Retroposed elements as archives for the evolutionary history of placental mammals

Abstract

Reconstruction of the placental mammalian (eutherian) evolutionary tree has undergone diverse revisions, and numerous aspects remain hotly debated. Initial hierarchical divisions based on morphology contained many misgroupings due to features that evolved independently by similar selection processes. Molecular analyses corrected many of these misgroupings and the superordinal hierarchy of placental mammals was recently assembled into four clades. However, long or rapid evolutionary periods, as well as directional mutation pressure, can produce molecular homoplasies, similar characteristics lacking common ancestors. Retroposed elements, by contrast, integrate randomly into genomes with negligible probabilities of the same element integrating independently into orthologous positions in different species. Thus, presence/absence analyses of these elements are a superior strategy for molecular systematics. By computationally scanning more than 160,000 chromosomal loci and judiciously selecting from only phylogenetically informative retroposons for experimental high-throughput PCR applications, we recovered 28 clear, independent monophyly markers that conclusively verify the earliest divergences in placental mammalian evolution. Using tests that take into account ancestral polymorphisms, multiple long interspersed elements and long terminal repeat element insertions provide highly significant evidence for the monophyletic clades Boreotheria (synonymous with Boreoeutheria), Supraprimates (synonymous with Euarchontoglires), and Laurasiatheria. More importantly, two retropositions provide new support for a prior scenario of early mammalian evolution that places the basal placental divergence between Xenarthra and Epitheria, the latter comprising all remaining placentals. Due to its virtually homoplasy-free nature, the analysis of retroposon presence/absence patterns avoids the pitfalls of other molecular methodologies and provides a rapid, unequivocal means for revealing the evolutionary history of organisms.

Figure 1. Two Examples of Presence/Absence Analyses

(A) Genomic PCR fragments. The L1MB3 element is present in all boreotherian species. The element is located between exon 20 and 21 of the human AP4E1 gene on human Chromosome 15 (q21.2). Small-size variations are due to random indels. The larger fragment for human is due to an additional insertion of an Alu Sx element. Smaller fragments in afrotherians and xenarthrans indicate the absence situation prior to insertion of the element (plesiomorph condition). (B) A schematic representation of the presence/absence loci of various taxa after sequence determination. Direct repeats and the unoccupied target sites are shaded gray. (C) A phylogenetic interpretation of the presence/absence pattern. The L1MB3 element is present (+) in representatives of boreotherians and absent (−) in afrotherians and xenarthrans. The ball indicates the integration time of the L1MB3 element prior to the common ancestor of all recent boreotherians, but after this lineage separated from other placentals. The relative time of this integration is represented by node 3 in Figure 2; ten other integrations confirm the boreotherian hypothesis. (D) Genomic PCR fragments. The L1MB5 element, in addition to its presence in all boreotherian species is also found in the afrotherian species. The smaller fragments in xenarthrans indicate its absence in these species. Its integration site corresponds to the human locus on Chromosome 15 (q23). (E) A schematic representation of the presence/absence loci of various taxa after sequence determination. Direct repeats and the unoccupied target sites are shaded gray. (F) The L1MB5 element is present (+) in representatives of boreotherians and afrotherians, grouping them in the clade Epitheria, and is absent (−) in xenarthrans. The ball indicates the integration time of the L1MB5 element prior to the common ancestor of all Epitheria, but after this lineage separated from other placentals. This integration time is the same as node 2 in Figure 2, and we have so far recovered one additional retroposon integration to support the Epitheria hypothesis. DR, direct repeats.

Figure 2. Positions of Retroposed Elements as Landmarks of Evolution on the Bayesian-Based Placental Evolutionary Tree from Murphy et al. [ 2]

The resultant tree is consistent with previous studies [ 1, 2, 4, 5, 7, 8, 10, 38, 39] in most aspects. Note that the positions of afrotherians and xenarthras have been reversed, based on the presence of two retroposon insertions at node 2. Gray balls represent single insertion events. Supported splitting points are labeled with Arabic numerals. Superordinal clades, in the order shown, were established by Waddell et al. [ 6] and supported by several major studies [ 1, 2, 7, 8], and are labeled with Roman numerals. The taxa shown represent only those from which we sampled LINEs and LTRs. Dotted lines indicate nodes in need of further confirmation. Asterisks represent retroposon evidence from the literature for monophyly of Afrotheria [ 27], Primates [ 18], Rodentia [ 45], and Cetartiodactyla [ 26].

Figure 3. Representative Alignments of the Presence/Absence Regions Indicating Support for the Five Investigated Evolutionary Divergences

Potential direct repeats are boxed. The 5′ and 3′ ends of the retroposon insertions are partially shown in lower case letters on a gray background. Node designations corresponding to Figure 2 and the names of the supported monophyletic groups are given above the inserted elements.