Genomics of post-bottleneck recovery in the northern elephant seal.

Populations and species are threatened by human pressure, but their fate is variable. Some depleted populations, such as that of the northern elephant seal (Mirounga angustirostris), recover rapidly even when the surviving population was small. The northern elephant seal was hunted extensively and taken by collectors between the early 1800s and 1892, suffering an extreme population bottleneck as a consequence. Recovery was rapid and now there are over 200,000 individuals. We sequenced 260 modern and 8 historical northern elephant seal nuclear genomes to assess the impact of the population bottleneck on individual northern elephant seals and to better understand their recovery. Here we show that inbreeding, an increase in the frequency of alleles compromised by lost function, and allele frequency distortion, reduced the fitness of breeding males and females, as well as the performance of adult females on foraging migrations. We provide a detailed investigation of the impact of a severe bottleneck on fitness at the genomic level and report on the role of specific gene systems.

Sequence capture identifies fastidious chytrid fungi directly from host tissue.

The chytrid fungus Batrachochytrium dendrobatidis (Bd) was discovered in 1998 as the cause of chytridiomycosis, an emerging infectious disease causing mass declines in amphibian populations worldwide. The rapid population declines of the 1970s-1990s were likely caused by the spread of a highly virulent lineage belonging to the Bd-GPL clade that was introduced to naïve susceptible populations. Multiple genetically distinct and regional lineages of Bd have since been isolated and sequenced, greatly expanding the known biological diversity within this fungal pathogen. To date, most Bd research has been restricted to the limited number of samples that could be isolated using culturing techniques, potentially causing a selection bias for strains that can grow on media and missing other unculturable or fastidious strains that are also present on amphibians. We thus attempted to characterize potentially non-culturable genetic lineages of Bd from distinct amphibian taxa using sequence capture technology on DNA extracted from host tissue and swabs. We focused our efforts on host taxa from two different regions that likely harbored distinct Bd clades: (1) wild-caught leopard frogs (Rana) from North America, and (2) a Japanese Giant Salamander (Andrias japonicus) at the Smithsonian Institution's National Zoological Park that exhibited signs of disease and tested positive for Bd using qPCR, but multiple attempts failed to isolate and culture the strain for physiological and genetic characterization. We successfully enriched for and sequenced thousands of fungal genes from both host clades, and Bd load was positively associated with number of recovered Bd sequences. Phylogenetic reconstruction placed all the Rana-derived strains in the Bd-GPL clade. In contrast, the A. japonicus strain fell within the Bd-Asia3 clade, expanding the range of this clade and generating additional genomic data to confirm its placement. The retrieved ITS locus matched public barcoding data from wild A. japonicus and Bd infections found on other amphibians in India and China, suggesting that this uncultured clade is widespread across Asia. Our study underscores the importance of recognizing and characterizing the hidden diversity of fastidious strains in order to reconstruct the spatiotemporal and evolutionary history of Bd. The success of the sequence capture approach highlights the utility of directly sequencing pathogen DNA from host tissue to characterize cryptic diversity that is missed by culture-reliant approaches.

Microbiomes associated with avian malaria survival differ between susceptible Hawaiian honeycreepers and sympatric malaria-resistant introduced birds.

Of the estimated 55 Hawaiian honeycreepers (subfamily Carduelinae) only 17 species remain, nine of which the International Union for Conservation of Nature considers endangered. Among the most pressing threats to honeycreeper survival is avian malaria, caused by the introduced blood parasite Plasmodium relictum, which is increasing in distribution in Hawai'i as a result of climate change. Preventing further honeycreeper decline will require innovative conservation strategies that confront malaria from multiple angles. Research on mammals has revealed strong connections between gut microbiome composition and malaria susceptibility, illuminating a potential novel approach to malaria control through the manipulation of gut microbiota. One honeycreeper species, Hawai'i 'amakihi (Chlorodrepanis virens), persists in areas of high malaria prevalence, indicating they have acquired some level of immunity. To investigate if avian host-specific microbes may be associated with malaria survival, we characterized cloacal microbiomes and malaria infection for 174 'amakihi and 172 malaria-resistant warbling white-eyes (Zosterops japonicus) from Hawai'i Island using 16S rRNA gene metabarcoding and quantitative polymerase chain reaction. Neither microbial alpha nor beta diversity covaried with infection, but 149 microbes showed positive associations with malaria survivors. Among these were Escherichia and Lactobacillus spp., which appear to mitigate malaria severity in mammalian hosts, revealing promising candidates for future probiotic research for augmenting malaria immunity in sensitive endangered species.

Museum Genomics Provide Evidence for Persistent Genetic Differentiation in a Threatened Seabird Species in the Western Atlantic.

Connectivity among wildlife populations facilitates exchange of genetic material between groups. Changes to historical connectivity patterns resulting from anthropogenic activities can therefore have negative consequences for genetic diversity, particularly for small or isolated populations. DNA obtained from museum specimens can enable direct comparison of temporal changes in connectivity among populations, which can aid in conservation planning and contribute to the understanding of population declines. However, museum DNA can be degraded and only available in low quantities, rendering it challenging for use in population genomic analyses. Applications of genomic methodologies such as targeted sequencing address this issue by enabling capture of shared variable sites, increasing quantity and quality of recovered genomic information. We used targeted sequencing of ultra-conserved Elements (UCEs) to evaluate potential changes in connectivity and genetic diversity of roseate terns (Sterna dougallii) with a breeding distribution in the northwestern Atlantic and the Caribbean. Both populations experienced range contractions and population declines due to anthropogenic activity in the 20th century, which has the potential to alter historical connectivity regimes. Instead, we found that the two populations were differentiated historically as well as contemporaneously, with little evidence of migration between them for either time period. We also found no evidence for temporal changes in genetic diversity, although these interpretations may have been limited due to sequencing artifacts caused by the degraded nature of the museum samples. Population structuring in migratory seabirds is typically reflective of low rates of divergence and high connectivity among geographically segregated subpopulations. Our contrasting results suggest the potential presence of ecological mechanisms driving population differentiation, and highlight the value of targeted sequencing on DNA derived from museum specimens to uncover long-term patterns of genetic differentiation in wildlife populations.

First mitochondrial genome of the Caucasian squirrel Sciurus anomalus (Rodentia, Sciuridae).

The Caucasian Squirrel, Sciurus anomalus, is the only representative of the Sciuridae family in the Eastern Mediterranean region. In this study, the mitochondrial genome of the Sciurus anomalus species was generated, and we investigate its phylogenetic position within the Sciuridae family. The generated mitogenome sequence is 16,234 bp. It is composed of a control region and a conserved set of 37 genes containing 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes.

First mitochondrial genome of the marbled polecat Vormela peregusna (Carnivora, Mustelidae).

The marbled polecat, Vormela peregusna, is one of the least studied species in the Mustelidae family, especially with regard to phylogeography and genetic diversity. In this study, we determined the mitochondrial genome sequence of V. peregusna and investigated its position within the Mustelidae phylogeny. The generated mitogenome is 15,982 bp in length; it consists of 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a control region.

First mitochondrial genome of the Egyptian mongoose Herpestes ichneumon (Carnivora, Herpestidae).

The Egyptian mongoose, Herpestes ichneumon, is the only extant mongoose in Europe, with populations still distributed in Africa and the Middle East. In this study, we present the first mitochondrial genome sequence of Herpestes ichneumon and we investigate its phylogenetic position within Feliformia suborder. The resultant mitogenome sequence is 16,775 bps, composed of a conserved set of 37 genes containing 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes, and a control region. Our results represent a valuable resource for further phylogeographical studies.

First DNA sequence reference library for mammals and plants of the Eastern Mediterranean Region.

The Mediterranean region is identified as one of the world's 36 biodiversity hotspots, with the Earth's most biologically rich yet threatened areas. Lebanon is a hub for Eastern Mediterranean Region (EMR) biodiversity with 9116 characterized plant and animal species (4486 fauna and 4630 flora). Using DNA barcoding as a tool has become crucial in the accurate identification of species in multiple contexts. It can also complement species morphological descriptions, which will add to our understanding of the biodiversity and richness of ecosystems and benefit conservation projects for endangered and endemic species. In this study, we create the first reference library of standard DNA markers for mammals and plants in the EMR, with a focus on endemic and endangered species. Plant leaves were collected from different nature reserves in Mount Lebanon, and mammal samples were obtained from taxidermized museum specimens or road kills. We generated the 12S rRNA sequences of 18 mammal species from 6 orders and 13 different families. We also obtained the trnL and rbcL barcode sequences of 52 plant species from 24 different families. Twenty-five plant species and two mammal species included in this study were sequenced for the first time using these markers.

Assessing changes in genomic divergence following a century of human-mediated secondary contact among wild and captive-bred ducks.

Along with manipulating habitat, the direct release of domesticated individuals into the wild is a practice used worldwide to augment wildlife populations. We test between possible outcomes of human-mediated secondary contact using genomic techniques at both historical and contemporary timescales for two iconic duck species. First, we sequence several thousand ddRAD-seq loci for contemporary mallards (Anas platyrhynchos) throughout North America and two domestic mallard types (i.e., known game-farm mallards and feral Khaki Campbell's). We show that North American mallards may well be becoming a hybrid swarm due to interbreeding with domesticated game-farm mallards released for hunting. Next, to attain a historical perspective, we applied a bait-capture array targeting thousands of loci in century-old (1842-1915) and contemporary (2009-2010) mallard and American black duck (Anas rubripes) specimens. We conclude that American black ducks and mallards have always been closely related, with a divergence time of ~600,000 years before present, and likely evolved through prolonged isolation followed by limited bouts of gene flow (i.e., secondary contact). They continue to maintain genetic separation, a finding that overturns decades of prior research and speculation suggesting the genetic extinction of the American black duck due to contemporary interbreeding with mallards. Thus, despite having high rates of hybridization, actual gene flow is limited between mallards and American black ducks. Conversely, our historical and contemporary data confirm that the intensive stocking of game-farm mallards during the last ~100 years has fundamentally changed the genetic integrity of North America's wild mallard population, especially in the east. It thus becomes of great interest to ask whether the iconic North American mallard is declining in the wild due to introgression of maladaptive traits from domesticated forms. Moreover, we hypothesize that differential gene flow from domestic game-farm mallards into the wild mallard population may explain the overall temporal increase in differentiation between wild black ducks and mallards, as well as the uncoupling of genetic diversity and effective population size estimates across time in our results. Finally, our findings highlight how genomic methods can recover complex population histories by capturing DNA preserved in traditional museum specimens.

Parthenogenesis in a captive Asian water dragon (Physignathus cocincinus) identified with novel microsatellites.

Reptiles show varying degrees of facultative parthenogenesis. Here we use genetic methods to determine that an isolated, captive female Asian water dragon produced at least nine offspring via parthenogenesis. We identified microsatellites for the species from shotgun genomic sequences, selected and optimized primer sets, and tested all of the offspring for a set of seven microsatellites that were heterozygous in the mother. We verified that the seven loci showed high levels of polymorphism in four wild Asian water dragons from Vietnam. In all cases, the offspring (unhatched, but developed eggs, or hatched young) had only a single allele at each locus, and contained only alleles present in the mother's genotype (i.e., were homozygous or hemizygous). The probability that our findings resulted from the female mating with one or more males is extremely small, indicating that the offspring were derived from a single female gamete (either alone or via duplication and/or fusion) and implicating parthenogenesis. This is the first documented case of parthenogenesis in the Squamate family Agamidae.

Concurrent visual encounter sampling validates eDNA selectivity and sensitivity for the endangered wood turtle (Glyptemys insculpta).

Environmental DNA (eDNA) has been used to record the presence of many different organisms in several different aquatic and terrestrial environments. Although eDNA has been demonstrated as a useful tool for the detection of invasive and/or cryptic and declining species, this approach is subject to the same considerations that limit the interpretation of results from traditional survey techniques (e.g. imperfect detection). The wood turtle is a cryptic semi-aquatic species that is declining across its range and, like so many chelonian species, is in-need of a rapid and effective method for monitoring distribution and abundance. To meet this need, we used an eDNA approach to sample for wood turtle presence in northern Virginia streams. At the same time, we used repeat visual encounter surveys in an occupancy-modelling framework to validate our eDNA results and reveal the relationship of detection and occupancy for both methods. We sampled 37 stream reaches of varying size within and beyond the known distribution of the wood turtle across northern Virginia. Wood turtle occupancy probability was 0.54 (0.31, 0.76) and while detection probability for wood turtle occupancy was high (0.88; 0.58, 0.98), our detection of turtle abundance was markedly lower (0.28; 0.21, 0.37). We detected eDNA at 76% of sites confirmed occupied by VES and at an additional three sites where turtles were not detected but were known to occur. Environmental DNA occupancy probability was 0.55 (0.29, 0.78); directly comparable to the VES occupancy estimate. Higher probabilities of detecting wood turtle eDNA were associated with higher turtle densities, an increasing number of days since the last rainfall, lower water temperatures, and lower relative discharges. Our results suggest that eDNA technology holds promise for sampling aquatic chelonians in some systems, even when discharge is high and biomass is relatively low, when the approach is validated and sampling error is quantified.

Mitogenomes and relatedness do not predict frequency of tool-use by sea otters.

Many ecological aspects of tool-use in sea otters are similar to those in Indo-Pacific bottlenose dolphins. Within an area, most tool-using dolphins share a single mitochondrial haplotype and are more related to each other than to the population as a whole. We asked whether sea otters in California showed similar genetic patterns by sequencing mitogenomes of 43 otters and genotyping 154 otters at 38 microsatellite loci. There were six variable sites in the mitogenome that yielded three haplotypes, one found in only a single individual. The other two haplotypes contained similar percentages (33 and 36%) of frequent tool-users and a variety of diet types. Microsatellite analyses showed that snail specialists, the diet specialist group that most frequently used tools, were no more related to each other than to the population as a whole. The lack of genetic association among tool-using sea otters compared with dolphins may result from the length of time each species has been using tools. Tool-use in dolphins appears to be a relatively recent innovation (less than 200 years) but sea otters have probably been using tools for many thousands or even millions of years.

Hidden in plain sight: Cryptic and endemic malaria parasites in North American white-tailed deer (Odocoileus virginianus).

Malaria parasites of the genus Plasmodium are diverse in mammal hosts, infecting five mammalian orders in the Old World, but were long considered absent from the diverse deer family (Cervidae) and from New World mammals. There was a description of a Plasmodium parasite infecting a single splenectomized white-tailed deer (WTD; Odocoileus virginianus) in 1967 but none have been reported since, which has proven a challenge to our understanding of malaria parasite biogeography. Using both microscopy and polymerase chain reaction, we screened a large sample of native and captive ungulate species from across the United States for malaria parasites. We found a surprisingly high prevalence (up to 25%) and extremely low parasitemia of Plasmodium parasites in WTD throughout the eastern United States. We did not detect infections in the other ungulate species nor in western WTD. We also isolated the parasites from the mosquito Anopheles punctipennis. Morphologically, the parasites resemble the parasite described in 1967, Plasmodium odocoilei. Our analysis of the cytochrome b gene revealed two divergent Plasmodium clades in WTD representative of species that likely diverged 2.3 to 6 million years ago, concurrent with the arrival of the WTD ancestor into North America across Beringia. Multigene phylogenetic analysis placed these clades within the larger malaria parasite clade. We document Plasmodium parasites to be common in WTD, endemic to the New World, and as the only known malaria parasites from deer (Cervidae). These findings reshape our knowledge of the phylogeography of the malaria parasites and suggest that other mammal taxa may harbor infection by endemic and occult malaria parasites.