Speciation in savanna birds in South America: The case of the Least Nighthawk Chordeiles pusillus (Aves: Caprimulgidae) in and out of the Amazon.

The Least Nighthawk Chordeiles pusillus is widespread wherever there are savannas in the South American tropics, often in isolated patches, such as white-sands savannas in the Amazon rainforest realm. Here, we investigate genetic relationships between populations of the Least Nighthawk to understand historical processes leading to its diversification and to determine dispersal routes between northern and southern savannas by way of three hypothesized dispersal corridors by comparing samples from white-sand savannas to samples from other savannas outside of the Amazon rainforest region. We use 32 mtDNA samples from the range of C. pusillus to infer a dated phylogeny. In a subset of 17 samples, we use shotgun sequences to infer a distance-based phylogeny and to estimate individual admixture proportions. We calculate gene flow and shared alleles between white-sand and non-Amazonian populations using the ABBA-BABA test (D statistics), and Principal Component Analysis (PCA) to examine genetic structure within and between lineages. Finally, we use species distribution modelling (SDM) of conditions during the Last Glacial Maximum (LGM), currently, and in the future (2050-2080) to predict potential species occurrence under a climate change scenario. Two main clades (estimated to have diverged around 1.07 million years ago) were recovered with mtDNA sequences and Single Nucleotide Polymorphism (SNPs) and were supported by NGSadmix and PCA: one in the Amazon basin white-sand savannas, the other in the non-Amazonian savannas. Possible allele sharing between these clades was indicated by the D-statistics between northern non-Amazonian populations and the white-sand savanna population, but this was not corroborated by the admixture analyses. Dispersal among northern non-Amazonian populations may have occurred in a dry corridor between the Guianan and the Brazilian Shield, which has since moved eastward. Our data suggest that the lineages separated well before the Last Glacial Maximum, consequently dispersal could have happened at any earlier time during similar climatic conditions. Subsequently, non-Amazonian lineages became more divergent among themselves, possibly connecting and dispersing across the mouth of the Amazon River across Marajó island during favourable climatic conditions in the Pleistocene.

When colors mislead: Genomics and bioacoustics prompt re-classification of Asian flycatcher radiation (Aves: Niltavinae).

Traditional classification of many animals, including birds, has been highly dependent on external morphological characters like plumage coloration. However, both bioacoustics and genetic or genomic data have revolutionized our understanding of the relationships of certain lineages and led to sweeping taxonomic re-organizations. In this study, we present a case of erroneous delimitation of genus boundaries in the species-rich flycatcher subfamily Niltavinae. Genera within this subfamily have historically been delineated based on blue versus brown male body plumage until recent studies based on a few mitochondrial and nuclear loci unearthed several cases of generic misclassification. Here we use extensive bioacoustic data from 43 species and genomic data from 28 species for a fundamental reclassification of species in the Niltavinae. Our study reveals that song is an important trait to classify these birds even at the genus level, whereas plumage traits exhibit ample convergence and have led to numerous historic misattributions. Our taxonomic re-organization leads to new biogeographic limits of major genera, such that the genus Cyornis now only extends as far east as the islands of Sulawesi, Sula, and Banggai, whereas Eumyias is redefined to extend far beyond Wallace's Line to the islands of Seram and Timor. Our conclusions advise against an over-reliance on morphological traits and underscore the importance of integrative datasets.

Phylogenetic conflict between species tree and maternally inherited gene trees in a clade of Emberiza buntings (Aves: Emberizidae).

Different genomic regions may reflect conflicting phylogenetic topologies primarily due to incomplete lineage sorting and/or gene flow. Genomic data are necessary to reconstruct the true species tree and explore potential causes of phylogenetic conflict. Here, we investigate the phylogenetic relationships of four Emberiza species (Aves: Emberizidae) and discuss the potential causes of the observed mitochondrial non-monophyly of Emberiza godlewskii (Godlewski's bunting) using phylogenomic analyses based on whole genome resequencing data from 41 birds. Analyses based on both the whole mitochondrial genome and ~39 kilobases from the non-recombining W chromosome reveal sister relationships between each the northern and southern populations of E. godlewskii with E. cioides and E. cia, respectively. In contrast, the monophyly of E. godlewskii is reflected by the phylogenetic signal of autosomal and Z chromosomal sequence data as well as demographic inference analyses, which - in combination - support the following tree topology: (((E. godlewskii, E. cia), E. cioides), E. jankowskii). Using D-statistics, we detected multiple gene flow events among different lineages, indicating pervasive introgressive hybridization within this clade. Introgression from an unsampled lineage that is sister to E. cioides or introgression from an unsampled mitochondrial + W chromosomal lineage of E. cioides into northern E. godlewskii may explain the phylogenetic conflict between the species tree estimated from genome-wide data versus mtDNA/W tree topologies. These results underscore the importance of using genomic data for phylogenetic reconstruction and species delimitation.

Genomic and phenotypic changes associated with alterations of migratory behaviour in a songbird.

The seasonal migration of birds is a fascinating natural wonder. Avian migratory behaviour changes are common and are probably a polygenic process as avian migration is governed by multiple correlated components with a variable genetic basis. However, the genetic and phenotypic changes involving migration changes are poorly studied. Using one annotated near-chromosomal level de novo genome assembly, 50 resequenced genomes, hundreds of morphometric data and species distribution information, we investigated population structure and genomic and phenotypic differences associated with differences in migratory behaviour in a songbird species, Yellow-throated Bunting Emberiza elegans (Aves: Emberizidae). Population genomic analyses reveal extensive gene flow between the southern resident and the northern migratory populations of this species. The hand-wing index is significantly lower in the resident populations than in the migratory populations, indicating reduced flight efficiency of the resident populations. Here, we discuss the possibility that nonmigratory populations may have originated from migratory populations though migration loss. We further infer that the alterations of genes related to energy metabolism, nervous system and circadian rhythm may have played major roles in regulating migration change. Our study sheds light on phenotypic and polygenic changes involving migration change.

A near-complete and time-calibrated phylogeny of the Old World flycatchers, robins and chats (Aves, Muscicapidae).

The Old World flycatchers, robins and chats (Aves, Muscicapidae) are a diverse songbird family with over three hundred species. Despite continuous efforts over the past two decades, there is still no comprehensive and well-resolved species-level phylogeny for Muscicapidae. Here we present a supermatrix phylogeny that includes all 50 currently recognized genera and ca. 92% of all the species, built using data from up to 15 mitochondrial and 13 nuclear loci. In addition to assembling nucleotide sequences available in public databases, we also extracted sequences from the genome assemblies and raw sequencing reads from GenBank and included a few unpublished sequences. Our analyses resolved the phylogenetic position for several previously unsampled taxa, for example, the Grand Comoro Flycatcher Humblotia flavirostris, the Collared Palm Thrush Cichladusa arquata, and the Taiwan Whistling-Thrush Myophonus insularis, etc. We also provide taxonomic recommendations for genera that exhibit paraphyly or polyphyly. Our results suggest that Muscicapidae diverged from Turdidae (thrushes and allies) in the early Miocene, and the most recent common ancestors for the four subfamilies (Muscicapinae, Niltavinae, Cossyphinae and Saxicolinae) all arose around the middle Miocene.

Cryptic species in a colorful genus: Integrative taxonomy of the bush robins (Aves, Muscicapidae, Tarsiger) suggests two overlooked species.

Several cryptic avian species have been validated by recent integrative taxonomic efforts in the Sino-Himalayan mountains, indicating that avian diversity in this global biodiversity hotspot may be underestimated. In the present study, we investigated species limits in the genus Tarsiger, the bush robins, a group of montane forest specialists with high species richness in the Sino-Himalayan region. Based on comprehensive sampling of all 11 subspecies of the six currently recognized species, we applied an integrative taxonomic approach by combining multilocus, acoustic, plumage and morphometric analyses. Our results reveal that the isolated north-central Chinese populations of Tarsiger cyanurus, described as the subspecies albocoeruleus but usually considered invalid, is distinctive in genetics and vocalisation, but only marginally differentiated in morphology. We also found the Taiwan endemic T. indicus formosanus to be distinctive in genetics, song and morphology from T. i. indicus and T. i. yunnanensis of the Sino-Himalayan mountains. Moreover, Bayesian species delimitation using BPP suggests that both albocoeruleus and formosanus merit full species status. We propose their treatment as 'Qilian Bluetail' T. albocoeruleus and 'Taiwan Bush Robin' T. formosanus, respectively.

Multiple species delimitation approaches applied to the avian lark genus Alaudala.

Species delimitation has advanced from a purely phenotypic exercise to a branch of science that integrates multiple sources of data to identify independently evolving lineages that can be treated as species. We here test species limits in the avian Lesser Short-toed Lark Alaudala rufesens-Sand Lark A. raytal complex, which has an intricate taxonomic history, ranging from a single to three recognised species, with different inclusiveness in different treatments. Our integrative taxonomic approach is based on a combination of DNA sequences, plumage, biometrics, songs, song-flights, geographical distributions, habitat, and bioclimatic data, and using various methods including a species delimitation program (STACEY) based on the multispecies coalescent model. We propose that four species should be recognised: Lesser Short-toed Lark A. rufescens (sensu stricto), Heine's Short-toed Lark A. heinei, Asian Short-toed Lark A. cheleensis and Sand Lark A. raytal. There is also some evidence suggesting lineage separation within A. cheleensis and A. raytal, but additional data are required to evaluate this. The species delimitation based on STACEY agrees well with the non-genetic data. Although computer-based species delimitation programs can be useful in identifying independently evolving lineages, we stress that whenever possible, species hypotheses proposed by these programs should be tested by independent, non-genetic data. Our results highlight the difficulty and subjectivity of delimiting lineages and species, especially at early stages in the speciation process.

Molecular Phylogeny of the Subgenus Karstomys Reveals Genetic Signature of Post-Glacial Colonization of Apodemus mystacinus (Rodentia: Muridae) in the Zagros Mountains from Different Refugia.

Eastern broad-toothed field mouse, Apodemus mystacinus, is a rocky habitat dwelling rodent distributed in Asia Minor, the Levant, the Caucasus, and the Zagros Mountains. In this study, we investigated the phylogenetic relationship between different populations of A. mystacinus throughout its range, based on the mitochondrial cytb marker. Phylogenetic analyses revealed the existence of five separately evolving lineages within A. mystacinus, of which two previously unrecognized lineages were identified in the Zagros Mountains and the Levant. Divergence between two major clades of the subgenus Karstomys, corresponding to A. mystacinus and Apodemus epimelas, is inferred to coincide with the Messinian Salinity Crisis (Late Miocene), whereas the splits between major lineages of A. mystacinus are inferred to have occurred during the Pleistocene. Colonization of the Zagros may have occurred from different refugia via eastward migration of the Turkish population and then again by a more recent colonization from the Caucasus, after reopening of the land corridor between the Caucasus and the Zagros Mountains during the Holocene drought.

Phylogenomics and biogeography of the world's thrushes (Aves, Turdus): new evidence for a more parsimonious evolutionary history.

To elucidate the relationships and spatial range evolution across the world of the bird genus Turdus (Aves), we produced a large genomic dataset comprising ca 2 million nucleotides for ca 100 samples representing 53 species, including over 2000 loci. We estimated time-calibrated maximum-likelihood and multispecies coalescent phylogenies and carried out biogeographic analyses. Our results indicate that there have been considerably fewer trans-oceanic dispersals within the genus Turdus than previously suggested, such that the Palaearctic clade did not originate in America and the African clade was not involved in the colonization of the Americas. Instead, our findings suggest that dispersal from the Western Palaearctic via the Antilles to the Neotropics might have occurred in a single event, giving rise to the rich Neotropical diversity of Turdus observed today, with no reverse dispersals to the Palaearctic or Africa. Our large multilocus dataset, combined with dense species-level sampling and analysed under probabilistic methods, brings important insights into historical biogeography and systematics, even in a scenario of fast and spatially complex diversification.

A comprehensive phylogeny and taxonomic evaluation of the waxbills (Aves: Estrildidae).

We present a revised taxonomy of Estrildidae based on the first time-calibrated phylogeny of the family Estrildidae estimated from a data set including the majority of the species, and all genera except the monospecific Paludipasser, using two mitochondrial and five nuclear markers. We find that most differences in current taxonomy reflect alternative opinions among authors regarding inclusiveness of genera, which are usually not in conflict with the phylogeny. The most notable exception is the current circumscriptions of the genera Neochmia, Nesocharis and Taeniopygia, which are incompatible with the phylogeny. Estrildidae is subdivided into six well supported subclades, which we propose be recognized as the subfamilies Amandavinae, Erythrurinae, Estrildinae, Lagonostictinae, Lonchurinae and Poephilinae.

Allele Phasing Greatly Improves the Phylogenetic Utility of Ultraconserved Elements.

Advances in high-throughput sequencing techniques now allow relatively easy and affordable sequencing of large portions of the genome, even for nonmodel organisms. Many phylogenetic studies reduce costs by focusing their sequencing efforts on a selected set of targeted loci, commonly enriched using sequence capture. The advantage of this approach is that it recovers a consistent set of loci, each with high sequencing depth, which leads to more confidence in the assembly of target sequences. High sequencing depth can also be used to identify phylogenetically informative allelic variation within sequenced individuals, but allele sequences are infrequently assembled in phylogenetic studies. Instead, many scientists perform their phylogenetic analyses using contig sequences which result from the de novo assembly of sequencing reads into contigs containing only canonical nucleobases, and this may reduce both statistical power and phylogenetic accuracy. Here, we develop an easy-to-use pipeline to recover allele sequences from sequence capture data, and we use simulated and empirical data to demonstrate the utility of integrating these allele sequences to analyses performed under the multispecies coalescent model. Our empirical analyses of ultraconserved element locus data collected from the South American hummingbird genus Topaza demonstrate that phased allele sequences carry sufficient phylogenetic information to infer the genetic structure, lineage divergence, and biogeographic history of a genus that diversified during the last 3 myr. The phylogenetic results support the recognition of two species and suggest a high rate of gene flow across large distances of rainforest habitats but rare admixture across the Amazon River. Our simulations provide evidence that analyzing allele sequences leads to more accurate estimates of tree topology and divergence times than the more common approach of using contig sequences.

Niche filling slows the diversification of Himalayan songbirds.

Speciation generally involves a three-step process--range expansion, range fragmentation and the development of reproductive isolation between spatially separated populations. Speciation relies on cycling through these three steps and each may limit the rate at which new species form. We estimate phylogenetic relationships among all Himalayan songbirds to ask whether the development of reproductive isolation and ecological competition, both factors that limit range expansions, set an ultimate limit on speciation. Based on a phylogeny for all 358 species distributed along the eastern elevational gradient, here we show that body size and shape differences evolved early in the radiation, with the elevational band occupied by a species evolving later. These results are consistent with competition for niche space limiting species accumulation. Even the elevation dimension seems to be approaching ecological saturation, because the closest relatives both inside the assemblage and elsewhere in the Himalayas are on average separated by more than five million years, which is longer than it generally takes for reproductive isolation to be completed; also, elevational distributions are well explained by resource availability, notably the abundance of arthropods, and not by differences in diversification rates in different elevational zones. Our results imply that speciation rate is ultimately set by niche filling (that is, ecological competition for resources), rather than by the rate of acquisition of reproductive isolation.

Evidence for mtDNA capture in the jacamar Galbula leucogastra/chalcothorax species-complex and insights on the evolution of white-sand ecosystems in the Amazon basin.

Jacamar species occur throughout Amazonia, with most species occupying forested habitats. One species-complex, Galbula leucogastra/chalcothorax, is associated to white sand ecosystems (WSE). Previous studies of WSE bird species recovered shallow genetic structure in mtDNA coupled with signs of gene flow among WSE patches. Here, we characterize diversification of the G. leucogastra/chalcothorax species-complex with dense sampling across its distribution using mitochondrial and genomic (Ultraconserved Elements, UCEs) DNA sequences. We performed concatenated likelihood and Bayesian analysis, as well as a species-tree analysis using ∗BEAST, to establish the phylogenetic relationships among populations. The mtDNA results recovered at least six geographically-structured lineages, with G. chalcothorax embedded within lineages of G. leucogastra. In contrast, both concatenated and species-tree analyses of UCE data recovered G. chalcothorax as sister to all G. leucogastra lineages. We hypothesize that the mitochondrial genome of one of the G. leucogastra lineage (Madeira) was captured into G. chalcothorax in the past. We discuss how WSE evolution and the coevolution of mtDNA and nuclear genes might have played a role in this apparently rare event.

Contrasting patterns of diversification in two sister species of martins (Aves: Hirundinidae): The Sand Martin Riparia riparia and the Pale Martin R. diluta.

Species not only responded idiosyncratically to past climate changes, there were also regionally contrasting effects on spatio-temporal diversification patterns. Studies of closely related species appear to be a particularly promising comparative approach to disentangle such regionally differential impacts. In this study, we undertook a comprehensive geographic sampling to investigate the evolutionary history of the Holarctic Sand Martin Riparia riparia and the chiefly Central and East Asian Pale Martin R. diluta. Previous phylogenetic studies using only a limited geographic sampling, particularly for the latter, revealed the two to be genetically distinct, with the former showing only a shallow genetic structure in mitochondrial DNA. Based on one mitochondrial, one autosomal and one Z-linked nuclear marker, we confirmed the shallow genetic structure in R. riparia even when including the morphologically relatively distinct subspecies R. r. shelleyi from the Nile Valley in Egypt and probably the Middle East. On the other hand the different subspecies of R. diluta, i.e. R. d. diluta from Central Asia, R. d. indica from the northwestern Indian Subcontinent, R. d. tibetana from the Tibetan Plateau and R. d. fohkienensis from southeastern China, were found to be genetically distinct. Their diversification started before the Early to Middle Pleistocene Transition, which was followed by a pronounced succession of glacial and interglacial periods. These rather old divergence events contrast with the lack of any strong phylogeographic structure in R. riparia. Strongly structured populations and regional diversification have been reported in different forest passerine families of South-East Asia. Here we demonstrate, however, that species characteristic of open-country habitats such as R. diluta might display a similar pattern. Morphometric analyses of 120 individuals revealed no clear differences between the different subspecies of R. diluta. Given their similarity also in plumage features, we refrain from proposing any splits despite their marked genetic differentiation, pending further studies and particularly the discovery of potential secondary contact zones.

Comprehensive molecular phylogeny of the grassbirds and allies (Locustellidae) reveals extensive non-monophyly of traditional genera, and a proposal for a new classification.

The widespread Old World avian family Locustellidae ('grassbirds and allies') comprises 62 extant species in 11 genera. In the present study, we used one mitochondrial and, for most species, four nuclear loci to infer the phylogeny of this family. We analysed 59 species, including the five previously unsampled genera plus two genera that had not before been analysed in a densely sampled dataset. This study revealed extensive disagreement with current taxonomy; the genera Bradypterus, Locustella, Megalurus, Megalurulus and Schoenicola were all found to be non-monophyletic. Non-monophyly was particularly pronounced for Megalurus, which was widely scattered across the tree. Three of the five monotypic genera (Amphilais, Buettikoferella and Malia) were nested within other genera; one monotypic genus (Chaetornis) formed a clade with one of the two species of Schoenicola; whereas the position of the fifth monotypic genus (Elaphrornis) was unresolved. Robsonius was confirmed as sister to the other genera. We propose a phylogenetically informed revision of genus-level taxonomy, including one new generic name. Finally, we highlight several non-monophyletic species complexes and deep intra-species divergences that point to conflict in taxonomy and suggest an underestimation of current species diversity in this group.

Complete species-level phylogeny of the leaf warbler (Aves: Phylloscopidae) radiation.

The leaf warbler radiation (Aves: Phylloscopidae) has undergone a c. 50% increase in the number of recognised species over the last three decades, mainly as a result of analyses of vocalisations and DNA. Using a multilocus dataset for all of the species in this family, and multispecies coalescent-based as well as concatenation methods, we provide the first complete species-level phylogeny for this important group, as well as an estimate of the timing of diversification. The most recent common ancestor for the family was dated at 11.7 million years ago (mya) (95% highest posterior density 9.8-13.7 mya), and divergence times between sister species ranged from 0.5 mya (0.3-0.8 mya) to 6.1 mya (4.8-7.5 mya). Based on our results, we support synonymising Seicercus with Phylloscopus, which results in a monogeneric Phylloscopidae. We discuss the pros and cons of this treatment,and we argue againstproliferation of taxonomic names,and conclude that a large monogeneric Phylloscopidae leads to the fewest taxonomic changes compared to traditional classifications. We briefly discuss morphological evolution in the light of the phylogeny. The time calibrated phylogeny is a major improvement compared to previous studies based on a smaller number of species and loci and can provide a basis for future studies of other aspects of phylloscopid evolution.

The Rusty-tailed Flycatcher (Muscicapa ruficauda; Aves: Muscicapidae) is a member of the genus Ficedula.

The phylogenetic relationships of the avian family Muscicapidae (Old World chats and flycatchers) have historically been enigmatic and remain an active area of study. Widespread instances of non-monophyly resulting from misleading morphological and behavioral similarities have merited numerous taxonomic revisions to the group. Here we report one such instance with regard to the Rusty-tailed Flycatcher Muscicapa ruficauda, which has recently been placed in the newly proposed monotypic genus Ripleyia, due to inferred sister relationship to the genus Muscicapa and related genera (Voelker et al., 2016a). This name was later replaced by Ripleyornis, as it was realized that Ripleyia is a junior homonym of a genus of Mollusca (Voelker et al., 2016b). Using a Bayesian phylogenetic assessment of the Muscicapidae with near-complete taxon sampling of the genus Ficedula for five loci, along with an acoustic comparison of M. ruficauda to a subset of other flycatcher species, we show that this species should be reassigned to the genus Ficedula and accordingly that the names Ripleyia and Ripleyornis are both junior synonyms of Ficedula.

Unexpected divergence and lack of divergence revealed in continental Asian Cyornis flycatchers (Aves: Muscicapidae).

The flycatcher genus Cyornis (Aves: Muscicapidae) comprises 25 species with Oriental distributions. Their relationships are poorly known. We analyzed the phylogenetic relationships of 70 individuals from 12 species and several subspecies of Cyornis based on three mitochondrial genes and five nuclear introns, with special focus on Chinese and Vietnamese populations of the monotypic C. hainanus and polytypic C. rubeculoides. We found no support for inclusion of C. concretus in Cyornis. Deep divergences were observed among different subspecies of C. banyumas and C. rubeculoides. C. rubeculoides glaucicomans was also shown to have a highly distinctive song, and we propose that it is treated as a distinctive Chinese endemic species, C. glaucicomans. In contrast, the south Vietnamese C. rubeculoides klossi, which has a disjunct distribution from the other subspecies of C. rubeculoides, along with a recently discovered population in Guangdong Province (China) with several plumage features reminiscent of C. r. klossi, were indistinguishable in all loci analyzed from the phenotypically markedly different C. hainanus. More research is needed to elucidate the reasons for this unexpected pattern.

Multiple instances of paraphyletic species and cryptic taxa revealed by mitochondrial and nuclear RAD data for Calandrella larks (Aves: Alaudidae).

The avian genus Calandrella (larks) was recently suggested to be non-monophyletic, and was divided into two genera, of which Calandrella sensu stricto comprises 4-5 species in Eurasia and Africa. We analysed mitochondrial cytochrome b (cytb) and nuclear Restriction-site Associated DNA (RAD) sequences from all species, and for cytb we studied 21 of the 22 recognised subspecies, with the aim to clarify the phylogenetic relationships within the genus and to compare large-scale nuclear sequence patterns with a widely used mitochondrial marker. Cytb indicated deep splits among the currently recognised species, although it failed to support the interrelationships among most of these. It also revealed unexpected deep divergences within C. brachydactyla, C. blanfordi/C. erlangeri, C. cinerea, and C. acutirostris. It also suggested that both C. brachydactyla and C. blanfordi, as presently circumscribed, are paraphyletic. In contrast, most of the many subspecies of C. brachydactyla and C. cinerea were unsupported by cytb, although two populations of C. cinerea were found to be genetically distinct. The RAD data corroborated the cytb tree (for the smaller number of taxa analysed) and recovered strongly supported interspecific relationships. However, coalescence analyses of the RAD data, analysed in SNAPP both with and without an outgroup, received equally strong support for two conflicting topologies. We suggest that the tree rooted with an outgroup - which is not recommended for SNAPP - is more trustworthy, and suggest that the reliability of analyses performed without any outgroup species should be thoroughly evaluated. We also demonstrate that degraded museum samples can be phylogenetically informative in RAD analyses following careful bioinformatic treatment. We note that the genus Calandrella is in need of taxonomic revision.