Leeches

Briefly, after the genomic DNA was randomly broken into fragments using a Covairs ultrasonic breaker, a PE400 genomic DNA library was constructed based on the Whole Genome Shotgun (WGS) strategy and sequenced utilizing an Illumina NovaSeq 6000 instrument (Illumina, USA). More than 2.58 Gb of raw reads were obtained, and at least 1.67 Gb of clean reads were obtained from the raw reads after removing adaptor sequences using AdapterRemoval v2 [25 (link)] and filtering low-quality sequences using SOAPdenovo2 [26 (link)]. The obtained clean data were assembled using A5-miseq v20150522 [27 (link)] and SPAdesv3.9.0 [28 (link)] to construct contigs and scaffolds. A colinear analysis for splicing sequences was conducted using mummer v3.1 [29 (link)] and the complete mitogenome sequence was then revised and verified using pilon v1.18 [30 (link)]. All these procedures were performed by Shanghai Personal Biotechnology Co., Ltd., China.
The locations of protein-coding genes (PCGs) and rRNA genes were preliminarily predicted using GeSeq [31 (link)] and MITOS [32 (link)], and the precise locations were confirmed using related Hirudinea mitogenomes from GenBank. The initiation and termination codons were identified using ORF finder and Blastn of NCBI, according to their alignment with other related species, and the rearrangements of mitochondrial genes in the Hirudinea species were examined simply by observation. MITOS [32 (link)] was used to predict and annotate the locations and secondary structures of tRNA genes, and these were then visualized using the online software TBI-forna (http://rna.tbi.univie.ac.at/forna/, accessed on 4 January 2022). The nucleotide composition and the relative synonymous codon usage (RSCU) were determined using MEGA 7 [33 (link)].
AT skew = (A − T)/(A + T) and GC skew = (G − C)/(G + C) were analyzed to describe base composition [34 (link)]. A graphical diagram of the complete mitogenome was drawn using the online mitochondrial visualization tool mtviz (http://pacosy.informatik.uni-leipzig.de/mtviz/mtviz, accessed on 4 January 2022). Finally, the complete mitochondrial DNA sequence was uploaded to the GenBank database with accession number OL779256.

Fishes were sampled throughout Yellowstone Lake (Fig 2) during the ice-free season in 2018 and 2019 using gillnetting methods established by the NPS (see [31 (link)] for specifics on gillnetting placement and design). Diet samples of cutthroat trout and lake trout were collected by season: pre-stratification (before 1 August), stratification (1 August– 20 September), and post-stratification (after 20 September)—identical to Syslo et al. [53 (link)]. We sampled multiple individuals of each species in 50-mm total length classes starting at 100 mm during each season to account for ontogenetic diet shifts. Stomachs from cutthroat trout and lake trout that inadvertently died from gillnetting events were extracted and preserved in 70% ethanol. We pooled diet data among stratification seasons for subsequent analyses to more accurately complement stable isotope analysis from Syslo et al. [53 (link)], where stable isotope samples were not collected based on stratification season.
Fish tissue samples (~10 g of dorsal muscle tissue) were collected for stable isotope analysis. Methods for tissue collection, storage, and preparation were consistent with Syslo et al. [53 (link)]. Samples were analyzed at the University of Wyoming Stable Isotope Facility using an elemental analyzer (Thermo Finnigan Delta Plus XP, Costech 4010 and Carlo Erba 1110 Elemental Analyzer, Costech Zero Blank Autosampler, and Finnigan Conflo III Interface). Liver was used as the quality assurance material. The quality assurance of the isotope analysis is based on the standard uncertainty of the known value of the quality control reference materials analyzed during the analytical run. The standard uncertainty (1-sigma) is calculated from multiple analyses of the quality control reference materials. Stable isotope ratios were calculated using standard procedures outlined in Vander Zanden et al. [65 (link)] and Hershey et al. [66 ].
Stomach contents were analyzed for proportion of diet by wet mass [67 ], prey items were identified and separated by taxon, and the blotted wet weights were measured using the same methods as Ruzycki et al. [54 (link)] and Syslo et al. [53 (link)], thus studies were directly comparable. Invertebrates were identified to order or family and fishes were identified to species. Taxonomic identification categories were selected to match methodology used by Jones et al. [46 ], Ruzycki et al. [54 (link)], and Syslo et al. [53 (link)] and were defined as: cladocerans, copepods, amphipods, leeches, chironomids, insects (which included Ephemeroptera, Trichoptera, Plecoptera, and non-chironomid Diptera), mollusks, cutthroat trout, and unidentified fish. All field and laboratory sampling was conducted under Yellowstone National Park permit 8048. This study was performed under the auspices of Institutional Animal Care and Use Protocol 2018–72 at Montana State University.