Pattern and timing of diversification of the mammalian order Carnivora inferred from multiple nuclear gene sequences

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Abstract

The mammalian order Carnivora has attracted the attention of scientists of various disciplines for decades, leading to intense interest in defining its supra-familial relationships. In the last few years, major changes to the topological structure of the carnivoran tree have been proposed and supported by various molecular data sets, radically changing the traditional view of family composition in this order. Although a sequence of molecular studies have established a growing consensus with respect to most inter-familial relationships, no analysis so far has included all carnivoran lineages (both feliform and caniform) in an integrated data set, so as to determine comparative patterns of diversification. Moreover, no study conducted thus far has estimated divergence dates among all carnivoran families, which is an important requirement in the attempt to understand the patterns and tempo of diversification in this group. In this study, we have investigated the phylogenetic relationships among carnivoran families, and performed molecular dating analyses of the inferred nodes. We assembled a molecular supermatrix containing 14 genes (7765 bp), most of which have not been previously used in supra-familial carnivoran phylogenetics, for 50 different genera representing all carnivoran families. Analysis of this data set led to consistent and robust resolution of all supra-familial nodes in the carnivoran tree, and allowed the construction of a molecular timescale for the evolution of this mammalian order.

Introduction

The mammalian order Carnivora exhibits a remarkable diversity of form and function, evolved as adaptations to widely different habitats, ranging from equatorial deserts and forests to temperate mountains and polar marine environments. Carnivorans are very widespread geographically, and demonstrate one of the most extreme cases of size variation among all mammalian orders (from a ∼45 g weasel to a 3700 kg elephant seal). Members of this group range from charismatic species well known to the general public (e.g. cats, dogs, bears) to mysterious organisms about which almost nothing is known beyond museum materials used in original taxon descriptions (MacDonald, 2001, Nowak, 1999).

There are currently 286 recognized living carnivoran species, classified into 125 different genera (Wilson and Mittermeier, 2009) traditionally placed in 11 families (Nowak, 1999, Wozencraft, 1993). Due to their diversity, public and scientific appeal, and rich fossil record, carnivorans have been historically the subject of extensive evolutionary studies, including numerous attempts to resolve phylogenetic relationships among some or all of their lineages (e.g. Bininda-Emonds et al., 1999, Flynn et al., 2000, Flynn et al., 2005, Wozencraft, 1989). Phylogenies of the order Carnivora have been used to make inferences on processes involved in taxon diversification patterns, tempo and mode of character evolution, and conservation-related issues. As many carnivoran species have suffered tremendous anthropogenic impact on their populations and habitats, many of them are endangered or likely to become so in the future (Gittleman et al., 2001). A phylogenetic framework (Bininda-Emonds et al., 1999) has been used to make assessments of biological and geographic features related to extinction vulnerability, which were proposed to serve as guides in the design of conservation strategies. For such purposes, as well as for other biological applications, it is critical to assess whether the underlying phylogenies are accurate, and to obtain a stable evolutionary framework for this group. Likewise, insights from the rich carnivoran fossil record can be greatly augmented by synergistic interaction with a well-established evolutionary timescale derived from molecular data.

The order Carnivora is divided into two main evolutionary lineages: the suborders Feliformia and Caniformia. The suborder Feliformia has traditionally comprised four families (Felidae, Herpestidae, Hyaenidae and Viverridae), while Caniformia would contain the terrestrial families Canidae, Mustelidae, Procyonidae and Ursidae, along with the marine carnivores (Pinnipedia, which include the families Otariidae, Odobenidae and Phocidae). Caniformia is further subdivided into the Cynoidea (containing the family Canidae, thought to be the deepest divergence in this group) and Arctoidea (with the six remaining families in this suborder). This taxonomic arrangement was first proposed by Flower (1869) on the basis of the form and structure of the auditory bulla, and has since been consistently supported by numerous phylogenetic studies employing other anatomical/morphological and molecular characters (e.g. Flynn and Wesley-Hunt, 2005). Despite this consistency with respect to higher level relationships, extensive controversy has dominated the evolutionary literature regarding the phylogenetic relationships among families in each suborder, the placement of enigmatic taxa (e.g. giant and red pandas, walrus, and the Malagasy fossa [Cryptoprocta]) and even the monophyly of several families. The extensive literature covering these controversies will not be described here in detail (see Flynn and Wesley-Hunt (2005) and Eizirik and Murphy (2009) for reviews), and we will focus only on the most recent developments regarding these issues.

Recent challenges to the monophyly of traditional families have included the following propositions: (i) the African Palm civet (Nandinia binotata) is a basal feliform, and not included in the Viverridae (Flynn, 1996, Hunt, 1987); (ii) Asian linsangs (genus Prionodon) are also removed from the Viverridae, and constitute the sister-group to felids (Gaubert and Veron, 2003); (iii) Malagasy carnivores usually placed in the Herpestidae and Viverridae actually form a separate feliform clade, not included in either family (Yoder et al., 2003); (iv) skunks do not belong in the Mustelidae, and form a separate arctoid clade (Dragoo and Honeycutt, 1997, Wayne et al., 1989). These hypotheses have been proposed on the basis of morphological (i) and molecular (i–iv) data, with the latter based on DNA–DNA hybridization results (iv), mtDNA sequences (i–iv) and DNA sequences from 1 to 3 nuclear loci (iii). Recent papers have corroborated one or more of these phylogenetic propositions (e.g. Arnason et al., 2007, Flynn et al., 2005, Fulton and Strobeck, 2006, Johnson et al., 2006, Perelman et al., 2008), but none has addressed all of them simultaneously, nor used independent, multi-gene data sets to specifically test these hypotheses.

Phylogenetic relationships among most families have remained unresolved or tentative for decades. Recent studies have clarified several portions of the tree (e.g. sister-group relationship between Procyonidae and Mustelidae – Flynn et al., 2000), primarily using DNA sequences from one or a few mtDNA or nuclear segments. Most of these papers have focused on a single or a few phylogenetic issues, and so far no molecular study has attempted to address all outstanding problems using a single data set that covers all living families. Such a data set would allow for simultaneous and comparable testing of multiple phylogenetic hypotheses, and could also be used in divergence dating analyses of the whole order. This joint assessment would permit the construction of a unified phylogenetic framework and molecular timescale for the order Carnivora.

In this study we aimed to resolve the phylogeny of living carnivoran families and to date all the included divergence events, by generating a large, multi-gene data set composed exclusively of segments from the nuclear genome. Nuclear sequences have been found to be more informative than mtDNA at different phylogenetic levels (e.g. Koepfli and Wayne, 2003, Springer et al., 2001), and have been successfully used to resolve various portions of the mammalian phylogeny (e.g. Amrine-Madsen et al., 2003, Eizirik et al., 2001, Eizirik et al., 2004, Johnson et al., 2006, Koepfli et al., 2006, Koepfli et al., 2007, Koepfli et al., 2008, Koepfli and Wayne, 2003, Murphy et al., 2001a, Murphy et al., 2001b, Sato et al., 2006, Yu et al., 2004, Janecka et al., 2007). Concatenation of multiple independent segments has been shown to produce an amplification of the phylogenetic signal, usually leading to well-resolved and supported trees (e.g. Rokas et al., 2003, de Queiroz and Gatesy, 2007). In particular, we selected a novel set of genes, most of which have not been used previously in higher-level carnivoran phylogenetics (e.g. Flynn et al., 2005, Gaubert and Veron, 2003, Yoder et al., 2003, Sato et al., 2004, Sato et al., 2006, Yu et al., 2004), thus providing an independent test for many recently proposed supra-familial hypotheses. Using this data set and multiple inferential approaches, we arrived at a well-resolved phylogeny presenting congruence among methods and high support for all higher-level nodes. Divergence dating analyses based on this data set produced an evolutionary timescale of living carnivoran lineages, and led to inferences on historical processes involved in the diversification of this mammalian order.

Section snippets

Taxon sampling

The major goal of our taxon-sampling scheme was to represent all extant carnivoran families, as well as the most basal divergence within each family (i.e. the base of each crown-group). For that purpose, we included divergent genera (one species for each) from all traditionally recognized carnivore families, as well as all additional lineages whose membership in traditional families had been questioned by previous studies (e.g. Dragoo and Honeycutt, 1997, Flynn and Nedbal, 1998, Gaubert and

Supermatrix characteristics

Nucleotide sequences were obtained for 14 nuclear gene segments in a sample of 50 carnivoran genera and one pangolin (Manis pentadactyla) used as the outgroup (Table 1, Table 3). This data set included 714 segments (gene-taxon combinations), of which only 55 (7.7%) were missing (i.e. data could not be collected). The taxon with the most missing data (Mydaus) was represented by only three out of the 14 segments, yet its phylogenetic position could be robustly inferred with all methods (see

Discussion

The evolutionary relationships among carnivoran families have been extensively investigated in the last three decades (see Flynn and Wesley-Hunt, 2005, Wozencraft, 1989, Wozencraft, 2005, Eizirik and Murphy, 2009 for reviews), and a growing consensus is currently emerging with respect to the supra-familial structure of this mammalian order. Some classical hypotheses have been corroborated by large molecular data sets, while new ones have recently emerged (e.g. Gaubert and Veron, 2003, Yoder et

Acknowledgments

We thank all collaborators who have helped obtain samples of biological materials employed in this study, as well as Michael Gough, Laura Eskander, John Page, Ali Wilkerson, Jill Pecon-Slattery and Felipe Grazziotin for assistance throughout several phases of the project. We also thank Sandro L. Bonatto, Maurício R. Bogo and José W. Thomé for access to computational resources employed in the phylogenetic and dating analyses, and John Flynn for stimulating discussions on carnivoran evolution.

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