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

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

Life in the intertidal: cellular responses, methylation and epigenetics

1.Phenotypic plasticity is essential for the persistence of organisms under changing environmental conditions but the control of the relevant cellular mechanisms including which genes are involved and the regulation of those genes remains unclear. One way to address this issue is to evaluate links between gene expression, methylation and phenotype using transplantation and common garden experiments within genetically homogeneous populations. 2.This approach was taken using the Antarctic limpet Nacella concinna. In this species, two distinct phenotypes are associated with the intertidal and subtidal zones. The in situ gene expression and methylation profiles of intertidal and subtidal cohorts were directly compared before and after reciprocal transplantation as well as after a common garden acclimation to aquarium conditions for 9 months. 3.Expression profiles showed significant modulation of cellular metabolism to habitat zone with the intertidal profile characterised by transcription modules for antioxidant production, DNA repair and the cytoskeleton reflecting the need to cope with continually fluctuating and stressful conditions including wave action, UV irradiation and desiccation. 4.Transplantation had an effect on gene expression. The subtidal animals transplanted to the intertidal zone modified their gene expression patterns towards that of an intertidal profile. In contrast, many of the antioxidant genes were still differentially expressed in the intertidal animals several weeks after transplantation into the relatively benign subtidal zone. 5.Furthermore, a core of genes involved in antioxidation was still preferentially expressed in intertidal animals at the end of the common garden experiment. Thus, acclimation in an aquarium tank for 9 months did not completely erase the intertidal gene expression profile. 6.Significant methylation differences were measured between intertidal and subtidal animals from the wild and after transplantation, which were reduced on common garden acclimation. This suggests that epigenetic factors play an important role in physiological flexibility associated with environmental niche

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

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

Impacts of herbivory by ecological replacements on an island ecosystem

The use of ecological replacements (analogue species to replace extinct taxa) to restore ecosystem functioning is a promising conservation tool. However, this approach is controversial, in part due to a paucity of data on interactions between analogue species and established taxa in the ecosystem. We conducted ecological surveys, comprehensively DNA barcoded an ecosystem's flora and inferred the diet of the introduced Aldabra giant tortoise, acting as an ecological replacement, to understand how it might have modified island plant communities on a Mauritian islet. Through further dietary analyses, we investigated consequential effects on the threatened endemic Telfair's skink. Dietary overlap between tortoises and skinks was greater than expected by chance. However, there was a negative correlation between tortoise and skink preferences in herbivory and minimal overlap in the plants most frequently consumed by the reptiles. Changes in the plant community associated with 7 years of tortoise grazing were characterised by a decrease in the percentage cover of native herbs and creepers, and an increase in the cover of an invasive herb when compared to areas without tortoises. However, tortoise dietary preferences themselves did not directly drive changes in the plant community. Tortoises successfully dispersed the seeds of an endemic palm, which in time may increase the extent of unique palm-rich habitat. We found no evidence that tortoises have increased the extent of plant species hypothesised to be part of a lost Mauritian tortoise grazed community. Synthesis and applications. Due to a negative correlation in tortoise and skink dietary preferences and minimal overlap in the most frequently consumed taxa, the presence of tortoises is unlikely to have detrimental impacts on Telfair's skinks. Tortoise presence is likely to be beneficial to skinks in the long term by increasing the extent of palm-rich habitat. Although tortoises are likely to play a role in controlling invasive plants, they are not a panacea for this challenge. After 7 years, tortoises have not resurrected a lost tortoise grazed community that we hypothesise might have existed in limited areas on the islet, indicating that further interventions may be required to restore this plant community

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, hybridising 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 (P. neglecta and P. heraldica). This study uses microsatellite genotyping and tracking data to address the possibility of between-species hybridisation occurring outside the Indian Ocean. Dispersal and gene flow spanning three oceans was 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 inter-specific 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 Procellariform species on a global scale, demonstrating the need for consideration of widespread migration and hybridisation in the conservation of threatened seabirds

Conservation genetics in protected populations

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Code for GLM model

R code used to test various ecological hypothesis using GLM model