Analysis of Canis mitochondrial DNA demonstrates high concordance between the control region and ATPase genes

Abstract

Background: Phylogenetic studies of wild Canis species have relied heavily on the mitochondrial DNA control region (mtDNA CR) to infer species relationships and evolutionary lineages. Previous analyses of the CR provided evidence for a North American evolved eastern wolf (C. lycaon), that is more closely related to red wolves (C. rufus) and coyotes (C. latrans) than grey wolves (C. lupus). Eastern wolf origins, however, continue to be questioned. Therefore, we analyzed mtDNA from 89 wolves and coyotes across North America and Eurasia at 347 base pairs (bp) of the CR and 1067 bp that included the ATPase6 and ATPase8 genes. Phylogenies and divergence estimates were used to clarify the evolutionary history of eastern wolves, and regional comparisons of nonsynonomous to synonomous substitutions (dN/dS) at the ATPase6 and ATPase8 genes were used to elucidate the potential role of selection in shaping mtDNA geographic distribution.

Results: We found high concordance across analyses between the mtDNA regions studied. Both had a high percentage of variable sites (CR = 14.6%; ATP = 9.7%) and both phylogenies clustered eastern wolf haplotypes monophyletically within a North American evolved lineage apart from coyotes. Divergence estimates suggest the putative red wolf sequence is more closely related to coyotes (DxyCR = 0.01982 +/- 0.00494 SD; DxyATP = 0.00332 +/- 0.00097 SD) than the eastern wolf sequences (DxyCR = 0.03047 +/- 0.00664 SD; DxyATP = 0.00931 +/- 0.00205 SD). Neutrality tests on both genes were indicative of the population expansion of coyotes across eastern North America, and dN/dS ratios suggest a possible role for purifying selection in the evolution of North American lineages. dN/dS ratios were higher in European evolved lineages from northern climates compared to North American evolved lineages from temperate regions, but these differences were not statistically significant.

Conclusions: These results demonstrate high concordance between coding and non-coding regions of mtDNA, and provide further evidence that the eastern wolf possessed distinct mtDNA lineages prior to recent coyote introgression. Purifying selection may have influenced North American evolved Canis lineages, but detection of adaptive selection in response to climate is limited by the power of current statistical tests. Increased sampling and development of alternative analytical tools will be necessary to disentangle demographic history from processes of natural selection.

Figure 1

Map of ATPase haplotype distribution. Map of sample locations (circles) showing ATPase haplotype distribution. Colours represent different major clades indicated in Figures 2a and 2b. Specific locations for Genbank samples from Eurasia, Colorado, and the solitary sample shown in the Northwest Territories were unavailable so a random location within the country or state of origin was chosen.

Figure 2

Cladograms of Canis mtDNA CR and ATPase sequences. Cladograms of Canis sequences from Bayesian analysis in BEAST of a) 347 bp of the mitochondrial DNA (mtDNA) control region and b) 1067 bp from the mtDNA ATPase region. ^fam is a Husky dog sample from Sweden; * is a wolf from Saudi Arabia; ^ruf is the red wolf sequence, NW represents New World evolved clades, OW represents Old World evolved clades, ^NA in b) identifies North American grey wolf samples. Node labels show posterior probabilities rounded to the nearest hundredth. For each genetic region the eastern wolf clade is shown in red (NW_CR1ew & NW_ATP1ew), coyote clades I and II are shown in yellow (NW_CR2coyI, NW_ATP2coyI) and orange (NW_CR3coyII and NW_ATP3coyII), and Old World (OW) clades from North America (OWCR4_gwNA) and Eurasia (OWCR5_gwEU, OWATP4_gwNAEU) are shown in blue. Insets show radial view of tree.

Figure 3

Divergence between populations. Divergence estimates between phylogenetic clades and putative species sequences at the mtDNA control region (347 bp) and ATPase region (1067 bp). Dxy (JC) = average number of nucleotide substitutions per site between populations (with Jukes and Cantor correction). Error bars are the standard deviation. NW = New World evolved sequences; OW = Old World evolved sequences, EW = eastern wolf sequences; RW = red wolf sequence; NWcoy = NWcoyI + NWcoyII; NWcoyI and NWcoyII represent the two coyote clades shown in Figures 2a and 2b; OWNA = Old World sequences from North America; OWEU = Old World sequences from Eurasia. Gaps were excluded in the analysis.