Vegetation is an underutilized medium for environmental DNA (eDNA) sampling.eDNA methods leveraging water as a substrate exclude application to many terrestrial species. The use of eDNA to detect small mammals can complement current survey approaches (live capturing, track plating, and camera trapping) while reducing risks to the animals. The endangered New Mexico meadow jumping mouse (Zapus hudsonius luteus) is specialized to herbaceous riparian zones, making it an ideal candidate for developing a terrestrial eDNA detection method. We developed a species-specific assay for quantitative real-time PCR, then tested the long-term persistence of jumping mouse eDNA on plant material using four herbaceous day nests collected three to six months after occupancy. We conducted a field trial using sterile cotton swabs at six locations along two occupied streams to evaluate our assay's capability to detect present-day eDNA. Each of 60 swabs was used to swab a 0.50 m 2 area along streamside transects that included vegetation such as forbs, grasses, and sedges. We also opportunistically swabbed plants (n = 9) following visual observation of jumping mice.We determined the limit of detection for both assays are fewer than eight copies per reaction. We detected eDNA in three of four nests. From field trial samples, we successfully detected the species from randomly swabbed vegetation (N = 3), and four of nine swabs from vegetation recently used by individuals. Further work is required to develop a robust survey method using this eDNA detection approach. Our study demonstrated that mammalian eDNA can persist on nest vegetation long after the animal was present, highlighting the promise of using eDNA from plants to detect rare or endangered terrestrial species.

1.ABSTRACTBackgroundDetermining the life-history traits of extinct species is often difficult from skeletal remains alone, limiting the accuracy of studies modeling past ecosystems. However, the analysis of the degraded endogenous bacterial DNA present in paleontological fecal matter (coprolites) may enable the characterization of specific traits such as the host’s digestive physiology and diet. An issue when evaluating the microbial composition of coprolites is the degree to which the microbiome is representative of the host’s original gut community versus the changes that occur in the weeks following deposition due to desiccation. Analyses of paleontological microorganisms are also relevant in the light of recent studies linking the Late Pleistocene and Early Holocene extinctions with modern-day zoonotic pathogen outbreaks.MethodsShotgun sequencing was performed on ancient DNA (aDNA) extracted from coprolites of the Columbian mammoth (Mammuthus Columbi), Shasta ground sloth (Nothrotheriops shastensis) and paleontological bison (Bison sp.) collected from caves on the Colorado Plateau, Southwestern USA. The novel metagenomic classifier MTSv, parameterized for studies of aDNA, was used to assign bacterial taxa to sequencing reads. The resulting bacterial community of coprolites was then compared to those from modern fecal specimens of the African savannah elephant (Loxodonta africana), the brown-throated sloth (Bradypus variegatus) and the modern bison (Bison bison). Both paleontological and modern bison fecal bacterial communities were also compared to those of progressively dried cattle feces to determine whether endogenous DNA from coprolites had a microbiome signal skewed towards aerobic microorganisms typical of desiccated fecal matter.ResultsThe diversity of phyla identified from coprolites was lower than modern specimens. The relative abundance of Actinobacteria was increased in coprolites compared to modern specimens, with fewer Bacteroidetes and Euryarchaeota. Firmicutes had a reduced relative abundance in the mammoth and bison coprolites, compared to the African savanna elephants and modern bison. There was a significant separation of samples in NMDS plots based on their classification as either paleontological or modern, and to a lesser extent, based on the host species. Increasingly dried cattle feces formed a continuum between the modern and paleontological bison samples.ConclusionOur results reveal that any coprolite metagenomes should always be compared to desiccated modern fecal samples from closely related hosts fed a comparable diet to determine the degree to which the coprolite metagenome is a result of desiccation versus true dissimilarities between the modern and paleontological hosts. Also, a large-scale desiccation study including a variety of modern species may shed light on life-history traits of extinct species without close extant relatives, by establishing the proximity of coprolite metagenomes with those from dried modern samples.

Hybridization capture approaches allow targeted high-throughput sequencing analysis at reduced costs compared to shotgun sequencing. Hybridization capture is particularly useful in analyses of genomic data from ancient, environmental, and forensic samples, where target content is low, DNA is fragmented and multiplex PCR or other targeted approaches often fail. Here, we describe a DNA bait synthesis approach for hybridization capture that we call Circular Nucleic acid Enrichment Reagent, or CNER (pronounced ‘snare’). The CNER method uses rolling-circle amplification followed by restriction digestion to discretize microgram quantities of hybridization probes. We demonstrate the utility of the CNER method by generating probes for a panel of 23 771 known sites of single nucleotide polymorphism in the horse genome. Using these probes, we capture and sequence from a panel of ten ancient horse DNA libraries, comparing CNER capture efficiency to a commercially available approach. With about one million read pairs per sample, CNERs captured more targets (90.5% versus 66.5%) at greater mean depth than an alternative commercial approach.

Background Salas y Gómez is a small, volcanic island largely untouched by humans due to its diminutive size and remoteness. Since the waters surrounding Salas y Gómez were established as Motu Motiro Hiva Marine Park in 2010, marine investigations have been the primary research focus. Secondarily, nesting seabird communities have been censused since 2011. Methods and findings In 2016, terrestrial arthropods were sampled on the island. Two observers sampled two locations for 30 min per site. Fifteen morphospecies were identified including at least one likely undescribed species. Conclusions Our work represents the most comprehensive terrestrial arthropod inventory of Salas y Gómez island to date. We are hopeful the recommendations provided will spur additional research to both characterize the island’s arthropod community, as well as identify species of management concern.