Abstract
The Trispot Darter (Etheostoma trisella) is an imperiled small-bodied freshwater fish that inhabits headwaters of the upper Coosa River watershed in Alabama, Georgia, and Tennessee. We sequenced the complete mitochondrial genome of this species to develop non-invasive environmental DNA (eDNA) surveillance protocols. A mitochondrial phylogenomic analysis reveals the Trispot Darter to have diverged from soon after the common ancestor of genus Etheostoma. The mitochondrial genome sequence of E. trisella is similar to other darter species in terms of GC content and gene order, but the sequence is sufficiently divergent to permit the design of species-specific eDNA primers.
The Trispot Darter (Etheostoma trisella) is an imperiled, small-bodied (<6 cm) freshwater fish with unique life history traits (NatureServe Citation2013). Unlike most members of the subfamily Etheostominae, E. trisella is a migratory species that utilizes ephemeral streams as spawning habitat (Ryon Citation1986). Ephemeral streams are not afforded the same environmental protections as other freshwater habitats, and they are frequently disturbed by land-use changes. Etheostoma trisella is endemic to headwaters of the upper Coosa River watershed, where it is estimated that 80% of the species historical habitat has been lost to conversion to agriculture. In 2019, the species gained protection by the U.S. Endangered Species Act, and is currently listed as ‘Threatened’. Governmental agencies and non-governmental organizations recognize a need for periodic updates on habitat use and population status of E. trisella. Mitochondrial environmental DNA (eDNA) surveillance is affordable and noninvasive, and has demonstrated utility for monitoring imperiled freshwater fishes (Piggott Citation2016; Schroeter et al. Citation2019).
We sequenced the complete mitogenome of E. trisella to facilitate the design and development of eDNA detection protocols. A single specimen was collected from the Coosawattee River, Georgia (34.5731 N, −84.87556 W), in August 2018. A fin clip was taken following euthanasia, and the specimen was accessioned at the Georgia Department of Natural Resources (BA10-042). DNA was extracted using a Qiagen DNeasy kit. DNA quality and quantity were evaluated with a Qiaxcel and Nanodrop 2000 instruments, respectively. Shotgun library preparation and high-throughput sequencing were performed at the UC Berkeley DNA Sequencing Center.
The sample was barcoded and sequenced on 2.9% of a pooled single Illumina Novoseq S4 lane acquiring 73 million paired-end reads (150 bp length). Two fastq files were used as input for mitogenome assembly with MITObim 1.7. The ‘-quick’ assembly option was implemented, and the mitogenome of Etheostoma nigrum (accession KT289926) was used as a reference sequence. All other parameters were left to default values. The assembly reached 500× coverage and yielded a complete genome sequence of 16,572 bp. The mitogenome was annotated with the MitoAnnotator online server (Iwasaki et al. Citation2013) and uploaded to NCBI (accession MN792799). Twenty-seven additional percidae mitogenomes were aligned using the MAFFT online server (Katoh et al. Citation2019). Alignments were checked by eye in Bioedit, revealing a gene order and GC content (45.7%) that is consistent with the mitogenomes of related species (Hall Citation1999; Huang et al. Citation2017; Jones et al. Citation2019; Kral and Watson Citation2019 Kumar et al. Citation2016). A maximum likelihood phylogenetic analysis was conducted in RAxML 8.1.12 using the GTR + G substitution model (Stamatakis Citation2014). The resulting phylogeny resolved the monophyly of Sander, Perca, Percina, and Etheostoma (). Within Etheostoma, relationships were generally poorly supported, but are comparable with previous studies (Lang and Mayden Citation2007; Near et al. Citation2011). Strong support was provided for a common ancestor between E. trisella and Etheostoma jessiae (subgenus Gemmaperca). Pairwise sequence comparisons revealed E. trisella to differ from congeners by an average of 15.2%, providing ample possibilities for developing specific eDNA protocols.
Acknowledgements
We thank Dominique Dawson and John Larrimore for laboratory assistance.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Additional information
Funding
References
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