The complete mitochondrial genome of Gunnison’s prairie dog subspecies (Cynomys gunnisoni gunnisoni) and phylogenetic relationship within the genus Cynomys

Abstract

The mitochondrial genome of the Gunnison’s prairie dog, Cynomys gunnisoni gunnisoni, is described and incorporated into a phylogenetic inference of the genus Cynomys based on seven new mitochondrial genomes and published genomes from Cynomys leucurus and Cynomys ludovicianus. The mitochondrial genome from Ictidomys tridecemlineatus, the sister genus, was included as an outgroup. Our results lend further support to published phylogenetic hypotheses.

Keywords:

• Prairie dog
• ground squirrels
• Cynomys
• gunnisoni
• mitochondria
• genome

Introduction

Prairie dogs are colonial, semi-fossorial members of the family Sciuridae. The two subgenera within Cynomys include five species of extant prairie dogs. Subgenus Leucocrossuromys, the white-tailed prairie dogs, includes the Gunnison’s (Cynomys gunnisoni), Utah (Cynomys parvidens), and white-tailed (Cynomys leucurus) prairie dogs (Pizzimenti and Hoffman 1973). The subgenus Cynomys, the black-tailed prairie dogs, includes the Mexican (Cynomys mexicanus) and black-tailed prairie dogs (Cynomys ludovicianus) (Clark et al. 1971; Pizzimenti 1975; Seglund et al. 2006).

In an effort to better characterize the divergence among lineages within the genus, we assembled complete mitochondrial genome sequences from seven individuals from three of the distinct species. These sequences include five novel genomes of montane subspecies of Gunnison’s prairie dog, C.g. gunnisoni, and a single individual each from C. leucurus and C. ludovicianus (Sackett et al. 2014; Li et al. 2016). Additionally, existing mitochondrial genomes of Cynomys and the putative sister genus (Ictidomys tridecemlineatus) were obtained for phylogenetic analysis.

Methods

Whole genomic DNA was extracted from frozen spleen samples using Qiagen DNEasy Blood and Tissue Kits. DNA libraries were prepared by the BioFrontiers Institute at the University of Colorado, Boulder using Nextera Library prep kits. DNA was sequenced on an Illumina NextSeq500 using paired-end 2 × 150 bp reads. Sequence data were assembled using SPAdes v. 3.11 (Nurk et al. 2013). One annotation for C.g. gunnisoni (approximate location UTM 13 S 4550** 4310***. Sample archived at Colorado Parks and Wildlife, Wildlife Health Lab, Fort Collins, CO, USA) was submitted to GenBank (accession MG450794) and oriented to start with the cytochrome c oxidase I gene (cox1).

The seven assembled mitochondrial prairie dog genomes were aligned using ClustalW with default parameters (Larkin et al. 2007). Additional genomes were obtained from NCBI’s GenBank database which include a single C. ludovicianus (NCBI accession KP326310), C. leucurus (KP326309), and a 13-lined ground squirrel (Ictidomys tridecemlineatus; KP698974). Alignments were curated in MEGA7 (Kumar et al. 2016) and a bootstrap consensus tree was created using maximum likelihood with I. tridecemlineatus as an out-group. The Maximum Parsimony method was used to develop an initial tree for the heuristic search and the tree was drawn to scale with branch lengths proportional to the number of substitutions per site (Figure 1).

Figure 1. The evolutionary history was inferred by using the maximum-likelihood method based on the General Time Reversible model. The bootstrap consensus tree inferred from 200 replicates is taken to represent the evolutionary history of the 10 sequences analysed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (200 replicates) is shown above the branches. Initial trees for the heuristic search were obtained automatically by applying the Maximum Parsimony method. A discrete Gamma distribution was used to model evolutionary rate differences among sites (five categories (+G, parameter =0.1697)). The rate variation model allowed for some sites to be evolutionarily invariable ([+I], 44.33% sites). All positions containing gaps and missing data were eliminated. There were a total of 16,318 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 (Tamura and Nei 1993; Kumar et al. 2016). GUPD represents Cynomys Gunnisoni Gunnisoni (1-3 are in the Gunnison watershed, 4 and 5 are in the South Platte watershead), WTPD are Cynomys leucurus and BTPD are Cynomys ludovicianus

Results

The new C. g. gunnisoni sequences are similar to the previously published mitochondrial genome sequences of two congeners from Li et al. (2016). The five C. g. gunnisoni mitochondrial genomes varied in length from 16,404 to 16,462 base pairs. Genetic distances, nucleotide composition, and phylogenetic trees were obtained using MEGA v.7.0.26. Percent divergence (p-distance ×100) ranged from 0.02 to 0.29 within Gunnison’s prairie dogs. The two C. leucurus are 0.05% divergent and the two C. ludovicianus are 0.54% divergent. The average percent divergence seen between C.g. gunnisoni and C. leucurus is 1.29%. The divergence between C.g. gunnisoni and C. ludovicianus and between C. leucurus and C. ludovicianus is 4.04% and 4.01%, respectively.

Acknowledgements

We thank the Colorado Division of Parks and Wildlife for samples, sample preparation, and funding support. We also thank the BioFrontiers Institute Next-Gen Sequencing Core Facility, which performed the Illumina sequencing and library construction. M Miller reviewed an earlier draft of this manuscript.

Disclosure statement

We declare no conflict of interest.

Additional information

Funding

This work was supported by a grant from the Colorado’s Species Conservation Trust Fund and Division of Colorado Park’s and Wildlife (no. 13-CU-83560).