Keywords: Laurasiatheria, phylogenomics, rapid divergence, placental mammals

Taxonomic Sampling

Laboratory Protocols

Methods for isolating genomic DNA and sequencing are the same as those described byZhou et al. (2011).

Alignment and Combination of Data Sets

Initial sequence assembly was carried out using the ContigExpress software (Invitrogen). Chromatograms for exon sequences were viewed individually to ensure accuracy and were aligned with the consensus sequence from public database (OrthoMaM). Individual alignments for data set A were produced using Clustal W (Thompson et al. 1997) on the translated protein sequences. All alignments were checked by Muscle (Edgar 2004), and poorly aligned regions were improved manually. The final concatenated sequence alignments used for subsequent analyses were chosen using Gblock software (Castresana 2000). Intergenomic phylogenetic congruence was tested using a likelihood ratio test (LRT) implemented in Concaterpillar 1.4 (Leigh et al. 2008). Concaterpillar performed per-genome maximum likelihood (ML) analyses on identical taxon sets (15 taxa) using the general time reversible (GTR) model implemented in RAxML 7.2.3 (Stamatakis 2006;Stamatakis et al. 2008) at$\alpha =0.05$ level.

Phylogenetic Analyses

The effects of mutational saturation at third codon positions in the 97-gene combined data set were investigated. Uncorrected pairwise distances for transitions and transversions among all taxa in the data set were plotted against the GTR model combined with a gamma distribution or with an inverse Gaussian distribution across invariant sites distances (Waddell and Steel 1997) to detect mutational saturation at combined positions of the first and second codons, as well as at third positions. Third codon position transitions displayed the strongest evidence of mutational saturation (Supplementary Information [SI]Fig. 1, available fromhttp://www.sysbio.oxfordjournals.org); thus, we performed phylogenetic analyses on amino acid sequences, combined first and second codon positions, and the third codon position for the 97-gene data set. In an attempt to reduce noise, phylogenetic analyses were performed by coding the nucleotides as purines and pyrimidines (RY coding) (Phillips et al. 2004) for these positions and the more extreme approach of removing all third codon position sites for data set B. Phylogenetic trees were reconstructed using ML (RAxML software, version 7.2.3) and Bayesian inference (BI) (MrBayes software, version 3.1.2;Huelsenbeck and Ronquist 2001). The best-scoring ML tree was inferred using the novel rapid BP algorithm and ML searches after conducting 1000 RAxML runs. Akaike information criterion (AIC) as calculated by MrModeltest software (Nylander 2004) was used to determine the most appropriate model for each of the 97 genes and for the full data matrix. In MrBayes 3.1.2, two independent sets of Markov chain Monte Carlo (MCMC) chains were run, each with three heated and one unheated chain for 10,000,000 generations. Trees were sampled every 1000 generations, and convergence was confirmed with the help of are we there yet? (AWTY) graphical analysis (Nylander et al. 2008). A consensus tree was obtained from BI trees for the 97-gene data set using themajrule command in Clann, version 3.0.0 (Creevey and McInerney 2005). Because standard Bayesian PP values can be very unreliable when an incorrect model is used, and nuclear mammalian genes show clear deviations in base composition and relative rates of substitution types from those of commonly employed models (Waddell et al. 2009), we also implemented gene partitions and specified a model for each gene included in the Bayesian analyses of the 97-gene data set. Additionally, approximately unbiased (AU) tests (Shimodaira 2002) were conducted in Consel version 0.1 (Shimodaira and Hasegawa 2001) between the best ML tree and alternative topologies to evaluate whether their likelihoods were significantly different. Kishino–Hasegawa (KH;Kishino and Hasegawa 1989) and Shimodaira–Hasegawa (SH) tests (Shimodaira and Hasegawa 1999) were performed to measure the congruence between single-gene trees.

Molecular Dating Analyses

Molecular dating analyses were conducted with BEAST v1.5.0 (Drummond and Rambaut 2007) using the 97 and 60 nuclear-gene data matrices, with a relaxed-clock MCMC approach under an uncorrelated lognormal model. In view of modern Bayesian methods that allow for the incorporation of a prior distribution of ages (“age constraints”) into these fossil calibrations, we chose prior age distributions so that the youngest age of the distribution corresponded to the youngest possible age of the fossil. We chose a standard deviation (SD) of the distribution so that 95% of the distribution fell within the geological time period of the fossil (i.e., 5% of the tail extended into older ages). Five fossils were used as calibration age constraints in the present study. The first calibration point is the age of the divergence between Hippopotamidae and Cetacea, which is at least Ypresian (Eocene: 55.8–48.6 Ma) in age based on the fossil cetaceanPakicetus (Thewissen et al. 2001). We chose a lognormal distribution so that the earliest possible sampled age corresponded to 48.6 Ma and the older 95% credible interval (CI) encompassed the beginning of the Ypresian (55.8 Ma) (SD = 1.2). Previous studies have also used this fossil as a calibration point, with a similar age and confidence interval (52 Ma with a confidence interval of 49–61 Ma) (Waddell et al. 2001,Waddell 2008). The second calibration point is the horse/rhino split, which has been updated to 58 Ma byWaddell (2008). This divergence for fossil calibration data close to the age of placental intraordinal divergences (Waddell, Okada, and Hasegawa 1999) and SD was 1.5 (normal distribution). The age of the root of crown Laurasiatheria was the third calibration point, and it is based on the earliest record of Eulipotyphla, the late CretaceousOtlestes (Averianov and Archibald 2005,Prasad et al. 2007). We chose a lognormal distribution so that the earliest possible sampled age corresponded to 93.5 Ma and the older 95% CI encompassed the beginning of the late Cretaceous (99.6 Ma) (SD = 1.099). The phylogenetic affiliation ofOtlestes is controversial (Archibald 2003), so we also performed an analysis in which the third calibration point was replaced by a calibration based on the origin of Rodentia. To do this, we used the 60-gene data set in which additional taxa (guinea pig, rabbit, and squirrel) are included. It appears that Rodentia most likely evolved in Asia and moved into North America, which yields an estimate of 60 Ma (SD = 1.5, normal distribution) (Waddell 2008). Each BEAST analysis consisted of$2\times 10^7$ generations with a random starting tree, birth–death default priors (with the exception that we used a uniform [0, 100] prior distribution for the GTR substitution rates), and sampled every 1000 generations. Cumulative PP plots for each clade were constructed to determine convergence with the help of AWTY (Nylander et al. 2008), and PPs ≥0.95 were considered to indicate strong support for a clade (Huelsenbeck and Rannala 2004).

TABLE 2.

TABLE 3.

Eulipotyphla: The Basal Branch of Laurasiatheria?

Resolution of the phylogenetic relationship among basal clades of laurasiatherian mammals is essential for addressing many important questions concerning the diversification and evolution of placental mammals. Despite the numerous, intensive phylogenetic studies (Onuma et al. 1998,Waddell, Okada, and Hasegawa 1999,Mouchaty et al. 2000,Madsen et al. 2001,Murphy, Eizirik, Johnson, et al. 2001,Murphy, Eizirik, O'Brien, et al. 2001,Narita et al. 2001,Waddell et al. 2001,Kriegs et al. 2006,Nishihara et al. 2006), considerable disagreement still persists for basal laurasiatherian relationships. The present phylogenetic analyses provide strong support for the position of Eulipotyphla as the most basal branch of Laurasiatheria and thus the sister group to the remaining extant orders (Waddell et al. 2001) (Fig. 2; SIFig. 2c). Resolution of this issue is clearly the result of the addition of more genes as compared with previous studies. For example,Narita et al. (2001) conducted maximum parsimony MP) and ML analyses with two cDNA sequences, which supported the Eulipotyphla + Chiroptera basal hypothesis. In the present study, trees derived from the BI and ML analyses of first and second codon positions from 97 genes (data set A) placed Eulipotyphla alone as the sister group to the remaining laurasiatherian mammals, whereas analyses that used RY coding supported the hypothesis of a basal position for Eulipotyphla + Chiroptera (data not shown). However, trees reconstructed by BI and ML methods for the 1608-gene data set fully support (100%/1.00/100%) a basal position for Eulipotyphla and a more apical position for Chiroptera. This result was obtained when first and the second codon positions are analyzed, as well as when RY coding analyses were employed. (Fig. 2f). In addition, an AU test rejected the Eulipotyphla + Chiroptera topology with statistical significance (Table 2), which further supports our preferred phylogenetic hypothesis (Fig. 2a–f).

Phylogenetic Relationships among OtherLaurasiatherian Orders

Despite extensive phylogenetic analyses of placental, phylogenetic relationships, especially within Scrotif-era (Waddell, Cao, Hauf, and Hasegawa 1999; Chiroptera + Pholidota + Carnivora + Perissodactyla + Cetartiodactyla), remain controversial. Morphological and molecular evidence supports several alternative positions for Chiroptera (Pettigrew 1986;Novacek 1992,Pumo et al. 1998,Shoshani and McKenna 1998,Nikaido et al. 2000), and as a consequence, a systematic assignment of this order was lacking in the most recently published review of Laurasiatheria (Nishihara et al. 2006). In the present study, Chiroptera was the sister group to Fereuungulata in almost all the ML and BI trees (except for the combined analyses of the 97-gene data set and RY coding, data not shown), which provides the strongest support so far for the placement of Chiroptera (Fig. 2; SIFig. 2). Furthermore, the monophyletic status of Chiroptera (megabats + microbats) also received high BP and PP support in analyses of data set B both at the nucleotide and at the amino acid levels (Fig. 2f; SIFig. 2c), which is consistent with previous studies on a large and diverse set of morphological features (Gunnell and Simmons 2005) as well as mitochondrial and nuclear nucleotide sequence data (Miyamoto 1996,Van den Bussche and Hoofer 2001). Interestingly, the nesting of bats within Fereuungulata was also recovered in some analyses of the present study. The 97- and 82-gene data sets, when analyzed with RY coding, suggest that Chiroptera + Eulipotyphla is the sister clade of Cetartiodactyla + Ferae, whereas MP analysis of first and second codon positions from data set B support Chiroptera as the sister group of Cetartiodactyla (data not shown). These analytical results disagree with some previous studies, which identified a “([(Perissodactyla, Carnivora), Chiroptera], Cetartiodactyla)” or “([(Perissodactyla, Artiodactyla), Chiroptera], Carnivora)” topology (Nishihara et al. 2006,Prasad et al. 2008) but agree to some extent with the finding of Hou et al. that a cow + bat clade was the sister group of a horse + dog clade (Hou et al. 2009; also found inHallström and Janke 2010). Considering the weak BP support and very narrow divergence times among Scrotifera lineages, we attribute little significance to these new arrangements and suspect that these patterns are more reflective of incomplete lineage sorting.

As described in the introduction, previous morphological and molecular studies have come to very different placements for the laurasiatherian order Perissodactyla.Waddell, Okada, and Hasegawa (1999) suggested a sister-group relationship between Perissodactyla and Carnivora, which together form the clade Zooamata. This hypothesis received support from subsequent analyses of 19 nuclear and 3 mitochondrial genes (Murphy, Eizirik, O'Brien, et al. 2001). In contrast, the present phylogenetic analyses provides strong support for the placement of Perissodactyla as the sister group to Cetartiodactyla (Fig. 2), and the AU test fully rejected alternative topologies (Table 2). Specifically, both BI and ML analyses of first and second codon positions from data sets A and B as well as RY coding of data set B all support a close phylogenetic relationship between Perissodactyla and Cetartiodactyla (Fig. 2). The AU test combined with the first and second codon positions of the data set B rejected a sister-group relationship between Cetartiodactyla and Carnivora (Prasad et al. 2008,Figure 1;Fig. 2) at a significance level of 0.1%, whereas no significant difference was found between two other alternative placements of Perissodactyla, as shown inFig. 1 (Waddell, Okada, and Hasegawa 1999,Waddell et al. 2001). In addition, when applied to data set A, the AU test rejected a sister-group relationship between Perissodactyla and Carnivora + Pholidota (an arrangement similar to that ofWaddell, Okada, and Hasegawa 1999) at 0.05 significance level (Table 2). A recent study (Hou et al. 2009) based on 2705 protein-coding genes (∼40 Mb) from the dog, cow, and horse also favored a close affinity between the cow and horse, although they were not able to reject a horse + dog sister-group relationship. Thus, this recent study provides further evidence to support Euungulata (or Cetungulata), which is also our preferred hypothesis.

Although retroposon analyses (Schwartz et al. 2003,Nishihara et al. 2006) discovered many L1 loci that occur in Carnivora and Perissodactyla but not in Cetartiodactyla (cow or/and pig), we consider it premature to conclude that Zooamata and Pegasoferae are monophyletic because it remains unclear whether the L1 loci are present or absent in the orthologs of Pholidota (pangolins) or other important cetartiodactyl lineages, such as whales and dolphins. It is noteworthy that one locus (INT 283) isolated byNishihara et al. (2006) supports the monophyly of Carnivora, Perissodactyla, and Cetartiodactyla, which is inconsistent with their preferred hypothesis, which includes monophyly of Pegasoferae. As suggested byShedlock et al. (2004), such inconsistency can result when species diverged over a short evolutionary time span and there is incomplete lineage sorting of ancestral polymorphisms. Considering the very short duration over which the divergence of main laurasiatherian lineages occurred (Table 3), extensive inconsistencies among retroposon insertions are a distinct possibility. If so, then it maybe difficult to use retroposon insertions to infer the phylogeny within Laurasiatheria as more lineages are sampled, similar to the problems encountered in a study on the phylogeny of baleen whales (Nikaido et al. 2006). Many efforts have been made to resolve conflict among retropsoson insertions, presumably as a result of incomplete lineage sorting of polymorphisms. For example,Waddell et al. (2001) have introduced a method that uses likelihood statistics to test the validity of a superordinal clade whose monophyly is based on retroposon data. However, whether this kind of method is suitable for rapid divergences has not been established.

Based on morphological data, some systematists (e.g.,Novacek 1992) have allied Pholidota with Xenarthra (e.g., armadillo and sloth) at the base of the eutherian tree. However, many other systematists have challenged this proposal and instead have advocated a sister-group relationship between Pholidota and Carnivora (Shoshani and McKenna 1998) based on morphological data, with Perissodactyla as the sister group of this clade (Waddell, Okada, and Hasegawa 1999,Murphy, Eizirik, O'Brien, et al. 2001) based on molecular data.Arnason et al. (2002) also suggested that the definition of Cetferungulata (which is in fact a synonym of Fereuungulata) be modified to include Pholidota. However, a topology with Pholidota as basal to other cetferungulates could not be statistically rejected byArnason et al. (2002). In the present study, we demonstrated strong support for a sister-group relationship between Pholidota and Carnivora, regardless of partitioning strategy or phylogenetic optimality criterion for the 97-, 67-, and 82-gene data sets. This was corroborated with an AU test, which rejected Pholidota as being basal to the remaining fereuungulates at a significance level of 0.1% (Table 2).

Relationships between Major Clades of Cetartiodactyla

Relationships among cetartiodactyl clades have been the focus of numerous phylogenetic studies during the past decade (reviewed inGatesy et al. 2002). A consensus has been reached on the placement and taxonomic content of some clades, and our results are strongly congruent with this consensus (Fig. 2; SIFig. 1a,b). In addition, our analyses provide resolution to some controversial issues in cetartiodactyl relationships.

The paraphyly of conventional Artiodactyla (i.e., terrestrial artiodactyls) is widely supported by recent phylogenetic analyses of molecular data, regardless if supermatrix or supertree methods are employed (Shimamura et al. 1997,Nikaido et al. 1999,Madsen et al. 2001,Murphy, Eizirik, Johnson, et al. 2001,Beck et al. 2006). Although morphological data now weakly support paraphyly of Artiodactyla as well, they continue to support a close relationship between Ruminantia and Camelidae (e.g.,Geisler and Uhen 2003). By contrast, molecular data support a sister relationship between Ruminantia and Whippomorpha (Waddell, Okada, and Hasegawa 1999; Cetacea + Hippopotamidae) (Nikaido et al. 1999,Matthee et al. 2001). The present phylogenomic analysis strongly supports cetaceans nested within artiodactyls as follows: (Camelidae, [Suina, (Ruminantia, Whippomorpha)]). This arrangement has also been supported by recent studies that combine morphological and molecular data (O'Leary and Gatesy 2008,Geisler and Theodor 2009,Spaulding et al. 2009). To corroborate the monophyly of Cetartiodactyla, we sequenced two cetartiodactylan exons (NFE2L2 exon 11 and PTPN22 exon 13) and then aligned them with the other laurasiatherian species in our data sets. The alignments indicated that there were several single amino acid insertion/deletion events specific to Cetartiodactyla (SIFig. 2). In addition, we found single and triple amino acid insertion and deletion events in NPAS4 exon 7 that suggest a close relationship between Whippomorpha and Ruminantia (Ursing and Arnason 1998) (SIFig. 2).

Phylogenetic placement of Suina and Camelidae is the sole unresolved problem regarding the basal relationships within Cetartiodactyla. Their positions have varied among different molecular studies of mitochondrial and/or nuclear genes (Ursing and Arnason 1998,Gatesy et al. 1999,Nikaido et al. 1999,Arnason et al. 2000,Murphy, Eizirik, Johnson, et al. 2001). Here, as mentioned above, the Camelidae was placed as the basal member of Cetartiodactyla, and an AU test rejected a basal position of Suina at a 5% level of significance. However, our AU test did not reject the basal placement of Suina + Camelidae that was supported byArnason et al. (2000).

Rapid Diversification of Laurasiatherian Lineages

Three models, that is, explosive, long fuse, and short fuse, have been suggested for placental mammal diversification, with a focus on the timing of the placental mammal radiation relative to the Cretaceous–Paleogene (Tertiary) boundary (Springer et al. 2003). Our study generally supports the long fuse model, placing most interordinal divergences of laurasiatherian mammals in the Cretaceous and intraordinal divergences in the Cenozoic. In particular, the divergence times we estimated using the first and second codon positions of two of our data sets (97- and 60-gene) suggest that interordinal splits of laurasiatherian mammals were concentrated in the Cretaceous, whereas basal cladogenesis within most of the orders, such as Perissodactyla and Cetartiodactyla, was in the Cenozoic (Table 3). Similar conclusions have been reached by several previous studies (Waddell, Okada, and Hasegawa 1999,Cao et al. 2000,Waddell et al. 2001,Douady and Douzery 2003,Springer et al. 2003,Kitazoe et al. 2007,Arnason et al. 2008). Moreover, late Cretaceous zhelestids, which may or may not be near the root of fereuungulates (Archibald 1996,Waddell, Cao, Hauf, and Hasegawa 1999,Wible et al. 2007), combined with the discovery of a possible late Cretaceous ungulate mammal (Kharmerungulatum vanvaleni) (Prasad et al. 2007), coupled with endemic perissodactyls (cambaytherids) and artiodactyls (raoellids) in the Early or Middle Eocene (55–50 Ma) rocks of India (Bajpai et al. 2005,Thewissen et al. 2007,Prasad 2009), suggest that ungulate-like characters evolved on multiple occasions and that “ungulates” diversified in the Cretaceous and early Palaeocene. However, as noted elsewhere (Benton and Donoghue 2007), most orders within Laurasiatheria do not have fossil records earlier than Eocene or Paleocene. Although a number of hypotheses have been advanced to explain this discrepancy, final resolution will only come when phylogenetic methods for converting sequence data to relative time on a clock-like tree get improved as increased accuracy of fossil calibration points (Waddell et al. 2001). And to improve the latter, if the molecular divergence estimates for laurasiatherian mammals are compatible with the long fuse model, intensive paleontological exploration should be conducted on Gondwanan continents, particularly in Africa and India.

Regardless of the absolute divergence time of each clade, our Bayesian relaxed clock analyses suggest a spread of 20 My from the emergence of Laurasiatheria to the emergence of Ferae, which is inconsistent with the short branches in phylogenetic trees (Fig. 2 andTable 3). This discrepancy may be caused by the model used in the BEAST software, which cannot fully adjust for a sharp rate slowdown right after a calibration point, as discussed byKitazoe et al. (2007). By contrast, branch lengths and our molecular clock analyses do agree that Fereuungulata diversified rapidly, probably over just a few million years (Fig. 2). In addition, most first fossil occurrences of laurasiatherian orders occur in the early Paleocene and early Eocene of Asia and/or North America, which is further evidence for the rapid diversification of fereuungulates. For this reason, the relationships among these clades were difficult to resolve with high BP support in the previous studies, even with the introduction of phylogenomic data. This persistent difficulty in resolving the phylogeny of Fereuungulata could be caused by their genomes being mosaics of conflicting genealogies that resulted from rapid speciation and incomplete lineage sorting (Pollard et al. 2006,Hallström and Janke 2010).

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