Network dynamics of eukaryotic LTR retroelements beyond phylogenetic trees

Llorens, Carlos; Muñoz-Pomer, Alfonso; Bernad, Lucia; Botella, Hector; Moya, Andrés

doi:10.1186/1745-6150-4-41

Research
Open Access
Published: 02 November 2009

Network dynamics of eukaryotic LTR retroelements beyond phylogenetic trees

Carlos Llorens^1,2,
Alfonso Muñoz-Pomer^2,3,
Lucia Bernad²,
Hector Botella^4,1 &
Andrés Moya^1,5,6

Biology Direct volume 4, Article number: 41 (2009) Cite this article

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Abstract

Background

Sequencing projects have allowed diverse retroviruses and LTR retrotransposons from different eukaryotic organisms to be characterized. It is known that retroviruses and other retro-transcribing viruses evolve from LTR retrotransposons and that this whole system clusters into five families: Ty3/Gypsy, Retroviridae, Ty1/Copia, Bel/Pao and Caulimoviridae. Phylogenetic analyses usually show that these split into multiple distinct lineages but what is yet to be understood is how deep evolution occurred in this system.

Results

We combined phylogenetic and graph analyses to investigate the history of LTR retroelements both as a tree and as a network. We used 268 non-redundant LTR retroelements, many of them introduced for the first time in this work, to elucidate all possible LTR retroelement phylogenetic patterns. These were superimposed over the tree of eukaryotes to investigate the dynamics of the system, at distinct evolutionary times. Next, we investigated phenotypic features such as duplication and variability of amino acid motifs, and several differences in genomic ORF organization. Using this information we characterized eight reticulate evolution markers to construct phenotypic network models.

Conclusion

The evolutionary history of LTR retroelements can be traced as a time-evolving network that depends on phylogenetic patterns, epigenetic host-factors and phenotypic plasticity. The Ty1/Copia and the Ty3/Gypsy families represent the oldest patterns in this network that we found mimics eukaryotic macroevolution. The emergence of the Bel/Pao, Retroviridae and Caulimoviridae families in this network can be related with distinct inflations of the Ty3/Gypsy family, at distinct evolutionary times. This suggests that Ty3/Gypsy ancestors diversified much more than their Ty1/Copia counterparts, at distinct geological eras. Consistent with the principle of preferential attachment, the connectivities among phenotypic markers, taken as network-represented combinations, are power-law distributed. This evidences an inflationary mode of evolution where the system diversity; 1) expands continuously alternating vertical and gradual processes of phylogenetic divergence with episodes of modular, saltatory and reticulate evolution; 2) is governed by the intrinsic capability of distinct LTR retroelement host-communities to self-organize their phenotypes according to emergent laws characteristic of complex systems.

Reviewers

This article was reviewed by Eugene V. Koonin, Eric Bapteste, and Enmanuelle Lerat (nominated by King Jordan)

Background

Mobile genetic elements (MGEs) are abundant selfish components of living organisms that have helped sculpt the complexity and size of their host genomes over the course of evolution [1]. In particular, retroelements (retrotransposons and retroviruses) constitute a widespread super-family of MGEs that are reverse-transcribed into a double-stranded DNA copy for insertion into their host genomes. Retroelements can be divided into four major systems: the long terminal repeat (LTR) retroelements; the tyrosine recombinase (YR) retroelements; the non-LTR retroelements; and the Penelope retrotransposons (for a review, see [2]). Our study is about LTR retroelements. These encompass the broad panoply of LTR retrotransposons and retroviruses circulating among plants, fungi and animals and can be classified into four major groups or families: the Ty3/Gypsy, the Retroviridae, the Ty1/Copia and the Bel/Pao families (see [2]). LTR retroelements share ancestry not only with other retroelement systems, but also with diverse host genes and with a variety of viruses [2]. The most obvious examples of viruses evolutionarily related to LTR retroelements are plant caulimoviruses (Caulimoviridae). These are circular double-stranded DNA pararetroviruses that replicate in plants via a RNA intermediate evolved from LTR retroelements [3]. Although caulimoviruses do not have LTRs, they have been included in this work because their study is essential for understanding the deep history of LTR retroelements. For simplicity's sake, we will use the term "LTR retroelement system" throughout the rest of this paper to refer collectively to all families investigated, including caulimoviruses.

An important aim of the post-genomic era is to classify all MGEs and viruses inhabiting (or circulating in) living organisms to understand their role not only as pathogenic agents but also as vectors of evolution. Regarding LTR retroelements, phylogenetic analyses usually show how families split into multiple distinct lineages (clades and genera), but it is yet unclear how deep evolution occurred in this system. The traditional notion in the origin and evolution of the distinct families is monophyletic, but such an assumption has been challenged by increasing evidence suggesting that natural evolution can proceed by gradual and vertical means, but also through distinct modular, saltatory, and reticulate events [3–14]. In general, phylogenetic analyses accumulate systematic and sampling errors when attempts are made to force natural evolution into a tree-like mode. In the words of WF Doolittle [15] - molecular phylogeneticists will have failed to find the true tree, not because their methods are inadequate or because they have chosen the wrong genes, but because the history of life cannot properly be represented as a tree. Our research is consistent with this perception. In a previous study [16] we discovered a network of relationships between Ty3/Gypsy and Retroviridae LTR retroelements on the basis of gag and pol polyproteins. Our results suggested a scenario of polyphyly, whereby we proposed three Ty3/Gypsy ancestors in the deep evolutionary history of the Retroviridae (that is the three kings hypothesis). Later [17], we found that on the basis of clan AA of aspartic proteases [18], this network extends not only to the Ty1/Copia, Bel/Pao and Caulimoviridae families but also to other host peptidases, described in both prokaryotes and eukaryotes (on this topic, see also [19, 20]). This triggered our interest in investigating the natural history of LTR retroelements, not only as a tree but also as a network system. In this regard, network biology is an emerging field of graph theory that does not provide all the answers to all evolutionary questions, but that offers an appropriate framework for the study of complex systems (for more information on this topic, see [21–24]). Taking this into primary consideration, we performed (and present in this paper) a comprehensive study investigating not only the phylogenetic diversity of LTR retroelements, but also their evolutionary dynamics beyond phylogenetic trees. In particular, we will show how eukaryotic LTR retroelements are not only a phylogenetic system but also a self-organized system of scale-free characteristics, as observed in all complex networks of major scientific and social interest.

Results and discussion

Phylogenetic patterns of LTR retroelements based on pol

LTR retroelements known to date can be classified into four major families, namely Ty3/Gypsy, Ty1/Copia, Bel/Pao and Retroviridae [2]. These share ancestry with the Caulimoviridae [3] through two common polyproteins, gag and pol. Gag usually contains three protein domains called matrix (MA), capsid (CA) and nucleocapsid (NC). Analyses based on this polyprotein are rarely reported because it evolves rapidly. In contrast, pol consists of four protein domains termed protease (PR), reverse transcriptase (RT), ribonuclease H (RH), and integrase (INT). RT shows a robust phylogenetic signal and it is commonly used for inferring the evolutionary history of LTR retroelements (see [2]). The RT evolutionary history comprises multiple distinct phylogenetic patterns and is further supported by other phylogenies inferred on the basis of RH [8], INT [4, 25] and PR [17]. However, increasing evidence suggests that this history is accurate only with respect to the clustering of OTUs into lineages (clades and genera) because evolution of transposable elements is in most cases modular (see [5]). Indeed, in attempts to investigate the deep history of LTR retroelements, the internal tree topology usually accumulates systematic errors and shows various important discrepancies depending on the pol protein domain evaluated. Upon this, in a previous approach [26] we found that when LTR retroelement phylogenies are inferred from the concatenation of two or more protein domains, the statistical power of the analysis is increased and diverse (but not all) single-gene discrepancies are corrected. This strategy enables taxonomy levels to be assigned to the different OTUs evaluated and provides an accurate perspective on the multiple distinct phylogenetic patterns of each family. With this aim, we used an alignment concatenation of the PR, RT, RH and INT pol protein domains to infer the phylogeny of each family using the Neighbor Joining (NJ) method of phylogenetic reconstruction [27]. A description of each family follows (for more details about sequences and lineages, see Additional file 1 or the section "Sequences" in Methods).

Ty3/Gypsy

These elements constitute a family of retroviruses and LTR retrotransposons widely distributed among the genomes of plants, fungi and animals. According to the International Committee on the Taxonomy of Viruses (ICTV), Ty3/Gypsy elements were originally classified into two major genera called Metaviridae and Errantiviridae (see [2]). For some time, this classification has been an important reference in LTR retroelement taxonomy, but it is now considered inconclusive by many authors because sequencing projects have revealed new lineages, updating the original perspective. As shown in Figure 1A, the inferred phylogeny of Ty3/Gypsy LTR retroelements reveals two large branches. The first branch (in red) encompasses all chromodomain-INT-containing LTR retrotransposons [4], usually called chromoviruses [28]. This branch includes at least two well-supported clusters of LTR retrotransposons, here named "Plants" and "Fungi/Vertebrates". The cluster of plant chromoviruses splits into six clades detailed in the figure, while that of fungi and vertebrates consists of seven clades. This includes a new clade, which we call V-clade, based on the name suggested by Oliver Piskurek (in personal communication) to describe Amn-like [29] and Sushi-like chromoviruses of vertebrates, collectively. The chromoviral branch includes other divergent fungal elements, which have unclear classification. The second Ty3/Gypsy branch (in black), referred to as Branch 2, encompasses the remaining lineages of non-chromoviral Ty3/Gypsy LTR retrotransposons and plant and animal retroviruses.

Ty1/Copia

This is a family of retroviruses and LTR retrotransposons abundantly represented in the genomes of plants, fungi and animals. The present ICTV classification [30] of the Ty1/Copia into three genera - Pseudovirus, Hemivirus and Sirevirus - has limitations identical to those of Ty3/Gypsy LTR retroelements. It was important for understanding the original family but it is not conclusive for managing the currently-available diversity of the Ty1/Copia family. As shown in Figure 1B, the inferred phylogeny of Ty1/Copia LTR retroelements based on pol reveals two major branches, respectively depicted in red and black. The first branch, which we call Branch 1, is supported by bootstrap and encompasses the original Pseudovirus genus (see [31]) together with a clade called GalEA [32], specifically found in marine bilaterians and all CoDi-like LTR retrotransposons described in diatoms. The wide variety of CoDi-like elements splits into four clades that we simply name A, B, C and D. One of the most exciting aspects of CoDi-like elements is that the different elements belonging to clade A encode INTs carrying a putative chromodomain at their C-terminus (see Additional File 2, AF2A). The second Ty1/Copia branch encompasses the remaining lineages of LTR retrotransposons and potential retroviruses, including the previously described Copia-like hemiviruses and sireviruses.

Bel/Pao

These constitute a family of LTR retrotransposons and retroviruses that have been described to date only in metazoan genomes. According to the ICTV classification, members of the Bel/Pao family are called semotiviruses (see [2]). A later study [33] showed that the Bel/Pao family can be divided into five clades called Pao, Sinbad, Bel, Tas and Suzu. Taking a step forward, we show in Figure 2A that on the basis of the inferred pol phylogeny, these five can be collected into three branches that we have numbered 1, 2 and 3. Branch 1 (in red) encompasses two clades - Tas and Bel - of LTR retrotransposons and retroviruses found in the genomes of cnidarians and protostomes; Branch 2 (in black) includes the Pao and Sinbad clades of LTR retrotransposons described in protostomes and deuterostomes; Branch 3 (in blue) consists of a single clade called Suzu, which includes only LTR retrotransposons that have so far been described exclusively in vertebrates.

Caulimoviridae

These comprise a retro-transcribing family of viruses that, in a yet unclear saltatory event, evolved from LTR retroelements to DNA pararetroviruses (see also [3, 13]). As shown in Figure 2B, the inferred pol phylogeny of caulimoviruses reveals four branches, which we term "classes" following a viral systematic similar to that of Retroviridae retroviruses (discussed below). The four caulimovirus classes may be divided into six lineages - Caulimo-, Soymo-, Cavemo-, Tungro-, Badna- and Petuvirus - according to the genera proposed by ICTV [34] and further corroborated by [3]. Class 1 (in red) includes two genera - Caulimovirus and Soymovirus; Class 2 (in black) consists of two other genera - Tungrovirus and Badnavirus - and it is the most abundant branch of Caulimoviridae elements; Class 3 (in blue) represents the genus Cavemovirus. The phylogeny also includes the Petunia Vein Clearing Virus (PVCV), which constitutes the genus Petuvirus. This element falls close to Class 1 but occupies the deepest position in the phylogeny (Class 4 in green).

Retroviridae

These are a family of retroviruses restricted to vertebrate animals. These retroviruses originally received attention when infectious representatives were characterized in humans. However, it is now known that any LTR retrotransposon capable of recruiting a third ORF envelope gene (env) is potentially capable of becoming a retrovirus (env is the most basic difference between LTR retrotransposons and retroviruses). Consistent with ICTV [34], the inferred phylogeny of Retroviridae based on pol shows seven genera - Alpha-, Beta-, Gamma-, Delta-, Epsilon-, Spumaretroviridae and Lentiviridae - that together with ERV-L elements we divide into three classes, 1, 2 and 3 (according to [16, 35] and references therein). As shown in Figure 2C, Class 1 comprises gamma- and epsilonretroviruses; Class 2 includes lentiviruses, delta-, alpha- and betaretroviruses; and Class 3 encompasses spumaretroviruses and ERV-L elements. This analysis includes an element called Snakehead retrovirus (SnRV) [36], which has unclear classification; but the common LTR retroelement phylogeny (shown in Additional file 1) places this sequence within Class 1. Finally, the analysis also shows a Spumaretroviridae-like element that we found in genome of Danio rerio and that we call Danio rerio Foamy Virus Type 1 (DrFV-1). This is a complex retrovirus displaying the typical gag and pol ORFs of LTR retroelements and three additional ORFs that show no similarity to any sequence currently known (including env). DrFV-1 is taxonomically important because; 1) it is unclear if it is a true retrovirus carrying a highly divergent env plus two accessory or additional genes or a LTR retrotransposon just carrying three additional genes; 2) its presence in Danio rerio suggests that spumaretroviruses are more widely distributed than previously thought. In Additional file 2 AF2B, we show diverse sequence comparisons using gag and pol ORFs of DrFV-1 as queries against the search of taxonomic HMMs available at the Gypsy Database (GyDB) [26]. These show that DrFV-1 is a spumaretrovirus, but also an intermediate sequence among the Retroviridae and other LTR retroelement families.

What does the post-genomic era suggest about LTR retroelement macroevolution?

Phylogenetic analyses help to classify the diversity of LTR retroelements by assigning taxonomy levels to each family. For evaluating the whole system, Additional file 1 shows the inferred phylogeny of the 268 LTR retroelements investigated based on pol using the NJ method of phylogenetic reconstruction. This tree reports a trichotomy that separates the five evaluated families into three major clusters - Ty1/Copia, Bel/Pao and Ty3/Gypsy-Caulimoviridae-Retroviridae (TCR). The latter suggests an ancestral node common to three families that probably corresponds to the Ty3/Gypsy family in light of its wide distribution. While this evidences an evolutionary history mainly driven by gradual means, LTR retroelement trees often show branching variations in the internal topology, due to diverse reticulate processes. These may be due to; A) the different rates of evolution among the distinct retroelement protein domains (i.e. modularity [4, 5]); B) the incidence of natural mechanisms such as gene recruitment [37], genome rearrangement [6, 7] and recombination [8–10]; C) horizontal transfer, caulimoviruses and exogenous retroviruses (and more rarely, endogenous retroviruses and many LTR retrotransposons) usually spread via infection or by horizontal means [11, 12, 14]; D) saltatory evolution, caulimoviruses are DNA viruses evolved from LTR retroelements (see [3, 13]). These processes support an alternative scenario more appropriately described as a network than as a tree (see the phylogenetic networks shown in Additional file 2 AF2C). In this regard, in a previous study [16] we explored the Ty3/Gypsy origins of Retroviridae retroviruses comparing the Ty3/Gypsy family with the three Retroviridae classes 1, 2 and 3. We found a network whereby Ty3/Gypsy lineages of plants and fungi, such as Tat and Athila elements and chromoviruses, can be related to the Retroviridae Class 1. In turn, other Ty3/Gypsy lineages of insects, such as Micropia/Mdg3 clade and errantiviruses, can be related to Classes 2 and 3. In light of this polyphyletic scenario we proposed the three kings hypothesis [16], according to which the three Retroviridae classes can potentially be tracing three Ty3/Gypsy ancestors, emerged at different evolutionary times (see, Additional file 2 AF2C). Therefore, while we can accept that the Retroviridae evolved from the Ty3/Gypsy family, the way in which their distinct lineages appeared or can be related with the Ty3/Gypsy family, is probably not monophyletic. This perspective stimulated our interest in evaluating the evolutionary dynamics of the five LTR retroelement families as a whole system. Upon this, sequencing projects continuously reveal multiple distinct communities of LTR retroelements overlapping in the genome of a single eukaryotic host (see [28, 38–40]). It is well known that eukaryotes usually transmit their retroelement communities via germ lines (i.e. by vertical means), except in the case of exogenous retroviruses and caulimoviruses which spread horizontally although only within the boundaries of the same biological supergroup. In other words, the host distribution of the distinct LTR retroelement communities is fairly vertically at the phylum level. Taking this into account, we superimposed the phylogenetic patterns of the LTR retroelement system over the tree of eukaryotes to investigate their macroevolutionary history, at distinct evolutionary times (Figure 3). Although we assume five major supergroups of eukaryotic hosts (Excavata, Rhizaria, Archeoplastida, Chromalveolata and Unikonta, according to [41]) no information about LTR retroelements in Excavata and Rhizaria has been published yet (as far as we know). Therefore, the framework depicted in Figure 3 focuses only on the host distributions of LTR retroelements described in Archeoplastida, Chromalveolata and Unikonta. These were divided in three major transitions by assuming times of emergence, according to the age and macroevolution patterns of their hosts, calibrated by prior molecular estimations and the fossil record. There follows a discussion of the three transitions based on their most parsimonious interpretation.

The first transition traces the most likely root of the LTR retroelement system and covers a long episode in the history of life. This is approximately 1,950-2,170 Millions years ago (Mya) between the Archean and the Proterozoic. The transition runs from the earliest eubacterial fossils and the first unicellular algae to the segregation of eukaryotes into the major supergroups (3,500-1,330 Mya [42–47]). According to this framework, the root of the LTR retroelement system is dichotomous as it consists of two families - Ty1/Copia and Ty3/Gypsy -, which segregated into two separate phylogenetic patterns. This is probably because of the genomic rearrangement of int that differentiates these two. Subsequently, Ty3/Gypsy chromoviruses acquired a GPY/F module and a chromodomain. The wide distribution of Ty3/Gypsy chromoviruses, not only in algae but also in land plants, amoebae, fungi and animals and chromalveolates, suggests that these constitute the oldest branch of Ty3/Gypsy LTR retrotransposons. In Figure 1, we distinguished two branches in regards of the Ty1/copia family. Branch 2 spreads among algae, plants, fungi and animals and Branch 1 among fungi, diatoms and marine animals. Also, a prior study [48] describes a Ty1/Copia taxon close to the GalEA clade (Ty1/Copia Branch 1) in red algae, suggesting that the two Ty1/Copia branches probably co-existed with Ty3/Gypsy chromoviruses before the split between plants and unikonts (1,550 Mya according to molecular estimations [46, 47]). These estimations are older than the earliest predictions of the fossil record (1330-720 Mya [43–45]), but the absence of fossil evidence for the early history of plants, fungi and animals might be because these were probably small or microscopic soft-bodied organisms. Another interesting aspect of this transition is that it seems to link the root of the LTR retroelement system with the evolutionary history of other retroelements. The similarity in both sequence and gag-RT-RH genomic architecture between LTR- and YR retrotransposons suggests an ancient gag-pol form, from which these two systems evolved, probably diverging from other retroelement systems. The segregation of LTR- and YR retroelements seems delineated by several genomic differences. YR retroelements captured a tyrosine recombinase (yr) gene (see [49]), while LTR retroelements recruited an int gene (probably from DNA transposons [50]) and a clan AA pr gene from the host genome [20]. Particularly, the history of clan AA gives additional support to the notion arguing that the history of life diversity is a network of networks, at distinct levels of complexity. This enzyme family spreads in both prokaryotes and eukaryotes and can be divided into two major structural forms; 1) the single-domain ORF typically found in LTR retroelements and several host genes [17, 20]; and 2) the two-domain pepsin monomers of eukaryotes classified as the Family A1 at MEROPS [18]. It is commonly assumed that the ancestral clan AA form is the single domain ORF and that pepsins evolved from this form by gene duplication [51]. While this is supported by the wide distribution of the single domain ORF in proteobacteria [17, 20] a recent issue has potentially extended the putative origin of pepsins (until now restricted to eukaryotes) by describing homologues in oceanic and plant symbiotic bacteria [19]. This finding re-opens debate about the origin of clan AA but it is, in a way or another, consistent with a major idea; certain genetic units (retroelements and viruses included) of eukaryotes may have evolved from the recombination and assembling of minor genetic elements during the prokaryotic-eukaryotic transition, as proposed in [52, 53].

The second transition covers approximately 1,000 million years in the history of life. It should be dated at the segregation of early eukaryotes into their major supergroups (1,330-380 Mya based on [43–45, 54]). The transition spans the interval from the Proterozoic to the Devonian Period within the Phanerozoic, during which all major groups of eukaryotes underwent dramatic diversification including key events such as the divergence between primitive animal phyla (e.g. Porifera, Cnidaria, Ctenophora) and higher phyla; the origin of Eumetazoa (1300-940 Mya [54, 55]; the Cambrian explosion 540-500 Mya [56]); the rise of vertebrates [57]; the emergence of land plants (probably back to the Ordovician or even the Cambrian Period [58, 59]); the origin of plant-fungal interactions [60]; and the divergence between arthropods and priapulids [61]. During this transition, the Ty1/Copia and Ty3/Gypsy radiated a variety of lineages, and the Bel/Pao and the Retroviridae families emerged in eukaryotes. To date, there has been no conclusive evidence of Ty1/Copia sibling lineages shared by plants and fungi, but Ty3/Gypsy phylogenies show two clusters of fungal and plant chromoviruses that are closely related to each other (see Figure 1). Chromoviruses are thus putative markers of ancient interactions between plants and fungi. Interestingly, while chromoviruses spread among vertebrates, they disappear from the genomic record of cnidarians, mollusks, protostomes and basal deuterostomes (according to current data). In contrast, what we find in these organisms are traces of Ty3/Gypsy Branch 2. We can therefore assume that Ty3/Gypsy Branch 2 elements inhabited bilaterian genomes before the split into protostomes and deuterostomes (761-531 Mya according to [54, 62]). Granted this assumption, the oldest Ty3/Gypsy lineage in metazoans is probably Mag cluster that spreads among cnidarians, protostomes and deuterostomes. Returning to the chromoviruses, it is unclear whether they were driven to extinction in protostomes, echinoderms and urochordates or were horizontally transmitted from plants or fungi to vertebrates. Horizontal transmission seems to be the simplest explanation in view of the strong similarity between V-clade and fungal chromoviruses, but if so, the event is at least as ancient as the split of Osteichthyes into actinopterygians and sarcopterygians. This is because V-clade is present in fishes such as Danio rerio but also in amphibians and amniotes (see also [29, 63]). Note that stem osteichthyan occurs in the fossil record in Upper Silurian times, 421 Mya [64], and the oldest unequivocal actinopterygians come from the Lochkovian (410-415 Mya [65]). Moreover, the fossil record indicates that the split between the Actinopterygii and the Sarcopterygii could have taken place between 421 and 416 Mya [62] (consistent with phylogenomic analyses based on expressed sequence tags (ESTs) [66]). The Ty1/Copia family is also spread among the genomes of cnidarians, arthropods and deuterostomes, but to our knowledge there is no current evidence of Ty1/Copia elements in platyhelminthes and nematodes. Based on the current availability of data, this suggests that Ty3/Gypsy ancestors diversified much more than their Ty1/Copia counterparts during the history of metazoans. The wide distribution of Bel/Pao LTR retroelements among metazoans and their absence from fungi and plants additionally suggests that their origin antedates the split between primitive animals and higher eumetazoans (1300-380 Mya according to [43–45, 54]). Bel/Pao LTR retroelements are spread among cnidarians, protostomes and deuterostomes and can be related to Ty3/Gypsy and Retroviridae elements in view of the same int order and the presence of a GPY/F module at the C-terminus of INT (see Additional file 2 AF2D). This supports the notion that LTR retrotransposons capable of becoming metazoan retroviruses (Ty3/Gypsy Branch 2, and Bel/Pao and Retroviridae LTR retroelements) might have emerged before the bilateria split into protostomes and deuterostomes (761-531 Mya according to [54, 62]). In regards of Retroviridae retroviruses, Figure 3 shows that the distribution of Retroviridae Class 1 overlaps with that of V-clade chromoviruses in Danio rerio (and probably in Salmonidae species [67]), amphibians and sauropsids ([29]). This suggests that the first true Retroviridae taxa probably appeared in the history of vertebrates before the Osteichthyes split into the actinopterygians and the sarcopterygians. The presence of DrFV-1 in Danio rerio suggests that Retroviridae Class 3 might be as old as Class 1. However, the remaining spumaretroviruses known so far are restricted to synapsids (see [68]). Further analyses are thus required to clarify whether spumaretroviruses also inhabit (or circulate in) the genomes of other fishes, amphibians and sauropsids, to calibrate the origin of Retroviridae Class 3.

The third transition covers the period from 330 million years (from the Paleozoic Era) to the present. This is concomitant with diverse events such as the origin of the first gymnosperms (360-248 Mya [59, 69]); the split of amniotes into sauropsids and synapsids (330.4-312.3 Mya [62]); the massive radiation of winged insects (380-325 Mya [70]); and the emergence of flowering plants (130-90 Mya [59, 71]). These evolutionary events generated new levels of complexity and new ecosystems that probably activated massive radiations of retroviruses and LTR retrotransposons, as well as the emergence of caulimoviruses. Evaluating information of amniotes, we find that the Ty3/Gypsy and the Retroviridae distributions overlap in the genomes of sauropsids, but there is no evidence of functional Ty3/Gypsy elements (nor Ty1/Copia and Bel/Pao) in mammals where the Retroviridae are in turn widely distributed. Despite this, mammalian genomes preserve traces of ancient Ty3/Gypsy inhabitants, which are not pseudogenic relics but neo-functionalized host genes evolved from LTR retrotransposons (see, [25, 72]). This suggests that Ty3/Gypsy LTR retroelements were co-opted by synapsid hosts (mammals and their extinct relatives) and that their extinction as selfish MGEs allowed the three Retroviridae classes to spread in these organisms (see also [73]). The two largest Retroviridae distributions in amniotes correspond to gamma- and betaretroviruses (see ICTV, online [74]). An important characteristic of Retroviridae retroviruses is that almost but not quite all of them carry additional genes. Briefly, gammaretroviruses (Class 1) display the simplest retrovirus organization, typical of Ty3/Gypsy and Bel/Pao retroviruses; betaretroviruses usually carry one additional gene plus a DUTPase similar to that of various fungal chromoviruses [75]; the remaining genera in classes 2 and 3 show diverse additional genes, which are preserved depending on the lineage (for more information, see [26]). This suggests that gammaretroviruses are probably molecular fossils still preserving the ancient gag-pol-env structure of Ty3/Gypsy retroviruses, and that the acquisition of additional genes by the remaining Retroviridae genera was an important step in their evolution. As suggested by Katzourakis et al. [68]; "retrovirus accessory genes and mammalian mechanisms of innate immunity will be best understood when considered as the join products of macroevolutionary conflicts played out over a geological scale". When evaluating the known genomes of gymnosperms and angiosperms, we find massive radiations of Athila/Tat-like Ty3/Gypsy retroviruses and LTR retrotransposons overlapping with their chromoviral counterparts and with several Ty1/Copia lineages (see [76–79]). The ancestors of Athila/Tat (i.e. all potential the Ty3/Gypsy retroviruses and LTR retrotransposons of plants) might have thus emerged before the divergence of conifers and angiosperms (360-248 Mya [59, 69]). The most likely age of these lineages is concomitant with the appearance of Retroviridae Class 1 gammaretroviruses in amniotes (consistent with the three kings hypothesis). A similar perspective is shown by currently available genomic information on winged insects such as flies, mosquitoes and others. In these organisms we find multiple distinct lineages of Ty1/Copia, Ty3/Gypsy and Bel/Pao LTR retroelements [39]. In view of the emergence of winged insects (380-325 Mya [70]), these LTR retroelements most probably emerged simultaneously with those of Retroviridae classes 2 and 3 in amniotes (again consistent with the three kings hypothesis). Interestingly, caulimoviruses can also be related with Ty3/Gypsy LTR retroelements on the basis of the gag-pol region and they show (like the Retroviridae) diverse additional genes, which are necessary for their viral life cycle and transmission (see [3, 74]). However, this does not relate caulimoviruses with Retroviridae retroviruses, as their distinct additional genes cannot be related through function or though similarity. In fact, prior trends relate caulimoviruses to other virus systems based on the common share of the movement protein [13], indicating that the most likely origin of caulimoviruses, was chimeric (an hybrid between Ty3/Gypsy retrotransposons and other RNA viruses). It is not yet clear whether the ancestors of caulimoviruses were inhabitants of plants or insects, but their most likely origin was at least simultaneous with the emergence of flowering plants (130-90 Mya [59, 71]). This third transition also covers the Ty1/Copia LTR retroelements called CoDi-like described in diatoms (origins of diatoms dated 164-166 Mya according to [80]). The existence of a Ty1/Copia lineage -CoDi-A - showing INTs with chromodomain makes of CoDi-like elements an interesting case study that merits further attention.

It is too early to draw more specific conclusions, but we think that CoDi-A elements might have captured their chromodomain independently from Ty3/Gypsy chromoviruses. Note that this feature is a ubiquitous component of many eukaryotic proteins and that no chromodomain has been to date detected in other Ty1/Copia elements. This finding is however important in the topic as prior to the present study the status of chromovirus was thought to be restricted to diverse Ty3/Gypsy lineages.

Mapping reticulate (network) evolution patterns

In the former section we presented an evolutionary framework showing how the history of the LTR retroelement diversity is shaped not only by phylogenetic patterns but also by epigenetic host factors. It is important to stress that no Retroviridae and Bel/Pao element has been described in the genomes of plants and fungi, and no caulimoviruses have been detected to replicate within animal cells or in their genomes (they act only as vectors). On the more diverse and widely-distributed families such as Ty3/Gypsy and Ty1/Copia we have an identical perspective. The lineages of these two families described in plants have more-or-less counterparts in fungi and animals but do not inhabit the animal and fungal genomes, and vice versa. The simplest interpretation of this hierarchy is that eukaryotes impose natural barriers to the distribution of LTR retrotransposons, retroviruses and caulimoviruses. This lends support to the idea of an inflationary mode of evolution, whereby we think that the various diversity explosions of eukaryotes induced massive LTR retroelement radiations, as well as the emergence of a network of LTR retroelements. This network co-evolves with the eukaryotic complexity. The first traces of this network are defined by the original genomic reorganization that segregated the Ty3/Gypsy and Ty1/Copia families into two different phylogenetic patterns. While these two diversified into multiple distinct lineages, our results suggest that the appearance of the Bel/Pao, Retroviridae and Caulimoviridae families in this network can be related with distinct inflations of the Ty3/Gypsy family, at different evolutionary times. This led us to speculate with the idea that Ty3/Gypsy LTR retroelements were more successful than their original Ty1/Copia counterparts during evolution, because their phenotypic plasticity is higher than that of Ty1/Copia LTR retroelements. This might have allowed the Ty3/Gypsy family to explore new adaptive strategies during its diversification from which not only lineages, but also families, evolved at distinct geological eras. Following this idea, it is reasonable to assume that the intrinsic evolution of LTR retroelements imprints diversity patterns into a variety of functional retroelement features that derive in the emergence and fixation of molecular phenotypes associated to ancient processes of divergence and convergence. Upon this, in a previous issue we described the existence of a network of relationships between Ty3/Gypsy and Retroviridae LTR retroelements (discussed in the former section). This network resides not only in sequence similarity and host distributions but also in the duplication-diversification-loss of three molecular features. These are the number of Cys-X2-Cys-X4-His-X4-Cys (CCHC) zinc finger arrays [81] at NC; the sequence flap motif of PR [82]; and the GPY/F motif of the INT module with an identical name [4]. As shown in Figure 4A, the number of CCHC arrays in NC varies depending on the Ty3/Gypsy lineage and the Retroviridae class (links in red), and both the flap and the GPY/F motifs delineate multiple variant motifs based on sequence polymorphisms preserved similarly (links in black and blue, respectively). These features are lineage-specific markers, suggesting a network scenario of poly- or paraphyly, since they occur not only in Retroviridae retroviruses of vertebrates but also in all Ty3/Gypsy LTR retrotransposons and retroviruses of plants, fungi and other animals. We have also noted that with respect to the PR marker, we surveyed the diversity of clan AA PRs in a subsequent issue [17]. In that work, we constructed a collection of HMMs as well as a major PR consensus template based on six amino acid patterns and found that not only Ty3/Gypsy and Retroviridae PRs, but also all other PRs, show specific motifs in sequence pattern 3 of this template. This extended the Ty3/Gypsy-Retroviridae network to all other LTR retroelements, but only based on PR. From that point onwards, our aim was to investigate all possible markers, not limited to the Ty3/Gypsy and Retroviridae families but including all other LTR retroelement families. In this particular, we identified eight features that can be classified as gene features (GFs) or as polymorphic amino acid motifs (PAMs). Additional file 3 AF3A provides a detailed "sequence-to-sequence" summary of all these markers and their distinct states, which are also illustrated in Figure 4. A description follows.

Figure 4B shows the CCHC array in NC, which is involved in virion assembly, RNA packaging, reverse transcription and integration processes [83]. This feature is a PAM with six states; zero, one, two or three CCHC arrays (detailed using sequence logos). Arrays based on two and three motifs vary with respect to the size of the sequence between motifs and zero arrays have no logos representation.

Figure 4C describes PR pattern 3 (i.e. the potential or real flap of PR), which confers specificity on the enzyme by carrying important substrate-binding functions [82]. We distinguished 20 states in this PAM including loss of the motif. Nineteen of these were grouped into four major variants: GANG, GIGG, TIHG and LPIV. This follows the nomenclature introduced in [17] and applies an ad hoc criterion of classification. That is, in its major consensus form, the GANG variant, encompasses a variety of motifs related by the predominance of an alanine (or a hydrophobic residue) and an aspartate/asparagine at the second and third positions, respectively. The TIHG variant describes the consensus for diverse motifs that show a threonine at the first position, followed by an isoleucine (or a hydrophobic residue) at the second position and a histidine at the third (or vice versa). The GIGG variant encompasses all sequence motifs with an isoleucine (or another hydrophobic residue) at the second position followed by two glycines. The LPIV variant describes an atypical motif found in only a few Ty1/Copia PRs. Finally, all motifs for which we were unable to resolve a consensus state were combined into a single state considering the loss of the motif. This classification updates the characterization performed in [17] for Ty1/Copia, Bel/Pao and caulimoviral PRs and revises various Ty3/Gypsy motifs resolved in [17].

Figure 4D shows the ILG motif located at the C-terminus of PR. This is one of the most well-preserved features of clan AA PRs [84], and, in the proteinase fold, corresponds with the structural loop that interacts with the catalytic motif (see [17]). On this PAM, we distinguished three states (plus the motif loss) detailed through sequence logos. These are the LLS motif found in Ty1/Copia PRs, the ILG motif predominant in all other PRs, and a PWL-like motif preserved by spumaretroviral PRs. Again, motifs for which we resolved no consensus were collected into a single state describing the motif as lost.

Figure 4E details the int gene position, on the basis of diverse positional rearrangements in the retroelement genome or its loss. This GF presents five states; first, downstream of pr, as in all known Ty1/Copia element genomes (see [7]) and Gmr-1 Ty3/Gypsy elements [6]; second, downstream to rh as usually found in Bel/Pao and Retroviridae LTR retroelements, and almost but not all Ty3/Gypsy LTR retroelements; third, truncated and in reverse frame, as in the REM1 Ty3/Gypsy chromovirus, which only preserves the GPY/F module and the chromodomain [85]; fourth, gene loss, as in almost all caulimovirus species except PVCV [86], which shows an INT in direct frame and upstream of gag (this is the fifth state).

Figure 4F describes the GPY/F motif of the module with the same name [4]. The role of the GPY/F module is not yet understood but it is located at the C-terminal end of INT. This trait coordinates the integration of the retroelement into the host genome [87, 88]. In regard to the GPY/F motif, we distinguish 13 states. 11 of these have been detailed through sequence logos, and were divided into five variants following a criterion similar to that used for pattern 3 of PR. The GPY/F variants are called GPx, KP/LT, GGw, GDY/F and GxV/I. The GPx variant includes motifs in which a glycine and a proline predominate at the third and fourth positions, respectively. The KP/LT variant collects motifs in which lysine and threonine respectively predominate in the third and fifth positions, while the fourth position is proline or isoleucine/leucine. The GGw variant represents a state based on these three residues. The GDY/F variant encompasses motifs with glycine and aspartate/glutamate (or relatives) at the third and fourth positions, respectively. The GxV/I variant comprises, three motifs commonly based on six residues and usually showing glycine and isoleucine-valine at the fourth and six positions. The two other states considered are motif loss plus the absence of the whole module (as in Ty1/Copia INTs and caulimoviruses, which have no INT). This description improves the previous evaluation in [16], which was based only on Ty3/Gypsy and Retroviridae INTs. In this update we include the GPY/F module we found in Bel/Pao INTs (see Additional file 2, AF2D) and revise the previous motif of the Mag-like GPY/F module.

Figure 4G illustrates the chromodomain (i.e. the chromatin organization modifier). The chromodomain is a small protein module involved in chromatin re-modeling and regulation of gene expression (see also [89]), it is found in a variety of host proteins but also at the C-terminus of INT coded by almost all Ty3/Gypsy chromoviruses and CoDi-A Ty1/Copia LTR retrotransposons (see Additional File 2, AF2A). A previous study showed that Ty3/Gypsy chromoviruses probably use this feature for chromatin integration [90]. We consider two states, presence and absence, for this GF.

Figure 4H corresponds to env, which potentially confers the capacity to become a retrovirus and is thus necessary for transferring retroviruses cell-to-cell. This is another GF in which we consider the presence or absence of the feature.

Figure 4I considers the additional (accessory) genes of many but not all LTR retroelements. These play a variety of roles in the life cycle and transmission of Retroviridae and Caulimoviridae viruses. It is now known that several Ty3/Gypsy elements belonging to the Tat clade and another clade of fungal chromoviruses are also carriers of additional genes (see [31, 75]). In this case, we consider the presence or absence of one or more additional genes as a GF.

The above described markers find functional arguments of reticulate evolution in the fact that lineages of distinct families can share not only a marker, but also distinct states of such a marker (for a more exhaustive detail, see Additional file 3). Certainly, the differential preservation of GFs normally derives from ancient processes of gene recruitment, recombination and genomic rearrangement. PAMs have a more intriguing interpretation, as their distinct states could be potentially tracing divergence, convergence, or just random evolution (mutational saturation). In regard to the ILG motif at PR, the LLS and ILG variants make an important taxonomical differentiation between Ty1/Copia PRs and the remaining PRs associated to ancient processes of divergence between Ty1/Copia and Ty3/Gypsy ancestors (with a very few exceptions all Ty1/Copia PRs preserve the LLS motif). To evaluate the three other PAMs, we designed a bipartite multigraph model (a graph based on two types of nodes) contemplating three networks analyses that relate the distinct genomic communities of LTR retroelements in eukaryotes with; A) number of CCHC arrays; B) PR pattern 3; C) GPY/F motif. As shown in Additional file 3 AF3B, these networks reveal how the distinct LTR retroelement communities of eukaryotes usually preserve particular PAM states depending on their host distributions. Subsequently, we designed a bipartite multigraph model that contemplates three other networks (Additional file 3, AF3C) relating the three PAMs with the distinct phylogenetic patterns of the LTR retroelement system. This second model illustrates how the multiple distinct states of each PAM are usually redundant among the distinct LTR retroelement families but lineage-specific within them. Certainly, the distribution of PAMs observed in the two discussed graph models cannot be explained as the result of a random association, as the incidence of edges over every PAM node in a random network is approximately the average between distribution edges and nodes [22]. PAMs are thus powerful indicators of divergence, within and among families but we do not dismiss convergent processes in this interpretation because the distinct PAM variants collect motifs and not all sequences belonging to a lineage, in which a variant is predominant, are necessarily carriers of such a variant. This reveals a high degree of phenotypic plasticity whereby evolution is capable of continuously developing, maintaining and recycling functional mechanisms. In other words, whether because of divergence or convergence, it is thus reasonable that PAM variations derive from the fact that the diversity of LTR retroelements is in continuous expansion, not only among families but also within families. We can thus assume that the history of LTR retroelements depends not only on phylogenetic patterns and epigenetic factors, but also on the phenotypic plasticity of LTR retroelements that enables them to adapt these phylogenetic patterns under any epigenetic change of the host environment. This means that the multiple distinct LTR retroelement phenotypes we have investigated are not arbitrary and as a consequence they are limited by the system as a whole. If such an intrinsic feature is in part responsible, to an observable extent, for the complexity of the system, this could result in self-organized, scale-free characteristics.

Indeed, one of the most important properties of scale-free networks is that the connections between nodes are distributed following a power law. Such a property derives from two facts; 1) real-world networks evolve over time and expand by addition of new nodes; 2) new nodes preferentially attach to nodes that are highly connected (according to [22, 24]). This directly results in the small-world phenomenon in which most nodes can be reached from any other in a small number of steps (the threshold is often regarded to be six). Taking this into primary consideration, we aimed to investigate the connectivity between complex phenotypic identities, within and between families. With this aim we combined the eight markers shown in Figure 4 into marker combinations (MCs) to construct a network model based on phenotypic neighbors. In total, we constructed 268 MCs (summarized in Additional file 3 AF3A), one for each LTR retroelement taxon. However, MCs determine phenotypic identities, which can be common to two or more lineages. This means that the number of MCs can be reduced to 76 (detailed in Additional file 3, AF3D), free of redundant combinations. By comparing MCs and detecting common subsets of features, we can trace neighbor relationships, which are shortcuts between lineages of different families. For example, several Ty3/Gypsy errantiviruses and Retroviridae spumaretroviruses share the following phenotype - "gag with zero CCHC arrays, plus TIHG PR plus INT after RH with GPY/F module carrying a KPT motif"- but differ in the state of the ILG motif and the presence or absence of additional genes. Errantiviruses and spumaretroviruses are related by their MCs by paths of length 2, two steps or trait changes. Following this strategy we constructed the global network of phenotypic neighbors (Figure 5) connecting all MCs, represented by 76 nodes (in white). Single changes are represented by red links, which attach two MCs if they share 7 of the 8 MC features. This produces seven single-node MCs with no links and seven additional components of connected neighbors (detailed in boxes). We then added 2 links and 1 intermediate node among the MCs of components that share 6 of the 8 MC features (in blue). Next, we added 3 links and 2 nodes (in green) to relate those MCs that share 5 of the 8 MC features (for more details, see Additional file AF3E). Connections between MCs in the same component are omitted. In our model, these would be redundant because any MC that can be reached through two or three red links can also be reachable through a blue or a green path, respectively. In doing so, all the initial components are connected in a global network that can be navigated under the relation "one link one change". Intermediate blue and green nodes (inserted between MCs separated by two and three changes, respectively), can be considered as combinations potentially describing lineages not yet characterized or extinguished forms, so that the model allows the evaluation of not only reticulate processes but also putative evolutionary gaps in the history of LTR retroelements. For instance, one can navigate the Ty1/Copia family through single changes, except to all CoDi-A and two sirevirus MCs. These combinations need two changes to reach other Ty1/Copia phenotypes as they differ in the presence of a chromodomain at INT (in the case of the CoDi-A combinations), or in the duplication or triplication of the CCHC array (in the case of the large C-terminus of sirevirus gags). For example, in the CoDi-A case an intermediate node represents any putative sequence halfway between the conventional Ty1/Copia and their CoDi-A counterparts (for instance a CoDi-A element without chromodomain), etc.

The network model of phenotypic neighbors supports the idea of shortcuts (phenotypic proximity) between the Ty3/Gypsy and Ty1/Copia families. Some Ty3/Gypsy combinations are as close to other Ty3/Gypsy as to Ty1/Copia phenotypes due to; 1) the presence of a chromodomain in CoDi-A elements; 2) the Ty1/Copia-like INT ORF order of the Ty3/Gypsy lineage called Gmr1. Another interesting aspect of this network is that it shows multiple distinct identities (MCs sharing the eight markers) and links among different Retroviridae and Ty3/Gypsy combinations, and among the latter and distinct Bel/Pao combinations. The proximity of these three families in terms of phenotypes and ORF organization, suggests that the Ty3/Gypsy family diversified much more than the Ty1/Copia family, during the course of evolution. Interestingly, the analysis supports the notion of a poly- or paraphyletic relationship between diverse Ty3/Gypsy phenotypes and those of Retroviridae retroviruses (in agreement with the three kings hypothesis). Similarly, distinct caulimoviral combinations link to each other via a single step but need three steps to connect with other phenotypes (because of differences as diverse as the absence of INT, GPY/F module, env, chromodomain, etc). Almost all the caulimovirus links observed in the network correspond with the changes needed to reach diverse Ty3/Gypsy combinations.

Finally, the model of phenotypic neighbors is consistent with the principle of preferential attachment; the high connectivity of a few Ty3/Gypsy phenotypes permits the navigation of the whole network step-by-step and from one family into another. Statistical evaluation of the network topology reveals that the connectivities between nodes are power-law distributed (see Additional file AF3E) confirming that the network is free-scale. This type of distribution cannot be explained as the result of a purely random process, as the edge distribution in a random network follows a Poisson distribution with a characteristic value, which is the number of edges divided by the number of nodes (for more details about random networks, see [22]). Moreover, the inference of mean shortest path (the average of the shortest distances between pairs of nodes) gives a value of 5.61 indicating that the network has also small-world characteristics. This is because the model is composed of phenotypic components characterized by the presence of shortcuts between almost any pair of combinations. In fact, the network is constituted by nodes which, in most cases, describe MCs common to two or more lineages. Consequently, if we trace as many links between a pair of nodes as lineages share each represented MC, we can observe that the clustering coefficient follows a power-law (see Additional file AF3E), as typically observed in hierarchical networks (see [22]). The emergence of this hierarchy reinforces the idea that the phenotypic combinations with high connectivity are widely distributed in the system, probably because they represent the most adaptive strategies in the phylogenetic history of LTR retroelements. This fact is not trivial and merits further attention, as it shows that phenotypic plasticity displays both structure and distribution. This structure is determined by properties that do not depend only on phylogenetic phenotype per se or by epigenetic effect of host evolution, but by the capability of the system to self-organize according to emergent laws characteristics of real complex systems.

Conclusion

The evolutionary history of LTR retroelements can be traced as a time-evolving network that depends on phylogenetic patterns, epigenetic host-factors and phenotypic plasticity. The Ty1/Copia and the Ty3/Gypsy families represent the oldest patterns in this network that we found mimics eukaryotic macroevolution. The emergence of the Bel/Pao, Retroviridae and Caulimoviridae families in this network can be related with distinct inflations of the Ty3/Gypsy family, at distinct evolutionary times. This suggests that Ty3/Gypsy ancestors diversified much more than their Ty1/Copia counterparts, at distinct geological eras. Consistent with the principle of preferential attachment, the connectivities among phenotypic markers, taken as network-represented combinations, are power-law distributed. This evidences an inflationary mode of evolution where the system diversity; 1) expands continuously alternating vertical and gradual processes of phylogenetic divergence with episodes of modular, saltatory and reticulate evolution; 2) is governed by the intrinsic capability of distinct LTR retroelement host-communities to self-organize their phenotypes according to emergent laws characteristic of complex systems.

Methods

We used distinct Ty3/Gypsy and Retroviridae LTR retroelements classified at GyDB [26] and diverse protein sequences of known Ty1/Copia, Bel/Pao and Caulimoviridae elements as queries to search online against the non-redundant database at NCBI [91]. The programs tBLASTn and BLASTp [92] were used for these searches. We collected the outputs involving non-redundant sequences with their available full-length genomes and built a database of 268 non-redundant LTR retroelements. Additional File 1 shows the inferred NJ tree of these sequences providing detailed information about the names, taxonomy, hosts and Genbank accessions of all taxa. Of this set, 28 sequences (summarized in Table 1) correspond to new sequences retrieved from diverse sequencing projects at NCBI.

Table 1 New LTR retroelement sequences retrieved from sequencing projects

Full size table

The most conserved parts (cores) of the PR, RT, RH and INT protein domains encoded by the investigated sequences were used to perform four multiple alignments, one for each domain, using Clustal X 2.09 [93]. Alignments were manually refined using GeneDoc [94] in shade mode assuming the following groups of amino acid similarity: [T, S - small nucleophile amino acids], [K, R, H - basic amino acids], [D, E, N, Q - acidic amino acids and relative amides], and [L, I, V, M, A, G, P, F, Y, W - hydrophobic amino acids]. Next, alignments were concatenated into a single pol alignment using the "Join Alignments" server [95]. For methodological reasons, pol alignment presents the INT domain of all Ty1/Copia elements and those of REM1 and Gmr-1 Ty3/Gypsy elements after RH, as in the remaining sequences aligned. Using a similar criterion, caulimoviruses are represented with a gap covering the entire INT region in this alignment. Pol alignment is available in various formats within the GyDB collection [96] in the section "Pol" in box "Multiple alignments" at [97].

Pol alignment was used to infer the phylogenetic reconstruction analysis shown in Additional File 1. Subsequently, this alignment was divided into five sub-alignments, one for each retroelement family. We then re-refined each family's alignment using GeneDoc and inferred five phylogenies, shown in Figures 1 and 2. All phylogenetic reconstruction analyses employed MEGA 4.1 [98] and the NJ method with 100 bootstrap replicates under the following conditions: uniform rates among sites and pairwise deletion of gaps. Additionally, we constructed phylogenetic networks based on pol using SplitsTree 4.10 [21]. Details on the methodology used are available in Additional file 2 AF2C.

We investigated diverse phenotypic features such as duplication and variability of amino acid motifs or several gene features that imply different genomic organizations. These were classified as PAMs or as GFs, respectively. We constructed alignments based on the following PAMs: 1) CCHC array at NC; 2) PR pattern 3 motif; 3) PR ILG motif; 4) INT-like GPY/F motif. For the CCHC array, we created a sequence database with all motifs found at the C-terminus of gag. As described in Figure 4, we found five different CCHC array motifs in this database (plus the motif lost), which was subsequently divided in five sub-databases one for each state, these were used as inputs to perform five multiple alignments. For the three other PAMs, we used GeneDoc to select and to split (as alignments) each trait from pol alignment. By position (gaps included), PR pattern 3 lies between positions 60-70 of pol alignment; PR ILG motif between positions 200-203; and GPY/F motif between positions 1568-1574. Next, each PAM alignment was divided into a number of sub-alignments (one for each PAM state as described in Figure 4), which were used as inputs to CheckAlign [99] to construct sequence logos [100]. Conditions used for constructing logos were those previously described in [16, 17]. All PAM alignments are available in various formats within the GyDB collection [96], in box "Polymorphic amino acid motifs" at [97]. As GFs we considered the following features: 1) presence/absence or ORF position of four genomic features; 2) INT ORF organization; 3) presence/absence of chromodomain at the C-terminus of INT; 4) env; 5) additional genes. Information of these features was retrieved from Genbank accessions or by comparing the sequences with the HMM search at GyDB. Finally, the eight markers were combined in a single MC as follows:

where "mx" is one of the eight markers and "i" the state of "mx".

An exhaustive summary of all markers, their states and the distinct MCs is available in Additional file 3, Sections AF3A.

PAMs' information (except that of the ILG motif) was used to design bipartite networks models capturing network relationships based on single markers under two different scenarios. Bipartite networks are multigraphs in which nodes can be partitioned into two disjoint sets, P and Q, so that each edge in E joins one node in P with one node in Q. These were constructed following steps 1 and 2 or steps 1 and 3 summarized in Additional file 4 using Mathematica 7.0 [101].

Finally, MCs' information was used to construct an undirected multigraph model to investigate the global network structure of the system, based of the phenotypic proximity of the distinct MCs, in single steps. Undirected multigraphs are graphs in which the same pair of nodes may be linked by more than one edge and where the order of the nodes is not relevant. This model was constructed following steps 1, 4a and 4b summarized in Additional file 4 using Mathematica 7.0 [101]. We calculated the degree distribution P(k), the average clustering coefficient C(k) and the mean shortest path of this graph using Mathematica 7.0 and the steps 5a and 5b summarized in Additional file 4. The degree distribution is the probability that a node in the network has a degree of value k (represented in the horizontal axis). This parameter represents the relative amount of hubs (highly connected nodes) in the network. The clustering coefficient is the proportion of edges connecting a node of degree k that form triplets. In short, this parameter is the probability that subsets of distance 2 nodes form highly connected clusters. The mean shortest path is the average of all the shortest distances between pairs of nodes.

Reviewers' comments

Reviewer's report 1

Eugene V. Koonin (National Institutes of Health, Bethesda, MD, USA)

Reviewer comments

A comprehensive study on the evolutionary dynamics of LTR retroelements, using several network methods in conjunction with standard, alignment-based phylogenies. I generally agree with the authors that phylogenetic trees are not suited to fully describe the modular evolution of mobile elements such as retroelements, so I think the use of all these network analyses and representations is welcome. The conclusion on the existence of "big bang-like" transitions in the evolution of retroelements (if I understood the authors' meaning rightly) is very appealing. The details on evolution of specific groups of retroelements will be of interest to researchers in the field.

Authors' response

We thank this referee for his positive evaluation. Yes, our results suggest inflationary mode of evolution in the evolutionary history of LTR retroelements. This gives support to the idea of that LTR retroelement evolution does not proceeds as a tree but rather a network. The history of LTR retroelements can be traced as network that evolves alternating gradual and vertical means, with diverse episodes of modular, saltational, and reticulate evolution. Our study gives additional support to; 1) the notion arguing that the history of life not as a tree but as a network of networks; 2) the existence of "big bang" patterns in the evolution of biological systems as modeled by this reviewer in prior studies [52, 53] (see also our answer to Reviewer 3).

Reviewer comments

Despite my overall positive impression of this manuscript, I do perceive several problems at different levels:

1.
The repeated and emphatic mention of "adaptive landscape", "functional landscapes", "landscapes of functional strategies" etc in this article does not refer to fitness landscape as a mathematical concept or even a concrete image but rather as a very general metaphor. Along the same lines, the discussion of the power laws and how these apply to evolutionary networks, I think, needs to be more careful and concrete (or just dropped altogether as it adds precious little to our understanding of the evolution of retroelements, or any other class of genetic elements). I think the manuscript would benefit from toning down this rhetoric that has the potential confuse the general audience and to annoy the experts.
2.
I do not think that the authors' representation of eukaryotic phylogeny is up to date. In particular, the notion of "crown group" is, I believe, obsolete, and should be abandoned. Neither is it appropriate to speak of a "three-way split" between animals, fungi, and plants. We know for a fact now that animals and fungi are sister groups. The appropriate representation of eukaryotic phylogeny at this time is a star of 5 supergroups (see Keeling et al. The tree of eukaryotes. Trends Ecol Evol. 2005 Dec;20(12):670-6). Actually, the representation of the eukaryotic phylogeny in Figure 4 (very appealing figure, indeed) seems to adhere to the supergroup concept and is unobjectionable to me but the text needs to be brought in sync with this.
3.
I think the article would be more complete if the authors at least touched upon the prokaryotic roots of the modules that comprise the eukaryotic retroelements, for instance, the origin of protease of these elements from a distinct family of bacterial aspartic proteases (Krylov DM, Koonin EV. Curr Biol. 2001 Aug 7;11(15):R584-7).

Authors' response

1) To avoid excess in both manuscript size and topics, we have removed terms such as "adaptive landscape", "functional landscapes", and "landscapes of functional strategies" from the final manuscript version (see also our response to reviewer 2). However, in our humble opinion the underlying relationship between these terms and the notion of "fitness landscape" merits further attention as it may be not just metaphoric. That is, we noted that the distinct markers and their states evidence variability of diverse functional features of LTR retroelements. This variability gives universality to the network and represents functional phenotypes, which at the molecular level, offer taxonomical differentiation. Note that one can distinguish a retrovirus from a retrotransposon based on the presence or absence of an env gene, or can differentiate at least two distinct strategies of integration based on the presence or absence of a chromodomain module at INT [87], etc. It is thus reasonable that, if the features we describe enclose phylogenetically relevant taxonomic states, these suggest variations over function probably related with retroelement speciation. An interesting discussion is if "homoplasy" (convergence) represents a better explanation than "ancestral homology" (ancient divergence) of such universality. The acquisition of GFs seems independent as the original acquisition of such functions is not by inheritance from a common ancestor. This implies that the acquisition of a new mechanism is potentially capable to give selective advantage to the original ancestral lineage that captured the feature. Regarding PAMs, the similarity of their distinct states, the host-dependent hierarchy of these states, and the synchrony among particular states of distinct PAMs, suggest that the diversity of the system expands continuously, within and between families. Taking the Retroviridae as an example, one can see that this family can be divided into a number of genera collected in three classes. With a few exceptions, we found that the distinct elements classified in each class show the same markers states. If we now take Class 1 and within it the genus gammaretrovirus, we will find that each gammaretrovirus sequence has a particular marker combination. We originally described this observation as "functional landscape", as the relationship between this and fitness landscape derives from the fact that markers are lineage-specific and can be constituted in phenotypic combinations. Hence, is there is a phenotypic combination that is representative for gammaretroviruses, that is probably because such a combination is the most "adaptive" phenotypic combination for gammaretroviruses. If so, any variation of the usual combination might be representing functional variations in the fitness of a gammaretrovirus, and this can be tested by both empirical and computational methods. On the other hand, the redundancy of marker states observed between certain families is clear evidence of reticulate (network) evolution. This is not trivial regarding the discussion of power laws properties in the diversity distribution of LTR retroelements. Big-bang means can be related with power-law patterns (this is a universal property of real world networks). Taking this into primary consideration, we have rewritten the manuscript and improved several analyses to clarify this context.

2) Done, we removed expressions such as "crown eukaryotes" or "three way split" from the current manuscript version, updated Figure 4 (which in the final manuscript version is Figure 3) and rewritten the related text following the line traced by this reviewer and by reviewer 2.

3) Done, the new manuscript version includes and discusses the clan AA topic at the prokaryotic-eukaryotic transition.

Reviewer's report 2

Eric Bapteste (Dalhousie University, Halifax, Canada)

Reviewer comments

Editorial changes to be considered:

The background section is very complex (with lots of information) and it is simply impossible to understand (for a non specialist) without a figure. This section needs extensive rewriting to clarify how the PAMs are defined and their nature. Interestingly, such a figure almost exists in the manuscript - it is figure 1-, and it should be introduced much earlier than page 8 (if possible by mapping on it the classes I, II, and III).

Authors' response

The whole manuscript has been completely rewritten and restructured. It includes a background section more accessible to any reader in broad terms. Much of the information mentioned by the expert has conveniently been moved to corresponding subsections under 'Results and discussion' and complemented by an improved version of Figure 1, although now it is Figure 3. The current manuscript is conducted appropriately to reach and understand the background of this figure.

Reviewer's comment to the revised manuscript

Reviewer comments

The paper by Llorens et al. entitled "Mapping the landscaping network principle in explaining the diversity and evolutionary patterns of eukaryotic LTR retroelements" is both too long and too complex (and in places hard to understand). It requires significant revisions. This manuscript should be:

1.
shortened
2.
clarified

To focus on its main original point, the proposition of a network framework to analyze the evolution of eukaryotic LTR retroelements and to show that, once a "good" combination of characters is obtained, then the LTR element mostly evolve within a lineage.

Authors' response

The final manuscript has been improved and reduced in size (we have removed 13 pages of the last version) to make it shorter and clearer than the former version as suggested by this reviewer.

Reviewer comments

To this end;

a) all the section entitled " Phylogenetic patterns of LTR retroelements based on pol" (p.3-11) could be significantly shortened (eventually removed or introduced as Supp. Mat). According to the authors, this part does not provide any really new perspective on the issue (cf. p.11), and truly the core argument of their paper does not start before p. 11 anyway

b) the text from "Under this scenario, " (p. 12) to 'in turn evolving in a tree-like fashion" (p.13), if summarizing ref. 19 can be removed.

c) almost all their figures, but figures 4 and 5, can be removed. Most of the networks presented here are too complex, in particular those with two kinds of nodes. It is not pedagogical. What the authors should do is reconstructing networks with one type of nodes (for instance the taxa) and connect them when two nodes (two taxa) share a common property (out of the 8 characteristics listed by them to define an LTR element). Alternatively, they could connect such nodes when they share a given combination of characteristics. This could be the only figure of their paper, and it would make their point. If they want to provide more in depth information, they could color the edges and the nodes of this single network according to various criteria (taxonomy, number of copies in taxa...) thus mapping any additional information they think is relevant.

d) there are many sentences, starting by the title, that are impossible to understand and should be rewritten. Sentences like:

"Here new strategies emerged passively and overlapped with prior strategies until configuring a complex network, whereby retroelement lineages co-opted the most adaptive landscape, depending on their molecular dynamics and host distribution. " (p.2)
"Here, it is important to emphasize that the three kings hypothesis does not intend to establish a direct relation between these lineages but that argues three Ty3/Gypsy ancestors in the evolutionary history of the Retroviridae common ancient poly- or paraphyletic scenario of diversity yet to be understood. » (p. 18)
"At the LTR retroelement level, the functional landscape of a LTR retroelement taxon can vary, or be more or less successful, if it gains or looses a marker or if a marker in this taxon evolves from one state into another." (p.24)
"Thus, new strategies emerged passively in the LTR retroelement system and overlapped prior strategies until configuring a network, whereby retroelement lineages co-opted the most adaptive landscape, depending on their molecular dynamics and host distribution. That is "the landscaping network principle" in explaining the diversity and evolutionary patterns of eukaryotic LTR retroelements. » (p.26) should be clarified or removed from the paper.

e) The notion of "landscape" is confusing and poorly explained. The analogy may fit or it may not. But the use of the word deserves more careful, direct, efficient explanations. The same criticism applies for the notion of "functional strategies". What does it mean? This lack of clarity entails that the notion of "landscaping network principle" is almost impossible to understand.

Authors' response

a) Phylogenetic analyses were performed in order to follow the line traced by reviewer 3. It would be conflictive if we remove such information. We have however reduced the text as much as possible, according to the criticisms of this reviewer, and particular care was taken in the process. In addition, it is important to highlight that the perspective that remains unchanged since 1998 (page 11) is the knowledge over the deep evolutionary history (the ancestral evolutionary relationships among families) and that phylogenetic reconstruction analyses cannot convincingly resolve this part of the LTR retroelement history. Actually, this is the foundational topic in this manuscript. Phylogenetic inference is essential as it is the most robust method to classify the OTU diversity into families and lineages. Our study makes an important update over prior knowledge of LTR retroelement diversity (and taxonomy). This is covered in the section entitled "Phylogenetic patterns of LTR retroelements based on pol", where we investigate the diversity patterns of all families and provide diverse and previously unpublished results.

b) Done in the manuscript.

c) Indeed, there are many methods to construct network models. Those we applied are well documented in graph theory, the area of mathematics that deals with the mathematical foundations of networks. Bipartite multigraphs (those with two types of nodes) are not complex; in fact, one of their properties is their simplicity. What is complex (because of its diversity) is the analyzed LTR retroelement system. Bipartite multigraphs are appropriate to evaluate the history of LTR retroelements based on two or more independent features, as this history does not depend only on a single feature. In this case, we considered markers (node 1), host distributions (node 2), and taxa or lineages (links). The obtained results can be quantitatively and qualitatively interpreted because the density of each set of links gives a clear vision of the frequency distribution of each PAM state under two independent scenarios. However, to simplify the final manuscript version (according to the general comment of this reviewer, "the manuscript is too long and too complex") bipartite multigraphs have been removed from results and are now provided within Additional file 3. On the other hand, we tested various graph methods. Constructing networks using the distinct taxa as nodes and the distinct states of a single maker as links only offers information regarding such a marker. By itself this kind of model is not very informative unless it integrates phylogenetic information. Note, for instance, that should we join "skipper" with "maggy" and "CoDi 7.1", based on a CCHC marker state, we have nothing in particular except that these sequences share that feature. For this reason it is important to resolve the multiple phylogenetic patterns. Constructing networks using taxa as nodes and using all markers as edges gives an impressive number of edges among all nodes. This results in a complete graph because the distinct markers have states, which are common not to two retroelements but to a number of them. Moreover, a systemic shortcoming with this model is that, in most cases, multiple distinct links among sequences within lineages mask other relationships which are a priori more interesting (e.g. those based on distinct families). In contrast, we found markers of reticulate evolution, which allowed us to overcome the mentioned obstacle. The most interesting aspect of these markers is their universality. Using combinations of markers we have phenotypes associated to phylogenetic identities. This allows a better interpretation of the network evolutionary dynamics, etc. Taking this into primary consideration and recognizing the contribution of this reviewer to the paper, we agree with him that the work needs a more explicative network framework addressing the main original point. This point is close to the feeling of this expert as noted in his phrase; "the proposition of a network framework to analyze the evolution of eukaryotic LTR retroelements and to show that, once a "good" combination of characters is obtained, then the LTR element mostly evolve within a lineage". With this aim we have rewritten the manuscript and revised, improved, and adapted undirected graphs to make them more comprehensible in the line traced by this reviewer.

d) The sentences addressed have been revised and restructured in order to better convey their intended meaning.

I have read the new manuscript and the responses made by the authors. They have indeed made a lot of modifications that make the manuscript very interesting and much more clear than the first version. I don't see any new comments and I think the article can be published.

Abbreviations

(AG):: Additional or accessory genes
(CA):: Capsid
(CCHC):: Cysteine zinc finger array
(CH):: Chromodomain
(DrFV-1):: Danio rerio Foamy Virus Type 1
(ERV-L):: Endogenous Retrovirus-Leucine
(GyDB):: Gypsy Database
(GF):: Gene Feature
(Gya):: Giga years ago
(HMM):: Hidden Markov model
(ICTV):: International Committee on Taxonomy of Viruses
(INT):: Integrase
(LTR):: Long terminal repeat
(MA):: Matrix
(MC):: Markers combination
(Mya):: Millions of years ago
(MGE):: Mobile genetic element
(NC):: Nucleocapsid
(NCBI):: National Center of Biotechnology Information
(NJ):: Neighbor joining
(ORF):: Open Reading Frame
(OTU):: Operative taxonomic unit
(PAM):: Polymorphic amino acid motif
(PR):: Protease
(PVCV):: Petunia Vein Clearing Virus
(RH):: Ribonuclease H
(RT):: Reverse transcriptase
(SnRV):: Snakehead retrovirus
(YR):: Tyrosine recombinase.

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Acknowledgements

We thank Horacio Naveira, Antonio Barbadilla, Antonio Marin, Francisco Silva, Juli Pereto, Amparo Latorre, Tim J. Goodwin, Cristina Vieira and Pierre Capy for early comments on the original research; and Oliver Piskurek for suggesting the names "Amn-ni" and "Amn-san" and "V-clade" proposed to describe two new sequences related to Amn-ichi as well as the clade collecting all "Amn-Sushi"-like elements. We also wish to thank Fernando Gonzalez, Rosario Gil and Pascual Asensi for hosting the GyDB project at ICBIBE, University of Valencia. We are particularly grateful to the three open reviewers of Biology Direct for critical reading and important comments. The research has been partly supported by financial grant 17092008 from ENISA (Empresa Nacional de Innovacion S.A), by grants IMIDTA/2008/103 and IMIDTA/2009/118 from IMPIVA, and by Torres-Quevedo grants PTQ-09-01-00020 and PTQ-09-01-00670 from MICINN. Funding to pay the Open Access publication charges for this article was provided by the University of Valencia.

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Affiliations

Institut Cavanilles de Biodiversitat i Biologia Evolutiva (ICBIBE), Universitat de València, Paterna, Valencia, Spain
Carlos Llorens, Hector Botella & Andrés Moya
Biotechvana, Parc Científic, Universitat de València, Paterna, Valencia, Spain
Carlos Llorens, Alfonso Muñoz-Pomer & Lucia Bernad
Departamento de Sistemas Informáticos y Computación (DSIC), Universitat Politècnica de València, Valencia, Spain
Alfonso Muñoz-Pomer
Área de Paleontología, Dpto. Geología, Universitat de València, Paterna, Valencia, Spain
Hector Botella
Centro Superior de Investigación en Salud Pública (CSISP), Valencia, Spain
Andrés Moya
CIBER de Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain
Andrés Moya

Authors

Carlos Llorens
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Alfonso Muñoz-Pomer
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Lucia Bernad
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Hector Botella
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Andrés Moya
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Corresponding author

Correspondence to Carlos Llorens.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

CL and AM conceived and designed the study; CL performed the analyses, characterized the markers, designed the models, and wrote the paper; AMP built the graphs, prepared Additional file 4, and co-wrote the paper; LB performed the phylogenetic reconstruction analyses; HB contributed interesting ideas and views and aged the tree of eukaryotes together with CL.

Electronic supplementary material

13062_2009_173_MOESM1_ESM.zip

Additional file 1: LTR retroelement phylogeny. Inferred based on pol using the 268 LTR retroelements used in this study. This tree includes information about names, Genbank accessions and hosts of all LTR retroelement taxa used. By clicking on each OTU in this tree, the user can download a GyDB file or Genbank accession of the requested element from GyDB or NCBI, respectively. (ZIP 224 KB)

Comparative analyses

Additional file 2: . Presented through an Excel file divided into four sections; AF2A) C-terminal module of CoDi-A-like Ty1/Copia INTs with significant similarity the chromodomain consensus; AF2B) hits of similarity using different core sequences of DrFV-1 as queries against HMM searches at GyDB; AF2C) phylogenetic networks; AF2D) GPY/F module multiple alignments between Bel/Pao and Ty3/Gypsy LTR retroelements. (ZIP 554 KB)

13062_2009_173_MOESM3_ESM.zip

Additional file 3: Network markers and graphs. Excel file containing five sections; AF3A) guide file summarizing all markers; AF3B) bipartite multigraphs between distributions and PAM-like markers; AF3C) bipartite multigraphs between retroelement phylogeny and PAM-like markers; AF3D) summary of non-redundant MCs; AF3E) Phenotypic neighbors' network. (ZIP 1 MB)

13062_2009_173_MOESM4_ESM.zip

Additional file 4: Building multigraphs. Zip-file containing all notebooks (Mathematica files) needed to visualize or reproduce graphs shown in this study. This is presented as a mini-web site containing three folders and two HTML files. Opening the HTML file called "Index.html" and following the steps summarized therein users can reproduce the analyses using Mathematica 7.0 or simply visualize them using the freely available Mathematica Player. (ZIP 1 MB)

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Llorens, C., Muñoz-Pomer, A., Bernad, L. et al. Network dynamics of eukaryotic LTR retroelements beyond phylogenetic trees. Biol Direct 4, 41 (2009). https://doi.org/10.1186/1745-6150-4-41

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Received: 26 October 2009
Accepted: 02 November 2009
Published: 02 November 2009
DOI: https://doi.org/10.1186/1745-6150-4-41

Keywords

Long Terminal Repeat
Long Terminal Repeat Retrotransposons
Phylogenetic Pattern
Long Terminal Repeat Element
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