Gene flow and an anomaly zone complicate phylogenomic inference in a rapidly radiated avian family (Prunellidae).

BACKGROUND: Resolving the phylogeny of rapidly radiating lineages presents a challenge when building the Tree of Life. An Old World avian family Prunellidae (Accentors) comprises twelve species that rapidly diversified at the Pliocene-Pleistocene boundary. RESULTS: Here we investigate the phylogenetic relationships of all species of Prunellidae using a chromosome-level de novo assembly of Prunella strophiata and 36 high-coverage resequenced genomes. We use homologous alignments of thousands of exonic and intronic loci to build the coalescent and concatenated phylogenies and recover four different species trees. Topology tests show a large degree of gene tree-species tree discordance but only 40-54% of intronic gene trees and 36-75% of exonic genic trees can be explained by incomplete lineage sorting and gene tree estimation errors. Estimated branch lengths for three successive internal branches in the inferred species trees suggest the existence of an empirical anomaly zone. The most common topology recovered for species in this anomaly zone was not similar to any coalescent or concatenated inference phylogenies, suggesting presence of anomalous gene trees. However, this interpretation is complicated by the presence of gene flow because extensive introgression was detected among these species. When exploring tree topology distributions, introgression, and regional variation in recombination rate, we find that many autosomal regions contain signatures of introgression and thus may mislead phylogenetic inference. Conversely, the phylogenetic signal is concentrated to regions with low-recombination rate, such as the Z chromosome, which are also more resistant to interspecific introgression. CONCLUSIONS: Collectively, our results suggest that phylogenomic inference should consider the underlying genomic architecture to maximize the consistency of phylogenomic signal.

Phylogenomic analysis points to a South American origin of Manihot and illuminates the primary gene pool of cassava.

The genus Manihot, with around 120 known species, is native to a wide range of habitats and regions in the tropical and subtropical Americas. Its high species richness and recent diversification only c. 6 million years ago have significantly complicated previous phylogenetic analyses. Several basic elements of Manihot evolutionary history therefore remain unresolved. Here, we conduct a comprehensive phylogenomic analysis of Manihot, focusing on exhaustive sampling of South American taxa. We find that two recently described species from northeast Brazil's Atlantic Forest were the earliest to diverge, strongly suggesting a South American common ancestor of Manihot. Ancestral state reconstruction indicates early Manihot diversification in dry forests, with numerous independent episodes of new habitat colonization, including into savannas and rainforests within South America. We identify the closest wild relatives to Manihot esculenta, including the crop cassava, and we quantify extensive wild introgression into the cassava gene pool from at least five wild species, including Manihot glaziovii, a species used widely in breeding programs. Finally, we show that this wild-to-crop introgression substantially shapes the mutation load in cassava. Our findings provide a detailed case study for neotropical evolutionary history in a diverse and widespread group, and a robust phylogenomic framework for future Manihot and cassava research.

Demography and evolutionary history of grey wolf populations around the Bering Strait.

Glacial and interglacial periods throughout the Pleistocene have been substantial drivers of change in species distributions. Earlier analyses suggested that modern grey wolves (Canis lupus) trace their origin to a single Late Pleistocene Beringian population that expanded east and westwards, starting c. 25,000 years ago (ya). Here, we examined the demographic and phylogeographic histories of extant populations around the Bering Strait with wolves from two inland regions of the Russian Far East (RFE) and one coastal and two inland regions of North-western North America (NNA), genotyped for 91,327 single nucleotide polymorphisms. Our results indicated that RFE and NNA wolves had a common ancestry until c. 34,400 ya, suggesting that these populations started to diverge before the previously proposed expansion out of Beringia. Coastal and inland NNA populations diverged c. 16,000 ya, concordant with the minimum proposed date for the ecological viability of the migration route along the Pacific Northwest coast. Demographic reconstructions for inland RFE and NNA populations reveal spatial and temporal synchrony, with large historical effective population sizes that declined throughout the Pleistocene, possibly reflecting the influence of broadscale climatic changes across continents. In contrast, coastal NNA wolves displayed a consistently lower effective population size than the inland populations. Differences between the demographic history of inland and coastal wolves may have been driven by multiple ecological factors, including historical gene flow patterns, natural landscape fragmentation, and more recent anthropogenic disturbance.

Low MSP-1 haplotype diversity in the West Palearctic population of the avian malaria parasite Plasmodium relictum.

BACKGROUND: Although avian Plasmodium species are widespread and common across the globe, limited data exist on how genetically variable their populations are. Here, the hypothesis that the avian blood parasite Plasmodium relictum exhibits very low genetic diversity in its Western Palearctic transmission area (from Morocco to Sweden in the north and Transcaucasia in the east) was tested. METHODS: The genetic diversity of Plasmodium relictum was investigated by sequencing a portion (block 14) of the fast-evolving merozoite surface protein 1 (MSP1) gene in 75 different P. relictum infections from 36 host species. Furthermore, the full-length MSP1 sequences representing the common block 14 allele was sequenced in order to investigate if additional variation could be found outside block 14. RESULTS: The majority (72 of 75) of the sequenced infections shared the same MSP1 allele. This common allele has previously been found to be the dominant allele transmitted in Europe. CONCLUSION: The results corroborate earlier findings derived from a limited dataset that the globally transmitted malaria parasite P. relictum exhibits very low genetic diversity in its Western Palearctic transmission area. This is likely the result of a recent introduction event or a selective sweep.

Robust geographical determinants of infection prevalence and a contrasting latitudinal diversity gradient for haemosporidian parasites in Western Palearctic birds.

Identifying robust environmental predictors of infection probability is central to forecasting and mitigating the ongoing impacts of climate change on vector-borne disease threats. We applied phylogenetic hierarchical models to a data set of 2,171 Western Palearctic individual birds from 47 species to determine how climate and landscape variation influence infection probability for three genera of haemosporidian blood parasites (Haemoproteus, Leucocytozoon, and Plasmodium). Our comparative models found compelling evidence that birds in areas with higher vegetation density (captured by the normalized difference vegetation index [NDVI]) had higher likelihoods of carrying parasite infection. Magnitudes of this relationship were remarkably similar across parasite genera considering that these parasites use different arthropod vectors and are widely presumed to be epidemiologically distinct. However, we also uncovered key differences among genera that highlighted complexities in their climate responses. In particular, prevalences of Haemoproteus and Plasmodium showed strong but contrasting relationships with winter temperatures, supporting mounting evidence that winter warming is a key environmental filter impacting the dynamics of host-parasite interactions. Parasite phylogenetic community diversities demonstrated a clear but contrasting latitudinal gradient, with Haemoproteus diversity increasing towards the equator and Leucocytozoon diversity increasing towards the poles. Haemoproteus diversity also increased in regions with higher vegetation density, supporting our evidence that summer vegetation density is important for structuring the distributions of these parasites. Ongoing variation in winter temperatures and vegetation characteristics will probably have far-reaching consequences for the transmission and spread of vector-borne diseases.

The niches of nuthatches affect their lineage evolution differently across latitude.

Ecological niche evolution can promote or hinder the differentiation of taxa and determine their distribution. Niche-mediated evolution may differ among climatic regimes, and thus, species that occur across a wide latitudinal range offer a chance to test these heterogeneous evolutionary processes. In this study, we examine (a) how many lineages have evolved across the continent-wide range of the Eurasian nuthatch (Sitta europaea), (b) whether the lineages' niches are significantly divergent or conserved and (c) how their niche evolution explains their geographic distribution. Phylogenetic reconstruction and ecological niche models (ENMs) showed that the Eurasian nuthatch contained six parapatric lineages that diverged within 2 Myr and did not share identical climatic niches. However, the niche discrepancy between these distinct lineages was relatively conserved compared with the environmental differences between their ranges and thus was unlikely to drive lineage divergence. The ENMs of southern lineages tended to cross-predict with their neighbouring lineages whereas those of northern lineages generally matched with their abutting ranges. The coalescence-based analyses revealed more stable populations for the southern lineages than the northern ones during the last glaciation cycle. In contrast to the overlapping ENMs, the smaller parapatric distribution suggests that the southern lineages might have experienced competitive exclusion to prevent them from becoming sympatric. On the other hand, the northern lineages have expanded their ranges and their current abutting distribution might have resulted from lineages adapting to different climatic conditions in allopatry. This study suggests that niche evolution may affect lineage distribution in different ways across latitude.

A test of the European Pleistocene refugial paradigm, using a Western Palaearctic endemic bird species.

Hewitt's paradigm for effects of Pleistocene glaciations on European populations assumes their isolation in peninsular refugia during glacial maxima, followed by re-colonization of broader Europe during interstadials. This paradigm is well supported by studies of poorly dispersing taxa, but highly dispersive birds have not been included. To test this paradigm, we use the dunnock (Prunella modularis), a Western Palaearctic endemic whose range includes all major European refugia. MtDNA gene tree, multilocus species tree and species delimitation analyses indicate the presence of three distinct lineages: one in the Iberian refugium, one in the Caucasus refugium, and one comprising the Italian and Balkan refugia and broader Europe. Our gene flow analysis suggests isolation of both the Iberian and Caucasus lineages but extensive exchange between Italy, the Balkans and broader Europe. Demographic stability could not be rejected for any refugial population, except the very recent expansion in the Caucasus. By contrast, northern European populations may have experienced two expansion periods. Iberia and Caucasus had much smaller historical populations than other populations. Although our results support the paradigm, in general, they also suggest that in highly dispersive taxa, isolation of neighbouring refugia was incomplete, resulting in large super-refugial populations.

Multi-locus reassessment of a striking discord between mtDNA gene trees and taxonomy across two congeneric species complexes.

Resolving relationships among members of the yellow and citrine wagtail species complexes is among the greatest challenges in avian systematics due to arguably the most dramatic disagreements between traditional taxonomy and mtDNA phylogeny. Each species complex is divided into three geographically cohesive mtDNA clades. Each clade from one species complex has a sister from the other complex. Furthermore, one cross-complex pair is more distantly related to the remaining two pairs than are several other wagtail species. To test mtDNA gene tree topology, we sequenced the mtDNA ND2 gene and 11 nuclear introns for seven wagtail species. Our mtDNA gene tree reconstruction supported the results of previous studies, thereby confirming the disagreement between mtDNA phylogeny and taxonomy. However, our multi-locus species tree which used mtDNA clades as "taxa" was consistent with traditional taxonomy regardless of whether mtDNA was included in the analysis or not. Our multi-locus data suggest that despite the presence of strongly supported, geographically structured mtDNA variation, the mtDNA gene tree misrepresents the evolutionary history of the yellow and citrine wagtail complexes. This mito-nuclear discord results from mtDNA representing the biogeographic, but not evolutionary history of these recently radiated Palearctic wagtails.

The roles of ecology, behaviour and effective population size in the evolution of a community.

Organismal traits such as ecological specialization and migratory behaviour may affect colonization potential, population persistence and degree of isolation, factors that determine the composition and genetic structure of communities. However, studies focusing on community assembly rarely consider these factors jointly. We sequenced 16 nuclear genes and one mitochondrial gene from Caucasian and European populations of 30 forest-dwelling avian species that represent diverse ecological (specialist-generalist) and behavioural (migratory-resident) backgrounds. We tested the effects of organismal traits on population divergence and community assembly in the Caucasus forest, a continental mountain island setting. We found that (i) there is no concordance in divergence times between the Caucasus forest bird populations and their European counterparts, (ii) habitat specialists tend to be more divergent than generalists and (iii) residents tend to be more divergent than migrants. Thus, specialists and residents contribute to the high level of endemism of Caucasus forest avifauna more than do generalists and migrants. Patterns of genetic differentiation are better explained by differences in effective population sizes, an often overlooked factor in comparative studies of phylogeography and speciation, than by divergence times or levels of gene flow. Our results suggest that the Caucasus forest avifauna was assembled through time via dispersal and/or multiple vicariant events, rather than originating simultaneously via a single isolation event. Our study is one of the first multilocus, multispecies analyses revealing how ecological and migratory traits impact the evolutionary history of community formation on a continental island.

Diversity and distribution of avian haemosporidians in sub-Saharan Africa: an inter-regional biogeographic overview.

The diversity of avian malaria parasites is much greater than 20th century morphologists realized and virtually every study in this field in the last 15 years has uncovered previously undocumented diversity at multiple levels within the taxonomic hierarchy. Despite this explosion of knowledge, there remain vast sampling gaps, both geographically and host-taxonomically, which makes characterizing patterns of diversity extremely challenging. Here, we summarize the current state of knowledge of sub-Saharan African avian malaria parasite diversity, focusing on avian hosts endemic to Africa. The relative proportions of the parasite genera included here, Plasmodium, Haemoproteus (including Parahaemoproteus) and Leucocytozoon, varied between regions, in part due to habitat preferences of the insect vectors of these genera, and in part we believe due to sampling bias. Biogeographic regions of sub-Saharan Africa harbour about the same proportion of endemic to shared parasite lineages, but there appears to be no phylogenetic structuring across regions. Our results highlight the sampling problem that must be addressed if we are to have a detailed understanding of parasite diversity in Africa. Without broad sampling within and across regions and hosts, using both molecular tools and microscopy, conclusions about parasite diversity, host-parasite interactions or even transmission dynamics remain extremely limited.

Matching loci surveyed to questions asked in phylogeography.

Although mitochondrial DNA (mtDNA) has long been used for assessing genetic variation within and between populations, its workhorse role in phylogeography has been criticized owing to its single-locus nature. The only choice for testing mtDNA results is to survey nuclear loci, which brings into contrast the difference in locus effective size and coalescence times. Thus, it remains unclear how erroneous mtDNA-based estimates of species history might be, especially for evolutionary events in the recent past. To test the robustness of mtDNA and nuclear sequences in phylogeography, we provide one of the largest paired comparisons of summary statistics and demographic parameters estimated from mitochondrial, five Z-linked and 10 autosomal genes of 30 avian species co-distributed in the Caucasus and Europe. The results suggest that mtDNA is robust in estimating inter-population divergence but not in intra-population diversity, which is sensitive to population size change. Here, we provide empirical evidence showing that mtDNA was more likely to detect population divergence than any other single locus owing to its smaller Ne and thus faster coalescent time. Therefore, at least in birds, numerous studies that have based their inferences of phylogeographic patterns solely on mtDNA should not be readily dismissed.

Does the niche breadth or trade-off hypothesis explain the abundance-occupancy relationship in avian Haemosporidia?

Two hypotheses have been proposed to explain the abundance-occupancy relationship (AOR) in parasites. The niche breadth hypothesis suggests that host generalists are more abundant and efficient at colonizing different host communities than specialists. The trade-off hypothesis argues that host specialists achieve high density across their hosts' ranges, whereas generalists incur the high cost of adaptation to diverse immuno-defence systems. We tested these hypotheses using 386 haemosporidian cytochrome-b lineages (1894 sequences) recovered from 2318 birds of 103 species sampled in NW Africa, NW Iberia, W Greater Caucasus and Transcaucasia. The number of regions occupied by lineages was associated with their frequency suggesting the presence of AOR in avian Haemosporidia. However, neither hypothesis provided a better explanation for the AOR. Although the host generalist Plasmodium SGS1 was over three times more abundant than other widespread lineages, both host specialists and generalists were successful in colonizing all study regions and achieved high overall prevalence.

Viral pathogen detection in U.S. game-farm mallard (Anas platyrhynchos) flags spillover risk to wild birds.

The threat posed by emerging infectious diseases is a major concern for global public health, animal health and food security, and the role of birds in transmission is increasingly under scrutiny. Each year, millions of mass-reared game-farm birds are released into the wild, presenting a unique and a poorly understood risk to wild and susceptible bird populations, and to human health. In particular, the shedding of enteric pathogens through excrement into bodies of water at shared migratory stop-over sites, and breeding and wintering grounds, could facilitate multi-species long-distance pathogen dispersal and infection of high numbers of naive endemic birds annually. The Mallard (Anas platyrhynchos) is the most abundant of all duck species, migratory across much of its range, and an important game species for pen-rearing and release. Major recent population declines along the US Atlantic coast has been attributed to game-farm and wild mallard interbreeding and the introduction maladaptive traits into wild populations. However, pathogen transmission and zoonosis among game-farms Mallard may also impact these populations, as well as wildlife and human health. Here, we screened 16 game-farm Mallard from Wisconsin, United States, for enteric viral pathogens using metatranscriptomic data. Four families of viral pathogens were identified - Picobirnaviridae (Genogroup I), Caliciviridae (Duck Nacovirus), Picornaviridae (Duck Aalivirus) and Sedoreoviridae (Duck Rotavirus G). To our knowledge, this is the first report of Aalivirus in the Americas, and the first report of Calicivirus outside domestic chicken and turkey flocks in the United States. Our findings highlight the risk of viral pathogen spillover from peri-domestically reared game birds to naive wild bird populations.

Recent allopatric divergence and niche evolution in a widespread Palearctic bird, the common rosefinch (Carpodacus erythrinus).

A previously published phylogeographic analysis of mtDNA sequences from the widespread Palearctic common rosefinch (Carpodacus erythrinus) suggested the existence of three recently diverged groups, corresponding to the Caucasus, central-western Eurasia, and northeastern Eurasia. We re-evaluated the mtDNA data using coalescence methods and added sequence data from a sex-linked gene. The mtDNA gene tree and SAMOVA supported the distinctiveness of the Caucasian group but not the other two groups. However, UPGMA clustering of mtDNA Φ(ST)-values among populations recovered the three groups. The sex-linked gene tree recovered no phylogeographic signal, which was attributed to recent divergence and insufficient time for sorting of alleles. Overall, coalescence methods indicated a lack of gene flow among the three groups, and population expansion in the central-western and northeastern Eurasia groups. These three groups corresponded to named subspecies, further supporting their validity. A species distribution model revealed potential refugia at the Last Glacial Maximum. These three groups, which we hypothesized are in the early stages of speciation, provided an opportunity for testing tenets of ecological speciation. We showed that the early stages of speciation were not accompanied by ecological niche divergence, consistent with other avian studies.

A parapatric propensity for breeding precludes the completion of speciation in common teal (Anas crecca, sensu lato).

Speciation is a process in which genetic drift and selection cause divergence over time. However, there is no rule dictating the time required for speciation, and even low levels of gene flow hinder divergence, so that taxa may be poised at the threshold of speciation for long periods of evolutionary time. We sequenced mitochondrial DNA (mtDNA) and eight nuclear introns (nuDNA) to estimate genomic levels of differentiation and gene flow between the Eurasian common teal (Anas crecca crecca) and the North American green-winged teal (Anas crecca carolinensis). These ducks come into contact in Beringia (north-eastern Asia and north-western North America) and have probably done so, perhaps cyclically, since the Pliocene-Pleistocene transition, ~2.6 Ma, when they apparently began diverging. They have diagnosable differences in male plumage and are 6.9% divergent in the mtDNA control region, with only 1 of 58 crecca and 2 of 86 carolinensis having haplotypes grouping with the other. Two nuclear loci were likewise strongly structured between these teal (Φ(st) ≥ 0.35), but six loci were undifferentiated or only weakly structured (Φ(st) = 0.0-0.06). Gene flow between crecca and carolinensis was ~1 individual per generation in both directions in mtDNA, but was asymmetrical in nuDNA, with ~1 and ~20 individuals per generation immigrating into crecca and carolinensis, respectively. This study illustrates that species delimitation using a single marker oversimplifies the complexity of the speciation process, and it suggests that even with divergent selection, moderate levels of gene flow may stall the speciation process short of completion.

Does solar irradiation drive community assembly of vulture plumage microbiotas?

BACKGROUND: Stereotyped sunning behaviour in birds has been hypothesized to inhibit keratin-degrading bacteria but there is little evidence that solar irradiation affects community assembly and abundance of plumage microbiota. The monophyletic New World vultures (Cathartiformes) are renowned for scavenging vertebrate carrion, spread-wing sunning at roosts, and thermal soaring. Few avian species experience greater exposure to solar irradiation. We used 16S rRNA sequencing to investigate the plumage microbiota of wild individuals of five sympatric species of vultures in Guyana. RESULTS: The exceptionally diverse plumage microbiotas (631 genera of Bacteria and Archaea) were numerically dominated by bacterial genera resistant to ultraviolet (UV) light, desiccation, and high ambient temperatures, and genera known for forming desiccation-resistant endospores (phylum Firmicutes, order Clostridiales). The extremophile genera Deinococcus (phylum Deinococcus-Thermus) and Hymenobacter (phylum, Bacteroidetes), rare in vertebrate gut microbiotas, accounted for 9.1% of 2.7 million sequences (CSS normalized and log2 transformed). Five bacterial genera known to exhibit strong keratinolytic capacities in vitro (Bacillus, Enterococcus, Pseudomonas, Staphylococcus, and Streptomyces) were less abundant (totaling 4%) in vulture plumage. CONCLUSIONS: Bacterial rank-abundance profiles from melanized vulture plumage have no known analog in the integumentary systems of terrestrial vertebrates. The prominence of UV-resistant extremophiles suggests that solar irradiation may play a significant role in the assembly of vulture plumage microbiotas. Our results highlight the need for controlled in vivo experiments to test the effects of UV on microbial communities of avian plumage.

Patchy distributions belie morphological and genetic homogeneity in rosy-finches.

DNA sequence data often appear to contradict low-level avian taxonomy, which is usually based on patterns of external phenotypic similarity. We examined such an apparent contradiction in the Nearctic rosy-finches. On the basis of several phenotypic characters the finches were divided into three species congeneric with three Asian species. When Nearctic taxa were analyzed in a principal components analysis, 66.9% of phenotypic variation was explained by differences between the Bering Sea and continental populations, sexual dimorphism and a latitudinal cline. Our phylogenetic analysis of mitochondrial ND2 sequences revealed four clades among six species of rosy-finches. Three clades corresponded to three Asian species. The fourth clade included all three Nearctic species. Their haplotypes were not reciprocally monophyletic and the combined genetic variability of all Nearctic taxa was lower than in two of their Asian congeners. A Z-specific intron (ACO1I9) and an autosomal coding locus (MC1R) provided little additional phylogenetic information, most likely because of the longer coalescence times relative to ND2. Phylogeographic analyses of ND2 data revealed significant gene flow among neighboring localities regardless of their taxonomic assignment. Our analyses showed that DNA and phenotypic data are not in conflict, but rather complement each other, and together help clarify species limits. Our data are consistent with a single species in North America, not three.

Distinct microbiotas of anatomical gut regions display idiosyncratic seasonal variation in an avian folivore.

BACKGROUND: Current knowledge about seasonal variation in the gut microbiota of vertebrates is limited to a few studies based on mammalian fecal samples. Seasonal changes in the microbiotas of functionally distinct gut regions remain unexplored. We investigated seasonal variation (summer versus winter) and regionalization of the microbiotas of the crop, ventriculus, duodenum, cecum, and colon of the greater sage-grouse (Centrocercus urophasianus), an avian folivore specialized on the toxic foliage of sagebrush (Artemesia spp.) in western North America. RESULTS: We sequenced the V4 region of the 16S rRNA gene on an Illumina MiSeq and obtained 6,639,051 sequences with a median of 50,232 per sample. These sequences were assigned to 457 bacterial and 4 archaeal OTUs. Firmicutes (53.0%), Bacteroidetes (15.2%), Actinobacteria (10.7%), and Proteobacteria (10.1%)were the most abundant and diverse phyla. Microbial composition and richness showed significant differences among gut regions and between summer and winter. Gut region explained almost an order of magnitude more variance in our dataset than did season or the gut region × season interaction. The effect of season was uneven among gut regions. Microbiotas of the crop and cecum showed the greatest seasonal differences. CONCLUSIONS: Our data suggest that seasonal differences in gut microbiota reflect seasonal variation in the microbial communities associated with food and water. Strong differentiation and uneven seasonal changes in the composition and richness of the microbiota among functionally distinct gut regions demonstrate the necessity of wider anatomical sampling for studies of composition and dynamics of the gut microbiota.

Geographic patterns of mtDNA and Z-linked sequence variation in the Common Chiffchaff and the 'chiffchaff complex'.

The Common Chiffchaff Phylloscopus collybita is an abundant, polytypic Palearctic bird. Validity of some of its subspecies is controversial and birds from some parts of the species range remain unclassified taxonomically. The relationships among populations from different geographic areas have not been sufficiently explored with molecular data. In this study we analyzed the relationships among the four species in the 'chiffchaff complex' (Common Chiffchaff, Iberian Chiffchaff P. ibericus, Canary Islands Chiffchaff P. canariensis and Mountain Chiffchaff P. sindianus), and the patterns of intraspecific geographic variation in the mtDNA ND2 gene and intron 9 of the Z-linked aconitase gene (ACO1I9) across the Common Chiffchaff range, including a recently discovered population breeding on Mt. Hermon (Anti-Lebanon mountains). Our data supported the monophyly of the chiffchaff complex and its current systematics at the species level. Within the Common Chiffchaff, the Siberian race P. c. tristis was the most differentiated subspecies and may represent a separate or incipient species. Other Common Chiffchaff subspecies also were differentiated in their mtDNA, however, lineages of neighboring subspecies formed wide zones of introgression. The Mt. Hermon population was of mixed genetic origin but contained some birds with novel unique lineage that could not be assigned to known subspecies. All Common Chiffchaff lineages diverged at the end of the Ionian stage of Pleistocene. Lineage sorting of ACO1I9 alleles was not as complete as that of mtDNA. Chiffchaff species were mostly distinct at ACO1I9, except the Common and Canary Islands Chiffchaffs that shared multiple alleles. An AMOVA identified geographic structure in Common Chiffchaff ACO1I9 variation that was broadly consistent with that of mtDNA ND2 gene. The genetic and other data suggest the chiffchaff complex to be a group of evolutionarily young taxa that represent a paradigm of 'species evolution in action' from intergrading subspecies through to apparently complete biological speciation.

Pleistocene evolution of closely related sand martins Riparia riparia and R. diluta.

Climatic fluctuations during the Quaternary resulted in a dynamic history of species' range shifts, fragmentations and expansions. Some of these events left traces in the genetic structures of plants and animals. Recent avian phylogeographic studies demonstrated that Holarctic birds responded idiosyncratically to Pleistocene climate fluctuations. We present phylogeographic analyses of the Holarctic collared sand martin (Riparia riparia) and the Asian pale sand martin (Riparia diluta), which were considered conspecific until recently. Mitochondrial and nuclear sequences confirm species status of the pale sand martin; the two species diverged sometime between late Pliocene and middle Pleistocene, but precise dates could not be provided without calibration of the substitution rate. Within the pale sand martin, we found two mitochondrial clades that are likely to have diverged in the Pleistocene, one from Central Siberia, and the other restricted to Mongolia. The two clades were sympatric with the collared sand martin in Buryatiya and Mongolia, respectively. The mitochondrial gene genealogy and phi(st) analysis of the collared sand martin haplotypes indicate recent, but not ongoing, gene exchange between North America and Eurasia, and restricted gene flow between western and eastern Siberia that likely resulted from historic fragmentation of the species' range during the last glacial maximum.