Does Size Matter? An Experimental Evaluation of the Relative Abundance and Decay Rates of Aquatic Environmental DNA.

Environmental DNA (eDNA) is increasingly used to monitor aquatic macrofauna. Typically, short mitochondrial DNA fragments are targeted because these should be relatively more abundant in the environment as longer fragments will break into smaller fragments over time. However, longer fragments may permit more flexible primer design and increase taxonomic resolution for eDNA metabarcoding analyses, and recent studies have shown that long mitochondrial eDNA fragments can be extracted from environmental water samples. Nuclear eDNA fragments have also been proposed as targets, but little is known about their persistence in the aquatic environment. Here we measure the abundance of mitochondrial eDNA fragments of different lengths and of short nuclear eDNA fragments, originating from captive fish in experimental tanks, and we test whether longer mitochondrial and short nuclear fragments decay faster than short mitochondrial fragments following fish removal. We show that when fish are present, shorter mitochondrial fragments are more abundant in water samples than both longer mitochondrial fragments and short nuclear eDNA fragments. However, the rate of decay following fish removal was similar for all fragment types, suggesting that the differences in abundance resulted from differences in the rates at which different fragment types were produced rather than differences in their decay rates.

Selection on MHC class II supertypes in the New Zealand endemic Hochstetter's frog.

BACKGROUND: The New Zealand native frogs, family Leiopelmatidae, are among the most archaic in the world. Leiopelma hochstetteri (Hochstetter's frog) is a small, semi-aquatic frog with numerous, fragmented populations scattered across New Zealand's North Island. We characterized a major histocompatibility complex (MHC) class II B gene (DAB) in L. hochstetteri from a spleen transcriptome, and then compared its diversity to neutral microsatellite markers to assess the adaptive genetic diversity of five populations ("evolutionarily significant units", ESUs). RESULTS: L. hochstetteri possessed very high MHC diversity, with 74 DAB alleles characterized. Extremely high differentiation was observed at the DAB locus, with only two alleles shared between populations, a pattern that was not reflected in the microsatellites. Clustering analysis on putative peptide binding residues of the DAB alleles indicated four functional supertypes, all of which were represented in 4 of 5 populations, albeit at different frequencies. Otawa was an exception to these observations, with only two DAB alleles present. CONCLUSIONS: This study of MHC diversity highlights extreme population differentiation at this functional locus. Supertype differentiation was high among populations, suggesting spatial and/or temporal variation in selection pressures. Low DAB diversity in Otawa may limit this population's adaptive potential to future pathogenic challenges.

Genetic structure and demographic history of the endangered and endemic schizothoracine fish Gymnodiptychus pachycheilus in Qinghai-Tibetan Plateau.

The endangered schizothoracine fish Gymnodiptychus pachycheilus is endemic to the Qinghai-Tibetan Plateau (QTP), but very little genetic information is available for this species. Here, we accessed the current genetic divergence of G. pachycheilus population to evaluate their distributions modulated by contemporary and historical processes. Population structure and demographic history were assessed by analyzing 1811-base pairs of mitochondrial DNA from 61 individuals across a large proportion of its geographic range. Our results revealed low nucleotide diversity, suggesting severe historical bottleneck events. Analyses of molecular variance and the conventional population statistic FST (0.0435, P = 0.0215) confirmed weak genetic structure. The monophyly of G. pachycheilus was statistically well-supported, while two divergent evolutionary clusters were identified by phylogenetic analyses, suggesting a microgeographic population structure. The consistent scenario of recent population expansion of two clusters was identified based on several complementary analyses of demographic history (0.096 Ma and 0.15 Ma). This genetic divergence and evolutionary process are likely to have resulted from a series of drainage arrangements triggered by the historical tectonic events of the region. The results obtained here provide the first insights into the evolutionary history and genetic status of this little-known fish.

Genomic Tools in Biological Invasions: Current State and Future Frontiers.

Human activities are accelerating rates of biological invasions and climate-driven range expansions globally, yet we understand little of how genomic processes facilitate the invasion process. Although most of the literature has focused on underlying phenotypic correlates of invasiveness, advances in genomic technologies are showing a strong link between genomic variation and invasion success. Here, we consider the ability of genomic tools and technologies to (i) inform mechanistic understanding of biological invasions and (ii) solve real-world issues in predicting and managing biological invasions. For both, we examine the current state of the field and discuss how genomics can be leveraged in the future. In addition, we make recommendations pertinent to broader research issues, such as data sovereignty, metadata standards, collaboration, and science communication best practices that will require concerted efforts from the global invasion genomics community.

Identification multiplex assay of 19 terrestrial mammal species present in New Zealand.

An identification assay has been developed that allows accurate detection of 19 of the most common terrestrial mammals present in New Zealand (cow, red deer, goat, dog, horse, hedgehog, cat, tammar wallaby, mouse, weasel, ferret, stoat, sheep, rabbit, Pacific rat, Norway rat, ship rat, pig, and brushtail possum). This technique utilizes species-specific primers that, combined in a multiplex PCR, target small fragments of the mitochondrial cytochrome b gene. Each species, except hedgehog, produces two distinctive species-specific fragments, making the assay self-confirmatory and enabling the identification of multiple species simultaneously in DNA mixtures. The multiplex assay detects as little as 100 copies of mitochondrial DNA, which makes it a very reliable tool for degraded and trace samples. Reliability, accuracy, reproducibility, and sensitivity tests to validate the technique were performed. The technique featured here enabled a prompt response in a predation specific event, but can also be useful for wildlife management and conservation, pest incursions detection, forensic, and industrial purposes in a very simple and cost-effective manner.

Detection of Khapra Beetle Environmental DNA Using Portable Technologies in Australian Biosecurity.

Khapra beetle, Trogoderma granarium Everts, 1898, is a serious pest of stored grain products globally. Environmental DNA (eDNA)-based methods offer sensitive detection tools used to inform biosecurity officers on the presence of high-risk pests. This study tested laboratory and portable molecular technologies to detect khapra beetle environmental DNA extracted from dust samples collected during biosecurity responses (Tuggeranong and Fyshwick) to khapra beetle incursions in Australia. Airborne and floor dust samples were collected opportunistically using handheld vacuum cleaners and eDNA was extracted using either field or laboratory-based extraction methods and analyzed using laboratory benchtop real time PCR machines and portable machines with two TaqMan and one LAMP-based assay. We successfully collected, extracted, and amplified khapra beetle eDNA from dust samples by qPCR, but failed to amplify T. granarium eDNA using LAMP. The Laboratory qPCR machine showed significantly higher mean Ct values (p < 0.001) and significantly higher positive detections for both assays (p < 0.001) compared to the portable thermocycler. DNA yield was significantly higher in samples extracted using laboratory-based kits compared to field kits (p < 0.001) for both vacuumed and airborne samples (Mean DNA ± S.D. = 5.52 ± 4.45 and 4.77 ± 1.68 ng/µL, respectively), compared to field kits, (1.75 ± 1.17 and 1.36± 1.29 ng/µL for vacuumed and airborne samples, respectively). There were no significant differences in DNA yield between collection methods or differences in amplification associated to extraction or collection methods in either platform tested in this study. Portable technologies tested in this study (Franklin™ Real Time Thermocycler and Genie III) accurately amplified all tissue derived DNA during assay optimisation and field testing, highlighting the capacity of these technologies to complement biosecurity in confirming specimen ID. There was a high incidence of positive detections in field negative controls (Tuggeranong = 12.3 % and Fyshwick = 50 %), mostly attributed to the use of contaminated vacuum cleaners. We discuss suitable methods to minimize sample cross-contamination, the potential of portable molecular technologies as tools for biosecurity applications, and the suitability of eDNA-based molecular detection methods to complement global trade biosecurity for one of the most invasive and important grain pests worldwide.

Experimental infection of self-cured Leiopelma archeyi with the amphibian chytrid Batrachochytrium dendrobatidis.

The susceptibility of Archey's frog Leiopelma archeyi to Batrachochytrium dendrobatidis (Bd) is unknown, although one large population is thought to have declined sharply due to chytridiomycosis. As primary infection experiments were not permitted in this endangered New Zealand species, 6 wild-caught L. archeyi that naturally cleared infections with Bd while in captivity were exposed again to Bd to assess their immunity. These frogs were from an infected population at Whareorino, which has no known declines. All 6 L. archeyi became reinfected at low intensities, but rapidly self cured, most by 2 wk. Six Litoria ewingii were used as positive controls and developed heavier infections and clinical signs by 3 wk, demonstrating that the zoospore inoculum was virulent. Six negative controls of each species remained uninfected and healthy. Our results show that L. archeyi that have self cured have resistance to chytridiomycosis when exposed. The pattern is consistent with innate or acquired immunity to Bd, and immunological studies are needed to confirm this.

An insight into the prey spectra and livestock predation by cheetahs in Kenya using faecal DNA metabarcoding.

Dietary composition is a fundamental part of animal ecology and an important component of population dynamics. Therefore, obtaining accurate information on what an animal consumes is important for conservation planning, especially for wild large carnivores that exist in human-dominated landscapes where they are prone to direct conflicts with local people. We used faecal DNA metabarcoding to identify the vertebrate taxa commonly predated on by cheetahs (Acinonyx jubatus) with an emphasis on domestic taxa and determine the drivers of livestock predation by cheetahs residing in the Maasai Mara and Amboseli ecosystems which are important population strongholds in southern Kenya. From 84 cheetah faeces that we analysed, a total of 14 prey taxa were identified, including birds, wild and domestic mammals. The livestock taxa identified in cheetah faeces occurred at moderate frequency (12.8%) and the results showed that livestock predation was influenced neither by the sex of the cheetah nor by season. In general, our study shows that cheetahs prey on a diverse range of prey taxa including birds, wild ungulates of various sizes and occasionally on domestic animals, and that the faecal DNA metabarcoding approach represents a valuable complement to traditional dietary analysis methods.

Behavioral and trophic segregations help the Tahiti petrel to cope with the abundance of wedge-tailed shearwater when foraging in oligotrophic tropical waters.

Two species breeding in sympatry are more likely to coexist if their ecological niches are segregated either in time, space or in trophic habits. Here, we combined GPS-tracking, stable isotope analysis and DNA metabarcoding analysis to understand how the rare Tahiti petrel Pseudobulweria rostrata (TP) copes with the very abundant (i.e. 500,000 breeding pairs) wedge-tailed shearwater Ardenna pacifica (WTS) when breeding in sympatry in a tropical area. WTS foraged in restricted areas along their path, while TP predominantly foraged using extensive search behavior, suggesting a more opportunistic foraging strategy. Interspecific overlap of foraging areas was higher than intraspecific overlap. Breeding seasons largely overlap between species during the study, but TP seems to be asynchronous breeders. TP fed upon prey with higher δ15N values than WTS, and their diet was mainly composed of deep-sea organisms. TP could feed upon dead prey floating at the surface while WTS preyed mainly upon fish species that generally move in schools. Our study highlights several segregating mechanisms (temporal, behavioral and trophic) that could facilitate the coexistence of the two species despite the predominant number of WTS, and provides the very first information on the foraging and trophic ecology of the poorly-known TP.

Incorporating genotype uncertainty into mark-recapture-type models for estimating abundance using DNA samples.

Sampling DNA noninvasively has advantages for identifying animals for uses such as mark-recapture modeling that require unique identification of animals in samples. Although it is possible to generate large amounts of data from noninvasive sources of DNA, a challenge is overcoming genotyping errors that can lead to incorrect identification of individuals. A major source of error is allelic dropout, which is failure of DNA amplification at one or more loci. This has the effect of heterozygous individuals being scored as homozygotes at those loci as only one allele is detected. If errors go undetected and the genotypes are naively used in mark-recapture models, significant overestimates of population size can occur. To avoid this it is common to reject low-quality samples but this may lead to the elimination of large amounts of data. It is preferable to retain these low-quality samples as they still contain usable information in the form of partial genotypes. Rather than trying to minimize error or discarding error-prone samples we model dropout in our analysis. We describe a method based on data augmentation that allows us to model data from samples that include uncertain genotypes. Application is illustrated using data from the European badger (Meles meles).

Food from faeces: Evaluating the efficacy of scat DNA metabarcoding in dietary analyses.

Scat DNA metabarcoding is increasingly being used to track the feeding ecology of elusive wildlife species. This approach has greatly increased the resolution and detection success of prey items contained in scats when compared with other classical methods. However, there have been few studies that have systematically tested the applicability and reliability of this approach to study the diet of large felids species in the wild. Here we assessed the effectiveness of this approach in the cheetah Acinonyx jubatus. We tested how scat degradation, meal size, prey species consumed and feeding day (the day a particular prey was consumed) influenced prey DNA detection success in captive cheetahs. We demonstrated that it is possible to obtain diet information from 60-day old scats using genetic approaches, but the efficiency decreased over time. Probability of species-identification was highest for food items consumed one day prior to scat collection and the probability of being able to identify the species consumed increased with the proportion of the prey consumed. Detection success varied among prey species but not by individual cheetah. Identification of prey species using DNA detection methods from a single consumption event worked for samples collected between 8 and 72 hours post-feeding. Our approach confirms the utility of genetic approaches to identify prey species in scats and highlight the need to account for the systematic bias in results to control for possible scat degradation, feeding day, meal size and prey species consumed especially in the wild-collected scats.

High variability in the MHC class II DA beta chain of the brushtail possum (Trichosurus vulpecula).

The diversity of class II major histocompatibility complex (MHC) loci was investigated in the brushtail possum, an important marsupial pest species in New Zealand. Immunocontraception, a form of fertility control that generates an autoimmune response, is being developed as a population control method for the possum. Because the immune response is partly under genetic control, an understanding of immunogenetics in possum will be crucial to the development of immunocontraceptive vaccines. MHC molecules are critical in the vertebrate immune response. Class II MHC molecules bind and present exogenously derived peptides to T lymphocytes and may be important in the presentation of immunocontraceptives. We used polymerase chain reaction primers designed to amplify the peptide binding region of possum class II MHC genes to isolate sequences from 49 animals. We have previously described 19 novel alleles from the DAB locus in the possum (Holland et al., Immunogenetics 60:449-460, 2008). Here, we report on another 11 novel alleles isolated from possum DAB, making this the most diverse marsupial locus described so far. This high level of diversity indicates that DAB is an important MHC locus in the possum and will need to be taken into account in the design of immunocontraceptive vaccines.

Toward an ecoregion scale evaluation of eDNA metabarcoding primers: A case study for the freshwater fish biodiversity of the Murray-Darling Basin (Australia).

High-throughput sequencing of environmental DNA (i.e., eDNA metabarcoding) has become an increasingly popular method for monitoring aquatic biodiversity. At present, such analyses require target-specific primers to amplify DNA barcodes from co-occurring species, and this initial amplification can introduce biases. Understanding the performance of different primers is thus recommended prior to undertaking any metabarcoding initiative. While multiple software programs are available to evaluate metabarcoding primers, all programs have their own strengths and weaknesses. Therefore, a robust in silico workflow for the evaluation of metabarcoding primers will benefit from the use of multiple programs. Furthermore, geographic differences in species biodiversity are likely to influence the performance of metabarcoding primers and further complicate the evaluation process. Here, an in silico workflow is presented that can be used to evaluate the performance of metabarcoding primers on an ecoregion scale. This workflow was used to evaluate the performance of published and newly developed eDNA metabarcoding primers for the freshwater fish biodiversity of the Murray-Darling Basin (Australia). To validate the in silico workflow, a subset of the primers, including one newly designed primer pair, were used in metabarcoding analyses of an artificial DNA community and eDNA samples. The results show that the in silico workflow allows for a robust evaluation of metabarcoding primers and can reveal important trade-offs that need to be considered when selecting the most suitable primer. Additionally, a new primer pair was described and validated that allows for more robust taxonomic assignments and is less influenced by primer biases compared to commonly used fish metabarcoding primers.

Did Lizards Follow Unique Pathways in Sex Chromosome Evolution?

Reptiles show remarkable diversity in modes of reproduction and sex determination, including high variation in the morphology of sex chromosomes, ranging from homomorphic to highly heteromorphic. Additionally, the co-existence of genotypic sex determination (GSD) and temperature-dependent sex determination (TSD) within and among sister clades makes this group an attractive model to study and understand the evolution of sex chromosomes. This is particularly so with Lizards (Order Squamata) which, among reptiles, show extraordinary morphological diversity. They also show no particular pattern of sex chromosome degeneration of the kind observed in mammals, birds and or even in snakes. We therefore speculate that sex determination sensu sex chromosome evolution is labile and rapid and largely follows independent trajectories within lizards. Here, we review the current knowledge on the evolution of sex chromosomes in lizards and discuss how sex chromosome evolution within that group differs from other amniote taxa, facilitating unique evolutionary pathways.

Methods to maximise recovery of environmental DNA from water samples.

The environmental DNA (eDNA) method is a detection technique that is rapidly gaining credibility as a sensitive tool useful in the surveillance and monitoring of invasive and threatened species. Because eDNA analysis often deals with small quantities of short and degraded DNA fragments, methods that maximize eDNA recovery are required to increase detectability. In this study, we performed experiments at different stages of the eDNA analysis to show which combinations of methods give the best recovery rate for eDNA. Using Oriental weatherloach (Misgurnus anguillicaudatus) as a study species, we show that various combinations of DNA capture, preservation and extraction methods can significantly affect DNA yield. Filtration using cellulose nitrate filter paper preserved in ethanol or stored in a -20°C freezer and extracted with the Qiagen DNeasy kit outperformed other combinations in terms of cost and efficiency of DNA recovery. Our results support the recommendation to filter water samples within 24hours but if this is not possible, our results suggest that refrigeration may be a better option than freezing for short-term storage (i.e., 3-5 days). This information is useful in designing eDNA detection of low-density invasive or threatened species, where small variations in DNA recovery can signify the difference between detection success or failure.

Vkorc1 sequencing suggests anticoagulant resistance in rats in New Zealand.

BACKGROUND: Anticoagulant toxins are used globally to control rats. Resistance of Rattus species to these toxins now occurs in at least 18 countries in Europe, America and Asia. Resistance is often associated with single nucleotide polymorphisms (SNPs) in the Vkorc1 gene. This study gives a first overview of the distribution and frequency of Vkorc1 SNPs in rats in New Zealand. New Zealand is unusual in having no native rodents but three species of introduced Rattus - norvegicus Berk., rattus L. and exulans Peale. RESULTS: Sequence variants occurred in at least one species of rat at all 30 of the sites sampled. Three new SNPs were identified, one in kiore and two in ship rats. No SNPs previously associated with resistance were found in Norway rats or kiore, but seven ship rats were heterozygous and one homozygous for the A74T variant. Its resultant Tyr25Phe mutation has previously been associated with resistance to both first- and second-generation anticoagulants in ship rats in Spain. CONCLUSIONS: This is the first evidence of potential resistance to anticoagulant toxins in rats in New Zealand. Further testing using blood clotting response times in dosed rats is needed to confirm resistance potentially conferred by the Tyr25Phe mutation. Assessment is also needed of the potential of the other non-synonymous variants (Ala14Val, Ala26Val) recorded in this study to confer resistance to anticoagulant toxins. © 2016 Society of Chemical Industry.

A framework for estimating the sensitivity of eDNA surveys.

Imperfect sensitivity, or imperfect detection, is a feature of all survey methods that needs to be accounted for when interpreting survey results. Detection of environmental DNA (eDNA) is increasingly being used to infer species distributions, yet the sensitivity of the technique has not been fully evaluated. Sensitivity, or the probability of detecting target DNA given it is present at a site, will depend on both the survey method and the concentration and dispersion of target DNA molecules at a site. We present a model to estimate target DNA concentration and dispersion at survey sites and to estimate the sensitivity of an eDNA survey method. We fitted this model to data from a species-specific eDNA survey for Oriental weatherloach, Misgurnus anguillicaudatus, at three sites sampled in both autumn and spring. The concentration of target DNA molecules was similar at all three sites in autumn but much higher at two sites in spring. Our analysis showed the survey method had ≥95% sensitivity at sites where target DNA concentrations were ≥11 molecules per litre. We show how these data can be used to compare sampling schemes that differ in the number of field samples collected per site and number of PCR replicates per sample to achieve ≥95% sensitivity at a given target DNA concentration. These models allow researchers to quantify the sensitivity of eDNA survey methods to optimize the probability of detecting target species, and to compare DNA concentrations spatially and temporarily.

Phylogenetic relationships of extant zokors (Myospalacinae) (Rodentia, Spalacidae) inferred from mitochondrial DNA sequences.

In this study, we use three mitochondrial markers, cytochrome b gene (Cyt b), NADH dehydrogenase subunit 4 (ND4) and control region (D-loop) to investigate the phylogenetic relationships of extant zokor species in Mysopalacinae. The phylogenetic tree constructed based on Cyt b strongly supports the monophyly genera Eospalax and Myospalax with E. fontanierii being the most ancient species in Eospalax. Further phylogenetic analyses of four species of Eospalax based on ND4 and D-loop sequences revealed two clades that correspond to two geographical distributions. The basal clade includes E. cansus which is mainly found on Loess Plateau (LP) and another clade including E. baileyi, E. smithii and E. rufescens that inhabits areas above 2000 m on Qinghai-Tibetan Plateau (QTP) and Qinling Mountains. Geographical events of QTP and LP may have played a major role in the diversification and evolution of Mysopalacinae.

Reliable discrimination of 10 ungulate species using high resolution melting analysis of faecal DNA.

Identifying species occupying an area is essential for many ecological and conservation studies. Faecal DNA is a potentially powerful method for identifying cryptic mammalian species. In New Zealand, 10 species of ungulate (Order: Artiodactyla) have established wild populations and are managed as pests because of their impacts on native ecosystems. However, identifying the ungulate species present within a management area based on pellet morphology is unreliable. We present a method that enables reliable identification of 10 ungulate species (red deer, sika deer, rusa deer, fallow deer, sambar deer, white-tailed deer, Himalayan tahr, Alpine chamois, feral sheep, and feral goat) from swabs of faecal pellets. A high resolution melting (HRM) assay, targeting a fragment of the 12S rRNA gene, was developed. Species-specific primers were designed and combined in a multiplex PCR resulting in fragments of different length and therefore different melting behaviour for each species. The method was developed using tissue from each of the 10 species, and was validated in blind trials. Our protocol enabled species to be determined for 94% of faecal pellet swabs collected during routine monitoring by the New Zealand Department of Conservation. Our HRM method enables high-throughput and cost-effective species identification from low DNA template samples, and could readily be adapted to discriminate other mammalian species from faecal DNA.

Complete mitochondrial genome of the Gansu zokor, Eospalax cansus (Rodentia, Spalacidae).

Mysopalacinae (zokors) is a group of fossorial rodents for which the taxonomy has yet to reach consensus. Furthermore, due to their fossorial lifestyle, little is known about their ecology. Molecular data are important to elucidate such aspects. In this paper, the complete mitochondrial DNA genome of Gansu zokor (Eospalax cansus) of the type found in Lintan, China was determined. The genome is 16,354 bp in length and consists of 13 protein-coding genes, 22 tRNA genes, two ribosomal RNA genes, and two main non-coding regions (the control region and the origin of the light strand replication), the gene composition and order of which are similar to most other mammals. The overall base composition is T 30.0%, C 24.2%, A 33.5%, and G 12.3%, with an A + T bias of 63.5%. These mitogenome sequence data are potentially important for evolutionary, population genetic, and ecological studies of the Mysopalacinae.