Environmental DNA (eDNA) is emerging as a potentially powerful tool for inferring species' presence, and hence occupancy, from DNA that is shed into environmental samples such as water. Although eDNA screening has been used to detect DNA from a variety of taxonomic groups, it has not yet been used to identify DNA from species with numerous potentially sympatric confamilial species, a situation that may preclude the development of species-specific markers. There are 41 native freshwater mussel species (Unionidae) in Ontario, Canada. Many of these are potentially sympatric, and 14 species have been formally assessed as endangered, threatened, or special concern. We investigated whether there was sufficient variation within the cytochrome oxidase region (COI) to develop species-specific eDNA markers for at-risk unionids. We developed 32 COI markers for eight unionid species, and tested each of these on the target species plus 29 potentially sympatric unionid taxa. Six of these markers amplified DNA only from the intended target species. We then extracted and amplified mussel eDNA from rearing-tank water samples. We conclude that despite high species diversity, it should be possible to develop eDNA COI markers and screen water samples for habitat occupancy by unionid mussels.
BackgroundThe microbiome affects the health of plants and animals, including humans, and has many biological, ecological and evolutionary consequences. Microbiome studies typically rely on sequencing ribosomal 16S RNA gene fragments, which serve as taxonomic markers for prokaryotic communities; however, for eukaryotic microbes this approach is compromised, because 18S rRNA gene sequences from microbial eukaryotes are swamped by contaminating host rRNA gene sequences. ResultsTo overcome this problem, we developed CRISPR-Cas Selective Amplicon Sequencing (CCSAS), a high-resolution and efficient approach for characterizing eukaryotic microbiomes. CCSAS uses taxon-specific single-guide RNA (sgRNA) to direct Cas9 to cut 18S rRNA gene sequences of the host, while leaving protistan and fungal sequences intact. We validated the specificity of the sgRNA on ten model organisms and an artificially constructed (mock) community of nine protistan and fungal pathogens. The results showed that >96.5% of host rRNA gene amplicons were cleaved, while 18S rRNA gene sequences from protists and fungi were unaffected. When used to assess the eukaryotic microbiome of oyster spat from a hatchery, CCSAS revealed a diverse community of eukaryotic microbes, typically with much less contamination from oyster 18S rRNA gene sequences than other methods using non-metazoan or blocking primers. However, each method revealed taxonomic groups that were not detected using the other methods, showing that a single approach is unlikely to uncover the entire eukaryotic microbiome in complex communities. To facilitate the application of CCSAS, we designed taxon-specific sgRNA for ~16,000 metazoan and plant taxa, making CCSAS widely available for characterizing eukaryotic microbiomes that have largely been neglected. ConclusionCCSAS provides a high-through-put and cost-effective approach for resolving the eukaryotic microbiome of metazoa and plants with minimal contamination from host 18S rRNA gene sequences. Keywords: Eukaryotic microbiome, 18S rRNA gene, Microeukaryote, CRISPR-Cas, Taxon-specific single-guide RNA, gRNA-target-site, CasOligo, CCSAS
Stramenopiles are a diverse but relatively well-studied eukaryotic supergroup with considerable genomic information available (Sibbald and Archibald, 2017). Nevertheless, the relationships between major stramenopile subgroups remain unresolved, in part due to a lack of data from small nanoflagellates that make up a lot of the genetic diversity of the group. This is most obvious in Bigyromonadea, which is one of four major stramenopile subgroups but represented by a single transcriptome. To examine the diversity of Bigyromonadea and how the lack of data affects the tree, we generated transcriptomes from seven novel bigyromonada species described in this study: Develocauda condao, Develocanicus komovi, Develocanicus vyazemskyi, Cubaremonas variflagellatum, Pirsonia chemainus, Feodosia pseudopoda, and Koktebelia satura. Both maximum likelihood and Bayesian phylogenomic trees based on a 247 gene-matrix recovered a monophyletic Bigyromonadea that includes two diverse subgroups, Developea and Pirsoniales, that were not previously related based on single gene trees. Maximum likelihood analyses show Bigyromonadea related to oomycetes, whereas Bayesian analyses and topology testing were inconclusive. We observed similarities between the novel bigyromonad species and motile zoospores of oomycetes in morphology and the ability to self- aggregate. Rare formation of pseudopods and fused cells were also observed, traits that are also found in members of labyrinthulomycetes, another osmotrophic stramenopiles. Furthermore, we report the first case of eukaryovory in the flagellated stages of Pirsoniales. These analyses reveal new diversity of Bigyromonadea, and altogether suggest their monophyly with oomycetes, collectively known as Pseudofungi, is the most likely topology of the stramenopile tree.
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