Negative effects of urbanisation on diurnal and nocturnal pollen-transport networks.

Pollinating insects are declining due to habitat loss and climate change, and cities with limited habitat and floral resources may be particularly vulnerable. The effects of urban landscapes on pollination networks remain poorly understood, and comparative studies of taxa with divergent niches are lacking. Here, for the first time, we simultaneously compare nocturnal moth and diurnal bee pollen-transport networks using DNA metabarcoding and ask how pollination networks are affected by increasing urbanisation. Bees and moths exhibited substantial divergence in the communities of plants they interact with. Increasing urbanisation had comparable negative effects on pollen-transport networks of both taxa, with significant declines in pollen species richness. We show that moths are an important, but overlooked, component of urban pollen-transport networks for wild flowering plants, horticultural crops, and trees. Our findings highlight the need to include both bee and non-bee taxa when assessing the status of critical plant-insect interactions in urbanised landscapes.

Evidence for strain-specific virulence of Trichomonas gallinae in African columbiformes.

Infection by parasites or pathogens can have marked physiological impacts on individuals. In birds, infection may affect moult and feather growth, which is an energetically demanding time in the annual cycle. Previous work has suggested a potential link between clinically visible Trichomonas gallinae infection and wing length in turtle doves Streptopelia turtur arriving on breeding grounds. First, T. gallinae infection was characterized in 149 columbids from 5 species, sampled on turtle dove wintering grounds in Senegal during the moulting period, testing whether infection by T. gallinae is linked to moult. Trichomonas gallinae prevalence was 100%, so rather than testing for differences between infected and uninfected birds, we tested for differences in moult progression between birds infected by different T. gallinae strains. Twelve strains of T. gallinae were characterized at the internal transcribed spacer 1 (ITS1)/5.8S/ITS2 region, of which 6 were newly identified within this study. In turtle doves only, evidence for differences in wing length by strain was found, with birds infected by strain Tcl-1 having wings nearly 6 mm longer than those infected with strain GEO. No evidence was found for an effect of strain identity within species on moult progression, but comparisons between infected and uninfected birds should be further investigated in species where prevalence is lower.

Assessing rates of parasite coinfection and spatiotemporal strain variation via metabarcoding: Insights for the conservation of European turtle doves Streptopelia turtur.

Understanding the frequency, spatiotemporal dynamics and impacts of parasite coinfections is fundamental to developing control measures and predicting disease impacts. The European turtle dove (Streptopelia turtur) is one of Europe's most threatened bird species. High prevalence of infection by the protozoan parasite Trichomonas gallinae has previously been identified, but the role of this and other coinfecting parasites in turtle dove declines remains unclear. Using a high-throughput sequencing approach, we identified seven strains of T. gallinae, including two novel strains, from ITS1/5.8S/ITS2 ribosomal sequences in turtle doves on breeding and wintering grounds, with further intrastrain variation and four novel subtypes revealed by the iron-hydrogenase gene. High spatiotemporal turnover was observed in T. gallinae strain composition, and infection was prevalent in all populations (89%-100%). Coinfection by multiple Trichomonas strains was rarer than expected (1% observed compared to 38.6% expected), suggesting either within-host competition, or high mortality of coinfected individuals. In contrast, coinfection by multiple haemosporidians was common (43%), as was coinfection by haemosporidians and T. gallinae (90%), with positive associations between strains of T. gallinae and Leucocytozoon suggesting a mechanism such as parasite-induced immune modulation. We found no evidence for negative associations between coinfections and host body condition. We suggest that longitudinal studies involving the recapture and investigation of infection status of individuals over their lifespan are crucial to understand the epidemiology of coinfections in natural populations.

Whole-genome sequencing and genome regions of special interest: Lessons from major histocompatibility complex, sex determination, and plant self-incompatibility.

Whole-genome sequencing of non-model organisms is now widely accessible and has allowed a range of questions in the field of molecular ecology to be investigated with greater power. However, some genomic regions that are of high biological interest remain problematic for assembly and data-handling. Three such regions are the major histocompatibility complex (MHC), sex-determining regions (SDRs) and the plant self-incompatibility locus (S-locus). Using these as examples, we illustrate the challenges of both assembling and resequencing these highly polymorphic regions and how bioinformatic and technological developments are enabling new approaches to their study. Mapping short-read sequences against multiple alternative references improves genotyping comprehensiveness at the S-locus thereby contributing to more accurate assessments of allelic frequencies. Long-read sequencing, producing reads of several tens to hundreds of kilobase pairs in length, facilitates the assembly of such regions as single sequences can span the multiple duplicated gene copies of the MHC region, and sequence through repetitive stretches and translocations in SDRs and S-locus haplotypes. These advances are adding value to short-read genome resequencing approaches by allowing, for example, more accurate haplotype phasing across longer regions. Finally, we assessed further technical improvements, such as nanopore adaptive sequencing and bioinformatic tools using pangenomes, which have the potential to further expand our knowledge of a number of genomic regions that remain challenging to study with classical resequencing approaches.

Subspecies hybridization as a potential conservation tool in species reintroductions.

Reintroductions are a powerful tool for the recovery of endangered species. However, their long-term success is strongly influenced by the genetic diversity of the reintroduced population. The chances of population persistence can be improved by enhancing the population's adaptive ability through the mixing of individuals from different sources. However, where source populations are too diverse the reintroduced population could also suffer from outbreeding depression or unsuccessful admixture due to behavioural or genetic barriers. For the reintroduction of Asiatic wild ass Equus hemionus ssp. in Israel, a breeding core was created from individuals of two different subspecies (E. h. onager & E. h. kulan). Today the population comprises approximately 300 individuals and displays no signs of outbreeding depression. The aim of this study was a population genomic evaluation of this conservation reintroduction protocol. We used maximum likelihood methods and genetic clustering analyses to investigate subspecies admixture and test for spatial autocorrelation based on subspecies ancestry. Further, we analysed heterozygosity and effective population sizes in the breeding core prior to release and the current wild population. We discovered high levels of subspecies admixture in the breeding core and wild population, consistent with a significant heterozygote excess in the breeding core. Furthermore, we found no signs of spatial autocorrelation associated with subspecies ancestry in the wild population. Inbreeding and variance effective population size estimates were low. Our results indicate no genetic or behavioural barriers to admixture between the subspecies and suggest that their hybridization has led to greater genetic diversity in the reintroduced population. The study provides rare empirical evidence of the successful application of subspecies hybridization in a reintroduction. It supports use of intraspecific hybridization as a tool to increase genetic diversity in conservation translocations.

Phylogenomics and species delimitation for effective conservation of manta and devil rays.

Practical biodiversity conservation relies on delineation of biologically meaningful units. Manta and devil rays (Mobulidae) are threatened worldwide, yet morphological similarities and a succession of recent taxonomic changes impede the development of an effective conservation strategy. Here, we generate genome-wide single nucleotide polymorphism (SNP) data from a geographically and taxonomically representative set of manta and devil ray samples to reconstruct phylogenetic relationships and evaluate species boundaries under the general lineage concept. We show that nominal species units supported by alternative data sources constitute independently evolving lineages, and find robust evidence for a putative new species of manta ray in the Gulf of Mexico. Additionally, we uncover substantial incomplete lineage sorting indicating that rapid speciation together with standing variation in ancestral populations has driven phylogenetic uncertainty within Mobulidae. Finally, we detect cryptic diversity in geographically distinct populations, demonstrating that management below the species level may be warranted in certain species. Overall, our study provides a framework for molecular genetic species delimitation that is relevant to wide-ranging taxa of conservation concern, and highlights the potential for genomic data to support effective management, conservation and law enforcement strategies.

Spatio-temporal processes drive fine-scale genetic structure in an otherwise panmictic seabird population.

When and where animals breed can shape the genetic structure and diversity of animal populations. The importance of drivers of genetic diversity is amplified in island populations that tend to have more delineated gene pools compared to continental populations. Studies of relatedness as a function of the spatial distribution of individuals have demonstrated the importance of spatial organisation for individual fitness with outcomes that are conditional on the overall genetic diversity of the population. However, few studies have investigated the impact of breeding timing on genetic structure. We characterise the fine-scale genetic structure of a geographically-isolated population of seabirds. Microsatellite markers provide evidence for largely transient within-breeding season temporal processes and limited spatial processes, affecting genetic structure in an otherwise panmictic population of sooty terns Onychoprion fuscatus. Earliest breeders had significantly different genetic structure from the latest breeders. Limited evidence was found for localised spatial structure, with a small number of individuals being more related to their nearest neighbours than the rest of the population. Therefore, population genetic structure is shaped by heterogeneities in collective movement in time and to a lesser extent space, that result in low levels of spatio-temporal genetic structure and the maintenance of genetic diversity.

Allelic diversity and patterns of selection at the major histocompatibility complex class I and II loci in a threatened shorebird, the Snowy Plover (Charadrius nivosus).

BACKGROUND: Understanding the structure and variability of adaptive loci such as the major histocompatibility complex (MHC) genes is a primary research goal for evolutionary and conservation genetics. Typically, classical MHC genes show high polymorphism and are under strong balancing selection, as their products trigger the adaptive immune response in vertebrates. Here, we assess the allelic diversity and patterns of selection for MHC class I and class II loci in a threatened shorebird with highly flexible mating and parental care behaviour, the Snowy Plover (Charadrius nivosus) across its broad geographic range. RESULTS: We determined the allelic and nucleotide diversity for MHC class I and class II genes using samples of 250 individuals from eight breeding population of Snowy Plovers. We found 40 alleles at MHC class I and six alleles at MHC class II, with individuals carrying two to seven different alleles (mean 3.70) at MHC class I and up to two alleles (mean 1.45) at MHC class II. Diversity was higher in the peptide-binding region, which suggests balancing selection. The MHC class I locus showed stronger signatures of both positive and negative selection than the MHC class II locus. Most alleles were present in more than one population. If present, private alleles generally occurred at very low frequencies in each population, except for the private alleles of MHC class I in one island population (Puerto Rico, lineage tenuirostris). CONCLUSION: Snowy Plovers exhibited an intermediate level of diversity at the MHC, similar to that reported in other Charadriiformes. The differences found in the patterns of selection between the class I and II loci are consistent with the hypothesis that different mechanisms shape the sequence evolution of MHC class I and class II genes. The rarity of private alleles across populations is consistent with high natal and breeding dispersal and the low genetic structure previously observed at neutral genetic markers in this species.

Rhizobacterial Community Assembly Patterns Vary Between Crop Species.

Currently our limited understanding of crop rhizosphere community assembly hinders attempts to manipulate it beneficially. Variation in root communities has been attributed to plant host effects, soil type, and plant condition, but it is hard to disentangle the relative importance of soil and host without experimental manipulation. To examine the effects of soil origin and host plant on root associated bacterial communities we experimentally manipulated four crop species in split-plot mesocosms and surveyed variation in bacterial diversity by Illumina amplicon sequencing. Overall, plant species had a greater impact than soil type on community composition. While plant species associated with different Operational Taxonomic Units (OTUs) in different soils, plants tended to recruit bacteria from similar, higher order, taxonomic groups in different soils. However, the effect of soil on root-associated communities varied between crop species: Onion had a relatively invariant bacterial community while other species (maize and pea) had a more variable community structure. Dynamic communities could result from environment specific recruitment, differential bacterial colonization or reflect broader symbiont host range; while invariant community assembly implies tighter evolutionary or ecological interactions between plants and root-associated bacteria. Irrespective of mechanism, it appears both communities and community assembly rules vary between crop species.

A diet rich in C3 plants reveals the sensitivity of an alpine mammal to climate change.

Plant-herbivore interactions provide critical insights into the mechanisms that govern the spatiotemporal distributions of organisms. These interactions are crucial to understanding the impacts of climate change, which are likely to have an effect on the population dynamics of alpine herbivores. The Royle's pika (Ochotona roylei, hereafter pika) is a lagomorph found in the western Himalaya and is dependent on alpine plants that are at risk from climate change. As the main prey of many carnivores in the region, the pika plays a crucial role in trophic interactions. We examined topographical features, plant genera presence and seasonal dynamics as drivers of the plant richness in the pika's diet across an elevational gradient (2,600-4,450 m). We identified 79 plant genera in the faecal pellets of pikas, of which 89% were forbs, >60% were endemic to the Himalaya, and 97.5% of the diet plant genera identified followed the C3 photosynthetic pathway. We found that, during the premonsoon season, the number of genera in the pika's diet decreased with increasing elevation. We demonstrate that a large area of talus supports greater plant diversity and, not surprisingly, results in higher species richness in the pika's diet. However, in talus habitat with deep crevices, pikas consumed fewer plant genera suggesting they may be foraging suboptimally due to predation risk. The continued increase in global temperature is expected to have an effect on the distribution dynamics of C3 plants and consequently influence pika diet and distribution, resulting in a significant negative cascading effect on the Himalayan ecosystem.

Genomic associations with bill length and disease reveal drift and selection across island bird populations.

Island species provide excellent models for investigating how selection and drift operate in wild populations, and for determining how these processes act to influence local adaptation and speciation. Here, we examine the role of selection and drift in shaping genomic and phenotypic variation across recently separated populations of Berthelot's pipit (Anthus berthelotii), a passerine bird endemic to three archipelagos in the Atlantic. We first characterized genetic diversity and population structuring that supported previous inferences of a history of recent colonizations and bottlenecks. We then tested for regions of the genome associated with the ecologically important traits of bill length and malaria infection, both of which vary substantially across populations in this species. We identified a SNP associated with variation in bill length among individuals, islands, and archipelagos; patterns of variation at this SNP suggest that both phenotypic and genotypic variation in bill length is largely shaped by founder effects. Malaria was associated with SNPs near/within genes involved in the immune response, but this relationship was not consistent among archipelagos, supporting the view that disease resistance is complex and rapidly evolving. Although we found little evidence for divergent selection at candidate loci for bill length and malaria resistance, genome scan analyses pointed to several genes related to immunity and metabolism as having important roles in divergence and adaptation. Our findings highlight the utility and challenges involved with combining association mapping and population genetic analysis in nonequilibrium populations, to disentangle the effects of drift and selection on shaping genotypes and phenotypes.

New universal ITS2 primers for high-resolution herbivory analyses using DNA metabarcoding in both tropical and temperate zones.

DNA metabarcoding is a rapidly growing technique for obtaining detailed dietary information. Current metabarcoding methods for herbivory, using a single locus, can lack taxonomic resolution for some applications. We present novel primers for the second internal transcribed spacer of nuclear ribosomal DNA (ITS2) designed for dietary studies in Mauritius and the UK, which have the potential to give unrivalled taxonomic coverage and resolution from a short-amplicon barcode. In silico testing used three databases of plant ITS2 sequences from UK and Mauritian floras (native and introduced) totalling 6561 sequences from 1790 species across 174 families. Our primers were well-matched in silico to 88% of species, providing taxonomic resolution of 86.1%, 99.4% and 99.9% at the species, genus and family levels, respectively. In vitro, the primers amplified 99% of Mauritian (n = 169) and 100% of UK (n = 33) species, and co-amplified multiple plant species from degraded faecal DNA from reptiles and birds in two case studies. For the ITS2 region, we advocate taxonomic assignment based on best sequence match instead of a clustering approach. With short amplicons of 187-387 bp, these primers are suitable for metabarcoding plant DNA from faecal samples, across a broad geographic range, whilst delivering unparalleled taxonomic resolution.

The decline of the Turtle Dove: Dietary associations with body condition and competition with other columbids analysed using high-throughput sequencing.

Dietary changes linked to the availability of anthropogenic food resources can have complex implications for species and ecosystems, especially when species are in decline. Here, we use recently developed primers targeting the ITS2 region of plants to characterize diet from faecal samples of four UK columbids, with particular focus on the European turtle dove (Streptopelia turtur), a rapidly declining obligate granivore. We examine dietary overlap between species (potential competition), associations with body condition in turtle doves and spatiotemporal variation in diet. We identified 143 taxonomic units, of which we classified 55% to species, another 34% to genus and the remaining 11% to family. We found significant dietary overlap between all columbid species, with the highest between turtle doves and stock doves (Columba oenas), then between turtle doves and woodpigeons (Columba palumbus). The lowest overlap was between woodpigeons and collared doves (Streptopelia decaocto). We show considerable change in columbid diets compared to previous studies, probably reflecting opportunistic foraging behaviour by columbids within a highly anthropogenically modified landscape, although our data for nonturtle doves should be considered preliminary. Nestling turtle doves in better condition had a higher dietary proportion of taxonomic units from natural arable plant species and a lower proportion of taxonomic units from anthropogenic food resources such as garden bird seed mixes and brassicas. This suggests that breeding ground conservation strategies for turtle doves should include provision of anthropogenic seeds for adults early in the breeding season, coupled with habitat rich in accessible seeds from arable plants once chicks have hatched.

Widespread gene flow between oceans in a pelagic seabird species complex.

Global-scale gene flow is an important concern in conservation biology as it has the potential to either increase or decrease genetic diversity in species and populations. Although many studies focus on the gene flow between different populations of a single species, the potential for gene flow and introgression between species is understudied, particularly in seabirds. The only well-studied example of a mixed-species, hybridizing population of petrels exists on Round Island, in the Indian Ocean. Previous research assumed that Round Island represents a point of secondary contact between Atlantic (Pterodroma arminjoniana) and Pacific species (Pterodroma neglecta and Pterodroma heraldica). This study uses microsatellite genotyping and tracking data to address the possibility of between-species hybridization occurring outside the Indian Ocean. Dispersal and gene flow spanning three oceans were demonstrated between the species in this complex. Analysis of migration rates estimated using bayesass revealed unidirectional movement of petrels from the Atlantic and Pacific into the Indian Ocean. Conversely, structure analysis revealed gene flow between species of the Atlantic and Pacific oceans, with potential three-way hybrids occurring outside the Indian Ocean. Additionally, geolocation tracking of Round Island petrels revealed two individuals travelling to the Atlantic and Pacific. These results suggest that interspecific hybrids in Pterodroma petrels are more common than was previously assumed. This study is the first of its kind to investigate gene flow between populations of closely related Procellariiform species on a global scale, demonstrating the need for consideration of widespread migration and hybridization in the conservation of threatened seabirds.

Evaluation of genetic isolation within an island flora reveals unusually widespread local adaptation and supports sympatric speciation.

It is now recognized that speciation can proceed even when divergent natural selection is opposed by gene flow. Understanding the extent to which environmental gradients and geographical distance can limit gene flow within species can shed light on the relative roles of selection and dispersal limitation during the early stages of population divergence and speciation. On the remote Lord Howe Island (Australia), ecological speciation with gene flow is thought to have taken place in several plant genera. The aim of this study was to establish the contributions of isolation by environment (IBE) and isolation by community (IBC) to the genetic structure of 19 plant species, from a number of distantly related families, which have been subjected to similar environmental pressures over comparable time scales. We applied an individual-based, multivariate, model averaging approach to quantify IBE and IBC, while controlling for isolation by distance (IBD). Our analyses demonstrated that all species experienced some degree of ecologically driven isolation, whereas only 12 of 19 species were subjected to IBD. The prevalence of IBE within these plant species indicates that divergent selection in plants frequently produces local adaptation and supports hypotheses that ecological divergence can drive speciation in sympatry.