A new phylogenetic marker, apolipoprotein B, provides compelling evidence for eutherian relationships

Abstract

Higher-level relationships within, and the root of Placentalia, remain contentious issues. Resolution of the placental tree is important to the choice of mammalian genome projects and model organisms, as well as for understanding the biogeography of the eutherian radiation. We present phylogenetic analyses of 63 species representing all extant eutherian mammal orders for a new molecular phylogenetic marker, a 1.3 kb portion of exon 26 of the apolipoprotein B (APOB) gene. In addition, we analyzed a multigene concatenation that included APOB sequences and a previously published data set (Murphy et al., 2001b) of three mitochondrial and 19 nuclear genes, resulting in an alignment of over 17 kb for 42 placentals and two marsupials. Due to computational difficulties, previous maximum likelihood analyses of large, multigene concatenations for placental mammals have used quartet puzzling, less complex models of sequence evolution, or phylogenetic constraints to approximate a full maximum likelihood bootstrap. Here, we utilize a Unix load sharing facility to perform maximum likelihood bootstrap analyses for both the APOB and concatenated data sets with a GTR + Γ + I model of sequence evolution, tree-bisection and reconnection branch-swapping, and no phylogenetic constraints. Maximum likelihood and Bayesian analyses of both data sets provide support for the superordinal clades Boreoeutheria, Euarchontoglires, Laurasiatheria, Xenarthra, Afrotheria, and Ostentoria (pangolins + carnivores), as well as for the monophyly of the orders Eulipotyphla, Primates, and Rodentia, all of which have recently been questioned. Both data sets recovered an association of Hippopotamidae and Cetacea within Cetartiodactyla, as well as hedgehog and shrew within Eulipotyphla. APOB showed strong support for an association of tarsier and Anthropoidea within Primates. Parsimony, maximum likelihood and Bayesian analyses with both data sets placed Afrotheria at the base of the placental radiation. Statistical tests that employed APOB to examine a priori hypotheses for the root of the placental tree rejected rooting on myomorphs and hedgehog, but did not discriminate between rooting at the base of Afrotheria, at the base of Xenarthra, or between Atlantogenata (Xenarthra + Afrotheria) and Boreoeutheria. An orthologous deletion of 363 bp in the aligned APOB sequences proved phylogenetically informative for the grouping of the order Carnivora with the order Pholidota into the superordinal clade Ostentoria. A smaller deletion of 237–246 bp was diagnostic of the superordinal clade Afrotheria.

Introduction

Since the advent of PCR, numerous studies have addressed the molecular phylogenetics of the living eutherian orders of mammals. Single gene studies with limited ordinal representation (Porter et al., 1996; Springer and Kirsch, 1993; Stanhope et al., 1992) have matured into multigene data sets representing all placental orders (Arnason et al., 2002; Madsen et al., 2001; Murphy et al., 2001a, Murphy et al., 2001b; Scally et al., 2001; Springer et al., in press). The largest of these contains over 16 kb of sequence from both nuclear exons, untranslated regions, and mitochondrial rRNA + tRNA genes (Murphy et al., 2001b; Springer et al., in press) and resolves many of the nodes on the placental tree with high maximum likelihood (ML) bootstrap support and Bayesian posterior probabilities. Paleontologists have also added to the wealth of data related to the evolutionary history of placental orders. Early cetacean fossils (Gingerich et al., 2001; Thewissen et al., 2001) corroborate molecular evidence uniting the orders Cetacea and Artiodactyla and dispel the traditional view that cetaceans are derived from an extinct group of mammals, the mesonychids. One of these reports (Gingerich et al., 2001) accords with molecular topologies (Gatesy, 1997; Gatesy et al., 1996; Gatesy et al., 1999; Ursing and Arnason, 1998) by suggesting Artiodactyla paraphyly.

Despite this intensive multidisciplinary effort, a well agreed upon phylogeny for placental orders does not yet exist. In some cases, morpholology is at odds with molecules. For example, one of the best supported associations based on morphology is the unification of elephants and sirenians into the superordinal group Tethytheria (Domning et al., 1986; McKenna, 1975; Shoshani and McKenna, 1998). In contrast, molecular studies have failed to resolve the paenungulate trifurcation and in some cases Tethytheria is not as well supported as competing hypotheses (Amrine and Springer, 1999; de Jong et al., 1993; Springer et al., 1997). Conversely, the superordinal clade Afrotheria (elephants, hyraxes, sirenians, elephant shrews, golden moles, and tenrecs) is well supported by a variety of molecular studies (Madsen et al., 1997, Madsen et al., 2001; Murphy et al., 2001a, Murphy et al., 2001b; Scally et al., 2001; Springer et al., 1997; Stanhope et al., 1998a; Stanhope et al., 1998b). However, Afrotheria has received little or no morphological support (Asher, 1999; Werdelin and Nilsonne, 1999; Whidden, 2002).

Furthermore, a sister-group relationship between hippopotomids and cetaceans has compelling molecular support from both DNA sequence data (Gatesy et al., 1996; Gatesy et al., 1999; Matthee et al., 2001; Ursing and Arnason, 1998) and SINES (Nikaido et al., 1999; Shimamura et al., 1997; Shedlock et al., 2000), but recent morphological reports based on fossil data (O’Leary and Geisler, 1999; Thewissen et al., 2001) and characters of the gastrointestinal tract (Langer, 2001) favor a monophyletic Artiodactyla. There is not only contention at the interordinal level but also within orders. One example is the placement of tarsiers within the order Primates. Competing hypotheses place tarsiers as sister-group to anthropoids (Goodman et al., 1998; Kay et al., 1997; Porter et al., 1995; Shoshani et al., 1996), a sister-group to lemurs (Gingerich, 1992; Murphy et al., 2001a), or at the base of the primate radiation (Arnason et al., 2002).

Along with the disagreement between morphology and molecules, there is occasional disagreement across morphological studies or across molecular studies. The superordinal grouping of pangolins and carnivores into Ostentoria (Springer et al., in press) is supported by some morphological studies (Rose and Emry, 1993; Shoshani and McKenna, 1998) and rejected by others in favor of a grouping of pangolins and xenarthrans into the clade Edentata (Novacek, 1992; Novacek and Wyss, 1986). On the molecular side, a recent report by Arnason et al. (2002), including 60 eutherian taxa for 12 mitochondrial protein coding genes (mtpcg) conflicts with results from nuclear and mitochondrial RNA genes (Eizirik et al., 2001; Madsen et al., 2001; Murphy et al., 2001a; Scally et al., 2001; Springer et al., in press). Mitochondrial protein coding genes consistently place hedgehog as the basal eutherian, rendering Eulipotyphla diphyletic (Janke et al., 1997; Krettek et al., 1995). In addition, a monophyletic Rodentia is difficult to recover with mtpcg; instead, a paraphyletic Rodentia generally occurs near the base of the placental tree, branching off after the hedgehog lineage (Arnason et al., 2002; Mouchaty et al., 2000; Penny et al., 1999). This arrangement for hedgehog and rodents disrupts the monophyly of both Laurasiatheria and Euarchontoglires, which are two of the four (Afrotheria and Xenarthra are the other two) main clades of placental mammals robustly supported by other data sets (Eizirik et al., 2001; Madsen et al., 2001; Murphy et al., 2001a, Murphy et al., 2001b; Scally et al., 2001; Springer et al., in press).

Most surprisingly, mtpcg studies place the order Dermoptera (flying lemurs) as sister to Anthropoidea, in a new clade, Dermosimii, rendering Primates paraphyletic. Primate monophyly is well established with both molecular and morphological work (Goodman et al., 1994; Goodman et al., 1998; Shoshani et al., 1996; Van Den Bussche et al., 2002). There are also many cases of agreement between molecular data sets including Cetartiodacytla, Hippopotamidae + Cetacea, Xenarthra, Afrotheria, and Ostentoria (Arnason et al., 2002; Delsuc et al., 2001; Murphy et al., 2001b; Scally et al., 2001; Ursing and Arnason, 1998).

In light of the above, new molecular markers are needed to test previous hypotheses and provide additional resolution. These new markers should include genetically independent loci with varying function (to avoid functional associations) as well as alternate phylogenetic characters such as SINES, indels, and protein sequence signatures (Ogiwara et al., 2002; Scally et al., 2001; van Dijk et al., 2001). SINEs provide evidence for Cetartiodactyla (Shimamura et al., 1997; Shimamura et al., 1999), Hippopotamidae + Cetacea (Nikaido et al., 1999; Shimamura et al., 1997), tarsiers + anthropoids (Schmitz et al., 2001), and Carnivora (Vassetzky and Kramerov, 2002). Indels (Scally et al., 2001) and protein sequence signatures (van Dijk et al., 2001) provide evidence for Afrotheria. These characters are appealing as they are more similar to conventional morphological synapomorphies (Ragg et al., 2001; Shedlock and Okada, 2000; van Dijk et al., 2001). In addition, these characters are either present or absent and do not require complex models of sequence evolution to explain their evolution (Griffiths and Gupta, 2002; Gupta and Griffiths, 2002; Miyamoto, 1999).

Here, we present an independent phylogenetic marker, a portion of the apolipoprotein B (APOB) gene, for 63 species representing all extant orders of Placentalia as well as outgroup taxa from both Marsupialia and Monotremata. APOB is one of the largest known mammalian genes. It contains 29 exons and spans 45 kb (Chen et al., 1986; Cladaras et al., 1986; Knott et al., 1986). APOB is a single copy nuclear locus producing a 4536 amino acid (AA) protein in humans, and is the sole protein component of LDL, the low-density lipoprotein particle (Young, 1990). As part of LDL, APOB binds to the LDL receptor (LDLr) through putative binding domains located in exon 26 (Ebert et al., 1988; Hospattankar et al., 1986; Maeda et al., 1988; Yang et al., 1986). We sequenced a portion of exon 26 of APOB containing the putative LDLr binding domains that resulted in an aligned data set of 1342 bp.

This paper reports the results of analyses of APOB on its own, as well as combined with the 16.4 kb data set of Murphy et al., 2001a, Murphy et al., 2001b for a concatenated alignment of approximately 17.7 kb for 44 taxa. Concatenation has proved invaluable for acquiring sufficient phylogenetic signal to resolve deep level divergences (Brown et al., 2001; Teeling et al., 2000). Computer simulations (e.g., Rosenberg and Kumar, 2001) have suggested that longer DNA sequences, rather than adding more taxa, tend to result in greater phylogenetic accuracy. Other authors (e.g., Hillis, 1996; Hillis et al., 2003; Zwickl and Hillis, 2002) contend that increased taxon sampling increases phylogenetic accuracy. Therefore, it is important to maintain adequate taxon sampling in addition to increasing the length of a data set. In addition to providing further details and corroborative evidence regarding the major placental clades, the results reported here include indel evidence pertaining to the evolutionary history of afrotherians, pangolins, and carnivores.