Summary of Laurasiatheria (Mammalia) Phylogeny

: Laurasiatheria is one of the richest and most diverse superorders of placental mammals. Because this group had a rapid evolutionary radiation, the phylogenetic relationships among the six orders of Laurasiatheria remain a subject of heated debate and several issues related to its phylogeny remain open. Reconstructing the true phylogenetic relationships of Laurasiatheria is a significant case study in evolutionary biology due to the diversity of this suborder and such research will have significant implications for biodiversity conservation. We review the higher-level (inter-ordinal) phylogenies of Laurasiatheria based on previous cytogenetic, morphological and molecular data, and discuss the controversies of its phylogenetic relationship. This review aims to outline future researches on Laurasiatheria phylogeny and adaptive evolution.

As a mammalian group bearing important evolutionary significance and conservation value, Laurasiatheria has long been a focus of researches.
Similar to the karyotype studies, the earliest divergence of Eulipotyphla and the close association between Carnivora and Pholidota were supported. Regarding the phylogenetic placements of the other three orders, however, morphological studies demonstrated more resolutions and debates than the karyotype studies.

Laurasiatheria phylogeny inferred from molecular evidence Phylogenetic estimate from mitochondrial genes
Due to relatively small effective population size and lack of recombination as well as easy obtainment, mitochondrial DNA (mtDNA) has often been chosen as a preferred molecular marker in early molecular phylogenetics (Bargelloni et al, 2000;Brito & Edwards, 2009;Phillips & Penny, 2003;Sánchez-Gracia & Castresana, 2012;Springer et al, 2001;Sturmbauer & Meyer, 1993;Yu et al, 2007), Pumo et al (1998) analyzed 12 complete mt genome sequences of five Laurasiatheria orders, placing Chiroptera as the sister group of Cetartiodactyla, Perissodactyla and Carnivora, while positioning Eulipotyphla as the most basal order of placental mammals, thus rejecting Laurasiatheria monophyly. Cao et al (1998) investigated the mt genome sequences of three orders, placing Cetartiodactyla as the sister group of Perissodactyla and Carnivora. By summarizing the results proposed in the "International Symposium on the Origin of Mammalian Orders", Waddell et al (1999b) suggested the Laurasiatheria phylogeny as (Eulipotyphla, (Chiroptera, (Cetartiodactyla, (Perissodactyla, (Carnivora, Pholidota))))) ( Figure 1B, Table 1). These relationships are consistent with those from the morphological study of Wildman et al (2006). Moreover, these relationships are also supported by later analysis of 13 important rare genomic changes (RGCs) (Springer et al, 2004). By adding the mtDNA genome sequences of other orders and by using different tree-building schemes, later studies obtained the same phylogenetic relationships as those from Waddell et al (1999b) and Wildman et al (2006) Cao et al, 2000;Gibson et al, 2005;Kern & Kondrashov, 2004;Kjer & Honeycutt, 2007;Lin et al, 2002;Reyes et al, 2004) ( Figure 1B, Table 1) with the exception of Eulipotyphla positioned as the most basal order of placental mammals, and Cetartiodactyla as the sister group of Perissodactyla and Carnivora (Waddell et al, 1999a).

Phylogenetic estimate from nuclear genes
Although analyses of mtDNA sequences have provided insights into the phylogeny and evolution of Laurasiatheria, the fact that all genes comprising mt genome are inherited as a single, haploid linkage unit is a well-known limitation on phylogenetic reconstruction because the resulting mt gene trees are unlikely to reflect one independent estimate of the species tree (Giannasi et al, 2001;Johnson & Clayton, 2000;Moore, 1995;Page, 2000;Yu et al, 2004;Yu & Zhang, 2006a). Therefore, when much longer nuclear DNA sequences become widely available, nuclear genes received more and more attention in molecular phylogenetic studies, and demonstrated superiority in resolving deep phylogenies (Springer et al, 1999. Since 2001, a series of Laurasiatheria studies based on analyses of nuclear genes made important contributions to the resolution of relationships among Laurasiatheria orders, entering into an unprecedented progress. Madsen et al (2001) first use three nuclear genes,combined with mt tRNA and rRNAs (~5 kb in total), to investigate Laurasiatheria relationships and proposed a tree topology of ((Carnivora, Perissodactyla), (Cetartiodactyla, (Pholidota, (Chiroptera, Eulipotyphla)))) ( Figure 1F, Table 1). When another nuclear gene (BRCA1) was added and the taxon of representatives was increased to test the stability of the resulting tree, the tree topology changed to (Eulipotyphla, ((Carnivora, Pholidota), (Chiroptera, (Cetartiodactyla, Perissodactyla)))) ( Figure  1G, Table 1), indicating that Laurasiatheria phylogeny varied with the genes and species used in the analyses.
Different from those protein-coding nuclear genes commonly used in previous studies, Matthee et al (2007) first utilize three nuclear introns (~6 kb) from 114 Laurasiatheria species to rebuild the relationships among the six orders. Their study supported the phylogeny of (Eulipotyphla, (Perissodactyla, (Cetartiodactyla, (Chiroptera, (Carnivora, Pholidota))))) ( Figure 1K, Table  1). However, these relationships were poorly supported. Asher (2007) reanalyzed 19 nuclear and three mt sequences data from Murphy et al (2001b) as well as 196 morphological characters from Asher et al (2003) (Table  1). By incorporating indels of sequence data, the study suggested that the phylogenetic relationships among the Laurasiatheria orders changed with the combinations of characters and the analytic methods.
Based on the above studies, Laurasiatheria phylogeny at the ordinal level remains an outstanding problem in mammalian systematics due to the contradicting conclusions reached under different datasets, especially in the case of the phylogenetic positions of Perissodactyla, Cetartiodactyla and Chiroptera.

Phylogenomics of Laurasiatheria
With the increasing availability of genomic data of more species, phylogenetic analysis, which use genomic data to infer evolutionary relationships, is entering a new era (Delsuc et al, 2005;Rokas & Chatzimanolis, 2008;Yu & Zhang, 2006a, b). By performing phylogenomic studies, a reliable phylogenetic tree can be reconstructed using many more characters than those in previous studies, including gene families, large insertion and deletion gene fragments, and gene rearrangement, etc. (Meslin et al, 2011;Wu et al, 2012;Yu et al, 2011). These characters might be useful for distinguishing nodes resulting from rapid radiation episodes such as the Laurasiatheria speciation events. Bashir et al (2005) attempted to reconstruct the Laurasiatheria phylogeny of four orders (Eulipotyphla, Cetartiodactyla, Perissodactyla, and Carnivora) based on more than 1 000 orthologous repetitive elements obtained from publically available genome sequence data. In their study, Perissodactyla and Carnivora were grouped together, whereas the placements of the other two orders remained unclear.
Based on 218 protein-coding genes (~200 kb) obtained from the analysis of 18 placental mammalian genomes, Nikolaev et al (2007) suggested that Eulipotyphla diverged first, followed by Chiroptera, and. Cetartiodactyla and Carnivora were sister-group ( Figure  1L, Table 1). Subsequently, by searching for informative coding indels within whole-genome sequence data and amplifying them in Laurasiatheria species, Murphy et al (2007) yielded a new tree topology as (Eulipotyphla, (Cetartiodactyla, ((Chiroptera, Perissodactyla), (Carnivora, Pholidota)))) ( Figure 1A, Table 1). The close relatedness of Chiroptera and Perissodactyla has been only recovered in karyotype research (Kulemzina et al, 2010). Prasad et al (2008) reconstructed the phylogeny of Laurasiatheria except for Pholidota based on 1.9 Mb gene regions. The protein-coding sequence analyses supported the tree as (Eulipotyphla, (Carnivora, (Chiroptera, (Perissodactyla, Cetartiodactyla)))) ( Figure  1N, Table 1), whereas the combination of coding and non-coding sequence analyses favored the tree as (Eulipotyphla, (Chiroptera, (Perissodactyla, (Carnivora, Cetartiodactyla)))) ( Figure 1O, Table 1), showing a lack of consistency in Laurasiatheria phylogeny under different kinds of characters used. The lack of consistency with such large amounts of data can be also evidenced from Hou et al (2009), in which 2705 orthologous genes (~40 Mb) from Cetartiodactyla, Perissodactyla, and Carnivora were used, and different tree topologies were produced under different treebuilding methods. When Chiroptera was added into the analysis, they found close relatedness of Perissodactyla with Carnivora, and that of Cetartiodactyla with Chiroptera ( Figure 1P, Table 1).
Hallström & Janke (2008) screened 3 012 genes (~280 kb) from four orders with available genome sequences (Eulipotyphla, Chiroptera, Carnivora, and Cetartiodactyla). Their results only recovered the basal placement of Eulipotyphla, and failed to resolve the relationships of the other three orders. The rapid radiation of Laurasiatheria within a narrow time scale was proposed to explain the irresolution using such large amounts of data. By utilizing the third condon position GC content (GC3) of 1 138 protein-coding orthologous genes (~300 kb), Romiguier et al (2010) revealed the Laurasiatheria phylogeny to be (Eulipotyphla, (Cetartiodactyla, (Chiroptera, (Perissodactyla, Carnivora)))) ( Figure 1M, Table 1).
Hallström et al (2011) analysed 4 775 proteincoding genes (~6 Mb) screened from the available genome sequences of five Laurasiatheria orders and determined the tree as (Eulipotyphla, (Chiroptera, (Cetartiodactyla, (Perissodactyla, Carnivora)))) ( Figure  1B, Table 1). Their results were more consistent with morphological and mt genome analyses as well as nuclear analyses of Murphy et al (2001b). However, Pholidota was not included in the analyses. When the retroposon insertions from these genome sequences were analysed, their study supported the earliest divergence of Eulipotyphla, but failed to resolve the relationships among the other four orders. Additionally, McCormack et al (2011) analysed 683 loci of ultraconserved elements, supporting the tree topology (Eulipotyphla, (Cetartiodactyla, (Chiroptera, (Perissodactyla, Carnivora)))), which was consistent with Nishihara et al (2006) (Figure 1M, Table  1).

Perspectives
The phylogeny of Laurasiatheria, which is characterized by rapid species radiations and short internal tree branches, has long been one of the most controversial and challenging problems in mammalian systematics. So far, only the earliest divergence of Eulipotyphla and the close relationship between Carnivora and Pholidota has been generally accepted. The main controversies are concentrated on the phylogenetic positions of the other three orders, i.e., Chiroptera, Cetartiodactyla and Perissodactyla.
Most current investigations of Laurasiatheria phylogeny at the ordinal level, especially phylogenomic studies, have been mainly conducted based on five orders with available genome sequences. The lack of Pholidota genome sequences in those studies may be one of the reasons causing inconsistent phylogenetic relationships (Bashir et al, 2005;Dos Reis et al, 2012;Hallström et al, 2011;Hou et al, 2009;McCormack et al, 2012;Nery et al, 2012;Nishihara et al, 2006;Prasad et al, 2008;Romiguier et al, 2010;Song et al, 2012), given that tree topology of phylogenetic trees can be biased by sampling errors, leading to an unreliable and unstable estimation. In addition, we note that previous understanding of Laurasiatheria phylogeny largely depended on the analyses of coding DNA sequences, with only a few studies concerning other characters. Therefore, in future work, more complete sampling of the Laurasiatheria species and of the various characters in use would be expected to clarify the phylogenetic puzzles in Laurasiatheria.