Divergent Selection in Low Recombination Regions Shapes the Genomic Islands in Two Incipient Shorebird Species.

Speciation in the face of gene flow is usually associated with a heterogeneous genomic landscape of divergence in nascent species pairs. However, multiple factors, such as divergent selection and local recombination rate variation, can influence the formation of these genomic islands. Examination of the genomic landscapes of species pairs that are still in the early stages of speciation provides an insight into this conundrum. In this study, population genomic analyses were undertaken using a wide range of sampling and whole-genome resequencing data from 96 unrelated individuals of Kentish plover (Charadrius alexandrinus) and white-faced plover (Charadrius dealbatus). We suggest that the two species exhibit varying levels of population admixture along the Chinese coast and on the Taiwan Island. Genome-wide analyses for introgression indicate that ancient introgression had occurred in Taiwan population, and gene flow is still ongoing in mainland coastal populations. Furthermore, we identified a few genomic regions with significant levels of interspecific differentiation and local recombination suppression, which contain several genes potentially associated with disease resistance, coloration, and regulation of plumage molting and thus may be relevant to the phenotypic and ecological divergence of the two nascent species. Overall, our findings suggest that divergent selection in low recombination regions may be a main force in shaping the genomic islands in two incipient shorebird species.

Ecological predictors of interspecific variation in bird bill and leg lengths on a global scale.

Bills and legs are two vital appendages for birds, and they exhibit huge interspecific variation in form and function, yet no study has examined the global predictors of this variation. This study examined global gradients in the relative lengths of bird bills and tarsi (i.e. exposed leg parts) to body size across non-migratory birds, while accounting for phylogeny. We found that relative bill length and tarsus length were related to diet, habitat density, latitude, annual mean temperature, temperature variability and hand-wing index (HWI), a proxy for birds' flight efficiency. Among these factors, diet played a primary role in predicting bill length, with nectar-feeding pollinators, vertivores, invertivores and omnivores having longer bills; HWI emerged as the predominant predictor of tarsus length, wherein species with higher HWI had shorter tarsi. However, the effects of these factors differed between passerines and non-passerines, with some temperature-related effects exhibiting opposite trends between these two groups. Our findings highlight the compromise in adaptations for feeding, thermoregulation and flight performance between the two distinct appendages.

Sex differences in immune gene expression in the brain of a small shorebird.

Males and females often exhibit differences in behaviour, life histories, and ecology, many of which are typically reflected in their brains. Neuronal protection and maintenance include complex processes led by the microglia, which also interacts with metabolites such as hormones or immune components. Despite increasing interest in sex-specific brain function in laboratory animals, the significance of sex-specific immune activation in the brain of wild animals along with the variables that could affect it is widely lacking. Here, we use the Kentish plover (Charadrius alexandrinus) to study sex differences in expression of immune genes in the brain of adult males and females, in two wild populations breeding in contrasting habitats: a coastal sea-level population and a high-altitude inland population in China. Our analysis yielded 379 genes associated with immune function. We show a significant male-biased immune gene upregulation. Immune gene expression in the brain did not differ in upregulation between the coastal and inland populations. We discuss the role of dosage compensation in our findings and their evolutionary significance mediated by sex-specific survival and neuronal deterioration. Similar expression profiles in the coastal and inland populations suggest comparable genetic control by the microglia and possible similarities in pathogen pressures between habitats. We call for further studies on gene expression of males and females in wild population to understand the implications of immune function for life-histories and demography in natural systems.

Prolactin concentrations predict parental investment and nest survival in a free-living shorebird.

The hormonal milieu that exists during reproduction is one of the key factors influencing the trade-off between reproductive investment and self-maintenance. Much previous work in birds has focused on prolactin as a physiological mediator since prolactin is involved in the onset and maintenance of parental care. However, how prolactin relates to reproductive success in terms of altering parental behavior in wild bird populations is not fully understood. Here, we report prolactin concentrations in breeding Kentish plovers (Charadrius alexandrinus), a small shorebird with variable mating systems and parental care, as an ecological model of mating system evolution. Throughout the breeding season, we estimated the circulating prolactin concentrations in male and female plovers during incubation. In addition, we monitored parental behavior and determined the fate of nests. We found that prolactin concentrations decreased during incubation but increased with clutch completion date. In addition, males and females with high prolactin concentrations spent more time on incubation than those with low prolactin concentrations. Importantly, higher prolactin concentrations in either males or females predict higher nest survival. Our results suggest that prolactin is an indicator of parental behavior in a wild shorebird population, although additional studies including experimental manipulation of prolactin concentrations are necessary to verify this relationship.

Genetic, phenotypic and ecological differentiation suggests incipient speciation in two Charadrius plovers along the Chinese coast.

BACKGROUND: Speciation with gene flow is an alternative to the nascence of new taxa in strict allopatric separation. Indeed, many taxa have parapatric distributions at present. It is often unclear if these are secondary contacts, e.g. caused by past glaciation cycles or the manifestation of speciation with gene flow, which hampers our understanding of how different forces drive diversification. Here we studied genetic, phenotypic and ecological aspects of divergence in a pair of incipient shorebird species, the Kentish (Charadrius alexandrinus) and the White-faced Plovers (C. dealbatus), shorebirds with parapatric breeding ranges along the Chinese coast. We assessed divergence based on molecular markers with different modes of inheritance and quantified phenotypic and ecological divergence in aspects of morphometric, dietary and climatic niches. RESULTS: Our integrative analyses revealed small to moderate levels of genetic and phenotypic distinctiveness with symmetric gene flow across the contact area at the Chinese coast. The two species diverged approximately half a million years ago in dynamic isolation with secondary contact occurring due to cycling sea level changes between the Eastern and Southern China Sea in the mid-late Pleistocene. We found evidence of character displacement and ecological niche differentiation between the two species, invoking the role of selection in facilitating divergence despite gene flow. CONCLUSION: These findings imply that ecology can indeed counter gene flow through divergent selection and thus contributes to incipient speciation in these plovers. Furthermore, our study highlights the importance of using integrative datasets to reveal the evolutionary history and assist the inference of mechanisms of speciation.

Quaternary climate and environmental changes have shaped genetic differentiation in a Chinese pheasant endemic to the eastern margin of the Qinghai-Tibetan Plateau.

The geological complexity generated by the uplift of the Qinghai-Tibetan Plateau and the resulting habitat heterogeneity have functioned together with climatic oscillations in the Quaternary to have a profound impact on the patterns of genetic diversity and demography of the fauna in this region. To understand the effect of the climatic and environmental shifts of the Quaternary on intraspecific genetic patterns and evolutionary history, we investigated the population genetic structure of the blue eared pheasant (Crossoptilon auritum), an endemic bird inhabiting the easternmost region of the plateau. Our phylogeographic analysis of mitochondrial DNA sequences and eight autosomal microsatellites reveals that the blue eared pheasant is subdivided into four distinct subpopulations: a central group (Huzhu and Taizi Mountains), a southern Zoige group, a southernmost Wanglang group and the northernmost Helan Mountain group. These groups are likely to have diverged in the Pleistocene, corresponding to geological changes and the interglacial-glacial climate oscillations that occurred at the eastern margin of the Qinghai-Tibetan Plateau. These subpopulations thus represent major conservation units, especially for the isolated Helan subpopulation. Our findings provide evidence of population divergence driven by complex Quaternary climate and environmental changes and, once more, highlight the importance of phylogeographic studies for conservation endeavours.

First breeding record of the black-headed penduline tit (Remiz macronyx) in China.

Black-headed penduline tit (Remiz macronyx) is a poorly known bird species mainly distributed in Iran, Turkmenistan, and Kazakhstan. The distribution of black-headed penduline tit is disjointed and fragmented, and it occurs only along lakes or rivers surrounded by extensive reedbeds. Four subspecies of R. macronyx have been recognized (macronyx, neglectus, nigricans, and ssaposhnikowi). The ssaposhnikowi subspecies was previously known to occur only around lakes in southeastern Kazakhstan. In this study, we reported the first confirmed breeding record of R. m. ssaposhnikowi in the Nalati wetland, Ili, Xinjiang, China, extending the distribution range of the black-headed penduline tit by 350 km to the east. We also obtained new information about the morphology and breeding behavior of R. m. ssaposhnikowi, which can be useful for the taxonomy of penduline tits, especially in distinguishing black-headed penduline tits from Eurasian penduline tits (R. pendulinus).

Mammalian predators and vegetated nesting habitat drive reduced protected area nesting success of Kentish plovers, Yellow Sea region, China.

Protected areas provide essential habitats for wildlife by conserving natural and semi-natural habitats and reducing human disturbance. However, whether breeding birds vulnerable to nest predation can benefit from strict land management in the protected area is unclear. Here, we compare the nesting performance of two groups of a ground-nesting shorebird, the Kentish plover (Charadrius alexandrinus), in the protected area (Liaohekou Natural Reserve, hereinafter PA), and the control non-protected area (non-PA) around the Liaohekou Natural Reserve, in the north of the Yellow Sea, China, and identify which environmental factors, such as nesting habitat and nest materials, influence the daily nest survival rate (DSR). We found similar nesting habitats in both study areas, dominated by bare land or Suaeda salsa grassland. However, DSR was lower in PA (0.91 ± 0.01) than in non-PA (0.97 ± 0.01). Kentish plovers nesting in areas with vegetation cover experienced lower DSR than in bare lands in both areas, and nests built with materials of S. salsa sticks had the lowest DSR in the bare land. Data from infrared cameras confirmed relatively higher predator abundances and nest predation rates by nocturnal mammals, such as Eurasian badgers (Meles meles), in PA than in non-PA, and this pattern was especially evident for plover nests located in S. salsa grassland. Our results suggest that Liaohekou Natural Reserve protected area may not necessarily provide safe nesting sites for Kentish plovers due to the abundance of generalist mammal nest predators. However, the PA includes about 80% of the nests from both locations. This means the contribution of the total number of successful nests continues to be much higher within PA, with the benefit for the species that this brings in terms of conservation. The variation and mechanisms underlying differences in the nest predator communities of PA and non-PA deserve further study.

Genomic status of yellow-breasted bunting following recent rapid population decline.

Global biodiversity is facing serious threats. However, knowledge of the genomic consequences of recent rapid population declines of wild organisms is limited. Do populations experiencing recent rapid population decline have the same genomic status as wild populations that experience long-term declines? Yellow-breasted Bunting (Emberiza aureola) is a critically endangered species that has been experiencing a recent rapid population decline. To answer the question, we assembled and annotated the whole genome of Yellow-breasted Bunting. Furthermore, we found high genetic diversity, low linkage disequilibrium, and low proportion of long runs of homozygosity in Yellow-breasted Bunting, suggesting that the populations following recent rapid declines have different genomic statuses from the population that experienced long-term population decline.

Mercury exposure in sedentary and migratory Charadrius plovers distributed widely across China.

Mercury pollution is a global problem and of particular concern in high emissions areas, such as China. We studied the migratory Kentish Plover, Charadrius alexandrinus, which breeds in coastal northern/central China and the inland Qinghai Lake, and the White-faced Plover C. dealbatus, a year-round resident of coastal southern China. We measured total mercury (THg) concentrations in feather and blood samples of breeding females. We expected low levels at the remote Qinghai Lake, but we found instead that feather THg concentrations were highest there (3.89 ± 1.53 [SD] µg/g DW [n = 34]; compared to 1.29 ± 0.61 µg/g of Kentish Plover elsewhere [n = 35] and 2.08 ± 1.45 µg/g for White-faced Plover [n = 56]), a result is consistent over 2 years. When including only coastal populations in the analysis, there were no differences in THg concentrations between the two species, although White-faced Plover had more variation. Feather THg concentrations for the coastal populations are similar to other studies on plovers and sandpipers globally, with most birds under the threshold of adverse effects (3 µg/g, an estimate that itself may be too low). Nevertheless, the Qinghai Kentish Plover population has mean feather concentrations above this threshold, indicating high exposure during the nonbreeding season, and some individuals have extreme values (e.g., a bird with a blood level of 7.63 µg/g DW from Zhanjiang, south China), so further research and monitoring are needed.

Successful breeding predicts divorce in plovers.

When individuals breed more than once, parents are faced with the choice of whether to re-mate with their old partner or divorce and select a new mate. Evolutionary theory predicts that, following successful reproduction with a given partner, that partner should be retained for future reproduction. However, recent work in a polygamous bird, has instead indicated that successful parents divorced more often than failed breeders (Halimubieke et al. in Ecol Evol 9:10734-10745, 2019), because one parent can benefit by mating with a new partner and reproducing shortly after divorce. Here we investigate whether successful breeding predicts divorce using data from 14 well-monitored populations of plovers (Charadrius spp.). We show that successful nesting leads to divorce, whereas nest failure leads to retention of the mate for follow-up breeding. Plovers that divorced their partners and simultaneously deserted their broods produced more offspring within a season than parents that retained their mate. Our work provides a counterpoint to theoretical expectations that divorce is triggered by low reproductive success, and supports adaptive explanations of divorce as a strategy to improve individual reproductive success. In addition, we show that temperature may modulate these costs and benefits, and contribute to dynamic variation in patterns of divorce across plover breeding systems.

Genome Assembly of the Common Pheasant Phasianus colchicus: A Model for Speciation and Ecological Genomics.

The common pheasant (Phasianus colchicus) in the order Galliformes and the family Phasianidae, has 30 subspecies distributed across its native range in the Palearctic realm and has been introduced to Europe, North America, and Australia. It is an important game bird often subjected to wildlife management as well as a model species to study speciation, biogeography, and local adaptation. However, the genomic resources for the common pheasant are generally lacking. We sequenced a male individual of the subspecies torquatus of the common pheasant with the Illumina HiSeq platform. We obtained 94.88 Gb of usable sequences by filtering out low-quality reads of the raw data generated. This resulted in a 1.02 Gb final assembly, which equals the estimated genome size. BUSCO analysis using chicken as a model showed that 93.3% of genes were complete. The contig N50 and scaffold N50 sizes were 178 kb and 10.2 Mb, respectively. All these indicate that we obtained a high-quality genome assembly. We annotated 16,485 protein-coding genes and 123.3 Mb (12.05% of the genome) of repetitive sequences by ab initio and homology-based prediction. Furthermore, we applied a RAD-sequencing approach for another 45 individuals of seven representative subspecies in China and identified 4,376,351 novel single nucleotide polymorphism (SNPs) markers. Using this unprecedented data set, we uncovered the geographic population structure and genetic introgression among common pheasants in China. Our results provide the first high-quality reference genome for the common pheasant and a valuable genome-wide SNP database for studying population genomics and demographic history.

Effects of migration and reproduction on the variation in persistent organic pollutant levels in Kentish Plovers from Cangzhou Wetland, China.

Migratory Birds have been considered biovectors of persistent organic pollutants (POPs) from sources to remote areas. In the present study, Kentish Plovers (Charadrius alexandrines) were collected in different periods, including immigration, breeding and emigration, to investigate the effects of migration and reproduction on POP variations in this bird species. Significant differences were found for dichlorodiphenyltrichloroethane (DDT) and hexachlorobenzene (HCB) concentrations in muscles between the immigration and emigration periods (p < 0.01 and p < 0.001, respectively), which could be attributed to the higher pesticide residues in the wintering grounds of plovers. Female plovers could excrete about 20.8-42.7% of POP load into eggs. Nevertheless, the POP levels didn't exhibit great reduction during the breeding period compared with other seasons, which suggested that the breeding status had little impact on POP levels in female plovers. The estimated mean transport masses of POPs driven by plover migration were at the milligram level (range: 0.02-7.05 mg), suggesting that the migration of plovers had limited impacts on the redistributions of POPs along their migratory routes.

Demographic Histories and Genome-Wide Patterns of Divergence in Incipient Species of Shorebirds.

Understanding how incipient species are maintained with gene flow is a fundamental question in evolutionary biology. Whole genome sequencing of multiple individuals holds great potential to illustrate patterns of genomic differentiation as well as the associated evolutionary histories. Kentish (Charadrius alexandrinus) and the white-faced (C. dealbatus) plovers, which differ in their phenotype, ecology and behavior, are two incipient species and parapatrically distributed in East Asia. Previous studies show evidence of genetic diversification with gene flow between the two plovers. Under this scenario, it is of great importance to explore the patterns of divergence at the genomic level and to determine whether specific regions are involved in reproductive isolation and local adaptation. Here we present the first population genomic analysis of the two incipient species based on the de novo Kentish plover reference genome and resequenced populations. We show that the two plover lineages are distinct in both nuclear and mitochondrial genomes. Using model-based coalescence analysis, we found that population sizes of Kentish plover increased whereas white-faced plovers declined during the Last Glaciation Period. Moreover, the two plovers diverged allopatrically, with gene flow occurring after secondary contact. This has resulted in low levels of genome-wide differentiation, although we found evidence of a few highly differentiated genomic regions in both the autosomes and the Z-chromosome. This study illustrates that incipient shorebird species with gene flow after secondary contact can exhibit discrete divergence at specific genomic regions and provides basis to further exploration on the genetic basis of relevant phenotypic traits.

The first set of universal nuclear protein-coding loci markers for avian phylogenetic and population genetic studies.

Multiple nuclear markers provide genetic polymorphism data for molecular systematics and population genetic studies. They are especially required for the coalescent-based analyses that can be used to accurately estimate species trees and infer population demographic histories. However, in avian evolutionary studies, these powerful coalescent-based methods are hindered by the lack of a sufficient number of markers. In this study, we designed PCR primers to amplify 136 nuclear protein-coding loci (NPCLs) by scanning the published Red Junglefowl (Gallus gallus) and Zebra Finch (Taeniopygia guttata) genomes. To test their utility, we amplified these loci in 41 bird species representing 23 Aves orders. The sixty-three best-performing NPCLs, based on high PCR success rates, were selected which had various mutation rates and were evenly distributed across 17 avian autosomal chromosomes and the Z chromosome. To test phylogenetic resolving power of these markers, we conducted a Neoavian phylogenies analysis using 63 concatenated NPCL markers derived from 48 whole genomes of birds. The resulting phylogenetic topology, to a large extent, is congruence with results resolved by previous whole genome data. To test the level of intraspecific polymorphism in these makers, we examined the genetic diversity in four populations of the Kentish Plover (Charadrius alexandrinus) at 17 of NPCL markers chosen at random. Our results showed that these NPCL markers exhibited a level of polymorphism comparable with mitochondrial loci. Therefore, this set of pan-avian nuclear protein-coding loci has great potential to facilitate studies in avian phylogenetics and population genetics.