Availability of suitable nectar and pollen resources is a limiting factor for pollinator survival, with both overall resource quantity and quality, along with provision throughout the season, being critical. Yet, our understanding of how the selection of floral resources changes over time, and how this relates to floral availability within the landscape is limited. To fill this knowledge gap, we characterise floral use by honeybee (Apis mellifera) colonies, in a diverse horticultural and agricultural landscape, from April to September, over two years, using pollen DNA metabarcoding of honey samples. We compared the pollen found to the availability of host plants within the surrounding landscape using floral surveys. Honeybees used a total of 143 plant taxa, but only 10 of these were determined as major sources (>10% of total sequence reads in any month) and total plant use represented a small proportion of the available floral resources (23% of genera). Distinct patterns of diet specialisation were identified in June and August, where colonies diverged in their floral preferences before re-aligning in July and September. Following optimal foraging theory, these patterns represent periods of resource limitation. Honeybees showed a preference for flowering trees in the spring, followed by shrubs and herbs in summer and used native and near-native plants more than horticultural plants, as major food sources. DNA metabarcoding allows an increased ecological insight into floral resource use by honeybees and highlights the importance of providing continuous and sufficient floral resources throughout the year.

Disseminated neoplasia (DN) is one of the most challenging and unrecognised diseases occurring in aquatic fauna. It has been diagnosed in four bivalve species from the Gulf of Gdańsk (Southern Baltic Sea) with the highest frequency in Limecola balthica (formerly Macoma balthica), reaching up to 94% in some populations. The aetiology of DN in the Baltic Sea has not yet been identified, with earlier studies trying to link its occurrence with environmental pollution. Taking into account recent research providing evidence that DN is horizontally transmitted as clonal cells between individuals in some bivalve species, we aimed to test whether DN is a transmissible cancer in the population of L. balthica from the Gulf of Gdańsk highly affected with cancer. We examined mitochondrial cytochrome c oxidase I (mtCOI) and elongation factor 1α (EF1α) sequences of genomes obtained from haemolymph and tissues of neoplastic and healthy individuals. Sequence analysis resulted in detection of an independent transmissible cancer lineage occurring in 4 neoplastic clams that is not present in healthy animals. This paper describes the first case of transmissible DN in the clam L. balthica providing further insights for studies on this disease.

We recently published a paper quantifying the genome-wide consequences of natural selection, including the effects of indirect selection due to the correlation of genetic regions (neutral or selected) with directly selected regions (Gompert et al. 2022). In their critique of our paper, Charlesworth & Jensen (2022) make two main points: (i) indirect selection is equivalent to hitchhiking and thus well documented (i.e., our results are not novel), and (ii) that we do not demonstrate the source of linkage disequilibrium (LD) between SNPs and the Mel-Stripe locus in the Timema cristinae experiment we analyze. As we discuss in detail below, neither of these are substantial criticisms of our work.

The gut microbiota of bees affect nutrition, immunity, and host fitness, yet the role of diet, sociality, and geographic variation in determining microbiome structure, including strain-level diversity and relatedness, remain poorly understood. Here, we use full-length 16S amplicon sequencing to compare the crop and gut microbiomes of two incipiently social carpenter bee species, Xylocopa sonorina and Xylocopa tabaniformis, from multiple geographic sites within each species’ range. We found that Xylocopa species share a set of core taxa consisting of Bombilactobacillus, Bombiscardovia, and Lactobacillus apis, found in >95% of all individual bees sampled, and Gilliamella and Apibacter were also detected in the gut of both species with high frequency. The crop bacterial community of both species was comprised nearly entirely of Apilactobacillus with occasionally abundant nectar bacteria. Despite sharing core taxa, Xylocopa species’ microbiomes were distinguished by multiple bacterial lineages, including species-specific strains of core taxa. In both bee species, bacterial species exhibited geographic patterns in the presence of specific sequence variants. The use of long-read amplicons revealed otherwise cryptic species and population-level differentiation in core microbiome members which was masked when a shorter fragment of the 16S (V4) was considered. We conclude that these Xylocopa species host a distinctive microbiome, similar to that of previously characterized social apids, which suggests that further investigation to understand the evolution of bee microbiome and its drivers is warranted.

Understanding the environmental impact on the assembly of local communities in relation to their spatial and temporal connectivity is still a challenge in metacommunity ecology. This study aims to unravel underlying metacommunity processes and environmental factors that result in observed zooplankton communities. Unlike most metacommuniy studies, we jointly examine active and dormant communities using a DNA metabarcoding approach to overcome limitations of morphological species identification. We applied two-fragment (COI and 18S) metabarcoding to monitor communities of 24 kettle holes over a two-year period to unravel (I) spatial and temporal connectivity of the communities, (II) environmental factors influencing local communities, and (III) dominant underlying metacommunity processes in this system. We found a strong separation of zooplankton communities from kettle holes of different hydroperiods (degree of permanency) throughout the season, while the community composition within single kettle holes did not differ between years. Species richness was primarily dependent on pH and permanency, while species diversity (Shannon Index) was influenced by kettle hole location. Community composition was impacted by kettle hole size and surrounding field crops. Environmental processes dominated temporal and spatial processes. Sediment communities showed a different composition compared to water samples, but did not differ between ephemeral and permanent kettle holes. Our results suggest that communities are mainly structured by environmental filtering based on pH, kettle hole size, surrounding field crops, and permanency. Environmental filtering based on specific conditions in individual kettle holes seems to be the dominant process in community assembly in the studied zooplankton metacommunity. Understanding the environmental impact on the assembly of local communities in relation to their spatial and temporal connectivity is still a challenge in metacommunity ecology. This study aims to unravel underlying metacommunity processes and environmental factors that result in observed zooplankton communities. Unlike most metacommunity studies, we jointly examine active and dormant zooplankton communities using a DNA metabarcoding approach to overcome limitations of morphological species identification. We applied two-fragment (COI and 18S) metabarcoding to monitor communities of 24 kettle holes over a two-year period to unravel (I) spatial and temporal connectivity of the communities, (II) environmental factors influencing local communities, and (III) dominant underlying metacommunity processes in this system. We found a strong separation of zooplankton communities from kettle holes of different hydroperiods (degree of permanency) throughout the season, while the community composition within single kettle holes did not differ between years. Species richness was primarily dependent on pH and permanency, while species diversity (Shannon Index) was influenced by kettle hole location. Community composition was impacted by kettle hole size and surrounding field crops. Environmental processes dominated temporal and spatial processes. Sediment communities showed a different composition compared to water samples, but did not differ between ephemeral and permanent kettle holes. Our results suggest that communities are mainly structured by environmental filtering based on pH, kettle hole size, surrounding field crops, and permanency. Environmental filtering based on specific conditions in individual kettle holes seems to be the dominant process in community assembly in the studied zooplankton metacommunity.    

Anthropogenic activities are leading to changes in the environment at global scales, and understanding these changes requires rapid, high-throughput methods of assessment. Pollen DNA metabarcoding and related methods provide advantages in throughput and efficiency over traditional methods, such as microscopic identification of pollen and visual observation of plant-pollinator interactions. Pollen DNA metabarcoding is currently being applied to assessments of plant-pollinator interactions and their responses to land-use change such as increased agricultural intensity and urbanisation, surveillance of ecosystem change, and monitoring of spatiotemporal distribution of allergenic pollen. In combination with historical specimens, pollen DNA metabarcoding can compare contemporary and past ecosystems. Current technical challenges with pollen DNA metabarcoding include the need to understand the relationship between sequence read and species abundance, develop methods for determining confidence limits for detection and taxonomic classification, increase method standardisation, and improve of gaps in reference databases. Future research expanding the method to intraspecific identification, analysis of DNA in ancient pollen samples, and increased use of museum and herbarium specimens could open further avenues for research. Ongoing developments in sequencing technologies can accelerate progress towards these goals. Global ecological change is happening rapidly, and we anticipate that high-throughput methods such as pollen DNA metabarcoding are critical for assessing these changes and providing timely management recommendations to preserve biodiversity and the evolutionary and ecological processes that support it.

Secondary contact of species that have evolved partial reproductive isolation in allopatry may result in several outcomes, which range from rampant hybridization to barrier reinforcement. Reinforcement arises from reduced hybrid fitness, which promotes assortative mating and hence speciation. In plants, self-fertilization and disjunction in reproductive phenology are often cited as evidence of reinforcement. However, local adaptation and pleiotropic effects during colonization can also lead to reproductive isolation without reinforcement. We explored these competing possibilities in a fir species complex (Abies flinckii - A. religiosa) distributed in ‘sky-islands’ along the Trans-Mexican Volcanic Belt (TMVB), in central Mexico. Despite co-occurring in two independent sympatric regions (west and center), these two taxa seem to rarely interbreed because of disjunct reproductive phenologies. We genotyped 1,147 SNPs, generated by GBS across 23 populations, and compared multiple demographic scenarios, built based on the geological history of the TMVB. The best-fitting model revealed one of the most rapid and complete speciation cases for a conifer species-pair, dating back to ~1.2 Ma, which included an early asymmetric gene flow (mostly from A. flinckii into A. religiosa) limited to the most ancient sympatric region. Coupled with the lack of support for colonization models, our demographic inferences point to an early development of reinforcement, as a putative mechanism for avoiding hybridization. Reinforcement should be considered as a key player for originating and maintaining species diversity in the tropics and subtropics and incorporated in studies with other model systems.

The takin (Budorcas taxicolor) is one of the largest bovid herbivores across caprinae subfamily. The takin is at high risk of extinction, however, its taxonomic status is still unclear. In this study, we constructed the first reference genome of B. taxicolor using PacBio long High-Fidelity reads and Hi-C technology. The assembled genome is ~2.95 Gb with a contig N50 of 68.05 Mb and a scaffold N50 of 101.27 Mb, which were anchored onto 25+XY chromosomes. Compared to the common ancestral karyotype of bovidae (2n=60), we found the takin (2n=52) experienced four chromosome fusions and one large translocation. We also found that the takin was most closely related to muskox, not other caprinae species. Further, we re-sequenced nine golden takins from the main distribution area, Qinling Mountains, and identified 3.3 million SNPs. The genetic diversity of takin was very low (θπ=0.00028 and heterozygosity=0.00038), which was among the lowest detected in the domestic and wild mammals. We also found takin genomes showed high inbreeding coefficient (FROH=0.217) suggesting severe inbreeding depression. The genome analysis show that the effective population size of takins declined significantly from ~100,000 years ago. Our results provide valuable information for protection of takins and insights into its evolution.

Most of our understanding of island diversity comes from the study of aboveground systems, while the patterns and processes of diversification and community assembly for belowground biotas remain poorly understood. Here we take advantage of a relatively young and dynamic oceanic island to advance our understanding of eco-evolutionary processes driving community assembly within soil mesofauna. Using whole organism community DNA (wocDNA) metabarcoding and the recently developed metaMATE pipeline, we have generated spatially explicit and reliable haplotype-level DNA sequence data for soil mesofaunal assemblages sampled across the four main habitats within the island of Tenerife. Community ecological and metaphylogeographic analyses have been performed at multiple levels of genetic similarity, from haplotypes to species and supraspecific groupings. Broadly consistent patterns of local-scale species richness across different insular habitats have been found, whereas local insular richness is lower than in continental settings. Our results reveal an important role for niche conservatism as a driver of insular community assembly of soil mesofauna, with only limited evidence for habitat shifts promoting diversification. Furthermore, support is found for a fundamental role of habitat in the assembly of soil mesofauna, where habitat specialism is mainly due to colonisation and the establishment of preadapted species. Hierarchical patterns of distance decay at the community level and metaphylogeographical analyses support a pattern of geographic structuring over limited spatial scales, from the level of haplotypes through to species and lineages, as expected for taxa with strong dispersal limitations. Our results demonstrate the potential for wocDNA metabarcoding to advance our understanding of biodiversity.

Bloom-forming marine gelatinous zooplankton, including the pelagic tunicate Dolioletta gegenbauri, occur circumglobally and have the potential to significantly influence the structure of pelagic marine food webs and biogeochemical cycling through interactions with microbial communities. Using targeted metabarcoding (16S rRNA genes recovering Bacteria/Archaea) and qPCR approaches associated with laboratory-based feeding experiments, we characterized patterns in doliolid gut microbiomes and microbial communities associated with doliolid fecal pellets and the surrounding seawater. The characterization of starved doliolids provides the first description of the doliolid gut microbiome. At the highest taxonomic levels, doliolid-associated bacterial communities are characteristic of marine bacterioplankton communities around the globe and were dominated by representatives of six major bacterial groups including Gammaproteobacteria, Alphaproteobacteria, Cyanobacteria, Planctomycetes, Bacteroidia and, Phycisphaerae. Comparison between sample types, however, revealed distinct patterns in diversity and biomass supporting the hypothesis that through their presence and trophic activity, doliolids influence the structure of pelagic food webs and biogeochemical cycling in subtropical continental shelf systems where doliolid blooms are common. Bacteria associated with starved doliolids (representative of the resident doliolid gut microbiome) possessed distinct communities, supporting the hypothesis that doliolids possess a unique but low diversity, low biomass microbiome optimized to support a detrital trophic mode. Among potential core microbiome taxa, the genera Pseudoalteromomas and Shimia were the most abundant, similar to patterns observed in other marine invertebrates. Exploratory bioinformatic analyses of predicted functional genes suggest that doliolids, via their interactions with bacterial communities, may affect important biogeochemical processes including nitrogen, sulfur, and organic matter cycling.

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 intra-strain variation and four novel sub-types 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.

We write to address recent claims by Gompert et al. (2021) about the potentially important and underappreciated phenomena of “indirect selection”, the observation that neutral regions may be affected by natural selection. We argue both that this phenomenon – generally known as genetic hitchhiking – is neither new nor poorly studied, and that the patterns described by the authors have multiple alternative explanations.

Emerging plant pathogens have been increasing exponentially over the last century. To address this issue, it is critical to determine whether these pathogens are native to ecosystems or have been recently introduced. Understanding the ecological and evolutionary processes fostering emergence can help to manage their spread and predict epidemics/epiphytotics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread, and evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia, and the Pacific Islands. We analyzed patterns of genetic variation using Bayesian inference, maximum likelihood phylogeny, populations splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent based theory using Approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from ghost population to Malaysia and the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate that P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that this pathogen was recently introduced to multiple geographic regions. Our results suggest that recent emergence of P. noxius in eastern Asia, Australia, and the Pacific Islands is likely driven by anthropogenic and natural disturbances, such as deforestation, land-use change, severe weather events, and/or introduction of exotic plants. This study provides a novel example of applying genome-wide allele frequency data to unravel dynamics of pathogen emergence under changing ecosystem conditions.

Methodological and technological improvements are continually revolutionizing the field of ancient DNA. Most ancient DNA extraction methods require the partial (or complete) destruction of finite museum specimens, which disproportionately impacts small or fragmentary subfossil remains, and future analyses. We present a minimally destructive ancient DNA extraction method optimized for small vertebrate remains. We applied these methods to detect lost mainland genetic diversity in the large New Zealand diplodactylid gecko genus Hoplodactylus, which is presently restricted to predator-free island sanctuaries. We present the first mitochondrial genomes for New Zealand diplodactylid geckos, recovered from 19 modern, six historic/archival (1898 to 2011) and 16 Holocene Hoplodactylus duvaucelii sensu latu specimens, and one modern Woodworthia sp. specimen. No obvious damage was observed in post-extraction micro-CT reconstructions. All ‘large gecko’ specimens examined from extinct populations were found to be conspecific with extant Hoplodactylus species, suggesting their large relative size evolved only once in the New Zealand diplodactylid radiation. Phylogenetic analyses of Hoplodactylus samples recovered two genetically (and morphologically) distinct North and South Island clades, probably corresponding to distinct species. Finer phylogeographic structuring within Hoplodactylus spp. highlighted the impacts of Late-Cenozoic biogeographic barriers, including the opening and closure of Pliocene marine straits, fluctuations in size and suitability of glacial refugia, and eustatic sea-level change. Recent mainland extinction obscured these signals from the modern tissue derived data. These results highlight the utility of minimally destructive DNA extraction in genomic analyses of less well studied small vertebrate taxa, and the conservation of natural history collections.

Understanding the mechanisms and genes that enable animal populations to adapt to pathogens is important from an evolutionary, health and conservation perspective. Berthelot’s pipit (Anthus berthelotii) experiences extensive and consistent spatial heterogeneity in avian pox infection pressure across its range of island populations, thus providing an excellent system with which to examine how pathogen-mediated selection drives spatial variation in immunogenetic diversity. Here we test for evidence of genetic variation associated with avian pox at both an individual and population-level. At the individual level, we find no evidence that variation in MHC class I and TLR4 (both known to be important in recognising viral infection) was associated with pox infection within two separate populations. However, using genotype-environment association (Bayenv) in conjunction with genome-wide (ddRAD-seq) data, we detected strong associations between population-level avian pox prevalence and allele frequencies of single nucleotide polymorphisms (SNPs) at a number of sites across the genome. These sites were located within genes involved in cellular stress signalling and immune responses, many of which have previously been associated with responses to viral infection in humans and other animals. Consequently, our analyses provide evidence that pathogen-mediated selection has shaped genomic variation among relatively recently colonised island bird populations, and highlights the utility of genotype-environment associations for identifying candidate genes involved in adaption to local pathogen pressures.

Habitat fragmentation is known to affect biodiversity, but the impact on pollinators and their interactions with plants is still unclear in anthropized landscapes. Islands are open-air laboratories for ecological studies with simplified communities and interactions, suitable to disentangle how land-use alteration impacts pollination ecology and its ecosystem service. Here, we used Maldives islands as model systems to investigate how pollinator richness, their mutualistic interactions with plants, and pollination efficiency are shaped by the degree of green area fragmentation (i.e., gardens, parks and semi-natural green covered patches), by considering both community- and species-level responses. To do this, we surveyed pollinators from 11 islands showing a gradient of green area fragmentation. In order to characterize the interactions between plants and pollinators and obtain a novel and comprehensive view of the key ecological dynamics, a DNA metabarcoding approach was adopted to identify the pollen carried by pollinators. We found that green area fragmentation at intermediate levels played positive effects on pollinator richness. However, fragmentation decreased interaction network complexity. Intriguingly, body size mediated the effect of landscape alteration on plant-pollinator interactions, as only the largest bee species expanded the foraging breath in terms of transported pollen richness at increasing fragmentation. In parallel, the pollination efficiency increased with pollinator species richness in two sentinel plants. This study shows that moderate landscape fragmentation of green areas shapes the ecosystem service of pollination, where in spite of interactions being less complex and mediated by pollinator body size, pollinator biodiversity and potential plant reproduction are supported.

Madagascar’s Central Highlands are largely composed of grasslands, interspersed with patches of forest. The pre-human extent of these grasslands is a topic of vigorous debate, with conventional wisdom holding that they are anthropogenic in nature and emerging evidence supporting that grasslands were a component of the pre-human Central Highlands vegetation. Here, we shed light on the temporal dynamics of Madagascar’s vegetative composition by conducting a population genomic investigation of Goodman’s mouse lemur (Microcebus lehilahytsara; Cheirogaleidae). These small-bodied primates occur both in Madagascar’s eastern rainforests and in the Central Highlands, which makes them a valuable indicator species. Population divergences among forest-dwelling mammals can serve as a proxy for habitat fragmentation and patterns of post-divergence gene flow can reveal potential migration corridors consistent with a wooded grassland mosiac. We used RADseq data to infer phylogenetic relationships, population structure, demographic models of post-divergence gene flow, and population size change through time. These analyses offer evidence that open habitats are an ancient component of the Central Highlands, and that wide-spread forest fragmentation occurred naturally during a period of decreased precipitation near the last glacial maximum. Models of gene flow suggest that migration across the Central Highlands has been possible from the Pleistocene through the recent Holocene via riparian corridors. Notably, though our findings support the hypothesis that Central Highland grasslands predate human arrival, we also find evidence for human-mediated population declines. This highlights the extent to which species imminently threatened by human-mediated deforestation may be more vulnerable from paleoclimatic changes.

Taxon-specific characteristics and extrinsic climatic and geological forces may both shape population differentiation and speciation. In geographically and taxonomically focused investigations, differentiation may occur synchronously as species respond to the same external conditions. Conversely, when evolution is investigated in taxa with largely varying traits, population differentiation and speciation is complex and shaped by interactions of Earth’s template and species-specific traits. As such, it is important to characterize evolutionary histories broadly across the tree of life, especially in geographic regions that are exceptionally diverse and under pressures from human activities such as in biodiversity hotspots. Here, using whole-genome sequencing data, we characterize genomic variation in populations of six Ethiopian Highlands forest bird species separated by a lowland biogeographic barrier, the Great Rift Valley (GRV). In all six species, populations on either side of the GRV exhibited significant but varying levels of genetic differentiation. Species’ dispersal ability was negatively correlated with levels of population differentiation. Isolation with migration models indicated varied patterns of population differentiation and connectivity among populations of the focal species. We found that demographic histories—estimated for each individual—varied by both species and population but were consistent between individuals of the same species and sampling region. We found that genomic diversity varied by half an order of magnitude across species, and that this variation could largely be explained by the harmonic mean of effective population size over the past 200,000 years. Overall, we found that even in highly dispersive species like birds, the GRV acts as a substantial biogeographic barrier.