DOI: 10.5061/dryad.jdfn2z3jv
title: Phylogenetic assessment within a species complex of a subterranean rodent (Geomys bursarius) with conservation implications for isolated subspecies

description: Range contraction and expansion from glaciation has led to genetic
 divergence that may be particularly pronounced in fossorial species with
 low dispersal. The plains pocket gopher (Geomys bursarius) is a fossorial
 species that ranges widely across North America but has a poorly
 understood phylogeny. We used mitogenomes (14,996 base pairs) from 56
 individuals across seven subspecies, plus two outgroup species, to assess
 genetic divergence from minimum spanning trees, measure genetic distances,
 and infer phylogenetic trees using BEAST. We found G. b. wisconsinensis
 was monophyletic with recent divergence. Further assessment is needed for
 G. b. major because it was paraphyletic and exhibited inconsistent
 groupings with other clades. Importantly, we identified G. b. illinoensis
 as being genetically distinct and monophyletic likely due to a unique
 colonization event eastward across the Mississippi River. Because G. b.
 illinoensis faces continued pressures from niche reduction and habitat
 loss, we recommend that G. b. illinoensis be considered an evolutionary
 significant unit warranting conservation actions to promote connectivity
 and restore suitable habitat. Such conservation efforts should benefit
 other grassland species including those originating from clades west of
 the Mississippi River that may also be evolutionary significant units.
description: We identified <em>G. bursarius </em>samples analyzed
 by Alexander et al. (2024) that had high DNA content based on
 microsatellite amplification to increase the likelihood of reconstructing
 complete mitogenomes. These samples included 41 toes from museum specimens
 from the Illinois Natural History Survey collected between 1934 and 1985,
 and 12 tissue samples from <em>G. bursarius illinoensis
 </em>live-trapped in 2018-2019, following appropriate guidelines
 (UIUC IACUC #17190). We sampled seven <em>G. bursarius</em>
 subspecies from across the range, excluding only <em>G. b.
 ozarkensis</em>. We included three samples from other
 <em>Geomys </em>species<em> </em>(one <em>G.
 breviceps dutcheri</em>, and two <em>Geomys
 jugossicularis</em>) as outgroups. <em>Geomys jugossicularis
 </em>was previously considered within the <em>G. bursarius
 </em>clade but was determined to be an independent
 species. Arbor Biosciences (arborbiosci.com; Ann Arbor,
 MI, USA) completed library preparation, mtDNA-bait enrichment and capture,
 and genome sequencing. Arbor Sciences created customized mtDNA-baits using
 a myBaits® kit to enrich for <em>Geomys</em> mtDNA prior to
 sequencing. Baits were customized using a <em>G.
 personatus</em> reference mitogenome (TK24928, 16,817bp in length;
 Greenia, 2020). To account for potential DNA degradation in the 44 museum
 samples, we used 4-plex reactions with dual-round enrichment and
 subsequent sequencing of ~3 million paired-end reads. The 12 live-trapped
 individuals with non-degraded DNA were enriched in a single-round using a
 12-plex reaction with subsequent sequencing of ~1 million paired-end
 reads. Enriched libraries were sequenced on the Illumina® NovaSeq platform
 to produce paired-end reads that were 150 base-pairs in length.
 We used AdapterRemoval v.2.3.1 (Schubert et al. 2016) to trim
 adapters and consecutive Ns from the 5’ and 3’ ends of our sequencing
 reads. We used Burrow-Wheeler Alignment with maximal exact matches
 (BWA-MEM) v. 0.7.17 (Li and Durbin 2010) to align samples to the
 <em>G. personatus </em>mitogenome (<em>G. personatus
 </em>TK24928, 16,817bp in length; Greenia 2020). We used SAMTools v.
 1.12 (Li et al. 2009) to remove duplicate and unmapped reads and reads
 with an alignment quality &lt;30, then sorted and converted the
 alignment files to Binary Alignment Map (BAM) format. We calculated the
 breadth of coverage (i.e., the percentage of the genome that had ≥1 X-fold
 read coverage) and the average depth coverage (the average X-fold number
 of reads that mapped at any location across the genome).
 We then imported the BAM files into Geneious Prime v. 2022.2.1
 (Biomatters, Ltd www.geneious.com)
 and created a mitogenome consensus sequence that contained sites with a
 minimum depth of 5X reads using a majority base-call approach. In Mega v.
 11.03 (Tamura et al. 2021), we manually curated our multiple-pairwise
 alignment by removing positions with high disagreement and low alignment
 quality that included portions of the D-loop and cyt-b regions. Because of
 the data-trimming and subsequent removal of cyt-b, we also individually
 aligned each read to the COX1 and cyt-b regions to a <em>G.
 bursarius </em>partial mitogenome (GenBank accession MZ030793.1;
 Greenia, 2020). We focused on these regions as COX1 had the lowest
 adaptive/synonymous replacement (~0), whereas cyt-b is commonly used in
 phylogenetic reconstruction and also has low adaptive/synonymous
 replacement in <em>Geomys </em>(Greenia 2020). We generated
 three datasets: (1) mitogenome-wide consensus sequences that were manually
 trimmed to remove disagreements, (2) consensus sequences based on reads
 that were independently aligned to the COX1 gene, and (3) consensus
 sequences based on reads independently aligned to the cyt-b
 region. Literature Cited Greenia, H.
 S. 2020. Comparative mitogenomics of the rodent suborder Castorimorpha.
 Thesis, Tarleton State University, Stephensville. Li,
 H. et al. 2009. The Sequence Alignment/Map format and SAMtools.
 Bioinformatics 25:2078–2079. Li, H., and R. Durbin.
 2009. Fast and accurate short read alignment with Burrows-Wheeler
 transform. Bioinformatics 25:1754–1760. Schubert, M.,
 S. Lindgreen, and L. Orlando. 2016. AdapterRemoval v2: Rapid adapter
 trimming, identification, and read merging. BMC Research Notes
 9:88. Tamura, K., G. Stecher, and S. Kumar. 2021.
 MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Molecular
 Biology and Evolution 38:3022–3027.
description: Manuscript Title: Phylogenetic assessment within a species complex of a
 subterranean rodent (Geomys bursarius) with conservation implications for
 isolated subspecies Authors: Nathan Alexander Alida de Flamingh Bradley J.
 Cosentino Robert L. Schooley The files contained herein include three
 alignments, a spreadsheet with sampling information (and is supplementary
 materials 1 in the published manuscript) Alignments: File Name:
 **Gopher_5_21_23_Subspecies_geo.nex** Description: trimmed and alinged
 partial mitogenomes of 53 Geomys bursarius, 2 Geomys jugossicularis, and 1
 Geomys breviceps dutcheri. This file can be opened in MEGA v 11.03, PopArt
 v. 1.7 File Name: **COX1_alignment_subspecies.nex** Description:
 individually aligned and compiled COX1 regions of 53 Geomys bursarius, 2
 Geomys jugossicularis, and 1 Geomys breviceps dutcheri. This file can be
 opened in PopArt v. 1.7 File Name:
 **Cytb_alignment_trimmed_subspecies.nexus** Description: individually
 aligned, compiled, and trimmed cytochrome b region of 53 Geomys bursarius,
 2 Geomys jugossicularis, and 1 Geomys breviceps dutcheri. This file can be
 opened in PopArt v. 1.7 File Name: **COX1_alignment_gb.nexus**
 Description: individually aligned and compiled COX1 regions of 53 Geomys
 bursarius, 2 Geomys jugossicularis, and 1 Geomys breviceps dutcheri nexus
 file submitted to GenBank. This file can be opened in MEGA v 11.03 File
 Name: **Cytb_alignment_trimmed_gb.nexus** Description: individually
 aligned, compiled, and trimmed cytochrome b region of 53 Geomys bursarius,
 2 Geomys jugossicularis, and 1 Geomys breviceps dutcheri. This file can be
 opened in MEGA v 11.03 File Name: **Sample_and_file_information.csv**
 Description: Spreadsheet with file name, specimen catalogue
 identification, species identification, sex, longitude, latitude, state,
 county, sampling location from catalogue tag, collection year, collection
 month, collector, SRA accession number, GenBank accession for cyt-b, BAM
 file names, index, total passing reads, percent of passing reads &gt; Q30,
 and coverage estimates for the min, max, and mean. This file can be opened
 in excel or programs that can read csv files.