Mitogenomic analyses of caniform relationships

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Abstract

Extant members of the order Carnivora split into two basal groups, Caniformia (dog-like carnivorans) and Feliformia (cat-like carnivorans). In this study we address phylogenetic relationships within Caniformia applying various methodological approaches to analyses of complete mitochondrial genomes. Pinnipeds are currently well represented with respect to mitogenomic data and here we add seven mt genomes to the non-pinniped caniform collection. The analyses identified a basal caniform divergence between Cynoidea and Arctoidea. Arctoidea split into three primary groups, Ursidae (including the giant panda), Pinnipedia, and a branch, Musteloidea, which encompassed Ailuridae (red panda), Mephitidae (skunks), Procyonidae (raccoons) and Mustelidae (mustelids). The analyses favored a basal arctoid split between Ursidae and a branch containing Pinnipedia and Musteloidea. Within the Musteloidea there was a preference for a basal divergence between Ailuridae and remaining families. Among the latter, the analyses identified a sister group relationship between Mephitidae and a branch that contained Procyonidae and Mustelidae. The mitogenomic distance between the wolf and the dog was shown to be at the same level as that of basal human divergences. The wolf and the dog are commonly considered as separate species in the popular literature. The mitogenomic result is inconsistent with that understanding at the same time as it provides insight into the time of the domestication of the dog relative to basal human mitogenomic divergences.

Introduction

Wyss and Flynn (1993) characterized the history of carnivoran classification as having been long and turbulent. When these authors and also Hunt and Tedford (1993) wrote their reviews in the same volume of “Mammal Phylogeny, Placentals”, carnivoran molecular data were still limited with respect to both taxonomic coverage and the amount available for individual taxa. Consequently, the discussion in the two papers was primarily based on classical approaches. The earliest carnivoran molecular study, that of Leone and Wiens (1956), was of a considerably earlier date, however. Among the issues that Leone and Wiens (1956) examined by applying serological approaches was the phylogenetic position of the giant panda, Ailuropoda melanoleuca, which the authors placed as a close relative of the bears among arctoid carnivores by explicitly expressing that “The results of this study definitely indicate that the giant panda belongs in the family Ursidae”. The findings of Leone and Wiens (1956) were corroborated by the likewise non-character state studies of Sarich, 1973, O’Brien et al., 1985 and later also in character state phylogenetic analyses based on complete mitochondrial (mt) genes (Ledje and Arnason, 1996a, Ledje and Arnason, 1996b). In a historical perspective it is interesting that the position of the giant panda was essentially never an issue among European phylogeneticists, who traditionally placed it within the Ursidae. Without doubt the German name of the giant panda, Bambusbär, combined with German systematic tradition played a role here.

Early molecular studies of caniform relationships were to a considerable extent concentrated on the pinnipeds. The question whether pinnipeds were mono- or diphyletic was for a long time a hotly debated issue as was also their position within the caniforms. Sarich, 1969a, Sarich, 1969b, applying immunological approaches, provided molecular evidence for pinniped monophyly. The results challenged the common morphological view at that time which postulated pinniped diphyly with Phocidae (true seals) originating from the Mustelidae, and Otarioidea (Odobenidae and Otariidae) from the Ursidae (but see Scheffer, 1958, who argued in favor of monophyletic Pinnipedia as a separate order). Pinniped monophyly was further supported in nuclear analyses in the 1980s (de Jong and Goodman, 1982, Arnason and Widegren, 1986, Miyamoto and Goodman, 1986), in the 1990s by analyses of complete mitochondrial (mt) genes (Arnason et al., 1995, Ledje and Arnason, 1996a, Ledje and Arnason, 1996b) and more recently in analyses of complete mt genomes (Arnason et al., 2002, Arnason et al., 2006, Arnason and Janke, 2002, Davis et al., 2004, Delisle and Strobeck, 2005). The accumulating molecular support for pinniped monophyly gradually led to a revision of the morphological view advocating pinniped diphyly. However, these morphological studies (e.g. Wyss, 1987, Wyss and Flynn, 1993, Berta and Wyss, 1994, Deméré, 1994) generally supported a sister group relationship between Phocidae and Odobenidae (=Phocomorpha) to the exclusion of Otariidae, a hypothesis, that in addition to being incongruent with essentially all molecular findings, had also been shown to be inconsistent with comparative pinniped karyology (Arnason, 1974, Arnason, 1977).

While basal relationships within recent Ursidae and Pinnipedia can be considered as well established, those within the Musteloidea have been less definite. This has also been the case with respect to the relationship between Ursidae, Pinnipedia and Musteloidea. The immunological work of Sarich, 1969a, Sarich, 1969b did not allow identification of the closest relative of the pinnipeds among the arctoids. Braunitzer and Hofmann (1987) examined the relationship of the giant and the red panda using hemoglobin data. The authors noted that the position of the Pinnipedia above the bears (Ursinae) in the tree was quite surprising, but did not discuss the topic further. However, the interpretation of the results is complicated by the fact that the giant panda did not group with the Ursinae but rather joined the red panda (Ailurus) on a common branch as the result of the great similarity of the sequences of the two species. The non-character state (DNA-hybridization) study of Arnason and Ledje (1993) showed “that the affinities between the pinnipeds and the mustelids [musteloids] were far greater than those between the pinnipeds and any other group of terrestrial carnivores [carnivorans]”. Although the approach applied by Arnason and Ledje (1993) had drawbacks compared to later character state analyses the findings were probably the first explicit molecular suggestion for a sister group relationship between Pinnipedia and Musteloidea to the exclusion of Ursidae. The recent studies of Delisle and Strobeck, 2005, Flynn et al., 2005, Fulton and Strobeck, 2006, Sato et al., 2006, Yu and Zhang, 2006 have all favored an arctoid tree with a sister group relationship between Pinnipedia and Musteloidea to the exclusion of Ursidae, even though the support for this relationship was not particularly strong in all instances.

With respect to the Musteloidea the questions of basal relationships have primarily been related to the position of the monotypic Ailuridae (represented by the red panda, Ailurus fulgens) and the relationship between Mephitidae, Mustelidae and Procyonidae. Analyses of complete single mt genes, cyt b (Ledje and Arnason, 1996a) and 12S (Ledje and Arnason, 1996b) and combination of these (Ledje and Arnason, 1996b), did not resolve the position of Ailurus, leaving it in an unresolved polytomy among other basal arctoid splits. Within the remaining Musteloidea the same data sets identified a sister group relationship between Mephitidae (skunks), and a branch encompassing Procyonidae and Mustelidae (sine Mephitidae), thereby making traditional Mustelidae (mustelids and skunks) paraphyletic. The paraphyly of traditional Mustelidae has been corroborated in essentially all subsequent molecular analyses (e.g. Dragoo and Honeycutt, 1997; and more recently in the studies of Sato et al., 2004, Sato et al., 2006, Delisle and Strobeck, 2005, Flynn et al., 2005, Fulton and Strobeck, 2006). Unless otherwise stated we will in the following refer to Mustelidae sensu stricto (s.s.), i.e. Mustelidae sine Mephitidae.

As discussed by Flynn et al. (2000) the phylogenetic position of the Ailuridae was for a long time difficult to establish. Analysis of complete mt genomes (Delisle and Strobeck, 2005) placed Ailurus within the Musteloidea as the sister group of the Mephitidae. A position of Ailurus within the Musteloidea was also obtained in the studies of Flynn et al. (2005) based on a combination of mt and nuclear genes, Sato et al. (2006) using three nuclear genes, and by Fulton and Strobeck (2006) in analyses of five nuclear data sets. One of these data sets, IRBP, was common to the three studies. Compared to Delisle and Strobeck (2005) the three studies favored other positions of Ailurus. Thus, Flynn et al. (2005) recovered Ailurus as the sister group of remaining Musteloidea in their Bayesian analyses (the MP results were inconclusive in this respect), while both Sato et al., 2006, Fulton and Strobeck, 2006 favored a basal Musteloidea split between Mephitidae and a branch that contained Ailurus, Procyonidae and Mustelidae. On this branch Procyonidae and Mustelidae joined as sister groups to the exclusion of Ailurus. Also Yu and Zhang (2006) examined various carnivoran molecular relationships. In the absence of mephitid species Ailurus was recovered as sister to Mustelidae/Procyonidae in that study.

The particular aim of the current study was to examine basal mitogenomic relationships within Arctoidea and Musteloidea and to compare the findings with previous studies that have addressed the same divergences. In order to extend the amount of data we have added seven new caniform mt genomes (one cynoid and six arctoid) to the preexisting data set. The arctoid extension includes inter alia the spotted skunk, Spilogale putorius, Ailurus fulgens and A. melanoleuca. The inclusion of Spilogale splits the mephitid branch, which was previously represented by a single species. This complementation was considered valuable for stabilizing the position of Mephitidae relative to other Musteloidea and for investigating the position of Ailurus in the tree. Similarly, the inclusion of Ailuropoda splits the ursid branch at deepest possible position, a circumstance that might be of importance for the examination of the relationship between Ursidae, Pinnipedia and Musteloidea. In addition to deeper divergences the study also addresses the mitogenomic distance between the wolf and the dog in relation to that between basal human divergences.

The current study has focused on molecular aspects of arctoid relationships. This has been at the expense of a discussion of morphological hypotheses. For an extended account of various morphological issues the reader should consult Bininda-Emonds et al., 1999, Flynn et al., 2005, Sato et al., 2006 and the comprehensive coverage of references provided in these studies.

Section snippets

Materials and methods

The 35 mt genomes included in the study are listed in Table 1 together with their accession numbers (when applicable). The accession numbers of the new genomes are shown in bold. The pinniped study of Arnason et al. (2006) included the mt genomes of all species of extant phocids and sea lions, three fur seals and the walrus. In order to avoid unnecessary redundancy only a selection of the pinniped mt genomes was included here. The sequences of the mt genomes of the wolf, the wolverine and the

The caniform mitogenomic tree

Fig. 1 shows the best tree identified in maximum likelihood (ML) analysis of the concatenated aa sequences of 12 mt protein-coding genes (3601 aa). Two perissodactyls, the Indian rhino and the donkey, were used as outgroup to root the tree. The choice of outgroup was based on the close ordinal relationship between Perissodactyla and Carnivora as demonstrated by Xu et al. (1996).

The basal carnivoran split between Feliformia and Caniformia was conclusively supported, as was also the traditionally

Molecular estimates of arctoid divergences

The molecular estimates of divergence times within the current data set are shown in Fig. 5. The estimates are based on the aa data set and the phylogeny shown in Fig. 1. Arnason et al. (2006) provided molecular estimates of pinniped divergences. The same general approach was applied in the current study. A comparison between the two studies shows some differences in the pinniped datings. Although the differences do not fall outside confidence limits the finding draws attention to the potential

Conclusions

The current study has corroborated the gradually established molecular understanding (Arnason and Ledje, 1993, Delisle and Strobeck, 2005, Flynn et al., 2005, Fulton and Strobeck, 2006, Sato et al., 2006, Yu and Zhang, 2006) of an arctoid sister group relationship between Musteloidea and Pinnipedia to the exclusion of Ursidae. The molecular results are inconsistent with the great majority of morphological studies of this particular relationship, which have rather advocated a sister group

Acknowledgments

We express our gratitude to Dr. Mieczyslaw Wolsan for comments on the manuscript and to persons and institutes that provided us with samples (see Table 1). The study was supported by the Swedish Research Council, The Nilsson-Ehle Foundation and the TMR program of the European Commission.

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