Incomplete Lineage Sorting and Hybridization Statistics for Large-Scale Retroposon Insertion Data

<div><p>Ancient retroposon insertions can be used as virtually homoplasy-free markers to reconstruct the phylogenetic history of species. Inherited, orthologous insertions in related species offer reliable signals of a common origin of the given species. One prerequisite for such a phylogenetically informative insertion is that the inserted element was fixed in the ancestral population before speciation; if not, polymorphically inserted elements may lead to random distributions of presence/absence states during speciation and possibly to apparently conflicting reconstructions of their ancestry. Fortunately, such misleading fixed cases are relatively rare but nevertheless, need to be considered. Here, we present novel, comprehensive statistical models applicable for (1) analyzing any pattern of rare genomic changes, (2) testing and differentiating conflicting phylogenetic reconstructions based on rare genomic changes caused by incomplete lineage sorting or/and ancestral hybridization, and (3) differentiating between search strategies involving genome information from one or several lineages. When the new statistics are applied, in non-conflicting cases a minimum of three elements present in both of two species and absent in a third group are considered significant support (p<0.05) for the branching of the third from the other two, if all three of the given species are screened equally for genome or experimental data. Five elements are necessary for significant support (p<0.05) if a diagnostic locus derived from only one of three species is screened, and no conflicting markers are detected. Most potentially conflicting patterns can be evaluated for their significance and ancestral hybridization can be distinguished from incomplete lineage sorting by considering symmetric or asymmetric distribution of rare genomic changes among possible tree configurations. Additionally, we provide an R-application to make the new KKSC insertion significance test available for the scientific community at <a href="http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/" target="_blank">http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/</a>.</p></div

Possible discrepancy between one- and two-sided (species) screenings.

<p>Screening for phylogenetic markers based on all possible tree topologies for three species A, B, C when only one reference genome A (a-c) or B (d-f) is available. The red lineage indicates the branches where markers can be detected. Screening from A reveals three markers. The two light red markers are artifacts from ancient incomplete lineage sorting (ILS) and the dark red marker is a phylogenetically informative marker. Screening from B reveals 11 markers with 8 markers supporting B plus C and one marker supporting A plus (B plus C). The two light red markers in (d) are the same detected from species A in (a). The correct topology is shown in tree (f). This correct tree would not be detectible by screening only from the genome of species A.</p

Schematic representation of all possible phylogenetic patterns.

<p>For the markers <i>n</i>₁—(AB)C, <i>n</i>_<i>2</i>—(AC)B, and <i>n</i>_<i>3</i>—(BC)A, their sum <i>n</i> is fixed (<i>n = n</i>_<i>1</i><i>+n</i>_<i>2</i><i>+n</i>_<i>3</i>). The triangle reflects all possible combinations of <i>n</i>₁, <i>n</i>_<i>2</i>, and <i>n</i>_<i>3</i>, whereby the values at the corners are (<i>n</i>_<i>1</i>:0:0), (0:<i>n</i>_<i>2</i>:0), and (0:0:<i>n</i>_<i>3</i>) (counterclockwise from the upper corner). The respective trees indicate supported tree configurations (<i>C-tree</i>, <i>A-tree</i>, and <i>B-tree</i>), red balls consolidate insertion support for the given branches. The grey scale arrowheads within the triangle indicate the statistically significant combinations of supporting tree configurations shown at the corners of the triangle; the darker the arrow the more significant support for the corresponding tree, the lighter the arrow the less support and the more the branching resembles a polytomy. The circular area at the center of the triangle denotes the <i>polytomy</i> zone (<i>ABC-tree</i>, where <i>n</i>_<i>1</i> = <i>n</i>_<i>2</i> = <i>n</i>_<i>3</i>). The trees on the outside edges of the central triangle indicate <i>hybridization</i> zones (<i>B-fusion</i>, <i>C-fusion</i>, and <i>A-fusion</i>, denoted as A(B)C hybridization (where <i>n</i>_<i>1</i>≥<i>n</i>_<i>2</i>, and <i>n</i>_<i>2</i><i>>n</i>_<i>3</i>), A(C)B hybridization (where <i>n</i>_<i>1</i>≥<i>n</i>_<i>3</i>, and <i>n</i>_<i>2</i><<i>n</i>₃), and B(A)C hybridization (where <i>n</i>_<i>2</i>≥<i>n</i>_<i>3</i>, and <i>n</i>_<i>1</i><<i>n</i>_<i>2</i>), respectively).</p

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

<p>The resultant tree is consistent with previous studies [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b001" target="_blank">1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b002" target="_blank">2</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b004" target="_blank">4</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b005" target="_blank">5</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b007" target="_blank">7</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b008" target="_blank">8</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b010" target="_blank">10</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b038" target="_blank">38</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b039" target="_blank">39</a>] 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. [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b006" target="_blank">6</a>] and supported by several major studies [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b001" target="_blank">1</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b002" target="_blank">2</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b007" target="_blank">7</a>, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b008" target="_blank">8</a>], 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 [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b027" target="_blank">27</a>], Primates [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b018" target="_blank">18</a>], Rodentia [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b045" target="_blank">45</a>], and Cetartiodactyla [<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-b026" target="_blank">26</a>]. </p

Two Examples of Presence/Absence Analyses

<div><p>(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 <i>AP4E1</i> 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 <i>Alu</i> Sx element. Smaller fragments in afrotherians and xenarthrans indicate the absence situation prior to insertion of the element (plesiomorph condition). </p> <p>(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.</p> <p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-g002" target="_blank">Figure 2</a>; ten other integrations confirm the boreotherian hypothesis. </p> <p>(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).</p> <p>(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.</p> <p>(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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-g002" target="_blank">Figure 2</a>, and we have so far recovered one additional retroposon integration to support the Epitheria hypothesis. </p> <p>DR, direct repeats.</p></div

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

<p>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 <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040091#pbio-0040091-g002" target="_blank">Figure 2</a> and the names of the supported monophyletic groups are given above the inserted elements. </p

Additional file 1: Tables S1–S4. of De-novo emergence of SINE retroposons during the early evolution of passerine birds

(PDF 312 kb

Additional file 3: Figures S1–S2. of De-novo emergence of SINE retroposons during the early evolution of passerine birds

(PDF 447 kb

Moller-Krull2007-MolBiolEvol.tre

Moller-Krull2007-MolBiolEvol.tr

Moller-Krull2007-MolBiolEvol.nex

Moller-Krull2007-MolBiolEvol.ne