Rapid shifts in environmental variables associated with elevational changes in montane ecosystems provide opportunities to test hypotheses regarding the effects of environmental heterogeneity on gene flow and genetic structure. In tropical mountains, spatial environmental heterogeneity combined with seasonal environmental stability is predicted to result in low dispersal across elevations. Few studies have investigated the genetic consequences of elevational environmental heterogeneity in tropical montane mammals. Here, we use a population genomics approach to test the hypothesis that mountain treeshrews (Tupaia montana) exhibit limited gene flow across elevational gradients and between two neighboring peaks within Kinabalu National Park (KNP) in Borneo. We sampled 83 individuals across elevations on Mt. Tambuyukon (MT) and Mt. Kinabalu (MK) and sequenced mitogenomes and 4,106 ultraconserved elements containing an average of 1.9 single nucleotide polymorphisms per locus. We detected high gene flow across elevations and between peaks. We found greater genetic differentiation on MT than MK despite its lower elevation and associated environmental variation. This implies that, contrary to our hypothesis, genetic structure in this system is not primarily shaped by elevation. We propose that this pattern may instead be the result of colonization history combined with restricted upslope gene flow on MT due to unique plant communities associated with its upper montane habitats. Our results serve as a foundation to identify and mitigate future effects of climate change on mountain treeshrews in KNP. Given predictions for 2100 CE, we predict that mountain treeshrews will maintain genetic connectivity in KNP, making it an important conservation stronghold.

Environmental DNA and metabarcoding have great potential for the biomonitoring of freshwater environments. However, successful application of metabarcoding to biodiversity monitoring requires universal primers with high taxonomic coverage that amplify highly-variable, short metabarcodes with high taxonomic resolution. Moreover, reliable and extensive reference databases are essential to match the outcome of metabarcoding analyses with the available taxonomy and biomonitoring indices. Benthic invertebrates, particularly insects, are key taxa for freshwater biomonitoring. Nevertheless, so far, no formal comparison has assessed primers for metabarcoding of freshwater macrobenthos. Here we combined in vitro and in silico analyses to test the performance of metabarcoding primers amplifying regions in the 18S rDNA (Euka02 metabarcode), 16S rDNA (Inse01), and COI (BF1_BR2-COI) genes, and developed an extensive database of benthic invertebrates of France and Europe, with a special focus on three key insect orders (Ephemeroptera, Plecoptera and Trichoptera). In vitro analyses on 1514 individuals, belonging to 578 different taxonomic units showed very different amplification rates across primer combinations. The Euka02 marker showed the highest universality, while the Inse01 marker showed excellent performance for the amplification of insects. The BF1_BR2-COI metabarcode showed the highest resolution, while the resolution of Euka02 was often limited. By combining in vitro data with GenBank information, we developed a curated database including sequences representing 822 genera. The heterogeneous performance of the different metabarcodes highlights the complexity of the identification of the best markers, and advocates for the integration of multiple metabarcodes for a more comprehensive and accurate understanding of ecological impacts on freshwater biodiversity.

Pacific lamprey (Entosphenus tridentatus) is a culturally important and imperiled anadromous fish with a parasitic ocean phase. Biological uncertainties challenge restoration efforts and life-history research is needed to explain observed trait variation and inform management actions. Using two new whole genome assemblies and genotypes from 7,716 single nucleotide polymorphism (SNP) loci in 518 individuals from across the species range, we identified four large regions of high genomic divergence (on chromosomes 01, 02, 04, and 22). We genotyped a subset of 302 broadly distributed SNPs in 2,145 individuals for genotype-by-phenotype trait associations for adult body size, sexual maturity, migration distance and timing, adult swimming ability, and larval growth. Body size traits were strongly associated with SNPs on chromosomes 02 and 04. Moderate associations also implicated SNPs on chromosome 01 as being associated with variation in female maturity. Using genotypic frequencies of candidate SNPs for female maturity and body size, we extrapolated a heterogeneous spatiotemporal distribution of these traits based on independent datasets of larval and adult collections. These maturity and body size results guide future studies to validate these predicted phenotypic distributions across the geographic range and elucidate factors driving regional optimization of these traits for fitness.

Biodiversity is under threat worldwide. Over the past decade, the field of population genomics has developed across non-model organisms, and the results of this research have begun to be applied in conservation and management of wildlife species. Genomics tools can provide precise estimates of basic features of wildlife populations, such as effective population size, inbreeding, demographic history, and population structure, that are critical for conservation efforts. Moreover, population genomics studies can identify particular genetic loci and variants responsible for inbreeding depression or adaptation to changing environments, allowing for conservation efforts to estimate the capacity of populations to evolve and adapt in response to environmental change and to manage for adaptive variation. While connections from basic research to applied wildlife conservation have been slow to develop, these connections are increasingly strengthening. Here we review the primary areas in which population genomics approaches can be applied to wildlife conservation and management, highlight examples of how they have been used, and provide recommendations for building on the progress that has been made in this field.

Monarch butterflies are known for their spectacular annual migration in eastern North America, with millions of monarchs flying up to 4,500 kilometers to overwintering sites in central Mexico. Monarchs also live west of the Rocky Mountains, where they travel shorter distances to overwinter along the Pacific Coast. It is often assumed that eastern and western monarchs form distinct evolutionary units, but genomic studies to support this notion are lacking. We used a tethered flight mill to show that migratory eastern monarchs have greater flight performance than western monarchs, consistent with their greater migratory distances. However, analyzing more than 20 million SNPs in 43 monarch genomes, we found no evidence for genomic differentiation between eastern and western monarchs. Genomic analysis also showed identical and low levels of genetic diversity, and demographic analyses indicated similar effective population sizes and ongoing gene flow between eastern and western monarchs. Gene expression analysis of a subset of candidate genes during active flight revealed differential gene expression related to non-muscular motor activity. Our results demonstrate that eastern and western monarchs maintain migratory differences despite ongoing gene flow, and suggest that migratory differences between eastern and western monarchs are not driven by select major-effects alleles. Instead, variation in migratory distance and destination may be driven by environmentally induced differential gene expression, or by many alleles of small effect.

Parallel evolution can occur through novel mutations, standing genetic variation, or adaptive introgression. Uncovering parallelism and introgressed populations can complicate management of threatened species, particularly as admixed populations are not generally considered under conservation legislations. We examined high coverage whole-genome sequences of 30 caribou (Rangifer tarandus) from across North America and Greenland, representing divergent intra-specific lineages, to investigate parallelism and levels of introgression contributing to the formation of ecotypes. Caribou are split into four subspecies and 11 extant conservation units, known as Designatable Units (DUs), in Canada. Using genomes from all four subspecies and six DUs, we undertake demographic reconstruction and confirm two previously inferred instances of parallel evolution in the woodland subspecies and uncover an additional instance of parallelism of the eastern migratory ecotype. Detailed investigations reveal introgression in the woodland subspecies, with introgressed regions found spread throughout the genomes encompassing both neutral and functional sites. Our comprehensive investigations using whole genomes highlight the difficulties in unequivocally demonstrating parallelism through adaptive introgression in non-model species with complex demographic histories, with standing variation and introgression both potentially involved. Additionally, the impact of parallelism and introgression on the designation of conservation units has not been widely considered, and the caribou designations will need amending in light of our results. Uncovering and decoupling parallelism and differential patterns of introgression will become prevalent with the availability of comprehensive genomic data from non-model species, and we highlight the need to incorporate this into conservation unit designations.

Accurate and testable species delimitation hypotheses are essential for measuring, surveying and managing biodiversity. Today, taxonomists often rely on mitochondrial DNA barcoding to complement morphological species delimitations. Although COI barcoding has largely proven successful in assisting identifications for most animal taxa, there are nevertheless numerous cases where mitochondrial barcodes do not necessarily reflect the species history. For instance, what is regarded as one single species can be associated with two distinct DNA barcodes, which can point either to cryptic diversity or to deep within-species mitochondrial divergences with no reproductive isolation. In contrast, two or more species can share barcodes, for instance due to mitochondrial introgression. These intrinsic limitations of mitochondrial DNA barcoding can only be addressed with nuclear genomic markers, which are expensive, labour intensive, poorly repeatable, and often require high-quality DNA. To overcome these limitations, we examined the use of ultraconserved nuclear genetic elements (UCEs) as a quick and robust genomic approach to address such problematic cases of species delimitation. This genomic method was assessed using six different bee species complexes suspected to harbour cryptic diversity, mitochondrial introgression, or mitochondrial paraphyly. The sequencing of UCEs recovered between 686 and 1860 homologous nuclear loci and provided explicit species delimitation hypotheses in all investigated species complexes. These results provide strong evidence for the suitability of UCEs as a fast method for species delimitation even in recently diverged lineages. Furthermore, this study provided the first conclusive evidence for both mitochondrial introgression among distinct species, and mitochondrial paraphyly within a single bee species.

Most new cryptic species are described using conventional tree- and distance-based species delimitation methods (SDMs), which rely on phylogenetic arrangements and measures of genetic divergence. However, although numerous factors such as spatial population structure and gene flow are known to confound phylogenetic and species delimitation inferences, the influence of these processes on species estimation is rarely evaluated. Using large amounts of exons, introns, and ultraconserved elements obtained using the FrogCap sequence-capture protocol, we compared conventional SDMs with more robust genomic analyses that assesses spatial population structure and gene flow to characterize species boundaries in a Southeast Asian frog complex (Pulchrana picturata). Our results showed that gene flow and introgression can produce phylogenetic patterns and levels of divergence that resemble distinct species (up to 10% divergent in mitochondrial DNA). Hybrid populations were inferred as independent (singleton) clades that were highly divergent from adjacent populations (7–10%) and unusually similar (<3%) to allopatric populations. Such anomalous patterns are not uncommon in Southeast Asian amphibians, which brings into question whether the high cryptic diversity observed in other amphibian groups reflect distinct cryptic species—or, instead, highly structured and admixed metapopulation lineages. Our results also provide an alternative explanation to the conundrum of divergent (sometimes non-sister) sympatric lineages―a pattern that has been celebrated as indicative of true cryptic speciation. Based on these findings, we recommend that species delimitation of continuously distributed “cryptic” groups should not rely solely on conventional SDMs but should necessarily examine spatial population structure and gene flow to avoid taxonomic inflation.

Genetic tools are increasingly used to identify and discriminate between species. One key transition in this process was the recognition of the potential of the ca 658bp fragment of the organelle cytochrome c oxidase I (COI) as a barcode region, which revolutionised animal bioidentification and lead, among others, to the instigation of the Barcode of Life database (BOLD), containing currently barcodes from >7.9 million specimens. Following this discovery, suggestions for other organellar regions and markers, and the primers with which to amplify them, have been continuously proposed. Most recently, the field has taken the leap from PCR based generation of DNA references into shotgun sequencing-based ‘genome skimming’ alternatives, which the ultimate goal of assembling organellar reference genomes. Unfortunately, in genome skimming approaches, much of the nuclear genome (as much as 99% of the sequence data) is discarded, which is not only wasteful but can also limit the power of discrimination at or below the species level. Here, we advocate that the full shotgun sequence data can be used to assign an identity (that we term for convenience its ‘DNA-mark’) for both voucher and query samples, without requiring any computationally intensive pretreatment (e.g., assembly) of reads. We argue that if reference databases are populated with such ‘DNA-marks’, it will enable future DNA-based taxonomic identification to complement, or even replace PCR of barcodes with genome skimming, and we discuss how such methodology ultimately could enable identification to population, or even individual, level.

Few phylogeographical studies exist for taxa inhabiting the Colorado Plateau province. We combined mitochondrial and genomic data with species distribution modeling to test Pleistocene hypotheses for Aphonopelma marxi, a large tarantula endemic to the plateau region. Mitochondrial and genomic analyses revealed that the species comprises at least three main clades that diverged in the Pleistocene. A clade distributed along the Mogollon Rim appears to have persisted in place during the last glacial maximum, whereas the other two clades probably colonized the central and northeastern portion of the species’ range from small refugial areas along river-carved canyons. Climate models support this hypothesis for the Mogollon Rim, but late glacial climate data appear too coarse to detect suitable areas in canyons. Locations of canyon refugia could not be inferred from genomic analyses due to missing data, encouraging us to explore the effect of missing loci in phylogeographical inferences using RADseq. In phylogenetic analyses, node support for major clades decreased with the addition of samples with significant amounts of missing data (more than 30%). Population genomic structure was greatly influenced by missing data, with the group membership of many taxa changing as samples with missing loci were added. Results from DAPC, a distance-based method, did not change as samples with significant amounts missing data were added. We conclude that the specific loci that are missing matters more than the number of missing loci, and that samples with missing data can still add information to RADseq-based analyses as long as results are interpreted cautiously.

Although species delimitation can be highly contentious, the development of reliable methods to accurately ascertain species boundaries is an imperative step in cataloguing and describing Earth’s quickly disappearing biodiversity. Spider species delimitation remains largely based on morphological characters; however, many mygalomorph spider populations are morphologically indistinguishable from each other yet have considerable molecular divergence. The focus of our study, Antrodiaetus unicolor species complex which contains two sympatric species, exhibits this pattern of relative morphological stasis with considerable genetic divergence across its distribution. A past study using two molecular markers, COI and 28S, revealed that A. unicolor is paraphyletic with respect to A. microunicolor. To better investigate species boundaries in the complex, we implement the cohesion species concept and employ multiple lines of evidence for testing genetic exchangeability and ecological interchangeability. Our integrative approach includes extensively sampling homologous loci across the genome using a RADseq approach (3RAD), assessing population structure across their geographic range using multiple genetic clustering analyses that include STRUCTURE, PCA, and a recently developed unsupervised machine learning approach (Variational Autoencoder). We evaluate ecological similarity by using large-scale ecological data for niche-based distribution modeling. Based on our analyses, we conclude that this complex has at least one additional species as well as confirm species delimitations based on previous less comprehensive approaches. Our study demonstrates the efficacy of genomic-scale data for recognizing cryptic species, suggesting that species delimitation with one data type, whether one mitochondrial gene or morphology, may underestimate true species diversity in morphologically homogenous taxa with low vagility.

Despite its inherent costs, sexual reproduction is ubiquitous in nature, and the mechanisms to protect it from a competitive displacement by asexuality remain unclear. Popular mutation-based explanations, like the Muller's ratchet and the Kondrashov's hatchet, assume that purifying selection may not halt the accumulation of deleterious mutations in the non-recombining genomes, ultimately leading to their degeneration. However, empirical evidence is scarce and it remains particularly unclear whether mutational degradation proceeds fast enough to ensure the decay of clonal organisms and to prevent them from outcompeting their sexual counterparts. To test this hypothesis, we jointly analyzed the exome sequences and the fitness-related phenotypic traits of the sexually-reproducing fish species and their clonal hybrids, whose evolutionary ages ranged from F1 generations to 300 ky. As expected, mutations tended to accumulate in the clonal genomes in a time-dependent manner. However, contrary to the predictions, we found no trend towards increased non-synonymity of mutations acquired by clones, nor higher radicality of their amino-acid substitutions. Moreover, there was no evidence for fitness degeneration in the old clones compared to that in the younger ones. In summary, although a purifying selection may still be relaxed in the asexual genomes, our data indicate that its efficiency is not drastically decreased. Even the oldest investigated clone was found to be too young to suffer fitness consequences from a mutation accumulation. This suggests that mechanisms other than mutation accumulation may be needed to explain the competitive advantage of sex in the short term.

Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. Compared to DNA sequencing that captures both alive and dead organisms, environmental RNA (eRNA) can be used to better target alive communities. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in 1) biodiversity of multiple microeukaryotic communities, and 2) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2–6.9 %), when compared to low OC sediments (1.2–2.8 %). The beta diversity for all three groups were different along the OC gradient, as well as the classified feeding type of nematode genera (with more non-selective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of living benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.

Parental effects influence offspring phenotypes through pre- and post-natal routes but little is known about their molecular basis, and therefore their adaptive significance. Epigenetic modifications, which control gene expression without changes in the DNA sequence and are influenced by the environment, may contribute to parental effects. Taking advantage of the self-fertilising and inbred nature of the mangrove killifish Kryptolebias marmoratus, we investigated the effects of the rearing environment on parents and offspring by comparing neophobia, metabolic rate and brain epigenetic (DNA methylation) patterns of genetically identical fish reared in enriched or barren environments. Parental fish reared in enriched environments had lower cortisol levels, lower metabolic rates and were more active and neophobic than those reared in barren environments. They also differed in 1,854 methylated cytosines (DMCs). Offspring activity and neophobia were determined by the parental environment and we also found evidence of, limited but significant, parental influence on the DNA methylation patterns of the offspring. Among the DMCs of the parents, 98 followed the same methylation patterns in the offspring, three of which were significantly influenced by parental environments irrespective of their own rearing environment. Our results suggest that the environment experienced by the parents influences the behaviour and, to some extent, brain DNA methylation patterns of the offspring in an environment-specific manner.

The field of landscape genetics has been rapidly evolving, adopting and adapting analytical frameworks to address research questions. As landscape genetic analyses have shifted away from Mantel-based analytical frameworks, studies are increasingly using regression-based frameworks to understand the individual contributions of landscape and habitat variables on genetic differentiation. This paper outlines appropriate and inappropriate uses of multiple regression for these purposes. Of concern is the prevalence of studies seeking to explain genetic differences by fitting regression models with effective distance variables calculated independently on separate landscape resistance surfaces. When moving across the landscape, organisms cannot respond independently and uniquely to habitat and landscape features. Therefore, independent resistance surfaces and their effective distance measures have no mechanistic meaning or relevant statistical interpretation. There are also tremendous challenges to fitting and interpreting regression models that include ‘independent’ effective distance measures as predictors, including statistical suppression. As such, regression analyses seeking to understand how landscape resistance affects gene flow should be univariate models, with the creation of a single resistance surface being a necessary precursor to the regression analysis. There are, however, important statistical advances underway that explicitly model the covariance of allele frequencies or genetic distances as functions of spatial landscape variables. The growth and evolution of landscape genetics as a field has been rapid and exciting. It is the goal of this opinion paper to highlight past missteps and to ensure that future use of regression models will appropriately consider the process being modeled, which will provide clarity to model interpretation.

Evaluating the factors that drive patterns of population differentiation in plants is critical for understanding several biological processes such as local adaptation and incipient speciation. Previous studies have given conflicting results regarding the significance of pollination mode, seed dispersal mode, mating system, growth form, and latitudinal region in shaping patterns of genetic structure, as estimated by FST values, and no study to date has tested their relative importance together across a broad scale. Here we assembled a 337-species dataset for seed plants from publications with data on FST from nuclear markers and species traits, including variables pertaining to the sampling scheme of each study. We used species traits, while accounting for sampling variables, to perform phylogenetic multiple regressions. Results demonstrated that FST values were higher for tropical, mixed-mating, non-woody species pollinated by small insects, indicating greater population differentiation, and lower for temperate, outcrossing trees pollinated by wind. Among the factors we tested, latitudinal region explained the largest portion of variance, followed by pollination mode, mating system and growth form, while seed dispersal mode did not significantly relate to FST. Our analyses provide the most robust and comprehensive evaluation to date of the main ecological factors predicted to drive population differentiation in seed plants, with important implications for understanding the basis of their genetic divergence. Our study is the first that we are aware of to robustly demonstrate greater population differentiation in tropical regions.