The Role of Hybridization in Species Formation and Persistence.

Hybridization, or interbreeding between different taxa, was traditionally considered to be rare and to have a largely detrimental impact on biodiversity, sometimes leading to the breakdown of reproductive isolation and even to the reversal of speciation. However, modern genomic and analytical methods have shown that hybridization is common in some of the most diverse clades across the tree of life, sometimes leading to rapid increase of phenotypic variability, to introgression of adaptive alleles, to the formation of hybrid species, and even to entire species radiations. In this review, we identify consensus among diverse research programs to show how the field has progressed. Hybridization is a multifaceted evolutionary process that can strongly influence species formation and facilitate adaptation and persistence of species in a rapidly changing world. Progress on testing this hypothesis will require cooperation among different subdisciplines.

Developing the Protocol Infrastructure for DNA Sequencing Natural History Collections.

Intentionally preserved biological material in natural history collections represents a vast repository of biodiversity. Advances in laboratory and sequencing technologies have made these specimens increasingly accessible for genomic analyses, offering a window into the genetic past of species and often permitting access to information that can no longer be sampled in the wild. Due to their age, preparation and storage conditions, DNA retrieved from museum and herbarium specimens is often poor in yield, heavily fragmented and biochemically modified. This not only poses methodological challenges in recovering nucleotide sequences, but also makes such investigations susceptible to environmental and laboratory contamination. In this paper, we review the practical challenges associated with making the recovery of DNA sequence data from museum collections more routine. We first review key operational principles and issues to address, to guide the decision-making process and dialogue between researchers and curators about when and how to sample museum specimens for genomic analyses. We then outline the range of steps that can be taken to reduce the likelihood of contamination including laboratory set-ups, workflows and working practices. We finish by presenting a series of case studies, each focusing on protocol practicalities for the application of different mainstream methodologies to museum specimens including: (i) shotgun sequencing of insect mitogenomes, (ii) whole genome sequencing of insects, (iii) genome skimming to recover plant plastid genomes from herbarium specimens, (iv) target capture of multi-locus nuclear sequences from herbarium specimens, (v) RAD-sequencing of bird specimens and (vi) shotgun sequencing of ancient bovid bone samples.

Sustained plumage divergence despite weak genomic differentiation and broad sympatry in sister species of Australian woodswallows (Artamus spp.).

Plumage divergence can function as a strong premating barrier when species come into secondary contact. When it fails to do so, the results are often genome homogenization and phenotypic hybrids at the zone of contact. This is not the case in the largely sympatric masked woodswallow and white-browed woodswallow species (Passeriformes: Artamidae: Artamus spp) complex in Australia where phenotypic integrity is sustained despite no discernible mitochondrial structure in earlier work. This lack of structure may suggest recent divergence, ongoing gene flow or both, and phenotypic hybrids are reported albeit rarely. Here, we further assessed the population structure and differentiation across the species' nuclear genomes using ddRAD-seq. As found in the mitochondrial genome, no structure or divergence within or between the two species was detected in the nuclear genome. This coarse sampling of the genome nonetheless revealed peaks of differentiation around the genes SOX5 and AXIN1. Both are involved in the Wnt/ß-catenin signalling pathway, which regulates feather development. Reconstruction of demographic history and estimation of parameters supports a scenario of secondary contact. Our study informs how divergent plumage morphs may arise and be sustained despite whole-genome homogenization and reveals new candidate genes potentially involved in plumage divergence.

A set of principles and practical suggestions for equitable fieldwork in biology.

Field biology is an area of research that involves working directly with living organisms in situ through a practice known as "fieldwork." Conducting fieldwork often requires complex logistical planning within multiregional or multinational teams, interacting with local communities at field sites, and collaborative research led by one or a few of the core team members. However, existing power imbalances stemming from geopolitical history, discrimination, and professional position, among other factors, perpetuate inequities when conducting these research endeavors. After reflecting on our own research programs, we propose four general principles to guide equitable, inclusive, ethical, and safe practices in field biology: be collaborative, be respectful, be legal, and be safe. Although many biologists already structure their field programs around these principles or similar values, executing equitable research practices can prove challenging and requires careful consideration, especially by those in positions with relatively greater privilege. Based on experiences and input from a diverse group of global collaborators, we provide suggestions for action-oriented approaches to make field biology more equitable, with particular attention to how those with greater privilege can contribute. While we acknowledge that not all suggestions will be applicable to every institution or program, we hope that they will generate discussions and provide a baseline for training in proactive, equitable fieldwork practices.

Punctuated evolution in the learned songs of African sunbirds.

Learned traits are thought to be subject to different evolutionary dynamics than other phenotypes, but their evolutionary tempo and mode has received little attention. Learned bird song has been thought to be subject to rapid and constant evolution. However, we know little about the evolutionary modes of learned song divergence over long timescales. Here, we provide evidence that aspects of the territorial songs of Eastern Afromontane sky island sunbirds Cinnyris evolve in a punctuated fashion, with periods of stasis of the order of hundreds of thousands of years or more, broken up by evolutionary pulses. Stasis in learned songs is inconsistent with learned traits being subject to constant or frequent change, as would be expected if selection does not constrain song phenotypes over evolutionary timescales. Learned song may instead follow a process resembling peak shifts on adaptive landscapes. While much research has focused on the potential for rapid evolution in bird song, our results suggest that selection can tightly constrain the evolution of learned songs over long timescales. More broadly, these results demonstrate that some aspects of highly variable, plastic traits can exhibit punctuated evolution, with stasis over long time periods.

Assortative mating and epistatic mating-trait architecture induce complex movement of the crow hybrid zone.

Hybrid zones provide a window into the evolutionary processes governing species divergence. Yet, the contribution of mate choice to the temporal and spatial stability of hybrid zones remains poorly explored. Here, we investigate the effects of assortative mating on hybrid-zone dynamics by means of a mathematical model parameterized with phenotype and genotype data from the hybrid zone between all-black carrion and gray-coated hooded crows. In the best-fit model, narrow clines of the two mating-trait loci were maintained by a moderate degree of assortative mating inducing pre- and postzygotic isolation via positive frequency-dependent selection. Epistasis between the two loci induced hybrid-zone movement in favor of alleles conveying dark plumage followed by a shift in the opposite direction favoring gray-coated phenotypes ∼1 200 generations after secondary contact. Unlinked neutral loci diffused near-unimpeded across the zone. These results were generally robust to the choice of matching rule (self-referencing or parental imprinting) and effects of genetic drift. Overall, this study illustrates under which conditions assortative mating can maintain steep clines in mating-trait loci without generalizing to genome-wide reproductive isolation. It further emphasizes the importance of the genetic mating-trait architecture for spatio-temporal hybrid-zone dynamics.

hiphop: Improved paternity assignment among close relatives using a simple exclusion method for biallelic markers.

Assignment of parentage with molecular markers is most difficult when the true parents have close relatives in the adult population. Here, we present an efficient solution to that problem by extending simple exclusion approaches to parentage analysis with single nucleotide polymorphic markers (SNPs). We augmented the previously published homozygote opposite test (hot), which counts mismatches due to the offspring and candidate parent having different homozygous genotypes, with an additional test. In this case, parents homozygous for the same SNP are incompatible with heterozygous offspring (i.e., "Homozygous Identical Parents, Heterozygous Offspring are Precluded": hiphop). We tested this approach in a cooperatively breeding bird, the superb fairy-wren, Malurus cyaneus, where rates of extra-pair paternity are exceptionally high, and where paternity assignment is challenging because breeding males typically have first-order adult relatives in their neighbourhood. Combining the tests and conditioning on the maternal genotype with a set of 1376 autosomal SNPs always allowed us to distinguish a single most likely sire from his relatives, and also to identify cases where the true sire must have been unsampled. In contrast, if just the hot test was used, we failed to identify a single most-likely sire in 2.5% of cases. Resampling enabled us to create guidelines for the number of SNPs required when first-order relatives coexist in the mating pool. Our method, implemented in the R package hiphop, therefore provides unambiguous parentage assignments even in systems with complex social organisation. We also identified a suite of Z- and W-linked SNPs that always identified sex correctly.

From molecules to populations: appreciating and estimating recombination rate variation.

Recombination is a central biological process with implications for many areas in the life sciences. Yet we are only beginning to appreciate variation in the recombination rate along the genome and among individuals, populations and species. Spurred by technological advances, we are now able to bring variation in this key biological parameter to centre stage. Here, we review the conceptual implications of recombination rate variation and guide the reader through the assumptions, strengths and weaknesses of genomic inference methods, including population-based, pedigree-based and gamete-based approaches. Appreciation of the differences and commonalities of these approaches is a prerequisite to formulate a unifying and comparative framework for understanding the molecular and evolutionary mechanisms shaping, and being shaped by, recombination.

Genome of an iconic Australian bird: High-quality assembly and linkage map of the superb fairy-wren (Malurus cyaneus).

The superb fairy-wren, Malurus cyaneus, is one of the most iconic Australian passerine species. This species belongs to an endemic Australasian clade, Meliphagides, which diversified early in the evolution of the oscine passerines. Today, the oscine passerines comprise almost half of all avian species diversity. Despite the rapid increase of available bird genome assemblies, this part of the avian tree has not yet been represented by a high-quality reference. To rectify that, we present the first high-quality genome assembly of a Meliphagides representative: the superb fairy-wren. We combined Illumina shotgun and mate-pair sequences, PacBio long-reads, and a genetic linkage map from an intensively sampled pedigree of a wild population to generate this genome assembly. Of the final assembled 1.07-Gb genome, 975 Mb (90.4%) was anchored onto 25 pseudochromosomes resulting in a final superscaffold N50 of 68.11 Mb. This high-quality bird genome assembly is one of only a handful which is also accompanied by a genetic map and recombination landscape. In comparison to other pedigree-based bird genetic maps, we find that the fairy-wren genetic map more closely resembles those of Taeniopygia guttata and Parus major maps, unlike the Ficedula albicollis map which more closely resembles that of Gallus gallus. Lastly, we also provide a predictive gene and repeat annotation of the genome assembly. This new high-quality, annotated genome assembly will be an invaluable resource not only regarding the superb fairy-wren species and relatives but also broadly across the avian tree by providing a novel reference point for comparative genomic analyses.

Current geography masks dynamic history of gene flow during speciation in northern Australian birds.

Genome divergence is greatly influenced by gene flow during early stages of speciation. As populations differentiate, geographic barriers can constrain gene flow and so affect the dynamics of divergence and speciation. Current geography, specifically disjunction and continuity of ranges, is often used to predict the historical gene flow during the divergence process. We test this prediction in eight meliphagoid bird species complexes codistributed in four regions. These regions are separated by known biogeographical barriers across northern Australia and Papua New Guinea. We find that bird populations currently separated by terrestrial habitat barriers within Australia and marine barriers between Australia and Papua New Guinea have a range of divergence levels and probability of gene flow not associated with current range connectivity. Instead, geographic distance and historical range connectivity better predict divergence and probability of gene flow. In this dynamic environmental context, we also find support for a nonlinear decrease of the probability of gene flow during the divergence process. The probability of gene flow initially decreases gradually after a certain level of divergence is reached. Its decrease then accelerates until the probability is close to zero. This implies that although geographic connectivity may have more of an effect early in speciation, other factors associated with higher divergence may play a more important role in influencing gene flow midway through and later in speciation. Current geographic connectivity may then mislead inferences regarding potential for gene flow during speciation under a complex and dynamic history of geographic and reproductive isolation.

Social selection parapatry in Afrotropical sunbirds.

The extent of range overlap of incipient and recent species depends on the type and magnitude of phenotypic divergence that separates them, and the consequences of phenotypic divergence on their interactions. Signal divergence by social selection likely initiates many speciation events, but may yield niche-conserved lineages predisposed to limit each others' ranges via ecological competition. Here, we examine this neglected aspect of social selection speciation theory in relation to the discovery of a nonecotonal species border between sunbirds. We find that Nectarinia moreaui and Nectarinia fuelleborni meet in a ∼6 km wide contact zone, as estimated by molecular cline analysis. These species exploit similar bioclimatic niches, but sing highly divergent learned songs, consistent with divergence by social selection. Cline analyses suggest that within-species stabilizing social selection on song-learning predispositions maintains species differences in song despite both hybridization and cultural transmission. We conclude that ecological competition between moreaui and fuelleborni contributes to the stabilization of the species border, but that ecological competition acts in conjunction with reproductive interference. The evolutionary maintenance of learned song differences in a hybrid zone recommend this study system for future studies on the mechanisms of learned song divergence and its role in speciation.

Resolving taxonomic uncertainty and historical biogeographic patterns in Muscicapa flycatchers and their allies.

Muscicapa flycatchers and their allies (Bradornis, Dioptornis, Empidornis, Fraseria, Myioparus, Namibornis, and Sigelus) are widely distributed in Africa, Europe and Asia. This broad distribution and the wide variety of habitats occupied by the group, ranging from arid to tropical forests, presents an interesting opportunity to explore the evolution of biogeographic patterns and habitat associations. Sequence data (up to 3310 base pairs from two mitochondrial and two nuclear genes) were generated for 36 of 42 species which comprise the assemblage. Complementary data from an additional species was retrieved from GenBank, as was an additional gene which was available for 21 of our included taxa. Using model-based phylogenetic methods and molecular clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the lineage. Ancestral area reconstructions were performed on the phylogeny using LaGrange and BioGeoBEARS. Our results indicate that Bradornis, Fraseria, and Muscicapa are each non-monophyletic, with the latter being shown to comprise five separate clades each more closely related to other genera. Two new genera (Chapinia and Ripleyia) are erected to account for these results. Muscicapa and allies originated c. 7.4 Ma, most likely in Africa given that their sister lineage is almost entirely from there, and rapidly achieved a Eurasian distribution by c. 7.1 Ma. A second divergence at c. 6.1 Ma resulted in two clades. The first is a largely Eurasian clade that subsequently recolonized Africa, perhaps as the result of the loss of migration. The second is an African clade, and ancestral reconstructions suggest a Congolian (e.g. tropical forest) origin for this clade, with several subsequent diversifications into more arid habitats. This is a unique result, as most tropical forest lineages are confined to that habitat. As with other studies of African bird lineages, Afrotropical forest dynamics appear to have played a significant role in driving diversification in Muscicapa and allies, and our results include just the second recorded case of southern to northern African colonization patterns.

Molecular phylogenetics suggests a New Guinean origin and frequent episodes of founder-event speciation in the nectarivorous lories and lorikeets (Aves: Psittaciformes).

The lories and lorikeets (Aves: Loriinae: Loriini) are a readily recognizable, discrete group of nectarivorous parrots confined to the Indo-Pacific region between Wallace's Line and the Pitcairn Island group in the central-east Pacific Ocean. We present the first phylogenetic analysis of all currently recognized genera in the group using two mitochondrial and five nuclear loci. Our analyses suggest a New Guinean origin for the group at about 10million years ago (95% HPD 4.8-14.8) but this origin must be interpreted within the context of that island's complicated, recent geological history. That is, the origin and early diversification of the group may have taken place as New Guinea's Central Cordillera arose and the final constituent terranes that form present-day New Guinea were accreted. The latter activity may have promoted dispersal as a key element in the group's history. We have detected several instances of dispersal out of New Guinea that we argue constitute instances of founder-event speciation. Some phenotypically cohesive genera are affirmed as monophyletic but other genera are clearly in need of taxonomic dismantlement and reclassification. We recognize Parvipsitta Mathews, 1916 for two species usually placed in Glossopsitta and we advocate transfer of Chalcopsitta cardinalis into Pseudeos Peters, 1935. Other non-monophyletic genera such as Charmosyna, Psitteuteles and, probably, Trichoglossus, require improved taxon sampling and further phylogenetic analysis before their systematics can be resolved. Cursory examination of trait mapping across the group suggests that many traits are ancestral and of little use in determining genus-level systematics.

Sequence capture using PCR-generated probes: a cost-effective method of targeted high-throughput sequencing for nonmodel organisms.

Recent advances in high-throughput sequencing library preparation and subgenomic enrichment methods have opened new avenues for population genetics and phylogenetics of nonmodel organisms. To multiplex large numbers of indexed samples while sequencing predominantly orthologous, targeted regions of the genome, we propose modifications to an existing, in-solution capture that utilizes PCR products as target probes to enrich library pools for the genomic subset of interest. The sequence capture using PCR-generated probes (SCPP) protocol requires no specialized equipment, is highly flexible and significantly reduces experimental costs for projects where a modest scale of genetic data is optimal (25-100 genomic loci). Our alterations enable application of this method across a wider phylogenetic range of taxa and result in higher capture efficiencies and coverage at each locus. Efficient and consistent capture over multiple SCPP experiments and at various phylogenetic distances is demonstrated, extending the utility of this method to both phylogeographic and phylogenomic studies.

Diversification in an Afro-Asian songbird clade (Erythropygia-Copsychus) reveals founder-event speciation via trans-oceanic dispersals and a southern to northern colonization pattern in Africa.

Erythropygia scrub-robins and their allies are distributed throughout Africa, Europe, Southeast Asia, India, Madagascar and the Seychelles. This broad distribution, as well as the distribution of Erythropygia taxa across Africa, presents an interesting opportunity to explore the mechanisms by which this biogeographic distribution was achieved. Multilocus sequence data (3310 base pairs from two mitochondrial and two nuclear genes) were generated for all species of Erythropygia and Cercotrichas scrub-robins, as well as from genera previously shown to render Erythropygia paraphyletic. Using model-based phylogenetic methods and molecular clock dating, we constructed a time-calibrated molecular phylogenetic hypothesis for the lineage. Ancestral area reconstructions were performed on the phylogeny using probabilistic approaches implemented in LaGrange and BioGeoBEARS. Our results confirm that Erythropygia is not monophyletic, and that one of the two Erythropygia clades is more closely related to a clade of Asian and Indian Ocean islands distributed species. Overall, the Erythropygia and allies clade originated in Africa in the late Miocene c. 6.9 Ma. Subsequently, a number of overwater dispersals occurred to include an initial colonization of Southeast Asia, and an ensuing progression of colonizations from Southeast Asia to the Seychelles, from there to Madagascar, and from these Indian Ocean islands back to Southeast Asia. Within the two clades of Erythropygia, ancestral area reconstructions within Africa indicate a Southern Africa origin, with subsequent lineage divergence in each clade indicating northward colonization. Overall, this clade of non-migratory songbirds shows a remarkable number of trans-oceanic colonization events, that were possibly facilitated by wind-driven dispersal; repeated Africa to Asia colonizations, two of which occur in this clade, are exceptionally rare in birds. Also rare is our finding that colonization patterns in Africa indicate a southern to northern progression.