Bayesian phylogeographic inferences reveal contrasting colonization dynamics among European groundwater isopods.

The potentially important role of northern microrefugia during postglacial dispersal is challenging the view of southern Europe as a refuge and source area of European biota. In groundwaters, large geographic ranges of presumably good dispersers are increasingly suspected to consist of assemblages of cryptic species with narrow ranges. Moreover, a large species range, even when confirmed by molecular evidence, tells us little about the spatiotemporal dynamics of dispersal. Here, we used phylogenetic inferences, species delineation methods and Bayesian phylogeographic diffusion models to test for the likelihood of postglacial colonization from distant refugia among five morphospecies of Proasellus (Isopoda, Asellidae). All morphospecies except one were monophyletic, but they comprised a total of 15-17 cryptic species. Three cryptic species retained ranges that spanned a distance >650 km, similar to that of the nominal morphospecies. Bayesian diffusion models based on mitochondrial markers revealed considerable spatiotemporal heterogeneity in dispersal rates, suggesting that short-time dispersal windows were instrumental in shaping species ranges. Only one species was found to experience a recent, presumably postglacial, range expansion. The Jura and Alpine foothills probably played a major role in maintaining diversity within Proasellus in northern regions by acting both as diversification hotspots and Pleistocene refugia. Gaining insight into the spatiotemporal heterogeneity of dispersal rates revealed contrasting colonization dynamics among species that were not consistent with a global postglacial colonization of Europe from distant refugia.

Thermal tolerance breadths among groundwater crustaceans living in a thermally constant environment.

The climate variability hypothesis assumes that the thermal tolerance breadth of a species is primarily determined by temperature variations experienced in its environment. If so, aquatic invertebrates living in thermally buffered environments would be expected to exhibit narrow thermal tolerance breadths (stenothermy). We tested this prediction by studying the thermal physiology of three isopods (Asellidae, Proasellus) colonizing groundwater habitats characterized by an annual temperature amplitude of less than 1°C. The species responses to temperature variation were assessed in the laboratory using five physiological variables: survival, locomotor activity, aerobic respiration, immune defense and concentrations of total free amino acids and sugars. The three species exhibited contrasted thermal physiologies, although all variables were not equally informative. In accordance with the climate variability hypothesis, two species were extremely sensitive even to moderate changes in temperature (2°C) below and above their habitat temperature. In contrast, the third species exhibited a surprisingly high thermal tolerance breadth (11°C). Differences in response to temperature variation among Proasellus species indicated that their thermal physiology was not solely shaped by the current temperature seasonality in their natural habitats. More particularly, recent gene flow among populations living in thermally constant yet contrasted habitats might explain the occurrence of eurytherm species in thermally buffered environments.

Testing dispersal and cryptic diversity in a widely distributed groundwater amphipod (Niphargus rhenorhodanensis).

Theories about colonization and evolution in groundwater have assumed that the fragmented structure of groundwater strongly limits dispersal. The high number of endemic and allopatric species in groundwater supports this hypothesis, but the occurrence of widely distributed groundwater taxa calls into question its universality. These widely distributed taxa might also be sets of cryptic species because extreme conditions of life in groundwater promote cryptic diversity by inducing convergent morphological evolution. Niphargus rhenorhodanensis is a widely distributed and ubiquitous groundwater amphipod which supposedly colonized the Alps after Quaternary glaciations. We tested the dispersal and the cryptic species hypotheses within this species using a phylogeographic approach based on two mitochondrial genes (COI and 16S) and a nuclear gene (28S). Results support the view that poor dispersal is a main evolutionary factor in groundwater. All genes independently supported the existence of numerous cryptic and mostly allopatric units within N. rhenorhodanensis, indicating that its apparently wide distribution range is an artefact generated by cryptic diversity. We reject the hypothesis of a recent and global colonization of the Alps and argue that some N. rhenorhodanensis lineages probably survived glaciations near or within the Alps.

Relationship between morphological taxonomy and molecular divergence within Crustacea: proposal of a molecular threshold to help species delimitation.

With today's technology for production of molecular sequences, DNA taxonomy and barcoding arose as a new tool for evolutionary biology and ecology. However, their validities still need to be empirically evaluated. Of most importance is the strength of the correlation between morphological taxonomy and molecular divergence and the possibility to define some molecular thresholds. Here, we report measurements of this correlation for two mitochondrial genes (COI and 16S rRNA) within the sub-phylum Crustacea. Perl scripts were developed to ensure objectivity, reproducibility, and exhaustiveness of our tests. Our analysis reveals a general correlation between molecular divergence and taxonomy. This correlation is particularly high for shallow taxonomic levels allowing us to propose a COI universal crustacean threshold to help species delimitation. At higher taxonomic levels this correlation decreases, particularly when comparing different families. Those results plead for DNA use in taxonomy and suggest an operational method to help crustacean species delimitation that is linked to the phylogenetic species definition. This pragmatic tool is expected to fine tune the present classification, and not, as some would have believed, to tear it apart.

Phylogeography of a subterranean amphipod reveals cryptic diversity and dynamic evolution in extreme environments.

Extreme conditions in subsurface are suspected to be responsible for morphological convergences, and so to bias biodiversity assessment. Subterranean organisms are also considered as having poor dispersal abilities that in turn generate a large number of endemic species when habitat is fragmented. Here we test these general hypotheses using the subterranean amphipod Niphargus virei. All our phylogenetic analyses (Bayesian, maximum likelihood and distance), based on two independent genes (28S and COI), revealed the same tripartite structure. N. virei populations from Benelux, Jura region and the rest of France appeared as independent evolutionary units. Molecular rates estimated via global or Bayesian relaxed clock suggest that this split is at least 13 million years old and accredit the cryptic diversity hypothesis. Moreover, the geographical distribution of these lineages showed some evidence of recent dispersal through apparent vicariant barrier. In consequence, we argue that future analyses of evolution and biogeography in subsurface, or more generally in extreme environments, should consider dispersal ability as an evolving trait and morphology as a potentially biased marker.

Phylogeography of a nematode (Heligmosomoides polygyrus) in the western Palearctic region: persistence of northern cryptic populations during ice ages?

This study establishes the continental phylogeographical pattern of a European nematode, Heligmosomoides polygyrus (Dujardin, 1845; Heligmosomoidea). We sequenced 687 base pairs of the mitochondrial DNA (mtDNA) cyt b gene for 136 individuals collected in 22 localities. The results revealed that H. polygyrus populations are separated into five major units corresponding to the Italian, northern European (Denmark and Ireland), Iberian, western European, and Balkan populations. Different subclades were also observed within the first two groups. Based on the rate of molecular evolution of H. polygyrus cyt b gene-estimated to 3.5%-3.7% divergence per million years (Myr) in a previous study--the isolation time of the five clades was estimated between 2.5 +/- 0.24 and 1.5 +/- 0.23 million years ago. Moreover, H. polygyrus presents a higher genetic variability in the Mediterranean peninsulas as compared to northwestern Europe, highlighting the role of these regions as refuge areas. Like its specific host, the wood mouse Apodemus sylvaticus, H. polygyrus' pattern of postglacial recolonization of northwestern Europe was initiated from Iberian populations, while Italian and Balkan populations did not expand to the north. The results also suggest the existence of forested and temperate refuges in the southern British Isles during the Quaternary. Finally, the genetic diversity as well as the level of genetic divergence between the lineages of H. polygyrus are compared to those observed in other vertebrate and invertebrate phylogeographical studies: the existence of highly differentiated lineages in H. polygyrus (5%-10% of genetic divergence) highlights that the effects of Pleistocene climate changes on free-living organisms are also reflected in their obligate parasites.

How big is the iceberg of which organellar genes in nuclear genomes are but the tip?

As more and more complete bacterial and archaeal genome sequences become available, the role of lateral gene transfer (LGT) in shaping them becomes more and more clear. Over the long term, it may be the dominant force, affecting most genes in most prokaryotes. We review the history of LGT, suggesting reasons why its prevalence and impact were so long dismissed. We discuss various methods purporting to measure the extent of LGT, and evidence for and against the notion that there is a core of never-exchanged genes shared by all genomes, from which we can deduce the "true" organismal tree. We also consider evidence for, and implications of, LGT between prokaryotes and phagocytic eukaryotes.

Resolution of the early placental mammal radiation using Bayesian phylogenetics.

Molecular phylogenetic studies have resolved placental mammals into four major groups, but have not established the full hierarchy of interordinal relationships, including the position of the root. The latter is critical for understanding the early biogeographic history of placentals. We investigated placental phylogeny using Bayesian and maximum-likelihood methods and a 16.4-kilobase molecular data set. Interordinal relationships are almost entirely resolved. The basal split is between Afrotheria and other placentals, at about 103 million years, and may be accounted for by the separation of South America and Africa in the Cretaceous. Crown-group Eutheria may have their most recent common ancestry in the Southern Hemisphere (Gondwana).

Compositional heterogeneity within and among isochores in mammalian genomes. I. CsCl and sequence analyses.

GC level distributions of a species' nuclear genome, or of its compositional fractions, encode key information on structural and functional properties of the genome and on its evolution. They can be calculated either from absorbance profiles of the DNA in CsCl density gradients at sedimentation equilibrium, or by scanning long contigs of largely sequenced genomes. In the present study, we address the quantitative characterization of the compositional heterogeneity of genomes, as measured by the GC distributions of fixed-length fragments. Special attention is given to mammalian genomes, since their compartmentalization into isochores implies two levels of heterogeneity, intra-isochore (local) and inter-isochore (global). This partitioning is a natural one, since large-scale compositional properties vary much more among isochores than within them. Intra-isochore GC distributions become roughly Gaussian for long fragments, and their standard deviations decrease only slowly with increasing fragment length, unlike random sequences. This effect can be explained by 'long-range' correlations, often overlooked, that are present along isochores.

Universal trees based on large combined protein sequence data sets.

Universal trees of life based on small-subunit (SSU) ribosomal RNA (rRNA) support the separate mono/holophyly of the domains Archaea (archaebacteria), Bacteria (eubacteria) and Eucarya (eukaryotes) and the placement of extreme thermophiles at the base of the Bacteria. The concept of universal tree reconstruction recently has been upset by protein trees that show intermixing of species from different domains. Such tree topologies have been attributed to either extensive horizontal gene transfer or degradation of phylogenetic signals because of saturation for amino acid substitutions. Here we use large combined alignments of 23 orthologous proteins conserved across 45 species from all domains to construct highly robust universal trees. Although individual protein trees are variable in their support of domain integrity, trees based on combined protein data sets strongly support separate monophyletic domains. Within the Bacteria, we placed spirochaetes as the earliest derived bacterial group. However, elimination from the combined protein alignment of nine protein data sets, which were likely candidates for horizontal gene transfer, resulted in trees showing thermophiles as the earliest evolved bacterial lineage. Thus, combined protein universal trees are highly congruent with SSU rRNA trees in their strong support for the separate monophyly of domains as well as the early evolution of thermophilic Bacteria.

Parallel adaptive radiations in two major clades of placental mammals.

Higher level relationships among placental mammals, as well as the historical biogeography and morphological diversification of this group, remain unclear. Here we analyse independent molecular data sets, having aligned lengths of DNA of 5,708 and 2,947 base pairs, respectively, for all orders of placental mammals. Phylogenetic analyses resolve placental orders into four groups: Xenarthra, Afrotheria, Laurasiatheria, and Euarchonta plus Glires. The first three groups are consistently monophyletic with different methods of analysis. Euarchonta plus Glires is monophyletic or paraphyletic depending on the phylogenetic method. A unique nine-base-pair deletion in exon 11 of the BRCA1 gene provides additional support for the monophyly of Afrotheria, which includes proboscideans, sirenians, hyracoids, tubulidentates, macroscelideans, chrysochlorids and tenrecids. Laurasiatheria contains cetartiodactyls, perissodactyls, carnivores, pangolins, bats and eulipotyphlan insectivores. Parallel adaptive radiations have occurred within Laurasiatheria and Afrotheria. In each group, there are aquatic, ungulate and insectivore-like forms.

Mitochondrial versus nuclear gene sequences in deep-level mammalian phylogeny reconstruction.

Both mitochondrial and nuclear gene sequences have been employed in efforts to reconstruct deep-level phylogenetic relationships. A fundamental question in molecular systematics concerns the efficacy of different types of sequences in recovering clades at different taxonomic levels. We compared the performance of four mitochondrial data sets (cytochrome b, cytochrome oxidase II, NADH dehydrogenase subunit I, 12S rRNA-tRNA-16S rRNA) and eight nuclear data sets (exonic regions of alpha-2B adrenergic receptor, aquaporin, ss-casein, gamma-fibrinogen, interphotoreceptor retinoid binding protein, kappa-casein, protamine, von Willebrand Factor) in recovering deep-level mammalian clades. We employed parsimony and minimum-evolution with a variety of distance corrections for superimposed substitutions. In 32 different pairwise comparisons between these mitochondrial and nuclear data sets, we used the maximum set of overlapping taxa. In each case, the variable-length bootstrap was used to resample at the size of the smaller data set. The nuclear exons consistently performed better than mitochondrial protein and rRNA-tRNA coding genes on a per-residue basis in recovering benchmark clades. We also concatenated nuclear genes for overlapping taxa and made comparisons with concatenated mitochondrial protein-coding genes from complete mitochondrial genomes. The variable-length bootstrap was used to score the recovery of benchmark clades as a function of the number of resampled base pairs. In every case, the nuclear concatenations were more efficient than the mitochondrial concatenations in recovering benchmark clades. Among genes included in our study, the nuclear genes were much less affected by superimposed substitutions. Nuclear genes having appropriate rates of substitution should receive strong consideration in efforts to reconstruct deep-level phylogenetic relationships.

Diversity and phylogenetic implications of CsCl profiles from rodent DNAs.

Buoyant density profiles of high-molecular-weight DNAs sedimented in CsCl gradients, i.e., compositional distributions of 50- to 100-kb genomic fragments, have revealed a clear difference between the murids so far studied and most other mammals, including other rodents. Sequence analyses have revealed other, related, compositional differences between murids and nonmurids. In the present study, we obtained CsCl profiles of 17 rodent species representing 13 families. The modal buoyant densities obtained for rodents span the full range of values observed in other eutherians. More remarkably, the skewness (asymmetry, mean - modal buoyant density) of the rodent profiles extends to values well below those of other eutherians. Scatterplots of these and related CsCl profile parameters show groups of rodent families that agree largely with established rodent taxonomy, in particular with the monophyly of the Geomyoidea superfamily and the position of the Dipodidae family within the Myomorpha. In contrast, while confirming and extending previously reported differences between the profiles of Myomorpha and those of other rodents, the CsCl data question a traditional hypothesis positing Gliridae within Myomorpha, as does the recently sequenced mitochondrial genome of dormouse. Analysis of CsCl profiles is presented here as a rapid, robust method for exploring rodent and other vertebrate systematics.