Phylogenetic analysis of the New Guinean forest wallabies, Dorcopsis and Dorcopsulus reveals cryptic divergent lineages impacted by highland and lowland barriers.

Phylogenetic analysis of the tree-kangaroos (Dendrolagus) reveals multiple divergent lineages within New Guinea

Reappraising adaptive radiation

This thesis describes the global molecular population structure of two shorebird species, in particular of the dunlin, Calidris alpina, by means of comparative sequence analysis of the most variable part of the mitochondrial DNA (mtDNA) genome. There are several reasons why mtDNA is the molecule of choice to probe the recent evolutionary history of a species. Most importantly, mtDNA accumulates substitutions at a high average rate that permits the tracing of genealogies within the time frame of speciation. The population structure of shorebirds, like that of arctic- breeding waterfowl (Ploeger, 1968), must have been influenced dramatically by the Pleistocene glaciations (mainly during the last one million years). The fastest evolving part of the mtDNA genome, the non-coding control region, offers sufficient genetic resolution to reveal differentiation of such recent origin. The typical mode of maternal inheritance, the absence of recombination, and the presumed neutrality of substitutions, are characteristics that add to the suitability of mtDNA for the construction of robust phylogenies ( Chapter 1 ).Cloning and sequencing of the control region of a turnstone ( Arenaria interpres ) facilitated subsequent amplification and direct sequencing of the homologous region in other turnstones, and dunlins as well. Comparison of this approximately 1200 basepairs (bp) region for several turnstones, dunlins and a chicken ( Gallusdomesticus ) revealed the presence of differentially evolving sequence blocks within the control region. Both shorebird species contain an AC repetitive sequence at the 3' end of the light strand, varying in size (around 100 bp) and composition between individuals. Sequence identity is highest in the central part of the control region, similar to the conservation of this part in other vertebrate species. Most single nucleotide substitutions, as well as insertions and deletions, are restricted to two segments, notably at the beginning and near the end of the control region. Overall, the organization of the avian control region resembles its human counterpart. Sequence comparison of the larger variable segment at the beginning of the control region (CR I) for worldwide samples of 25 tumstones and 25 dunlins demonstrated the utility of this region for the detection of intraspecific differentiation. The turnstone reveals few differences worldwide and identity of clones from distant regions, whereas the dunlin reveals divergent clusters of genotypes that are geographically restricted. It is concluded that the turnstone has been confined historically to one Pleistocene refugium, from which it has dispersed around the world to establish its current biogeography. The dunlin, on the other hand, became divided into several isolated populations during the Pleistocene and has retained a significant amount of intraspecific genetic diversity until the present ( Chapter 2 ).This remarkable difference in population genetic structure between the two shorebird species may be explained by their differing ecologies. The turnstone is a high arctic breeder, and depends mainly on cold tundra habitat, whereas the dunlin breeds mostly in the lower arctic and even in temperate zones. Cold tundra habitat may have disappeared almost entirely during the last interglacial (Eemian: around 125,000 years ago) that was characterized by high temperature peaks (Anklin et al., 1993). A very similar lack of global mtDNA differentiation has been observed in the knot ( Calidris canutus ) , another shorebird that is a typical breeder of the high arctic tundra (A.J. Baker and T. Piersma, personal communication).Representative samples of dunlin populations from four major regions in the world were analysed for 910 bases of mtDNA sequence from the control region and the cytochrome b gene. The regions comprised the Pacific coast of North America, the Atlantic coast of North America, the Atlantic coast of Europe and arctic central Siberia. Sequence comparison of the three amplified DNA fragments showed that most substitutions are located in the CR I fragment, and substantially less in another control region segment (CR II) and part of the coding sequence of the cytochrome b gene. The 50 substitutions that were found together defined 35 different genotypes. A genealogical tree relating these genotypes revealed five major clusters. Each cluster has high geographic specificity. The cluster containing the most divergent sequences is present along the Atlantic coast of North America and represents the dunlin population breeding in arctic central Canada. Two clusters of genotypes are located principally in western Europe and central Siberia. Evidence for a low level of gene flow between these latter two populations was provided by three individuals whose genotypes suggested they were immigrants. Two other clusters are found along the Pacific coast of North America. Whereas dunlins from southern Alaska assorted to one cluster, dunlins from the southerly wintering population revealed genotypes of both clusters.The genetic divergence of these major mtDNA lineages can be dated to the late Pleistocene based on a molecular clock for the control region of birds. Genotypic diversity within the population samples is extensive and the calculated long term effective (female) population sizes argue against strong historical bottlenecks. Overall, there is a negative correlation of mtDNA variation and previously defined morphometric variation in dunlins. This discordance is induced largely by the morphometrical similarity of the genetically most divergent populations from both North American coasts.A plausible scenario for the genetic divergence of the major dunlin lineages is the ancestral fragmentation of populations over tundra refugia, that were created by the extensive glaciations of the northern hemisphere during the Pleistocene. Prolonged isolation of populations of reduced size increased the effect of genetic drift and this may have led to the observed mtDNA monophyly. The different lineages continuously diverged by the process of mutation. This ancient subdivision has been retained after retreat of the icesheets, most likely as a result of the strong site-fidelity of dunlins to their breeding ground. Dunlin populations could thus not become homogenized genetically because gene flow is not extensive enough between them ( Chapter 3 ).The generally observed lack of genetic population differentiation in birds, in contrast to other vertebrate groups, has been interpreted as a sign of panmixia, caused by the high dispersive capabilities of birds (Cooke and Buckley, 1987). This conclusion is mainly based on the analysis of allozyme data, but more recently also on the analysis of mtDNA restriction polymorphism (Ball et al. 1988). However, allozymes are relatively conserved genetic markers, and thus do not provide resolution at shorter time scales of evolution. The dunlin is not exceptional in its degree of natal philopatry. Rather, the findings in dunlin indicate that population structure in this species is of recent evolutionary origin, that could be detected by virtue of the high rate of nucleotide substitution in the selected mtDNA sequences. In addition, the global coverage of this study is beyond the geographical scope of most avian studies, and thus had a better perspective for detecting major phylogenetic splits within a species.To elucidate the geographical distribution of mtDNA lineages over the circumpolar breeding range of the dunlin (intraspecific phylogeography: Avise et al., 1987), many additional samples from interspersed populations were analysed for both control region segments. No additional major lineages turned up among 155 breeding dunlins, but one lineage previously found among wintering dunlins in western North America could be located to the eastern Siberian breeding ground. Samples from breeding birds in Greenland, Iceland, the Baltic, southern Norway, northern Norway, and western Siberia revealed genotypes that cluster together in the major European lineage. The central Siberian lineage was found in northern Russia from the Lena river delta in the east, across the Taymyr peninsula in the middle, to the Yamal Peninsula in the west. A few of these 'central Siberian' genotypes were retrieved from dunlins breeding in Norway and eastern Siberia, indicating a restricted amount of gene flow between these populations. A zone of geographical overlap between the European and the central Siberian phylogeographic groups is present at the Yamal peninsula, where equal numbers of dunlins assorted to these respective major lineages. Dunlins captured in northern, western and southern Alaska all belonged to the same mtDNA lineage and thus constitute one genetic population.A large fraction of the total mtDNA variance in dunlins is distributed between the five major phylogeographic regions (76%). Extensive diversity also exists, however, among the individuals of a local population. This is induced by the high rate of substitution in CR I and renders the traditional population genetic correlation measure GST less applicable. Time estimates for the corrected sequence divergence of each phylogeographic group on the basis of a molecular clock indicate a repeated fragmentation of populations, and coincide well with the onset of glacial periods. The ancestral population in central Canada may have been separated from all other dunlin populations for over 200,000 years.Phylogeographic groups can be correlated to the global geography of morphometrically defined subspecies in the dunlin. Whereas several disputed subspecies gain support from the genetic data (i.e. C . a. hudsonia in central Canada and C . a. centralis in central Siberia), other subspecies merge into the same phylogeographic group. No major phylogenetic divisions are apparent among the morphometrically dissimilar populations in north-eastern Greenland, Iceland, the Baltic Sea, and Norway (recognized until now as three to four different subspecies). Gauged by the depth of the other phylogenetic splits in dunlins, they can jointly be referred to as C . a. alpina, Similarly, the dunlins from northern and southern Alaska can be merged under C. a. pacifica.Detailed comparison of populations in Europe reveals a developing geographic specificity of slightly divergent genotypes of the European genetic cluster. Intermediate genotypic correlation measures between locales are supported by measures of restricted gene flow, particularly for the Icelandic and Baltic populations. The genetic differences between European populations have likely evolved after retreat of the ice sheets, approximately 10,000 years ago. Post-Pleistocene colonization of newly exposed breeding grounds combined with the habit of strong site-fidelity can explain the population differentiation within Europe ( Chapter 4 ).It is thus revealed how morphology lacks an evolutionary perspective in the determination of intraspecific taxonomy. For the dunlin, a parallel morphological evolution of genetically divergent populations, as well as the opposing process of morphological divergence of evolutionary closely related populations, is observed. Morphometric characters employed in intraspecific avian taxonomy are suffering from homoplasy, either as a result of character plasticity and environmental induction (James, 1983), or because of very high mutation rates and strong directive selection acting on phenotypes (Turelli et al., 1988). Because morphometrically different dunlin populations are often mixed outside the short breeding season, environmental induction of morphology seems unlikely, although this possibility remains to be investigated. Although the concept of a molecular clock is debatable, general agreement exists as to the neutrality of most nucleotide substitutions in DNA and the cumulative character of the mutation process. On the basis of statistically reliable amounts of substitution, the phylogenetic branching order of intraspecific lineages can therefore be inferred with precision. This applies even more so to the non-coding mitochondrial control region. Although the oldest split in the dunlin mtDNA phylogeny is dated at approximately 200,000 years ago, the species itself is probably much older, in the range of a million years (Baker, 1992). This time discrepancy could imply that many populations have been transient in the intraspecific history of the dunlin. Only populations that radiated during the later part of the Pleistocene have survived until the present. The observed genetic differentiation within Europe thus represents the shallow branch tips in the phylogenetic tree of dunlins. The mtDNA assays suggest that measures to protect declining breeding populations in Europe, like the dunlins breeding around the Baltic Sea, cannot be argued for on the basis of a subspecific status of these populations. Rather, subspecies should be reserved for groups that represent a major source of intraspecific genetic diversity.Limited numbers of migratory and wintering dunlins from around the world were sequenced for both control region segments to trace lineages away from the breeding grounds. The mtDNA lineages detected in these birds were identical to those already known from the breeding grounds. Mixtures of major mtDNA lineages are present in different regions of the world. Samples of dunlins from both sides of the Pacific Ocean comprised two lineages that were found separately on the breeding grounds in eastern Siberia and Alaska. The two lineages identified in population samples from the western Palearctic (western Europe and western Asia) correspond to those present on the breeding grounds in Europe and central Siberia. Overall, it appears that dunlin populations breeding in different circumpolar regions occupy overlapping areas on migration and in winter through much of their southern range. Dunlins wintering along the North American west coast can be assigned to the Alaskan as well as to the eastern Siberian breeding grounds. In parallel, it is likely that dunlins migrating along the eastern Pacific coast of Asia originate from northern Siberia as well as from Alaska. Because the Alaskan genotype found in some eastern Asian dunlins occurs in high frequency only in birds breeding in northern Alaska, it appears that the northern and southern Alaskan populations migrate in different directions. The allocation of individual dunlins to their breeding population on the basis of their mtDNA genotype, can only be certain for those lineages that are geographically separated on the breeding grounds. Because of the limited gene flow between the European and central Siberian breeding populations, uncertainty exists in the population assignment of western Palearctic dunlins. Additional characters such as body mass and time of passage during spring migration or the presence of a particular moult pattern during fall migration can be instructive for the discrimination of dunlins of Siberian origin at European staging posts. These characters seem to be correlated with the possession of a central Siberian genotype by individual dunlins. Larger sample sizes remain to be tested, however, to obtain a better estimate of the diagnostic value of each of these methods ( Chapter 5 ).It is not clear what underlies the different genetic compositions of dunlin populations at the breeding grounds versus the wintering regions. Dunlins probably have also migrated during the Pleistocene, under the influence of seasonal temperature fluctuations. Their wintering quarters may have been fragmented by extensive glaciations, just as the breeding grounds were. Sharing of wintering grounds would likely have opened a route for exchange of individuals between the different populations. Such gene flow would have hampered the process of stochastic lineage sorting under the influence of genetic drift. The five major flyways that are recognized for dunlins around the world today, may still partially reflect the separate ranges that were occupied by populations throughout the last glacial period. Only the population migrating along the Atlantic coast of North America has remained geographically fully separate. What causes the mixing of the other populations outside their breeding range? The question might simply be reversed. Why does the subdivided population structure still exist over the northern breeding range? This can be explained by the imprinting of natal site-fidelity in the juvenile dunlin. Juvenile dunlins leave the breeding grounds indepently of the adult birds in a rough general direction, that may also be imprinted. Their exact direction of southward migration, however, is likely a learned behaviour and this is more prone to error or change (Rösner, 1990).This thesis demonstrates the utility of mtDNA in elucidating the population genetic structure of a bird species. By sequencing the most variable part of the mtDNA genome the major gene pools within a species can be detected together with their phylogenetic relationships. On this basis important insights into the evolutionary history and also the life history of the dunlin Calidris alpina were gained. This method should prove highly valuable not only in the detection and preservation of genetic diversity in dunlins but also in other (endangered) animal species.

Accurate species identification and assessment of species diversity are essential for studies on phylogeny and phylogeography, adaptation and ecological function. The development of molecular methods triggered the discovery of cryptic species, i.e., genetically distinct lineages in morphologically undifferentiated species. Collembola (Arthropoda, Hexapoda) are one of the most numerous soil-living animals occurring in virtually all terrestrial ecosystems and habitats. Species delimitation is particularly difficult in Collembola due to considerable morphological conservatism, and many Collembola species comprise high genetic divergence and high cryptic species diversity. DNA-based methods provide useful tools for species delimitation, phylogenetic reconstruction, and lineage divergence time estimation. By analyzing two mitochondrial and two nuclear genes from three morphospecies of the European Lepidocyrtus lanuginosus species group (Collembola: Entomobryidae) from different geographic regions of Europe, this thesis focuses on exploring cryptic species / lineages diversity, their phylogeny, and the effects of historical geographic and climatic changes on the divergence and distribution of this species group in Europe.
\nIn chapter II, I investigated phylogenetic relationships and genetic distances between populations of the morphospecies L. lanuginosus (Gmelin, 1788) from three different habitats in Central Europe, i.e. arable fields, grasslands, and forests replicated at six locations. Geographic distances between sampling locations were considerably larger than between habitat types. All four genes clearly separated the morphospecies L. lanuginosus into three major genetic lineages, with one of these lineages being close to Lepidocyrtus cyaneus Tullberg, 1871. The three lineages were genetically as distant to each other as well separated species. Selective colonization of the three habitats by these lineages indicate that they are sorted by habitats: one lineage was common and occurred in each of the three habitat types but preferentially in arable land; the second was restricted to forest; the third, although rare, preferentially occurred in grassland. The results indicate that genetic markers are a reliable and fast method to detect cryptic species, which may facilitate taxonomic research on Collembola species and the identification of possible species-specific morphological characters.
\nIn Chapter III, I delimited species boundaries of the L. lanuginosus species group sampled across Central and Southern Europe (north and south of the Alps) by utilizing three DNA-based methods, ABGD, PTP, and BPP. Species diversity delimited by morphology was compared with that delimited by genes. Three methods based on mitochondrial COI and COII congruently identified ten and nine distinct genetic lineages in the morphospecies L. cyaneus and L. lanuginosus, respectively. ABGD delimitated species barcoding gaps with K2P distances of 0.055–0.095 and 0.06–0.115 for COI and COII, respectively, within the species group. EF1-α separated 89% of these lineages, showing a higher resolution than 28S rDNA D1–2 in distinguishing closely related genetic lineages of the species group. The phylogenetic analysis based on the four genes showed that both morphospecies L. cyaneus and L. lanuginosus are polyphyletic, suggesting that body color is insufficient for delimiting morphospecies and lineages in this Collembola species group. This study challenges the current morphology-based species delimitation in the L. lanuginosus species group and suggests that molecular approaches are needed for accurate determination of Collembola species in both taxonomic and ecological studies. Overall, the results suggest that wide geographic sampling combined with molecular phylogenetic approaches is necessary to delimit species, understand the full range of cryptic diversity, and analyze phylogenetic relationships in Collembola.
\nIn Chapter IV, I studied the phylogeography of 18 lineages of the L. lanuginosus species group using a multi-locus molecular approach. The genetic diversity and population structure of all lineages were analyzed using COII, while all four above-mentioned genes were concatenated to infer the phylogeographic origin of these lineages. Results showed that the 18 lineages did not overlap in their distribution ranges in Central, Southern, and Southeastern Europe, suggesting high genetic structure and limited gene flow between these three regions. The major lineages diverged in the Late Miocene and Pliocene (17–2.59 million years ago, Mya), i.e. before Quaternary ice ages, indicating that distinct lineages survived in multiple refugia in each sampling region during Quaternary glacial periods. The genetic structure of the 18 lineages of the group supported a model of sequential allopatric diversification within each sampling region. Further, three distinct lineages which diverged during the Pleistocene and Holocene were widely distributed across Central Europe, suggesting that glacial cycles in the Quaternary affected the spread of these lineages. Identical haplotypes of both of these lineages, occurring in localities hundreds of kilometers apart, suggest recent human-mediated dispersal across Central Europe. These results indicate that distribution patterns of Collembola in Europe are more complex than previously assumed.
\nBy utilizing DNA-based analyses, my thesis highlights a novel view of the diversity, ecology, phylogeny, and phylogeography of the three morphospecies of the European L. lanuginosus species group. DNA-based analyses rejected the three-species hypothesis based on the morphological species concept and the monophyly of each species based on the phylogenetic species concept. This suggests that historical geographic and climatic changes dating to the late Miocene as well as recent human-mediated dispersal caused the lineage divergence and shaped the present-day distribution of this species group. Environmental factors other than geographic distances likely impede gene flow among lineages. Overall, the results of this thesis suggest that soil animals likely experienced different and more complex evolutionary forces than aboveground animals and plants in shaping their genetic diversification and biogeographic distribution. Future studies need to explore the physiological characters responsible for habitat sorting of cryptic species / lineages of the L. lanuginosus species group, and global scale phylogeographic studies on a number of Collembola species are needed for a deeper understanding of the dispersal, speciation, and evolution of Collembola and of soil animals in general.

Background. Australian scorpions have received far less attention from researchers than their overseas counterparts. Here we provide the first insight into the molecular variation and evolutionary history of the endemic Australian scorpion Urodacus yaschenkoi. Also known as the inland robust scorpion, it is widely distributed throughout arid zones of the continent and is emerging as a model organism in biomedical research due to the chemical nature of its venom. Methods. We employed Bayesian Inference (BI) methods for the phylogenetic reconstructions and divergence dating among lineages, using unique haplotype sequences from two mitochondrial loci (COXI, 16S) and one nuclear locus (28S). We also implemented two DNA taxonomy approaches (GMYC and PTP/dPTP) to evaluate the presence of cryptic species. Linear Discriminant Analysis was used to test whether the linear combination of 21 variables (ratios of morphological measurements) can predict individual’s membership to a putative species. Results. Genetic and morphological data suggest that U. yaschenkoi is a species complex. High statistical support for the monophyly of several divergent lineages was found both at the mitochondrial loci and at a nuclear locus. The extent of mitochondrial divergence between these lineages exceeds estimates of interspecific divergence reported for other scorpion groups. The GMYC model and the PTP/bPTP approach identified major lineages and several sub-lineages as putative species. Ratios of several traits that approximate body shape had a strong predictive power (83–100%) in discriminating two major molecular lineages. A time-calibrated phylogeny dates the early divergence at the onset of continental-wide aridification in late Miocene and Pliocene, with finer-scale phylogeographic patterns emerging during the Pleistocene. This structuring dynamics is congruent with the diversification history of other fauna of the Australian arid zones. Discussion. Our results indicate that the taxonomic status of U. yaschenkoi requires revision, and we provide recommendations for such future efforts. A complex evolutionary history and extensive diversity highlights the importance of conserving U. yaschenkoi populations from different Australian arid zones in order to preserve patterns of endemism and evolutionary potential.

BackgroundAustralian scorpions have received far less attention from researchers than their overseas counterparts. Here we provide the first insight into the molecular variation and evolutionary history of the endemic Australian scorpion Urodacus yaschenkoi. Also known as the inland robust scorpion, it is widely distributed throughout arid zones of the continent and is emerging as a model organism in biomedical research due to the chemical nature of its venom.MethodsWe employed Bayesian Inference (BI) methods for the phylogenetic reconstructions and divergence dating among lineages, using unique haplotype sequences from two mitochondrial loci (COXI, 16S) and one nuclear locus (28S). We also implemented two DNA taxonomy approaches (GMYC and PTP/dPTP) to evaluate the presence of cryptic species. Linear Discriminant Analysis was used to test whether the linear combination of 21 variables (ratios of morphological measurements) can predict individual’s membership to a putative species.ResultsGenetic and morphological data suggest that U. yaschenkoi is a species complex. High statistical support for the monophyly of several divergent lineages was found both at the mitochondrial loci and at a nuclear locus. The extent of mitochondrial divergence between these lineages exceeds estimates of interspecific divergence reported for other scorpion groups. The GMYC model and the PTP/bPTP approach identified major lineages and several sub-lineages as putative species. Ratios of several traits that approximate body shape had a strong predictive power (83–100%) in discriminating two major molecular lineages. A time-calibrated phylogeny dates the early divergence at the onset of continental-wide aridification in late Miocene and Pliocene, with finer-scale phylogeographic patterns emerging during the Pleistocene. This structuring dynamics is congruent with the diversification history of other fauna of the Australian arid zones.DiscussionOur results indicate that the taxonomic status of U. yaschenkoi requires revision, and we provide recommendations for such future efforts. A complex evolutionary history and extensive diversity highlights the importance of conserving U. yaschenkoi populations from different Australian arid zones in order to preserve patterns of endemism and evolutionary potential.

The California Floristic Province contains numerous ecological regions and a complex geological and geographical history that make it one of the worlds biodiversity hotspots. A number of wide-ranging taxa span across these regions and show complex patterns of dispersal, vicariance and lineage diversification, making localized small ranged species with lower levels of vagility essential to understanding the overall region. Here, we investigate the biogeography and population structure of the California Giant Salamander (Dicamptodon ensatus) (Eschscholtz 1833), an endemic species localized to a narrow coastal region between two areas of biological significance in the California Floristic Province, the North Coast Divide and Monterey Bay. We sequenced one mtDNA fragment (control region) for 133 individuals and a subset of 38 individuals for the anonymous nuclear locus E16C7. We analyzed these sequences with phylogenetic, coalescent, Bayesian clustering, and population genetic approaches in order to infer population structure, phylogenetic structure, and biogeographic history. Additionally, we examined occurrence data with species distribution modeling to generate a habitat suitability map to aid our interpretation of geographic structure. Our analyses recovered 4 major mtDNA lineages, two of which are combined into 3 major lineages when nuDNA is examined. These 3 major lineages are bounded by 4 major current or past geological features; the North Coast Divide, the former Wilson Grove Embayment/current Petaluma Gap, San Francisco Bay, and Monterey Bay. Other low-vagility species linked to moist microclimates and forest habitat do share similarities with the genetic patterns of D. ensatus hinting at a larger role for the past Wilson Grove embayment and modern Petaluma Gap in California biogeography.

Cryptic diversity in Brazilian endemic monkey frogs (Hylidae, Phyllomedusinae, Pithecopus) revealed by multispecies coalescent and integrative approaches

Incomplete or geographically biased sampling poses significant problems for research in phylogeography, population genetics, phylogenetics, and species delimitation. Despite the power of using genome-wide genetic markers in systematics and related fields, approaches such as the multispecies coalescent remain unable to easily account for unsampled lineages. The Empidonax difficilis/Empidonax occidentalis complex of small tyrannid flycatchers (Aves: Tyrannidae) is a classic example of widely distributed species with limited phenotypic geographic variation that was broken into two largely cryptic (or "sibling") lineages following extensive study. Though the group is well-characterized north of the US Mexico border, the evolutionary distinctiveness and phylogenetic relationships of southern populations remain obscure. In this article, we use dense genomic and geographic sampling across the majority of the range of the E. difficilis/E. occidentalis complex to assess whether current taxonomy and species limits reflect underlying evolutionary patterns, or whether they are an artifact of historically biased or incomplete sampling. We find that additional samples from Mexico render the widely recognized species-level lineage E. occidentalis paraphyletic, though it retains support in the best-fit species delimitation model from clustering analyses. We further identify a highly divergent unrecognized lineage in a previously unsampled portion of the group's range, which a cline analysis suggests is more reproductively isolated than the currently recognized species E. difficilis and E. occidentalis. Our phylogeny supports a southern origin of these taxa. Our results highlight the pervasive impacts of biased geographic sampling, even in well-studied vertebrate groups like birds, and illustrate what is a common problem when attempting to define species in the face of recent divergence and reticulate evolution.

Origin and diversification of Cristaria (Malvaceae) parallel Andean orogeny and onset of hyperaridity in the Atacama Desert

Phylogenetic relationships among 8 subspecies of Neotoma albigula and sister species from the United States and Mexico were examined using DNA sequence data from the mitochondrial DNA cytochrome-b gene. Parsimony, likelihood, and neighbor-joining analyses revealed a strong dichotomy between populations of N. albigula from Texas and eastern Mexico (eastern form) and those from New Mexico, Arizona, and northwestern Mexico (western form). These analyses indicate presence of 2 cryptic species within this taxon that are paraphyletic under current taxonomy. A sister-group relationship was found between N. albigula from Texas and eastern Mexico and N. micropus, whereas populations of N. albigula from New Mexico, Arizona, and northwestern Mexico formed a sister-group relationship with N. floridana. That latter group in turn formed a sister-taxon relationship to the Texas-eastern Mexico N. albigula and N. micropus clade. The Rio Grande and Rio Conchos seem to have been the major barriers restricting gene flow between ancestral populations of a N. floridana-like woodrat. Populations of N. floridana were further isolated geographically by reduction of suitable habitat brought about by changing climatic patterns that allowed formation of xeric plant communities soon after the end of the Late Wisconsin.

Aim The evolution of avian speciation patterns across much of Eurasia is under‐explored. Excepting phylogeographic patterns of single species, or speciation involving the Himalayas, there has been no attempt to understand the evolution of avian distributional patterns across the rest of the continent. Within many genera there is a pattern of (presumed) sister species occurring in adjacent areas (western, eastern or southern Eurasia), yet this pattern cannot be explained by existing biogeographic barriers. My aim was to examine the possible role of climate‐driven vicariance events in generating avian distributions.Location Eurasia.Methods I constructed a molecular phylogeny of Phoenicurus redstarts, and assembled phylogenetic data from published studies of seven other Eurasian bird genera. On each phylogeny, I assessed the distributional patterns of species and clades relative to refugial areas in western, eastern and southern Eurasia. I also estimated the timing of lineage divergences via a molecular clock, to determine whether distributional patterns can be explained by well‐defined periods of climate change in Eurasia that are recorded from dated sediments in the Chinese Loess Plateau.Results Species relationships in a well‐supported phylogeny of Phoenicurus show a pattern of distributions consistent with repeated speciation in major refugial areas, where one lineage is isolated in a single area of Eurasia relative to its sister lineage. This same pattern is evident in Eurasian Turdus thrushes, and six additional avian genera distributed across Eurasia. Molecular clock dating indicates that divergences within each genus are the result of multiple rounds of speciation in refugia through time, during major climate‐driven episodes of vicariance.Main conclusions Analyses revealed substantial evidence supporting a repeated, non‐random pattern of speciation within and across eight songbird lineages since the Late Miocene. The pattern of speciation supports a model of isolation in refugia during major episodes of vicariance, specifically periods of either intensified desertification of Central Asia or Eurasian glacial cycles. The densely sampled clades used here preclude inter‐continental dispersal as an alternative explanation for distributions. The signature of climate‐driven vicariance across epochs is, given the absence of extant biogeographic barriers, a suitable hypothesis to explain major lineage divergences in widely distributed Eurasian songbird lineages.

Does reproductive assurance explain the incidence of polyploidy in plants and animals?

Micropathogens (viruses, bacteria, fungi, parasitic protozoa) share a common trait, which is partial clonality, with wide variance in the respective influence of clonality and sexual recombination on the dynamics and evolution of taxa. The discrimination of distinct lineages and the reconstruction of their phylogenetic history are key information to infer their biomedical properties. However, the phylogenetic picture is often clouded by occasional events of recombination across divergent lineages, limiting the relevance of classical phylogenetic analysis and dichotomic trees. We have applied a network analysis based on graph theory to illustrate the relationships among genotypes of Trypanosoma cruzi, the parasitic protozoan responsible for Chagas disease, to identify major lineages and to unravel their past history of divergence and possible recombination events. At the scale of T. cruzi subspecific diversity, graph theory-based networks applied to 22 isoenzyme loci (262 distinct Multi-Locus-Enzyme-Electrophoresis -MLEE) and 19 microsatellite loci (66 Multi-Locus-Genotypes -MLG) fully confirms the high clustering of genotypes into major lineages or “near-clades”. The release of the dichotomic constraint associated with phylogenetic reconstruction usually applied to Multilocus data allows identifying putative hybrids and their parental lineages. Reticulate topology suggests a slightly different history for some of the main “near-clades”, and a possibly more complex origin for the putative hybrids than hitherto proposed. Finally the sub-network of the near-clade T. cruzi I (28 MLG) shows a clustering subdivision into three differentiated lesser near-clades (“Russian doll pattern”), which confirms the hypothesis recently proposed by other investigators. The present study broadens and clarifies the hypotheses previously obtained from classical markers on the same sets of data, which demonstrates the added value of this approach. This underlines the potential of graph theory-based network analysis for describing the nature and relationships of major pathogens, thereby opening stimulating prospects to unravel the organization, dynamics and history of major micropathogen lineages.

Identification of six Trypanosoma cruzi phylogenetic lineages by random amplified polymorphic DNA and multilocus enzyme electrophoresis