A 4-lineage Statistical Suite to Evaluate the Support of Large-Scale Retrotransposon Insertion Data to Reconstruct Evolutionary Trees.

Retrophylogenomics makes use of genome-wide retrotransposon presence/absence insertion patterns to resolve questions in phylogeny and population genetics. In the genomics era, evaluating high-throughput data requires the associated development of appropriately powerful statistical tools. The currently used KKSC 3-lineage statistical test for estimating the significance of retrophylogenomic data is limited by the number of possible tree topologies it can assess in one step. To improve on this, we have extended the analysis to simultaneously compare four lineages, enabling us to evaluate ten distinct presence/absence insertion patterns for 26 possible tree topologies plus 129 trees with different incidences of hybridization or introgression. The new tool provides statistics for cases involving multiple ancestral hybridizations/introgressions, ancestral incomplete lineage sorting, bifurcation, and polytomy. The test is embedded in a user-friendly web R application (http://retrogenomics.uni-muenster.de:3838/hammlet/) and is available for use by the scientific community. [ancestral hybridization/introgression; ancestral incomplete lineage sorting (ILS); empirical distribution; KKSC-statistics; 4-lineage (4-LIN) insertion polymorphism; polytomy; retrophylogenomics.].

Euarchontoglires Challenged by Incomplete Lineage Sorting.

Euarchontoglires, once described as Supraprimates, comprise primates, colugos, tree shrews, rodents, and lagomorphs in a clade that evolved about 90 million years ago (mya) from a shared ancestor with Laurasiatheria. The rapid speciation of groups within Euarchontoglires, and the subsequent inherent incomplete marker fixation in ancestral lineages, led to challenged attempts at phylogenetic reconstructions, particularly for the phylogenetic position of tree shrews. To resolve this conundrum, we sampled genome-wide presence/absence patterns of transposed elements (TEs) from all representatives of Euarchontoglires. This specific marker system has the advantage that phylogenetic diagnostic characters can be extracted in a nearly unbiased fashion genome-wide from reference genomes. Their insertions are virtually free of homoplasy. We simultaneously employed two computational tools, the genome presence/absence compiler (GPAC) and 2-n-way, to find a maximum of diagnostic insertions from more than 3 million TE positions. From 361 extracted diagnostic TEs, 132 provide significant support for the current resolution of Primatomorpha (Primates plus Dermoptera), 94 support the union of Euarchonta (Primates, Dermoptera, plus Scandentia), and 135 marker insertion patterns support a variety of alternative phylogenetic scenarios. Thus, whole genome-level analysis and a virtually homoplasy-free marker system offer an opportunity to finally resolve the notorious phylogenetic challenges that nature produces in rapidly diversifying groups.

The multicomparative 2-n-way genome suite.

To effectively analyze the increasing amounts of available genomic data, improved comparative analytical tools that are accessible to and applicable by a broad scientific community are essential. We built the "2-n-way" software suite to provide a fundamental and innovative processing framework for revealing and comparing inserted elements among various genomes. The suite comprises two user-friendly web-based modules. The 2-way module generates pairwise whole-genome alignments of target and query species. The resulting genome coordinates of blocks (matching sequences) and gaps (missing sequences) from multiple 2-ways are then transferred to the n-way module and sorted into projects, in which user-defined coordinates from reference species are projected to the block/gap coordinates of orthologous loci in query species to provide comparative information about presence (blocks) or absence (gaps) patterns of targeted elements over many entire genomes and phylogroups. Thus, the 2-n-way software suite is ideal for performing multidirectional, non-ascertainment-biased screenings to extract all possible presence/absence data of user-relevant elements in orthologous sequences. To highlight its applicability and versatility, we used 2-n-way to expose approximately 100 lost introns in vertebrates, analyzed thousands of potential phylogenetically informative bat and whale retrotransposons, and novel human exons as well as thousands of human polymorphic retrotransposons.

The Volcano Rabbit in the Phylogenetic Network of Lagomorphs.

The order Lagomorpha unifies pikas (Ochotonidae) and the hares plus rabbits (Leporidae). Phylogenetic reconstructions of the species within Leporidae based on traditional morphological or molecular sequence data provide support for conflicting hypotheses. The retroposon presence/absence patterns analyzed in this study revealed strong support for the broadly accepted splitting of lagomorphs into ochotonids and leporids with Pronolagus as the first divergence in the leporid tree. Furthermore, the retroposon presence/absence patterns nested the rare volcano rabbit, Romerolagus diazi, within an unresolved network of deeper leporid relationships and provide the first homoplasy-free image of incomplete lineage sorting and/or ancestral hybridization/introgression in rapidly radiated Leporidae. At the same time, the strongest retroposon presence/absence signal supports the volcano rabbit as a separate branch between the Pronolagus junction and a unified cluster of the remaining leporids.

The Beaver's Phylogenetic Lineage Illuminated by Retroposon Reads.

Solving problematic phylogenetic relationships often requires high quality genome data. However, for many organisms such data are still not available. Among rodents, the phylogenetic position of the beaver has always attracted special interest. The arrangement of the beaver's masseter (jaw-closer) muscle once suggested a strong affinity to some sciurid rodents (e.g., squirrels), placing them in the Sciuromorpha suborder. Modern molecular data, however, suggested a closer relationship of beaver to the representatives of the mouse-related clade, but significant data from virtually homoplasy-free markers (for example retroposon insertions) for the exact position of the beaver have not been available. We derived a gross genome assembly from deposited genomic Illumina paired-end reads and extracted thousands of potential phylogenetically informative retroposon markers using the new bioinformatics coordinate extractor fastCOEX, enabling us to evaluate different hypotheses for the phylogenetic position of the beaver. Comparative results provided significant support for a clear relationship between beavers (Castoridae) and kangaroo rat-related species (Geomyoidea) (p < 0.0015, six markers, no conflicting data) within a significantly supported mouse-related clade (including Myodonta, Anomaluromorpha, and Castorimorpha) (p < 0.0015, six markers, no conflicting data).

Speciation network in Laurasiatheria: retrophylogenomic signals.

Rapid species radiation due to adaptive changes or occupation of new ecospaces challenges our understanding of ancestral speciation and the relationships of modern species. At the molecular level, rapid radiation with successive speciations over short time periods-too short to fix polymorphic alleles-is described as incomplete lineage sorting. Incomplete lineage sorting leads to random fixation of genetic markers and hence, random signals of relationships in phylogenetic reconstructions. The situation is further complicated when you consider that the genome is a mosaic of ancestral and modern incompletely sorted sequence blocks that leads to reconstructed affiliations to one or the other relative, depending on the fixation of their shared ancestral polymorphic alleles. The laurasiatherian relationships among Chiroptera, Perissodactyla, Cetartiodactyla, and Carnivora present a prime example for such enigmatic affiliations. We performed whole-genome screenings for phylogenetically diagnostic retrotransposon insertions involving the representatives bat (Chiroptera), horse (Perissodactyla), cow (Cetartiodactyla), and dog (Carnivora), and extracted among 162,000 preselected cases 102 virtually homoplasy-free, phylogenetically informative retroelements to draw a complete picture of the highly complex evolutionary relations within Laurasiatheria. All possible evolutionary scenarios received considerable retrotransposon support, leaving us with a network of affiliations. However, the Cetartiodactyla-Carnivora relationship as well as the basal position of Chiroptera and an ancestral laurasiatherian hybridization process did exhibit some very clear, distinct signals. The significant accordance of retrotransposon presence/absence patterns and flanking nucleotide changes suggest an important influence of mosaic genome structures in the reconstruction of species histories.

Genome sequence of the basal haplorrhine primate Tarsius syrichta reveals unusual insertions.

Tarsiers are phylogenetically located between the most basal strepsirrhines and the most derived anthropoid primates. While they share morphological features with both groups, they also possess uncommon primate characteristics, rendering their evolutionary history somewhat obscure. To investigate the molecular basis of such attributes, we present here a new genome assembly of the Philippine tarsier (Tarsius syrichta), and provide extended analyses of the genome and detailed history of transposable element insertion events. We describe the silencing of Alu monomers on the lineage leading to anthropoids, and recognize an unexpected abundance of long terminal repeat-derived and LINE1-mobilized transposed elements (Tarsius interspersed elements; TINEs). For the first time in mammals, we identify a complete mitochondrial genome insertion within the nuclear genome, then reveal tarsier-specific, positive gene selection and posit population size changes over time. The genomic resources and analyses presented here will aid efforts to more fully understand the ancient characteristics of primate genomes.

Genomic analysis reveals hidden biodiversity within colugos, the sister group to primates.

Colugos are among the most poorly studied mammals despite their centrality to resolving supraordinal primate relationships. Two described species of these gliding mammals are the sole living members of the order Dermoptera, distributed throughout Southeast Asia. We generated a draft genome sequence for a Sunda colugo and a Philippine colugo reference alignment, and used these to identify colugo-specific genetic changes that were enriched in sensory and musculoskeletal-related genes that likely underlie their nocturnal and gliding adaptations. Phylogenomic analysis and catalogs of rare genomic changes overwhelmingly support the contested hypothesis that colugos are the sister group to primates (Primatomorpha), to the exclusion of treeshrews. We captured ~140 kb of orthologous sequence data from colugo museum specimens sampled across their range and identified large genetic differences between many geographically isolated populations that may result in a >300% increase in the number of recognized colugo species. Our results identify conservation units to mitigate future losses of this enigmatic mammalian order.

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

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 http://retrogenomics.uni-muenster.de:3838/KKSC_significance_test/.

Exploring Massive Incomplete Lineage Sorting in Arctoids (Laurasiatheria, Carnivora).

Freed from the competition of large raptors, Paleocene carnivores could expand their newly acquired habitats in search of prey. Such changing conditions might have led to their successful distribution and rapid radiation. Today, molecular evolutionary biologists are faced, however, with the consequences of such accelerated adaptive radiations, because they led to sequential speciation more rapidly than phylogenetic markers could be fixed. The repercussions being that current genealogies based on such markers are incongruent with species trees.Our aim was to explore such conflicting phylogenetic zones of evolution during the early arctoid radiation, especially to distinguish diagnostic from misleading phylogenetic signals, and to examine other carnivore-related speciation events. We applied a combination of high-throughput computational strategies to screen carnivore and related genomes in silico for randomly inserted retroposed elements that we then used to identify inconsistent phylogenetic patterns in the Arctoidea group, which is well known for phylogenetic discordances.Our combined retrophylogenomic and in vitro wet lab approach detected hundreds of carnivore-specific insertions, many of them confirming well-established splits or identifying and solving conflicting species distributions. Our systematic genome-wide screens for Long INterspersed Elements detected homoplasy-free markers with insertion-specific truncation points that we used to distinguish phylogenetically informative markers from conflicting signals. The results were independently confirmed by phylogenetic diagnostic Short INterspersed Elements. As statistical analysis ruled out ancestral hybridization, these doubly verified but still conflicting patterns were statistically determined to be genomic remnants from a time of ancestral incomplete lineage sorting that especially accompanied large parts of Arctoidea evolution.

The genome of the vervet (Chlorocebus aethiops sabaeus).

We describe a genome reference of the African green monkey or vervet (Chlorocebus aethiops). This member of the Old World monkey (OWM) superfamily is uniquely valuable for genetic investigations of simian immunodeficiency virus (SIV), for which it is the most abundant natural host species, and of a wide range of health-related phenotypes assessed in Caribbean vervets (C. a. sabaeus), whose numbers have expanded dramatically since Europeans introduced small numbers of their ancestors from West Africa during the colonial era. We use the reference to characterize the genomic relationship between vervets and other primates, the intra-generic phylogeny of vervet subspecies, and genome-wide structural variations of a pedigreed C. a. sabaeus population. Through comparative analyses with human and rhesus macaque, we characterize at high resolution the unique chromosomal fission events that differentiate the vervets and their close relatives from most other catarrhine primates, in whom karyotype is highly conserved. We also provide a summary of transposable elements and contrast these with the rhesus macaque and human. Analysis of sequenced genomes representing each of the main vervet subspecies supports previously hypothesized relationships between these populations, which range across most of sub-Saharan Africa, while uncovering high levels of genetic diversity within each. Sequence-based analyses of major histocompatibility complex (MHC) polymorphisms reveal extremely low diversity in Caribbean C. a. sabaeus vervets, compared to vervets from putatively ancestral West African regions. In the C. a. sabaeus research population, we discover the first structural variations that are, in some cases, predicted to have a deleterious effect; future studies will determine the phenotypic impact of these variations.

Ancient traces of tailless retropseudogenes in therian genomes.

Transposable elements, once described by Barbara McClintock as controlling genetic units, not only occupy the largest part of our genome but are also a prominent moving force of genomic plasticity and innovation. They usually replicate and reintegrate into genomes silently, sometimes causing malfunctions or misregulations, but occasionally millions of years later, a few may evolve into new functional units. Retrotransposons make their way into the genome following reverse transcription of RNA molecules and chromosomal insertion. In therian mammals, long interspersed elements 1 (LINE1s) self-propagate but also coretropose many RNAs, including mRNAs and small RNAs that usually exhibit an oligo(A) tail. The revitalization of specific LINE1 elements in the mammalian lineage about 150 Ma parallels the rise of many other nonautonomous mobilized genomic elements. We previously identified and described hundreds of tRNA-derived retropseudogenes missing characteristic oligo(A) tails consequently termed tailless retropseudogenes. Additional analyses now revealed hundreds of thousands of tailless retropseudogenes derived from nearly all types of RNAs. We extracted 2,402 perfect tailless sequences (with discernible flanking target site duplications) originating from tRNAs, spliceosomal RNAs, 5S rRNAs, 7SK RNAs, mRNAs, and others. Interestingly, all are truncated at one or more defined positions that coincide with internal single-stranded regions. 5S ribosomal and U2 spliceosomal RNAs were analyzed in the context of mammalian phylogeny to discern the origin of the therian LINE1 retropositional system that evolved in our 150-Myr-old ancestor.

GPAC-genome presence/absence compiler: a web application to comparatively visualize multiple genome-level changes.

Our understanding of genome-wide and comparative sequence information has been broadened considerably by the databases available from the University of California Santa Cruz (UCSC) Genome Bioinformatics Department. In particular, the identification and visualization of genomic sequences, present in some species but absent in others, led to fundamental insights into gene and genome evolution. However, the UCSC tools currently enable one to visualize orthologous genomic loci for a range of species in only a single locus. For large-scale comparative analyses of such presence/absence patterns a multilocus view would be more desirable. Such a tool would enable us to compare thousands of relevant loci simultaneously and to resolve many different questions about, for example, phylogeny, specific aspects of genome and gene evolution, such as the gain or loss of exons and introns, the emergence of novel transposed elements, nonprotein-coding RNAs, and viral genomic particles. Here, we present the first tool to facilitate the parallel analysis of thousands of genomic loci for cross-species presence/absence patterns based on multiway genome alignments. This genome presence/absence compiler uses annotated or other compilations of coordinates of genomic locations and compiles all presence/absence patterns in a flexible, color-coded table linked to the individual UCSC Genome Browser alignments. We provide examples of the versatile information content of such a screening system especially for 7SL-derived transposed elements, nuclear mitochondrial DNA, DNA transposons, and miRNAs in primates (http://www.bioinformatics.uni-muenster.de/tools/gpac, last accessed October 1, 2014).

Multiple lineages of ancient CR1 retroposons shaped the early genome evolution of amniotes.

Chicken repeat 1 (CR1) retroposons are long interspersed elements (LINEs) that are ubiquitous within amniote genomes and constitute the most abundant family of transposed elements in birds, crocodilians, turtles, and snakes. They are also present in mammalian genomes, where they reside as numerous relics of ancient retroposition events. Yet, despite their relevance for understanding amniote genome evolution, the diversity and evolution of CR1 elements has never been studied on an amniote-wide level. We reconstruct the temporal and quantitative activity of CR1 subfamilies via presence/absence analyses across crocodilian phylogeny and comparative analyses of 12 crocodilian genomes, revealing relative genomic stasis of retroposition during genome evolution of extant Crocodylia. Our large-scale phylogenetic analysis of amniote CR1 subfamilies suggests the presence of at least seven ancient CR1 lineages in the amniote ancestor; and amniote-wide analyses of CR1 successions and quantities reveal differential retention (presence of ancient relics or recent activity) of these CR1 lineages across amniote genome evolution. Interestingly, birds and lepidosaurs retained the fewest ancient CR1 lineages among amniotes and also exhibit smaller genome sizes. Our study is the first to analyze CR1 evolution in a genome-wide and amniote-wide context and the data strongly suggest that the ancestral amniote genome contained myriad CR1 elements from multiple ancient lineages, and remnants of these are still detectable in the relatively stable genomes of crocodilians and turtles. Early mammalian genome evolution was thus characterized by a drastic shift from CR1 prevalence to dominance and hyperactivity of L2 LINEs in monotremes and L1 LINEs in therians.

Retrophylogenomics place tarsiers on the evolutionary branch of anthropoids.

One of the most disputed issues in primate evolution and thus of our own primate roots, is the phylogenetic position of the Southeast Asian tarsier. While much molecular data indicate a basal place in the primate tree shared with strepsirrhines (prosimian monophyly hypothesis), data also exist supporting either an earlier divergence in primates (tarsier-first hypothesis) or a close relationship with anthropoid primates (Haplorrhini hypothesis). The use of retroposon insertions embedded in the Tarsius genome afforded us the unique opportunity to directly test all three hypotheses via three pairwise genome alignments. From millions of retroposons, we found 104 perfect orthologous insertions in both tarsiers and anthropoids to the exclusion of strepsirrhines, providing conflict-free evidence for the Haplorrhini hypothesis, and none supporting either of the other two positions. Thus, tarsiers are clearly the sister group to anthropoids in the clade Haplorrhini.

Ancestry of the Australian termitivorous numbat.

The Australian numbat, Myrmecobius fasciatus, is the only marsupial that feeds almost exclusively on termites and that has a life following the diurnally restricted and dynamic geographical distribution of termites. The millions of years of this adaptation led to unique morphological and anatomical features, especially basicranial and dental characteristics, that make it difficult to identify a clear phylogenetic affiliation to other marsupials. From DNA sequence analyses, the family Myrmecobiidae is placed within the dasyuromorph marsupials, but the exact position varies from study to study, and support values are mostly rather modest. Here, we report the recovery and analysis of approximately 110,000 quasifossilized traces of mobile element insertions into the genome of a dasyurid marsupial (Tasmanian devil), 25 of which are phylogenetically informative for early dasyuromorphial evolution. Fourteen of these ancient retroposon insertions are shared by the 16 Dasyuromorphia species analyzed, including the numbat, but are absent in the outgroups. An additional 11 other insertions are present in all Dasyuridae but are absent in the numbat. These findings place numbats as the sister group to all living Dasyuridae and show that the investigated Dasyuromorphia, including the Myrmecobiidae, constitutes a monophyletic group that is separated from Peramelemorphia, Notoryctemorphia, and other marsupials.

Retroposon insertion patterns of neoavian birds: strong evidence for an extensive incomplete lineage sorting era.

More than 150 Ma, the avian lineage separated from that of other dinosaurs and later diversified into the more than 10,000 species extant today. The early neoavian bird radiations most likely occurred in the late Cretaceous (more than 65 Ma) but left behind few if any molecular signals of their archaic evolutionary past. Retroposed elements, once established in an ancestral population, are highly valuable, virtually homoplasy-free markers of species evolution; after applying stringent orthology criteria, their phylogenetically informative presence/absence patterns are free of random noise and independent of evolutionary rate or nucleotide composition effects. We screened for early neoavian orthologous retroposon insertions and identified six markers with conflicting presence/absence patterns, whereas six additional retroposons established before or after the presumed major neoavian radiation show consistent phylogenetic patterns. The exceptionally frequent conflicting retroposon presence/absence patterns of neoavian orders are strong indicators of an extensive incomplete lineage sorting era, potentially induced by an early rapid successive speciation of ancestral Neoaves.

Mesozoic retroposons reveal parrots as the closest living relatives of passerine birds.

The relationships of passerines (such as the well-studied zebra finch) with non-passerine birds is one of the great enigmas of avian phylogenetic research, because decades of extensive morphological and molecular studies yielded highly inconsistent results between and within data sets. Here we show the first application of the virtually homoplasy-free retroposon insertions to this controversy. Our study examined ~200,000 retroposon-containing loci from various avian genomes and retrieved 51 markers resolving early bird phylogeny. Among these, we obtained statistically significant evidence that parrots are the closest and falcons the second-closest relatives of passerines, together constituting the Psittacopasserae and the Eufalconimorphae, respectively. Our new and robust phylogenetic framework has substantial implications for the interpretation of various conclusions drawn from passerines as model organisms. This includes insights of relevance to human neuroscience, as vocal learning (that is, birdsong) probably evolved in the psittacopasseran ancestor, >30 million years earlier than previously assumed.

A novel web-based TinT application and the chronology of the Primate Alu retroposon activity.

BACKGROUND: DNA sequences afford access to the evolutionary pathways of life. Particularly mobile elements that constantly co-evolve in genomes encrypt recent and ancient information of their host's history. In mammals there is an extraordinarily abundant activity of mobile elements that occurs in a dynamic succession of active families, subfamilies, types, and subtypes of retroposed elements. The high frequency of retroposons in mammals implies that, by chance, such elements also insert into each other. While inactive elements are no longer able to retropose, active elements retropose by chance into other active and inactive elements. Thousands of such directional, element-in-element insertions are found in present-day genomes. To help analyze these events, we developed a computational algorithm (Transpositions in Transpositions, or TinT) that examines the different frequencies of nested transpositions and reconstructs the chronological order of retroposon activities. RESULTS: By examining the different frequencies of such nested transpositions, the TinT application reconstructs the chronological order of retroposon activities. We use such activity patterns as a comparative tool to (1) delineate the historical rise and fall of retroposons and their relations to each other, (2) understand the retroposon-induced complexity of recent genomes, and (3) find selective informative homoplasy-free markers of phylogeny. The efficiency of the new application is demonstrated by applying it to dimeric Alu Short INterspersed Elements (SINE) to derive a complete chronology of such elements in primates. CONCLUSION: The user-friendly, web-based TinT interface presented here affords an easy, automated screening for nested transpositions from genome assemblies or trace data, assembles them in a frequency-matrix, and schematically displays their chronological activity history.