Complete mitochondrial genome of the imperiled Trispot Darter (Etheostoma trisella)

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.

Keywords:

• Etheostoma trisella
• percidae phylogeny
• mitochondrial genome
• mitogenome

The Trispot Darter (Etheostoma trisella) is an imperiled, small-bodied (<6 cm) freshwater fish with unique life history traits (NatureServe 2013). Unlike most members of the subfamily Etheostominae, E. trisella is a migratory species that utilizes ephemeral streams as spawning habitat (Ryon 1986). 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 2016; Schroeter et al. 2019).

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. 2013) and uploaded to NCBI (accession MN792799). Twenty-seven additional percidae mitogenomes were aligned using the MAFFT online server (Katoh et al. 2019). 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 1999; Huang et al. 2017; Jones et al. 2019; Kral and Watson 2019 Kumar et al. 2016). A maximum likelihood phylogenetic analysis was conducted in RAxML 8.1.12 using the GTR + G substitution model (Stamatakis 2014). The resulting phylogeny resolved the monophyly of Sander, Perca, Percina, and Etheostoma (Figure 1). Within Etheostoma, relationships were generally poorly supported, but are comparable with previous studies (Lang and Mayden 2007; Near et al. 2011). 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.

Figure 1. The evolutionary history of Percidae mitogenomes was inferred by using the Maximum Likelihood method based on the General Time Reversible model (GTR + I + G). The tree with the highest log likelihood score (−123285.9361) is shown. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1958)). All positions containing gaps and missing data were eliminated. There were a total of 16,419 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Kumar et al. 2016).

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

This publication was made possible through a grant from the National Fish and Wildlife Foundation (NFWF), with funding provided by the U.S. Fish and Wildlife Service, the U.S. Forest Service, and Southern Company. The views and conclusions contained in this document are those of the authors and should not be interpreted as representing the opinions or policies of the U.S. Government, NFWF or Southern Company. Mention of trade names or commercial products does not constitute their endorsement by the U.S. Government or NFWF or its funding sources.