Ilex probably experienced frequent lineage sorting and interlineage hybridization with subsequent nuclear or cytoplasmic introgression, making the study of its history very complex. Received September 24, 2001; accepted August 19, 2002 Published online: November 28, 2002 Addresses of the authors: Jean-Fran?ois Manen (e-mail: manen@cjb.ville-ge.ch), Yamama Naciri-Graven, Conservatoire et Jardin Botaniques, Impératrice 1, CH-1292 Chambésy/Genève, Switzerland. Michael C. Boulter, Palaeobiology Research Unit, University of East London, Romford Road, London E15 4LZ, UK.