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Advances in environmental DNA (eDNA) methodologies have led to improvements in the ability to detect species and communities in aquatic environments, yet the majority of studies emphasize biological diversity at the species level by targeting variable sites within the mitochondrial genome. Here, we demonstrate that eDNA approaches also have the capacity to detect intraspecific diversity in the nuclear genome, allowing for assessments of population‐level allele frequencies and estimates of the number of genetic contributors in an eDNA sample. Using a panel of microsatellite loci developed for the round goby (Neogobius melanostomus), we tested the similarity between eDNA‐based and individual tissue‐based estimates of allele frequencies from experimental mesocosms and in a field‐based trial. Subsequently, we used a likelihood‐based DNA mixture framework to estimate the number of unique genetic contributors in eDNA samples and in simulated mixtures of alleles. In both mesocosm and field samples, allele frequencies from eDNA were highly correlated with allele frequencies from genotyped round goby tissue samples, indicating nuclear markers can be reliably amplified from water samples. DNA mixture analyses were able to estimate the number of genetic contributors from mesocosm eDNA samples and simulated mixtures of DNA from up to 58 individuals, with the degree of positive or negative bias dependent on the filtering scheme of low‐frequency alleles. With this study we document the application of eDNA and multiple amplicon‐based methods to obtain intraspecific nuclear genetic information and estimate the absolute abundance of a species in eDNA samples. With proper validation, this approach has the potential to advance noninvasive survey methods to characterize populations and detect population‐level genetic diversity.

Genetic data can help elucidate the dynamics of biological invasions, which are fueled by the constant expansion of international trade. The introduction of European gypsy moth (Lymantria dispar dispar) into North America is a classic example of human‐aided invasion that has caused tremendous damage to North American temperate forests. Recently, the even more destructive Asian gypsy moth (mainly L. d. asiatica and L. d. japonica) has been intercepted in North America, mostly transported by cargo ships. To track invasion pathways, we developed a diagnostic panel of 60 DNA loci (55 nuclear and 5 mitochondrial) to characterize worldwide genetic differentiation within L. dispar and its sister species L. umbrosa. Hierarchical analyses supported strong differentiation and recovered five geographic groups that correspond to (1) North America, (2) Europe plus North Africa and Middle East, (3) the Urals, Central Asia, and Russian Siberia, (4) continental East Asia, and (5) the Japanese islands. Interestingly, L. umbrosa was grouped with L. d. japonica, and the introduced North American population exhibits remarkable distinctiveness from contemporary European counterparts. Each geographic group, except for North America, shows additional lower‐level structures when analyzed individually, which provided the basis for inference of the origin of invasive specimens. Two assignment approaches consistently identified a coastal area of continental East Asia as the major source for Asian invasion during 2014–2015, with Japan being another source. By analyzing simulation and laboratory crosses, we further provided evidence for the occurrence of natural Asian–North American hybrids in the Pacific Northwest, raising concerns for introgression of Asian alleles that may accelerate range expansion of gypsy moth in North America. Our study demonstrates how genetic data contribute to bio‐surveillance of invasive species with results that can inform regulatory management and reduce the frequency of trade‐associated invasions.

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high-throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high-resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high-throughput genotyping data can elucidate subtle genetic structure at previously-undetected scales in a dispersive marine fish.

This study applies an intervention based on autonomy support through dialogic learning to examine effects on motivation and the promotion of physical activity and sport involvement during schoolchildren's leisure time. One hundred and two primary school students, aged 11-13 years, participated (M = 10.93, SD = 0.75). The sample was divided into two groups: an experimental group (49 students) and a control group (53 students). A quasi-experimental study was conducted in physical education classes over the course of 9 months. Assessments of autonomy support from teachers, family, and peers; basic psychological needs; self-determined motivation in physical education and during leisure time; planned behavior variables; physical activity intention; and estimated and actual physical exercise behavior time were included in the model. Following the intervention, the results revealed that greater autonomy support by the teacher, the family and peers, satisfaction of the basic psychological needs, self-determined motivation during physical education class and during free time, greater perceived control, favorable attitude and intention to engage in physical activity contributed to the explanation of student physical activity involvement. These results suggest that when students receive extensive social support from diverse social agents that essential precursors to physical activity are strengthened and contribute to increased motivation and actual physical activity involvement. Efforts to promote effective communications with students and other pedagogical efforts, such as providing dialogic learning opportunities, should be considered in order to strengthen physical activity motivation and involvement in students.

Long-distance dispersal (LDD) is consequential to metapopulation ecology and evolution. In systems where dispersal is undertaken by small propagules, such as larvae in the ocean, documenting LDD is especially challenging. Genetic parentage analysis has gained traction as a method for measuring larval dispersal, but such studies are generally spatially limited, leaving LDD understudied in marine species. We addressed this knowledge gap by uncovering LDD with population assignment tests in the coral reef fish Elacatinus lori, a species whose short-distance dispersal has been well-characterized by parentage analysis. When adults (n = 931) collected throughout the species' range were categorized into three source populations, assignment accuracy exceeded 99%, demonstrating low rates of gene flow between populations in the adult generation. After establishing high assignment confidence, we assigned settlers (n = 3,828) to source populations. Within the settler cohort

Spread of nonindigenous organisms by shipping is one of the largest threats to coastal ecosystems. Limited monitoring and understanding of this phenomenon currently hinder development of effective prevention policies. Surveying ports in North America, South America, Europe, Southeast Asia, and Australia we explored environmental DNA community profiles evident of ship-born species spread. We found that community similarities between ports increased with the number of ship voyages, particularly if the ports had similar environments, and when indirect stepping-stone connections were considered. We also found 57 known non-indigenous taxa, some in hitherto unreported locations. We demonstrate the usefulness of eDNA-based tools for global biodiversity surveys, and highlight that shipping homogenizes biodiversity in predictable that could inform policy and management.

Invasions can be genetically diverse, and that diversity may have implications for invasion management in terms of resistance or tolerance to control methods. We analyzed the population genetics of Russian-olive (Elaeagnus angustifoliaL.), an ecologically important and common invasive tree found in many western U.S. riparian areas. We found three cpDNA haplotypes and, using 11 microsatellite loci, identified three genetic clusters in the 460 plants from 46 populations in the western United States. We found high levels of polymorphism in the microsatellites (5 to 15 alleles per locus; 106 alleles total). Our native-range sampling was limited, and we did not find a genetic match for the most common cpDNA invasive haplotype or a strong confirmation of origin for the most common microsatellite genetic cluster. We did not find geographic population structure (isolation by distance) across the U.S. invasion, but we did identify invasive populations that had the most diversity, and we suggest these as choices for initial biological control–release monitoring. Accessions from each genetic cluster, which coarsely represent the range of genetic diversity found in the invasion, are now included in potential classical biological control agent efficacy testing.

Anthropogenic changes to the environment challenge animal populations to adapt to new conditions and unique threats. While the study of adaptation has focused on genetic variation, epigenetic mechanisms may also be important. DNA methylation is sensitive to environmental stressors, such as parasites and pesticides, which may affect gene expression and phenotype. We studied the effects of an invasive ec toparasite, Philornis downsi, on DNA methylation of Galápagos mockingbirds (Mimus parvulus). We used the insecticide permethrin to manipulate P. downsi presence in nests of free-living mockingbirds and tested for effects of parasitism on nestling mockingbirds using epiGBS, a reduced-representation bisulfite sequencing (RRBS) approach. To distinguish the confounding effects of insecticide exposure, we con ducted a matching experiment exposing captive nestling zebra finches (Taeniopygia guttata) to permethrin. We used zebra finches because they were the closest model organism to mockingbirds that we could breed in controlled conditions. We identi fied a limited number of differentially methylated cytosines (DMCs) in parasitized versus nonparasitized mockingbirds, but the number was not more than expected by chance. In contrast, we saw clear effects of permethrin on methylation in captive zebra finches. DMCs in zebra finches paralleled documented effects of permethrin exposure on vertebrate cellular signaling and endocrine function. Our results from captive birds indicate a role for epigenetic processes in mediating sublethal nontar get effects of pyrethroid exposure in vertebrates. Environmental conditions in the field were more variable than the laboratory, which may have made effects of both parasitism and permethrin harder to detect in mockingbirds. RRBS approaches such as epiGBS may be a cost-effective way to characterize genome-wide methylation profiles. However, our results indicate that ecological epigenetic studies in natural populations should consider the number of cytosines interrogated and the depth of sequencing in order to have adequate power to detect small and variable effects

Aposematic organisms advertise their defensive toxins to predators using a variety of warning signals, including bright coloration. While most Neotropical poison frogs (Dendrobatidae) rely on crypsis to avoid predators, Oophaga poison frogs from South America advertise their chemical defenses, a complex mix of diet-derived alkaloids, by using conspicuous hues. The present study aimed to characterize the skin transcriptomic profiles of South American Oophaga poison frogs. Our analyses showed very similar transcriptomic profiles for these closely related species in terms of functional annotation and relative abundance of gene ontology terms expressed. Analyses of expression profiles of Oophaga and available skin transcriptomes of cryptic anurans allowed us to propose initial hypotheses for the active sequestration of alkaloid-based chemical defenses and to highlight some genes that may be potentially involved in resistance mechanisms to avoid self-intoxication and skin coloration. In doing so, we provide an important molecular resource for the study of warning signals that will facilitate the assembly and annotation of future poison frog genomes.

In this study, we present an iterative method for delimiting species under the general lineage concept (GLC) based on the multivariate clustering of morphological, ecological and genetic data. Our rationale is that distinct multivariate groups correspond to evolutionarily independent metapopulation lineages because they reflect the common signal of different secondary defining properties (environmental and genetic distinctiveness, phenotypic diagnosability, etc.) that imply the existence of barriers preventing or limiting gene exchange. We applied this method to study a group of endangered poison frogs, the Oophaga histrionica complex. In our study case, we used next-generation targeted amplicon sequencing to obtain a robust genetic data set that we combined with patterns of morphological and ecological features. Our analyses revealed the existence of at least five different species in the histrionica complex (three, new to science), some of them, occurring in small isolated populations outside any protected areas. The lineage delimitation proposed here has important conservation implications as it revealed that some of the Oophaga species should be considered among the most vulnerable of the Neotropical frogs. More broadly, our study exemplifies how multiple-amplicon and multivariate statistical techniques can be integrated to successfully identify species and their boundaries.

Aposematic signals represent one of the classical systems to study evolution and, as such, they have received considerable empirical and theoretical investigation. Despite the extensive literature on aposematic coloration, much uncertainty remains about genetic changes responsible for the repeated evolution of similar signals in multiple lineages. Here, we study the diversification and convergence of coloration among lineages of aposematic harlequin poison frogs (Oophaga histrionica complex). Our results suggest that different background phenotypes, showing different color and/or luminance contrast, have evolved independently at least twice in this group. We suggest that cellular arrangements are behind the striking diversity of color and patterns in this group and propose that differences in dorsal background color may be related to either or both, the presence/absence of xanthophores and the dispersion of melanosomes. Our genetic analyses support a role for the melanocortin receptor MC1R in melanosome aggregation, and we show evidence that two different mutations (∆433 and C432A) are responsible for the darker phenotypes that may display a more detectable, easier to learn, aposematic signal.

Hybridization and introgression can have complex consequences for both species evolution and conservation. Here, we investigated the origin and characteristics of a putative hybrid zone between two South American poison dart frog species, Oophaga anchicayensis and the critically endangered Oophaga lehmanni, which are heavily sought after on the illegal pet market. Using a combination of phenotypic (49 traits) and genomic (ddRADseq) data, we found that the putative hybrids are morphologically distinct from their parental species and confirmed genomic signatures of admixture in these populations. Several lines of evidence (hybrid indices, interspecific hybrid heterozygosity, genomic clines, comparisons with simulated hybrids and demographic modelling) support the conclusion that these populations are not comprised of early-generation hybrids and thus, they probably did not arise as a result of illegal translocations associated with wildlife trafficking. Instead, they probably represent an independent lineage which has persisted through isolation and has only relatively recently re-established gene flow with both parental species. Furthermore, we detected signals of differential introgression from parental species into these hybrid populations which suggest relaxed stabilizing selection on these aposematic colour morphs, potentially via context-dependent female choice. These populations thus provide a fascinating window into the role of hybridization, isolation and female choice in the diversification of South American poison dart frogs. In addition, our results underline the importance of landscape conservation measures to protect, not only known localities of nominal species, but also the phenotypic and genomic variation harbored by admixed lineages which represent crucial repositories for the impressive diversity in this system.

Aposematic organisms advertise their defensive toxins to predators using a variety of warning signals, including bright coloration. While most Neotropical poison frogs (Dendrobatidae) rely on crypsis to avoid predators, Oophaga poison frogs from South America advertise their chemical defenses, a complex mix of diet-derived alkaloids, by using conspicuous hues. The present study aimed to characterize the skin transcriptomic profiles of the South American clade of Oophaga poison frogs (O. anchicayensis, O. solanensis, O. lehmanni and O. sylvatica). Our analyses showed very similar transcriptomic profiles for these closely related species in terms of functional annotation and relative abundance of gene ontology terms expressed. Analyses of expression profiles of Oophaga and available skin transcriptomes of cryptic anurans allowed us to propose possible mechanisms for the active sequestration of alkaloid-based chemical defenses and to highlight some genes that may be potentially involved in resistance mechanisms to avoid self-intoxication and skin coloration. In doing so, we provide an important molecular resource for the study of warning signals that will facilitate the assembly and annotation of future poison frog genomes.

Eleven viral haemorrhagic septicaemia virus (VHSV) genotype IVb isolates were sequenced, and their genetic variation explored to determine the source of a VHS outbreak on the eastern shore of Cayuga Lake. An active fish kill of round gobies (Neogobius melanostomus, Pallas) was intensively sampled at King Ferry, NY and nearby Long Point State Park in May 2017. Gross lesions observed on 67 moribund round gobies and two rock bass (Ambloplites rupestris, Rafinesque) included moderately haemorrhagic internal organs and erythematous areas on the head, flank, and fins. RT-qPCR tests for VHSV were positive for all 69 fish. Viral isolation on epithelioma papulosum cyprinid cells showed cytopathic effect characteristic of VHSV for six round goby samples from King Ferry. The complete nucleotide sequence of the VHSV IVb genomes of five Cayuga Lake round goby isolates were derived on an Illumina platform along with 2017 VHSV IVb isolates from round gobies collected from the following: Lake Erie near Dunkirk, NY; the St. Lawrence River near Clayton and Cape Vincent, NY; and Lake St. Lawrence near Massena, NY. The phylogenetic tree created from these aligned sequences and four other complete VHSV IVb genomes shows Cayuga Lake isolates are closely related to the Lake Erie isolates.

Aim Despite the incredible diversity of lowland tropical rain forests, we still have limited understanding of the drivers of speciation in these ecoregions. Here, we investigated the relative contribution of geographical and environmental factors to the diversification of a Neotropical genus of poison frogs (Oophaga). Location Central and South America, including regions from southern Nicaragua to northern Ecuador. Methods We generated gene genealogies (12S, 16S, COI, CytB and tRNA-val, SIAH1, H3 and Rag1) and used species phylogenetic methods (MDC and *beast) to generate a robust phylogeny of Oophaga frogs. Then, we combined the resulting phylogenetic hypothesis with detailed geographical data and environmental niche modelling (ENM) to test the role of geographical isolation, climatic niche divergence and altitudinal gradients. Results Gene genealogies were discordant and did not show exclusive genealogical patterns; however, species tree resolved the phylogenetic relationship among Oophaga species with strong node support (> 0.9 ML/BPP). Geographical ranges showed little overlap between distantly related species. However, within the South American and Central American clades, sister taxa showed substantially overlapping ranges. Analyses of ecological disparity (DTT) indicated a departure from a neutral (Brownian) model of evolution, and age-range correlations, predicted niche occupancy profiles, and Seeva analyses showed that different species tend to evolve under different potential climatic niches. Main conclusions Oophaga frogs originated in Central America and reached South America after the closure of the Panama Isthmus. The South- and Central-American clades of this genus have convergently evolved to similar patterns of geographical distribution and niche occupancy. Within clades, sister taxa showed parapatric distributions replacing each other along elevational gradients as predicted by the models of divergence along continuous ecological gradients. Accordingly, we found strong shifts in climatic niches throughout the history of these two clades. However, the largest niche shifts seem to post-date the final elevation of the Talamanca and northern Andes. Overall, our data suggest that speciation along climatic gradients on a structured landscape has been a major evolutionary force behind the diversification of Oophaga poison frogs.

Four viral hemorrhagic septicemia virus (VHSV) genotype IVb isolates were sequenced, their genetic variation explored, and comparative virulence assayed with experimental infections of northern pike Esox lucius fry. In addition to the type strain MI03, the complete 11183 bp genome of the first round goby Neogobius melanostomus isolate from the St. Lawrence River, and the 2013 and 2014 isolates from gizzard shad Dorosoma cepedianum die-offs in Irondequoit Bay, Lake Ontario and Dunkirk Harbor, Lake Erie were all deep sequenced on an Illumina platform. Mutations documented in the 11 yr since the MI03 index case from Lake St. Clair muskellunge Esox masquinongy showed 87 polymorphisms among the 4 isolates. Twenty-six mutations were non-synonymous and located at 18 different positions within the matrix protein, glycoprotein, non-virion protein, and RNA polymerase genes. The same 4 isolates were used to infect northern pike fry by a single 1 h bath exposure. Cumulative percent mortality varied from 42.5 to 62.5%. VHSV was detected in 57% (41/72) of the survivors at the end of the 21-d trial, suggesting that the virus was not rapidly cleared. Lesions were observed in many of the moribund and dead northern pike, such as hemorrhaging in the skin and fins, as well as hydrocephalus. Mean viral load measured from the trunk and visceral tissues of MI03-infected pike was significantly higher than the quantities detected in fish infected with the most recent isolates of genotype IVb, but there were no differences in cumulative mortality observed.

We propose a iterative protocol for delimiting species under the generalized lineage concept (GLC) based on the multivariate clustering of morphological, ecological, and genetic data. Our rationale is that the resulting groups should correspond to evolutionarily independent metapopulation lineages because they reflect the common signal of different secondary defining properties (ecological and genetic distinctiveness, morphological diagnosability, etc.), implying the existence of barriers preventing or limiting gene exchange. We applied this method to study a group of highly endangered poison frogs, theOophaga histrionicacomplex. In our study case, we use next generation targeted amplicon sequencing to obtain a robust genetic dataset that we then combined with patterns of morphological and ecological divergence. Our analyses revealed the existence of at least five different species in the histrionica complex (three of them new to science) occurring in very small isolated populations outside any protected areas. More broadly, our study exemplifies how transcriptome-based reduction of genomic complexity and multivariate statistical techniques can be integrated to successfully identify species and their boundaries.In memoriam“I propose that each species has a distinctive life history, which include a series of stages that correspond to some of the named species concepts”Richard G. Harrison1945-2016

Background Seminal fluid proteins control many aspects of fertilization and in turn, they play a key role in post-mating sexual selection and possibly reproductive isolation. Because effective proteome profiling relies on the availability of high-quality DNA reference databases, our knowledge of these proteins is still largely limited to model organisms with ample genetic resources. New advances in sequencing technology allow for the rapid characterization of transcriptomes at low cost. By combining high throughput RNA-seq and shotgun proteomic profiling, we have characterized the seminal fluid proteins secreted by the primary male accessory gland of the migratory grasshopper (Melanoplus sanguinipes), one of the main agricultural pests in central North America. Results Using RNA sequencing, we characterized the transcripts of ~ 8,100 genes expressed in the long hyaline tubules (LHT) of the accessory glands. Proteomic profiling identified 353 proteins expressed in the long hyaline tubules (LHT). Of special interest are seminal fluid proteins (SFPs), such as EJAC-SP, ACE and prostaglandin synthetases, which are known to regulate female oviposition in insects. Conclusions Our study provides new insights into the proteomic components of male ejaculate in Orthopterans, and highlights several important patterns. First, the presence of proteins that lack predicted classical secretory tags in accessory gland proteomes is common in male accessory glands. Second, the products of a few highly expressed genes dominate the accessory gland secretions. Third, accessory gland transcriptomes are enriched for novel transcripts. Fourth, there is conservation of SFPs’ functional classes across distantly related taxonomic groups with very different life histories, mating systems and sperm transferring mechanisms. The identified SFPs may serve as targets of future efforts to develop species- specific genetic control strategies. Electronic supplementary material The online version of this article (doi:10.1186/s12864-015-2327-1) contains supplementary material, which is available to authorized users.

In damselflies, sexual colour dimorphism is commonly explained as a consequence of selection on traits that increase male attractiveness to females. However, while many species in the damselfly family Coenagrionidae (Insecta: Odonata) are sexually dimorphic, the males do not engage in displays, and male competition for mates resembles a “scramble”. An alternative explanation for the sexual differences in coloration within these species is that sexual dimorphism has evolved as a sex-related warning signal, with males signalling their uprofitability as mates to other males, thereby avoiding harassment from conspecifics. We evaluated an underlying assumption of the theory that male-male harassment rate is influenced by colour by comparing harassment of males of the species Nehalennia irene that had been painted to make them appear: (i) similar to an unaltered male (blue), (ii) different from a male (orange) and (iii) more similar to a female (black). When caged together we found that blue-painted males experienced significantly lower harassment than black-painted males. When unpainted males were caged with each type of painted male we found that blue-painted males and the unpainted males housed in the same cages experienced lower rates of harassment than males housed in cages where some males were painted black, suggesting that a single, reliable signal of unprofitability may benefit the individuals that carry it. While our results do not in themselves demonstrate that sexual colour dimorphism originally evolved as an intra-specific warning signal, they do show that harassment is influenced by coloration, and that such selection could conceivably maintain male coloration as a warning signal.