Performing Department
Natural Resources and the Environment
Non Technical Summary
Throughout the northeastern US, habitat restoration efforts are ongoing on behalf of declining young forest-dependent wildlife species. This project develops and applies genomic tools to monitor population and individual-level responses to these management efforts. The findings of this research will inform managers about the effectiveness of habitat restoration efforts and contribute to the conservation of declining wildlife species of concern.
Animal Health Component
25%
Research Effort Categories
Basic
30%
Applied
70%
Developmental
(N/A)
Goals / Objectives
The overarching goals of this research are to leverage genomic tools to measure the response of a young forest specialist to habitat restoration and improve understanding of the factors that promote successful restoration. Ultimately, these research goals are intended to guide decision-making in conservation management efforts for this fragile ecosystem.Specific objectives of this proposal are to:Develop genomic tools for noninvasive monitoring of New England cottontail population status from fecal pellets.Evaluate genome-wide inbreeding levels for individuals in vulnerable cottontail populations across the species range.Determine cottontail diets using metabarcoding to evaluate hypotheses about habitat quality.Assess the relationship of diet and forage availability and variation in cottontail gut microbiome.Through these objectives, this research will evaluate the effectiveness of extensive ongoingmanagement actionsaimed at creating, restoring and protecting young forest habitats on behalf of declining young-forest dependent wildlife species, including the New England cottontail.Understanding the success of these management actions and identifying the factors that lead to succesful restoration is crucial feedback for wildlife conservation decision making. This knowledge will increase not only the effectiveness of future conservation of the New England cottontail, but also benefit biodiversity conservation more broadly.
Project Methods
New England cottontail samples are collected by collaborating partners and a project PhD student during winter monitoring surveys of the species; this project will focus primarily on populations in central and seacoast New Hampshire and southern Maine. Fecal pellet samples are collected, following established protocols, into unique, sterile 15 ml vials and stored frozen. Additional live-trapping efforts of the captive breeding program will be leveraged to obtain fresh tissue samples (small ear tissue biopsy obtained by off-campus project partners in the New England Cottontail Conservation Initiative) to be used for the genomic protocol development phase of this proposal.To develop noninvasive genomic monitoring tools (Obj. 1), we will evaluate the efficacy of the FecalSeq protocolfor enrichment of cottontail genomes in fecal DNA extract.This methoduses magnetic beads bound with methyl-CpG-binding domain proteins to selectively bind and isolate DNA from fecal extract with high CpG-methylation density. For this enrichment step, we will use the NEB Next Microbiome DNA Enrichment Kit (New England Biolabs, Ipswich MA), following the manufacturer protocol and the modification of Chiou and Bergey (2018) for retaining the bead-bound host DNA. We anticipate some protocol optimization will be required, and that ultimately this will result in enrichment of the cottontail DNA in the fecal sample for higher DNA yield and better success with downstream genome sequencing, as evaluated by recovered reads that map to theOryctolagusgenome and sequencing coverage.We will then develop a panel of SNPs using targeted sequence capture, following the "RADCap" methodology of Hoffberg et al. (2016). In brief,DNA libraries will be developed from enriched cottontail DNA extracts for 3RAD Sequencing, using the method of Graham et al. (2015), and sequenced with paired-end 150-bp reads on an Illumina HiSeq 2500. SNP markers will be identified using the STACKS pipeline,which allows forde novoalignment(Catchen et al. 2013). We will select a panel of ~500 polymorphic SNPs, as this number of markers will enable tracking founder individuals, reconstructing parentage and pedigrees, as well as population genetic studies. We will develop custom biotinylated RNA baits for select loci using the commercially available MYbaits kit (MYcroarray, Ann Arbor, MI). These baits will then be used to selectively amplify SNP loci in individual cottontail samples via incubation of pooled RAD libraries with the baits, followed by PCR and Illumina Sequencing. Hundreds of samples will be multiplexed in a single sequencing lane, using unique sample-specific indexing (Ali et al. 2016).To track the success of restoration efforts on behalf of cottontail populations (Obj. 2), we will apply our noninvasive population monitoring survey and genetic-mark-recapture approaches that we developed in a previous NHAES-supported project (Kristensen and Kovach 2018; Bauer et al. 2020). In collaboration with New England Cottontail Conservation Initiative partners, we will receive samples collected from cottontails on management sites of interest, including sites where rabbits have been released from the captive breeding program (Dover, NH and Wells and Scarborough, ME). This will allow us to 1) identify individuals and thereby track the survival and, through parentage analysis, the reproductive contribution of captive releases; and 2) estimate the abundance of cottontails; and 3) evaluate genetic diversity on each management site. Initially, we will continue to use the microsatellite DNA markers that we have optimized for this objective (King et al. 2017; Kristensen and Kovach 2018). Once the SNP markers have been developed (in Obj. 1), we will incorporate these into our approach.We will develop genomic approaches to evaluate genome-wide inbreeding levels for wild cottontails. First, we will establish methodologies with tissue-sample extracted DNA from individuals born in captivity, with known pedigrees. Then we will use the enriched fecal DNA sampling approach (Obj. 1) to apply this new methodology to evaluate inbreeding of wild-caught individuals in at-risk populations across the species range. Genomic measures of inbreeding will include multi-locus heterozygosity (MLH) and the proportion of the genome in runs of homozygosity (continuous stretches of DNA comprised of homozygous alleles; fROH). MLH can be measured from a subset of SNPs in the genome, and therefore will be amenable for estimation from the 3RAD Sequencing data generated using the method outlined in Obj. 1. Because MLH calculated from as few as 500 loci have been shown to correlate with 88% accuracy with genome-wide MLH (Gagnon et al. 2019), we will also evaluate this approach on the 500 RADCap loci alone. We will use the R package inbreedR (Stoffel et al. 2016) to calculate standardized multi-locus heterozygosity across these RAD-Seq-identified SNPs. Calculating runs of homozygosity typically rely on continuous DNA sequences of considerable length (1000s of base pairs; Kardos et al. 2016), however, recently, researchers have calculated runs of homozygosity from RAD Sequencing data using windows as small as 25 kb (Grossens et al. 2019). In this proposal we will use the RAD Seq data to calculate runs of homozygosity, using the metric fROH in vcftools (Dancek et al. 2011). Pending additional funding, we will ground-truth this methodology with shotgun whole genome sequencing conducted at the UNH Hubbard Center for Genome Sequencing, which will enable generating longer continuous sequences for calculating runs of homozygosity.To investigate linkages between habitat heterogeneity, cottontail diet, and cottontail gut microbiome (Obj. 3 & 4), we will use fecal samples from cottontails collected from sites with varying habitat composition, including invasive and native shrub-dominated patches, across the species' range. To inform our diet determinations in the wild, we will also conduct feeding trials of captive rabbits, working with our conservation partners. To determine cottontail diets, we will use a meta-barcoding approach to amplify plant DNA sequences in the cottontail fecal extracts, using the primersgandhof Taberlet et al. (2007), which target a 146-bp fragment of the P6 loop in the chloroplast trnL intron. This is a standardly used barcode for identifying plant taxa in herbivore diet studies (e.g., Bergman et al. 2015, Kowalczyk et al. 2019). The short fragment length makes it amenable to amplification of plant material that has passed through an herbivore's gut and the variability of the target sequence enables differentiating plant species. For microbial sequencing, we will use 16S RNA primers, which are the standard for this work. Individual cottontail samples will be labeled with unique six-nucleotide sequences, allowing indexing of samples to enable assigning different sequence reads to individual samples and enabling multiplexing (96-384, depending on the combinations of indices used) in a single lane of Illumina sequencing. Sequencing will be performed on a fraction (5-15%) of an Illumina Hi Seq 2500 lane at the UNH Hubbard Center for Genome Studies. Bioinformatics analysis of both diet and microbial sequences will be conducted with the QIME pipeline (Caporaso et al. 2010), which will be used to identify distinct sequence variants as operational taxonomic units (OTUs). Using these OTUs, we will test for differences in diets of cottontails occupying habitats of various quality and forage availability and examine relationships with the gut microbiome. These factors will then be evaluated against site-specific cottontail abundance and response to reintroduction.