A genomic survey of transposable elements in the choanoflagellate Salpingoeca rosetta reveals selection on codon usage
Unicellular species make up the majority of eukaryotic diversity, however most studies on transposable elements (TEs) have centred on multicellular host species. Such studies may have therefore provided a limited picture of how transposable elements evolve across eukaryotes. The choanoflagellates, as the sister group to Metazoa, are an important study group for investigating unicellular to multicellular transitions. A previous survey of the choanoflagellate Monosiga brevicollis revealed the presence of only three families of LTR retrotransposons, all of which appeared to be active. Salpingoeca rosetta is the second choanoflagellate to have its whole genome sequenced and provides further insight into the evolution and population biology of transposable elements in the closest relative of metazoans.
Screening the genome revealed the presence of a minimum of 20 TE families. Seven of the annotated families are DNA transposons and the remaining 13 families are LTR retrotransposons. Evidence for two putative non-LTR retrotransposons was also uncovered, but full-length sequences could not be determined. Superfamily phylogenetic trees indicate that vertical inheritance and, in the case of one family, horizontal transfer have been involved in the evolution of the choanoflagellates TEs. Phylogenetic analyses of individual families highlight recent element activity in the genome, however six families did not show evidence of current transposition. The majority of families possess young insertions and the expression levels of TE genes vary by four orders of magnitude across families. In contrast to previous studies on TEs, the families present in S. rosetta show the signature of selection on codon usage, with families favouring codons that are adapted to the host translational machinery. Selection is stronger in LTR retrotransposons than DNA transposons, with highly expressed families showing stronger codon usage bias. Mutation pressure towards guanosine and cytosine also appears to contribute to TE codon usage.
S. rosetta increases the known diversity of choanoflagellate TEs and the complement further highlights the role of horizontal gene transfer from prey species in choanoflagellate genome evolution. Unlike previously studied TEs, the S. rosetta families show evidence for selection on their codon usage, which is shown to act via translational efficiency and translational accuracy.
KeywordsMutation pressure Natural selection Opisthokonta Optimal codons Pol Tnpase
American Type Culture Collection
Bayesian inference posterior probability
Frequency of optimal codons
Guanine+Cytosine content at synonymous third codon positions
Inverted terminal repeat
last common ancestor
Long terminal repeat
Miniature inverted repeat transposable element
maximum likelihood bootstrap percentage
Effective number of codons
Effective population size
Open reading frame
operational taxonomic unit
Sequence Read Archive
Target site duplication
Transposable elements (TEs) are repetitive mobile DNA sequences that are components of the majority of eukaryotic genomes. TEs can be categorised into two classes, distinguished by their method of transposition. Class I elements are retrotransposons, which transpose via an RNA intermediate; the retrotransposons can be further categorised by the presence or absence of long terminal repeats (LTRs), which flank the 5’and 3′ end of the element. Autonomous retrotransposons contain gag and pol open reading frames (ORFs) which respectively encode structural and enzymatic proteins that facilitate transposition. LTR retrotransposons may also exist as solo LTRs, due to recombination between the two LTR sequences of an individual element which results in the removal of an LTR sequence and the internal, coding DNA. Class II TEs are DNA transposons which transpose as a DNA copy, often by a “cut and paste” mechanism that results in the entire element being removed from the host chromosome and reinserted into a new position in the genome. This process is facilitated by a Transposase (Tnpase) protein for most DNA transposons, which binds to flanking inverted terminal repeats (ITRs) creating double-stranded breaks and allowing the integration of the transposon at a new genomic location.
TEs are major drivers of mutation within their host populations and, as such, individual copies may be subject to negative selection. Selection may operate against insertion mutations or ectopic recombination rearrangements [1, 2]. In addition, as a result of the metabolic burden of TE activity, selection may also operate against the transposition process itself .
To date TEs have predominantly been studied in multicellular organisms, with comparatively limited research into unicellular eukaryotes. A number of studies have investigated aspects of TE evolution within species across known eukaryotic diversity, including unicellular opisthokonts, amoebozoans, alveolates, stramenopiles and excavates [4, 5, 6, 7, 8, 9, 10]. Such studies have concentrated on the diversity of TEs present in the host genome and the use of phylogenetics to determine the evolutionary origin of the families. The population biology of the TEs in unicellular eukaryotes has received less attention. However phylogenetic evidence for recent transposition has been shown in the filasterean Capsaspora owczarzaki  and purifying selection on the amino acid sequences of TE proteins has been reported in the parabasalid Trichomonas vaginalis  as well as the the choanoflagellate Monosiga brevicollis . In addition, gene expression studies have also shown that TE proteins are transcribed in the latter two species.
TEs in holozoan protists
The eukaryotic supergroup Opisthokonta is composed of two major lineages in the Holozoa and Holomycota [11, 12]. The holozoans include Metazoa, as well as their unicellular relatives Choanoflagellatea, Filasteria, Ichthyosporea and Pluriformea ; Fungi and the nuclearioid amoebae make up the Holomycota . Within Holozoa, choanoflagellates are the closest living known relatives to the metazoans, and provide insight into the origin of animals . All choanoflagellates have unicellular stages in their lifecycle, however many species can develop ephemeral multicellular colonies [see 16 for a thorough review of the group].
M. brevicollis was the first unicellular holozoan to have its genome sequenced , allowing a study of its TEs. Only three families were identified, all of which were LTR retrotransposons . The same study also screened available EST sequences from a second choanoflagellate, Mylnosiga fluctuans (erroneously described as Monosiga ovata ), uncovering LTR and non-LTR retrotransposons in addition to DNA transposons, suggesting that M. brevicollis may be atypical in having a limited diversity of TE families . Further insight into the evolution of TEs in holozoan protists was provided by the genome analysis of C. owczarzaki . The genome harboured 23 TE families, all of which were identified as having orthologous families in Metazoa and Fungi, indicating that the common ancestor of the opisthokonts had a diverse repertoire of mobile elements. Despite the difference in the number of families identified within the genomes of M. brevicollis and C. owczarzaki, copy numbers for individual families were similar across both species with all families being reported as possessing fewer than 100 copies. One notable difference between M. brevicollis and C. owczarzaki was the finding that all families in the former are active, whereas the latter contains at least one family that is no longer functional.
Codon usage bias in S. rosetta
The degenerate nature of the genetic code results in all amino acids, with the exceptions of methionine and tryptophan, being encoded by more than one synonymous codon. Clark  predicted that a variety of forces may interact to dictate the non-random usage of codons and subsequent studies in a broad range of eukaryotes, prokaryotes and viruses have shown that codon usage tends to show a bias in most genes [20, 21]. The degree of bias in any given gene can be calculated using the “effective number of codons” (Nc) statistic . Values range from 20, where all amino acids only use a single synonymous codon, to 61 where each amino acid uses all synonymous codons equally. Any direction of bias may then be determined using GC3s, which is the measure of the proportion of guanosine and cytosine at synonymous third positions.
Codon usage bias may be determined by genetic drift, mutational pressure, selection for translationally optimal codons [23, 24], or a combination of the three forces. In species with a large effective population size (Ne), selection tends to be the dominant force; however, in species with smaller effective population sizes, mutational pressure and drift may act to swamp any selective advantage for optimal codons . Selection for translationally optimal codons may operate through efficiency, allowing the rapid synthesis of polypeptides , and accuracy, which reduces the likelihood of misincorporated amino acids . The degree to which the codon usage of a gene shows adaptation to the host optimal codons can be calculated using the frequency of optimal codons (Fop), which is the total number of optimal codons divided by the total number of codons in the sequence .
Patterns of relative synonymous codon usage for genes in S. rosetta and M. brevicollis, both of which are craspedid choanoflagellates , were established by Southworth et al. . This study identified the suite of 23 translationally optimal codons for S. rosetta and 24 optimal codons for M. brevicollis, which are predominantly GC-ending. Selection was shown to operate at the level of both translational accuracy and efficiency.
Codon usage bias in TEs
Past studies on TE codon usage bias have mainly failed to detect the action of natural selection; rather transposon and retrotransposon families, as well as LTR retroviruses, in a range of organisms show codon usage patterns similar to weakly expressed host genes and a slight excess of AT-ending codons [30, 31, 32, 33]. One reported exception is the LTR retrotransposons of the stramenopile genus Phytophthora . TE families within this group of unicellular eukaryotes were shown to have a preference for GC-ending codons that mirrors host genes; furthermore, high copy number families tended to show stronger codon usage bias than families with lower copy number. The study did not determine which, if any, of the Phytophthora codons were optimal codons or if the higher frequency of GC-ending codons was a result of the use of optimal codons. Due to the emphasis on multicellular eukaryotes within TE research, it is unknown if the putative selection observed on codon usage in Phytophthora families is commonplace or highly unusual within unicellular eukaryotes.
As the translationally optimal codons in S. rosetta mainly show guanosine or cytosine at synonymous sites, any selection for codon usage in the TE families should contrast sharply with the weak bias towards AT-ending codons previously observed in the majority of eukaryotic TEs. S. rosetta therefore provides an opportunity to determine if selection for codon usage is present in the TE families of unicellular eukaryotes other than those previously recorded in Phytophthora.
The presented study screened the genome of S. rosetta in order to identify the species’ TE complement. The TE families were phylogenetically analysed in an attempt to determine their evolutionary history. Evidence for recent element activity was gained through phylogenetic analyses of individual element insertions within the S. rosetta genome. The forces controlling codon usage within the TE genes were analysed to determine if selection for optimal codons was operating and if there was a contribution due to mutation pressure.
S. rosetta harbours a greater diversity of TE families than M. brevicollis
Characterisation of the LTR retrotransposon families identified within the S. rosetta genome
Copy Number (FLE/Solo LTR/ Truncated)
No. of Identical Paralogous Copies
Number of Sequence Reads
Intra-element LTR Identity
LTR Nucleotide Diversity ( π - Total/FLE/Solo)
Characterisation of the DNA transposon families identified in the S. rosetta genome
Copy Number (5′ ITR/3′ ITR)
No. of Identical Paralogous Copiesb
Number of Sequence Readsc
ITR Nucleotide Diversity (π)d
The predicted full-length LTR retrotransposons elements range from 4.8 to 11.2 kb in length (Table 1, Additional files 1 and 2). Sroscv4 and Sroscv5 both encode gag and pol in separate frames, with 6 bp slippage motifs facilitating the ribosomal frameshift (Additional file 2); the other LTR retrotransposon families all encode gag and pol within the same reading frame. Srospv6 possesses a gag-pol pseudogene and appears to be the only clearly non-functional family within the sequenced strain (Additional files 1 and 2). The gag-pol pseudogene contains five regions of trinucleotide repeats (Additional file 1) which contribute in part to the much greater length of this family (11.2 kb) compared the other identified TE families in the sequenced genome. Upon integration, all copia-like and chromoviral families create a 5 bp target site duplication (TSD), whereas the two non-chromoviral gypsy-like families generate 4 bp TSDs. Of the 13 LTR retrotransposon families, eight were present as both full-length elements (FLEs) and solo LTRs; Sroscv2, Srosgyp2, Srospv1, Srospv5 and Srospv6 all appeared to lack solo LTRs within the sequenced strain of S. rosetta. Ten of the families also showed truncated insertions, with deletions disrupting their sequences (Table 1).
The DNA transposon families ranged from 2.1 to 8.3 kb in length (Additional files 1 and 2). The transposon families each possessed a single gene, which encoded a putative Tnpase. Four of the seven genes contained introns, with SrosTm and the two Tigger-like families lacking introns. The helitron transposon family, SrosH, did not possess terminal repeats, whilst all other DNA transposon families had flanking ITRs. TSD length varied between 2 and 9 bp in length; however two families, SrosH and SrosTm, which were both multicopy, did not generate identifiable TSDs upon integration (Table 2).
Although S. rosetta exhibits a far greater diversity of TE families than M. brevicollis, the copy numbers of individual families were broadly similar within the two choanoflagellates. With the exception of two high-copy number families of copia-like LTR retrotransposon (Srospv2 and Srospv3), all families in both species have less than 50 copies within the sequenced genomes (Tables 1 and 2, ). Similar copy numbers were also reported for the filasterean C. owczarzaki , raising the possibility that low TE copy numbers are prevalent in holozoan protist genomes.
S. rosetta TE families have diverse evolutionary origins
In contrast to the copia-like phylogeny, the tree for the Tc1/mariner transposon SrosTm did not cluster the S. rosetta protein with those from other opisthokonts (Fig. 2). BLAST screening uncovered putative Tnpase sequences from metazoans, fungi, stramenopiles and bacteria, with the SrosTm Tnpase protein nested with strong support within the stramenopile proteins (85% ML/1.00 biPP). Furthermore, the SrosTm Tnpase is separated from the metazoan Tnpase sequences by two strongly supported branches (98% mlBP/1.00 biPP and 85% mlBP /1.00 biPP).
A predicted protein (XP_001743358) was also uncovered from M. brevicollis through BLAST, however an inspection of the annotated sequence indicates the predicted gene may in fact be a pseudogene. The gene annotation predicts an in-frame intron between nucleotides 984–1049, between the predicted second and third exons; however the translation of the putative intron sequence possesses 45.5% identity and 68.2% similarity to the homologous SrosTm amino acids if it is translated as part of the second exon (Additional file 4). Furthermore, the predicted intron sequence contains an in-frame premature stop codon. If the intron is genuine, then ten conserved residues would be absent from the truncated Tnpase. However, if the intron is a mis-annotation, then the Tnpase would be shortened by 315 amino acid residues due to the in-frame stop codon. Including the putative M. brevicollis protein in the Tc1/mariner superfamily phylogeny has limited effect on the support for the choanoflagellate Tnpase being nested within the stramenopile proteins (84% mlBP/1.00 biPP, Additional file 3). The presence of tnpase sequences from two craspedid choanoflagellates nested with strong support within stramenopile sequences is consistent with the horizontal transfer of a transposon from the latter group into the former.
Neither the chromoviral nor the non-chromoviral gypsy-like family phylogenies were robustly resolved, however in both trees the choanoflagellate Pol sequences were recovered as monophyletic (Additional file 3). In the chromoviral phylogeny the S. rosetta families clustered with Mbcv, previously identified in M. brevicollis , however the grouping lacks phylogenetic support (< 50% mlBP/< 0.70 biPP). As with the copia-like families, the closest relatives of the S. rosetta gypsy-like families are predominantly present in the genomes of other opisthokont species, consistent with their vertical inheritance since the opisthokonts last shared a common ancestor.
With the exception of SrosTm, the transposon family phylogenies could not robustly place the S. rosetta Tnpase sequences (Additional file 3). The Tnpase proteins of SrosH, SrosM, SrosS, SrosHar and SrosTig2 clustered with weak to moderate support (< 70%mlBP, < 0.70biPP) with families mainly uncovered from other opisthokont genomes. SrosTig1 also clustered with Tnpases recovered from opisthokont genomes, however sequences from stramenopiles were the closest proteins in the phylogeny (< 50%mlBP, 0.79biPP). The inheritance of SrosTig1 is therefore unresolved.
TE activity and expression in the S. rosetta genome
All three TE families identified in the M. brevicollis genome appeared to be active , however TEs are considered to have their own life cycles within their host genome  resulting in some families eventually being extinct relics. Each family in the S. rosetta genome was therefore examined for evidence of recent activity.
LTR sequences are identical upon the integration of a daughter element, and subsequently accumulate mutations over time . Intra-element LTR identity ranged from 94.2 to 100% across the retrotransposon families (Table 1), highlighting their recent transposition. Srospv3 was the only family where insertions showed lower than 99% intra-element LTR identity. The majority of mismatches between LTRs from the same insertions within Srospv3 are due to indels in and around a highly repetitive region of CA repeats (bases 303–406 in the annotated sequence in Additional file 2). When the indels are excluded, intra-element LTR nucleotide identity is over 99% for 6 out of the 7 elements where comparisons could be made (data not shown). The lower nucleotide identity between LTRs from the same insertions of Srospv3 may therefore not reflect their greater age, but perhaps is a result of slippage mutations occurring during reverse transcription. Srospv6 lacks a functional gag-pol ORF, however it appears that the single copy in the genome has integrated recently as it possesses both 100% identical LTRs and target site duplications.
Intra-element LTR identity as a method for detecting recent transposition is restricted to LTR retrotransposons, however phylogenies of individual copies within a family can also provide insights into the recent evolutionary history of a family [4, 39]. In particular, identical sequences at different locations within the genome highlight on-going transposition, whilst sequences placed on short terminal branches indicate recent transposition events. Phylogenies were generated for 16 of the 20 families where full-length sequences have been reconstructed (Additional file 5). Due to either low copy number or poor sequencing quality phylogenies could not be created for SrosH, SrosHar, Srospv6 and SrosS .
All of the gypsy-like families showed multiple identical sequences at different genomic locations, as do four of the copia-like families (Table 1, Additional file 5). Srospv6, as mentioned above, is a single copy, non-functional family. Srospv1 does not possess identical paralogous copies; the greatest LTR identity between paralogous copies is 98.97% (Additional files 5 and 6), indicating no copies in the genome show very recent common ancestry.
In contrast to the retrotransposons, only two DNA transposon families, SrosM and SrosTm, were found to possess identical paralogous copies (Table 2). Phylogenies could not be created however for SrosH, SrosS and SrosHar. The quality of sequencing reads for the flanking DNA was inadequate for the creation of reliable phylogenies in the case of SrosH whilst SrosHar appears to be a single copy family. SrosS only has two copies in the genome and the terminal 300 bp 5′ ITR/UTRs share 98.3% nucleotide identity (Additional file 6), suggesting that the family has not transposed recently within the sequenced strain of S. rosetta.
Nucleotide diversity values, based upon Nei’s π  were generated for all LTR retrotransposon families other than the single copy Srospv6. Values were produced for all insertions, using only a single LTR from FLEs, as well as the FLEs and the solo LTRs (Table 1). Diversity was lower for the LTRs sequences from full-length elements when compared to solo LTRs in Sroscv1, Sroscv3 and Srospv2, however in Srospv3 the FLE LTRs harboured greater diversity than the solo LTRs. The Srospv3 phylogeny shows deep population structure, with full-length elements falling into two distinct subgroups (labelled Srospv3A and Srospv3B), explaining the high pairwise nucleotide identity within the family (Additional file 5). The two subfamilies harbour identical copies at different genomic locations, indicating that both are currently transposing within the S. rosetta population. Nucleotide identity between the two subfamilies varies sharply across the length of their LTRs. Representative LTRs from full-length elements share 98.8% nucleotide identity over the terminal 166 bp at the 3′ end of the Srospv3A LTR, however the same LTRs only share 40.7% identity at the upstream 167 bp of the 5′ end (Additional file 6). The observed abrupt change in nucleotide identity across an LTR sequence is similar to the situation observed between Ty1 and the hybrid Ty1/2 elements in Saccharomyces cerevisiae  and is consistent with one of the subfamilies being composed of chimeric, recombinant elements. Within the DNA transposons, SrosTm and SrosTig1 have low nucleotide diversity (Table 2), consistent with their populations being composed of mainly of short-branched, presumably young, insertions (Additional file 5). SrosM and SrosTig2 both exhibit higher levels of nucleotide diversity, due to the presence of a greater proportion of long-branched copies that have accumulated unique mutations.
S. rosetta RNA-Seq reads (SRX042046–54, SRX3432761–2), with a combined total dataset of 74,845,386 reads, were screened for the 19 TE families with full-length consensus pol or tnpase sequences. Srospv6 was also shown to be expressed (data not shown), however the single copy is a pseudogene so it was not analysed further. Sequencing reads could be mapped onto all families in the S. rosetta genome (Tables 1 and 2). Absolute numbers of mapped reads for all pol and tnpase could not be determined, due to the presence of highly repetitive sequences in the families Srosgyp1, Sroscv2, Sroscv5 and SrosS. The repetitive regions mapped to a much greater number of sequencing reads in comparison to the remaining regions of the same families, presumably due to their additional presence in non-TE transcripts. The presented number of sequencing reads in Tables 1 and 2 are those generated once the repetitive regions had been omitted from the CDS regions. As the edited sequences are incomplete, they can therefore only be considered to provide an approximation of the genuine expression levels. Within the LTR retrotransposons, gene expression varied by two orders of magnitude, with Srospv4 exhibiting less than 50 reads across the 11 SRA transcriptome runs (Table 1) and Srospv3 mapping to over 5000 reads (Table 1). An even greater range of expression was observed within the DNA transposon families, with only single read mapping onto SrosHar whilst 19,480 reads were mapped to SrosM (Table 2).
S. rosetta transposable element families show a similar pattern of codon usage bias to host genes
Codon usage statistics for the TE families identified in the S. rosetta genome
Non-Coding GC Content
LTR Retrotransposon Families
44.82 ± 5.45
0.621 ± 0.089
0.697 ± 0.083
0.494 ± 0.028
52.14 ± 4.47
0.581 ± 0.077
0.63 ± 0.08
0.518 ± 0.027
Mutation pressure influences codon usage bias in S. rosetta LTR retrotransposon families
The codon usage of TE families in S. rosetta is likely to be driven by one, or a combination, of genetic drift, mutation pressure, or selection for translationally optimal codons. Genetic drift cannot be ruled out, but appears unlikely as it is a random process and the 19 examined TE genes show an excess of GC-ending codons.
Natural selection influences codon usage bias in S. rosetta TE families
The most abundant codon for each amino acid was determined for pol in LTR retrotransposons and tnpase in DNA transposons (Additional file 8). The identified codon was then compared to the S. rosetta major tRNA genes and optimal codons, identified in Southworth et al. . In all LTR retrotransposon families the majority of amino acids preferred codons complementary to the major tRNA genes for the pol ORFs. When host optimal codons were also considered, 17 out of 18 amino acids in the pol ORFs of Srospv2 and Srospv3, as well as the two non-chromoviral gypsy-like families, were found to prefer codons that should result in efficient translation.
SrosHar was the only DNA transposon that did not have a majority of amino acids where the most abundant codon complemented the major tRNA genes. As noted above, SrosHar appears to be transcriptionally silent, as only a single read, spanning 20 bp, was mapped to the tnpase gene in 11 transcriptome runs. However when the S. rosetta optimal codons were also considered, the majority of amino acids preferred translationally efficient codons in all tnpase genes. The TE families’ preferences for codons employed by highly expressed host genes are reflected by their Fop values (Table 3). Across the 19 families, Fop ranges from 0.448 to 0.746, with LTR retrotransposons showing higher mean Fop values (0.621 ± 0.089) than the DNA transposons (0.581 ± 0.077).
The strength of codon usage bias (using Fop) and family copy number show a very weak positive relationship for the LTR retrotransposons (R2 = 0.391, Additional file 9), however no such relationship was recovered for the DNA transposon families (R2 = 0.210). Investigating families within the two classes of TE individually shows greater heterogeneity within the relationships between Fop and copy number. The copia-like families show a strong positive relationship (R2 = 0.879) between copy number and use of optimal codons, whereas no relationship is recovered when the chromovirus families are considered in isolation (R2 = 0.295). Within the two non-chromoviral gypsy-like families, the family with the higher copy number (Srosgyp1) exhibits the higher value of Fop. The higher use of translationally optimal codons therefore is consistent with them conferring an advantage in transposition for some families over others within the S. rosetta genome.
Frequency of optimal codons (Fop) in non-domain and domain regions of S. rosetta TE genes
Significance (Fisher’s Exact Test)a
LTR Retrotransposon Families
GC-codon usage bias is present in the M. brevicollis LTR retrotransposon families
The three LTR retrotransposon families of M. brevicollis  also exhibit a positive association between Fop and copy number (R2 = 0.663, Additional file 10), raising the possibility that selection on codon usage may be a phenomenon common to many choanoflagellates TEs.
Mean Fop values for the three pol ORFs were similar, albeit lower, than those of the LTR retrotransposon families of S. rosetta (0.579 ± 0.008), mirroring the weaker codon usage bias also observed in the host genes in M. brevicollis . All three families possessed a majority of amino acids which preferred to use codons that either complemented the M. brevicollis major tRNA genes or were designated optimal codons  (Additional file 10). The current lack of expression data from M. brevicollis means that it is not possible to determine if selection for translational efficiency contributes to the observed relationship between copy number and Fop.
The three LTR retrotransposon families in M. brevicollis show elevated, significantly so in the case of Mbpv1, values of Fop in functional domain codons compared to non-domain codons (Additional file 10), consistent with selection operating on translational accuracy. Mutation pressure towards guanosine and cytosine appears to play a role in codon usage bias in the LTR retrotransposons, as non-coding GC content shows a positive relationship with GC3s (R2 = 0.958, Additional file 10). It therefore appears that the codon usage of the pol ORFs of M. brevicollis LTR retrotransposons is determined by a combination of selection for optimal codons and mutation pressure.
Much of our current understanding of TE biology comes from Drosophila melanogaster and S. cerevisiae and the families from both species have been reported to show no evidence for selection on codon usage . Mean Fop values, generated using species-specific optimal codons, of a sample of 26 LTR retrotransposon pol ORFs from D. melanogaster was 0.298 ± 0.067, whilst the four active families from S. cerevisiae had a mean Fop value of 0.444 ± 0.014 (Additional file 11). Accordingly, values of Nc for TE ORFs were also higher in both organisms when compared to those from S. rosetta (Table 3, Additional file 11). The data presented here show that the TEs of S. rosetta and M. brevicollis have evolved to utilize their host translation machinery more efficiently than either of the model organisms, D. melanogaster and S. cerevisiae, previously used extensively to study TE biology.
TE family diversity in the S. rosetta genome
The genome of S. rosetta contains a far wider repertoire of elements than M. brevicollis, with a minimum of 20 TE families, from both Class I and Class II uncovered. The diversity of families is similar to that in C. owczarzaki, which harbours 23 families from both TE classes . It is clear that unicellular eukaryotes are highly variable in terms of the diversity of TE families present. Species such as S. cerevisiae , M. brevicollis  and the alveolate Plasmodium falciparum  harbour a limited number of families, with fewer than five active families present in each species, whereas S. rosetta, C. owczarzaki  and T. vaginalis  all possess in excess of 20 families. Changes in TE diversity may evolve rapidly within eukaryotic lineages. In addition to the contrasting TE complements of the two craspedid choanoflagellates presented here, previous studies have highlighted large variations in TE family diversity in Plasmodium species and microsporidian fungi [5, 44].
The TEs in C. owczarzaki all had closely related orthologous families in other opisthokonts, highlighting a putatively diverse TE complement present in the last common ancestor of Opisthokonta. The TEs of S. rosetta show a more complex evolutionary history however, with most families appearing to be vertically inherited since the origin of opisthokonts whilst SrosTm appears to have been acquired horizontally from a stramenopile donor. Horizontal transfer is a well documented phenomenon in choanoflagellates [45, 46], with donor species, such as bacteria and small unicellular eukaryotes, being prey items of choanoflagellates . The close proximity of the nucleus to food vacuoles within the cell may facilitate the passage of foreign DNA between organelles and its integration into the choanoflagellate chromosomes. Transferred genes must confer a selective advantage to their recipient in order to be retained, however the replicative, selfish nature of TEs may allow invading families to proliferate in a naïve genome in the face of negative selection. The presence of a SrosTm orthologue, albeit a putatively non-functional one, in the M. brevicollis genome indicates that the transfer of this transposon family was an ancient one. The family has continued to successfully transpose in S. rosetta, however the M. brevicollis family now appears to have lost function.
The copia-like family from St. diplocostata, which clusters phylogenetically with homologous families in both S. rosetta and M. brevicollis, highlights the antiquity of copia-like retrotransposons within choanoflagellates. The monophyly of choanoflagellate copia-like elements, from both craspedid and acanthoecid species, strongly points to this form of TE being present in the ancestor of the choanoflagellates. The vertical inheritance of copia-like elements since the LCA of opisthokonts is also suggested here as the closest relatives of the choanoflagellate elements are predominantly present in opisthokont genomes.
TE activity in the S. rosetta genome
Similar to the TE families found in C. owczaraki , as well as the more distantly related S. cerevisiae and T. vaginalis [39, 43], the families in the S. rosetta genome show evidence for both current or recent activity. With the exception of SrosHar, which only had a single 20 bp SRA read mapped to it, all of the families, including the non-functional Srospv6, are expressed. Family phylogenies of individual insertions present sequences on short terminal branches, consistent with recent transposition events. The LTR sequences of both the copia-like and gypsy-like families show high (> 99%) intra-element identity (Table 1), further highlighting the recent activity of all retrotransposon families. One notable difference between the Class I and Class II families was the abundance of identical paralogous sequences in the former compared to the latter (Tables 1 and 2), with only SrosM and SrosTm of the transposons possessing identical copies at different locations. As identical copies are generated through transposition, this finding is consistent with a lower transposition rate for transposons in comparison to LTR retrotransposons, however this cannot be confirmed due to the current lack of information on direct transposition rates.
Further similarity can be drawn with C. owczarzaki and S. cerevisiae, in that all full-length LTR retrotransposons are presented on short branch lengths within phylogenies, and therefore can be presumed to be young elements [4, 39]. This finding supports the rapid elimination of full-length LTR retrotransposons, either through LTR recombination or their loss as a result of selection. In both the DNA transposon and retrotransposon families the ancient copies are mainly non-functional, as they are truncated and partial sequences, which can no longer transpose.
The TEs found in S. rosetta, M. brevicollis and C. owczarzaki all have low copy number (Tables 1 and 2, [4, 10]), with only one family, Srospv3, found to harbour over 100 copies. Many protist species possess large effective population sizes  and this may facilitate the efficient elimination of individual deleterious TE insertions. An alternative, although not mutually exclusive, explanation may be that the holozoan protists are under strong selection pressure for compact genomes, to enable rapid cell division and therefore reproduction, which would favour individuals with lower numbers of TE insertions.
The TE families of S. rosetta show evidence for selection on their codon usage
Past studies on codon usage within TE ORFs have highlighted a consistent pattern of a weak bias toward AT-ending codons. This trend has been observed within both classes of TE in a broad range of host species ([30, 31, 32, 33], Additional file 11). None of the TE families within the S. rosetta genome follow the commonly reported TE trend of AT-bias within their codon usage. In contrast, all families are GC-rich at synonymous third positions, with mean GC3s being greater than 0.60 for both Class I and Class II families (Table 3). The mean non-coding GC content of both classes is close to 0.50 (Table 3), which does not point to mutation pressure driving neutral base composition to guanosine and cytosine. Furthermore, each family shows a preference for translationally optimal codons, as defined by the S. rosetta host genes , in the majority of their encoded amino acids, with a preference for codons which complement the products of the major tRNA genes observed for most amino acids in most families (Table 3 and Additional file 8).
Unlike the S. rosetta host genes, where no clear evidence for mutation pressure was uncovered by Southworth et al. , the forces controlling codon usage within the TE families appear to be more complex. Both chromoviruses and the copia-like families show a positive relationship between GC content at non-coding DNA and synonymous third codon positions, indicating that mutation pressure towards GC makes a contribution to their codon usage bias. The non-chromoviral gypsy-like families and transposon families however failed to show a positive relationship between GC3s and non-coding GC content. In sharp contrast to the findings of past studies, the copia-like and transposon families of S. rosetta show clear evidence for selection for translational efficiency, with the pol and tnpase genes of highly expressed families being enriched for optimal codons in comparison to weakly expressed families. In addition to selection for translational efficiency, three of the copia-like families also exhibited significantly elevated use of optimal codons in functional domain regions of their pol ORFs when compared to the inter-domain codons, indicating that selection for translational accuracy also drives their codon usage. Non-significant enrichment of optimal codons in functional domain regions of genes was also observed in five other LTR retrotransposon families as well as four of the six tested transposon families, raising the possibility that this is a mechanism that influences codon usage patterns in the majority of TE families with the S. rosetta genome.
The LTR retrotransposon families have, on average, higher values of both GC3s and Fop than the DNA transposon families (Table 3). As noted earlier, the evidence from identical paralogous sequences indicates that the retrotransposons may be more active than the transposons. Selection is mainly expected to occur on TE sequences during transposition, since individual copies are, in effect, pseudogenes with respect to their host and expected to evolve neutrally as they accumulate random mutations. The higher use of optimal codons observed in retrotransposons may therefore be a product of their putatively higher transposition rate.
The presented evidence strongly points toward selection, for translationally optimal codons, playing an important role in the codon usage of the identified TE families in two species of choanoflagellate. The possession of a higher enrichment of translationally optimal codons is likely to provide a selective advantage between copies of the same family. Following Brookfield’s analogy of viewing the genome as an ecological community , it can also be speculated that increased use of optimal codons will allow some families to outcompete other families within a species’ genome. If “resources”, such as integration sites in a compact genome or interactions with host proteins to facilitate transposition, are limited, then families enriched for optimal codons may be able to synthesize Pol and Tnpase enzymes more rapidly and have an increased transposition rate and therefore a competitive advantage. Consistent with this, Srospv2 and Srospv3 have the highest Fop values of all TE families in the S. rosetta genome and also have the highest overall copy number, as well as the highest number of identical paralogous copies (Tables 1, 2 and 3).
The seven gypsy-like families (Sroscv1–5 and Srosgyp1–2) exhibit similar values of Fop to the five copia-like families (Table 3); the gypsy-like families also exhibit a preference for codons that complement the products of major tRNA genes (Additional file 8). However, unlike the copia-like families, they do not show any strong signature for selection operating upon codon usage at the level of translational accuracy or efficiency. One possibility is that the presence of highly repetitive regions in some gypsy-like families has resulted in inaccurate read values, thereby obscuring any putative evidence for selection at the level of translational efficiency. It may also be possible that the gypsy-like families are no longer evolving under selection for their codon usage and the use of optimal codons observed reflects the signature of past selection that has not had time to be erased from extant element copies. Long range sequencing reads, which span entire TE insertions, would allow patterns of nucleotide variation at synonymous and non-synonymous sites to be determined within copies of the same family, potentially highlighting the presence or absence of on-going selection on codon usage. However, choanoflagellate genome sequencing has yet to be undertaken with long read technologies, preventing a more in-depth exploration of on-going selection on codon usage.
Further work, on a broader range of unicellular eukaryotes, is required to determine if selection on TE codon usage is present outside of the choanoflagellates. The work of Jiang and Govers  on Phytophthora gypsy-like families mirrors the results here, with evidence consistent with selection on codon usage and stronger bias observed in high copy number families. The Phytopthora study did not however determine whether the codons preferentially utilized by high copy TE families were host optimal codons and also did not assess the role of mutation bias in TE codon usage. As unicellular species make up the majority of eukaryotic diversity , the past emphasis on multicellular species for TE research may have resulted in a clouded picture of how TE codon usage evolves within most eukaryotes. It is however clear that not all unicellular eukaryotes possess TEs that show selection on their codon usage, as no evidence for selection was found in the ORFs of the Ty elements of S. cerevisiae . The forces and host population biology that promote selection on TE codon usage may only be identified once the range of eukaryotes that exhibit such selection is identified. The transition to multicellularity in Metazoa may have resulted in a considerable reduction in Ne  and potentially also reduced the Ne of the TE families present in metazoan populations to a level where selection could no longer efficiently operate upon TE codon usage. Investigating TE codon usage at other unicellular/multicellular boundaries may allow the impact of host Ne upon TE codon usage to be evaluated.
The choanoflagellate S. rosetta harbours a diverse complement of TEs, which are mainly orthologous to families present within metazoan genomes. One DNA transposon family however appears to have been acquired by choanoflagellates via an ancient horizontal transfer event from a stramenopile. All identified families appear to have been active recently, however the sequenced strain of S. rosetta possesses one family no longer capable of transposition. The TEs of S. rosetta and a second choanoflagellate M. brevicollis show the clear signature of natural selection operating upon their codon usage. In contrast to previous findings in multicellular organisms, which have failed to find evidence for selection acting upon TE codon usage, the choanoflagellate TEs appear to be evolving under selection for translational efficiency and accuracy. Patterns of codon usage bias differ between DNA transposon, copia-like and gypsy-like families, with transposons showing weaker bias than retrotransposons. The use of host gene optimal codons is greater in highly expressed families and appears to provide a competitive advantage for some families enriched for optimal codons.
Identification of TE families in the S. rosetta genome
The 154 genomic supercontigs of S. rosetta deposited in the Origins of Multicellularity Project at the Broad Institute (https://www.broadinstitute.org/scientific-community/data/origins-multicellularity) were screened for TE sequences using two methodologies. The supercontigs were screened with the Protein Based RepeatMasker server hosted by the Institute for Systems Biology (http://www.repeatmasker.org/cgi-bin/RepeatProteinMaskRequest). Successful hits (≤E-05) for copia-like and gypsy-like LTR retrotransposon families; non-LTR retrotransposon families and DNA transposon families were recovered. Successful nucleotide hits were then translated into conceptual amino acid sequences and subjected to BLASTp analysis in order to confirm their identity as TE proteins. A second approach used the BLAST protocol and query sequences of Carr et al. . To identify putative MITE derivatives of DNA tranposon families, the supercontigs were screened with MITE-Hunter  and MITE Tracker . Both programs were used with default parameters.
The RepeatMasker and BLAST hits failed to recover full-length TE sequences. The Carr et al.  protocol of increasing consensus sequence coverage using overlapping sequencing reads from the NCBI Trace Archive, in this case for “Proterospongia sp. Atcc 50818” allowed the generation of full-length consensus sequences for all families, with the exception of two non-LTR retrotransposons. Consensus sequences for all full-length families are provided in Additional Data File 1. Individual families were defined on the basis of the 80–80-80 rule , showing less than 80% similarity over 80% of an aligned sequence covering a minimum of 80 bp. For retrotransposons, similarity was determined between LTRs, whilst for DNA transposons similarity was calculated between 5’ITR/UTRs. Srospv3A and Srospv3B were classified as subfamilies, rather than distinct families, as their LTRs share over 98% identity at the terminal 160 bp of the 3′ ends.
The St. diplocostata transcriptome assembly of Marron et al.  was screened with the Protein Based RepeatMasker server to identify putative TE sequences. Six contigs were recovered as potential copia-like sequences (Additional file 6) and aligned in MAFFT on the EMBI EBI server with default parameters  to produce the amino acid sequence used in the phylogenetic analyses. The St. diplocostata copia-like nucleotide sequences were translated using EMBOSS Transeq tool .
Datasets for superfamily phylogenies were created for all of the S. rosetta families by using translated amino acid query sequences of Transposase for DNA transposons, and Pol for LTR retrotransposons. The S. rosetta TE nucleotide consensus sequences were translated using EMBOSS Transeq. Sequence similarity searches of whole genome sequences were performed with BLAST (tBLASTn and BLASTp) through NCBI to identify closely related TE families in a diverse taxonomic range of eukaryotes (Alveolata, Amoebozoa, Apusozoa, Breviatea, Centroheliozoa, Cryptophyta, Rhodophyta, Stramenopiles, Choanoflagellatea, Ichthyosporea; Metazoa (screens divided into Deuterostomia, Gnathostomulida, Platyhelminthes, Protostomia, Cnidaria, Ctenophora, Mesozoa, Placozoa, Porifera); Fungi (screens divided into Blastocladiomycota, Chytridiomycota, Cryptomycota, Ascomycota, Basidiomycota, Entomophthoromycota, Glomeromycota, Microsporida, Neocallimastigomycota); Archaeplastida: (screens divided into Chlorophyta, Mesostigmata).
Recovered amino acid sequences were aligned using MAFFT, along with the choanoflagellate TE sequences. Alignments were manually edited by eye to reduce indel regions. Bayesian inference phylogenies were constructed using a mixed amino acid model with MrBayes 3.2.6 on XSEDE  using the CIPRES Science Gateway server . The MCMC analyses consisted of 5,000,000 generations, a sampling frequency of 1000, with a burnin value of 1250.
Maximum likelihood phylogenies were ran using raxmlGUI 1.5 beta . The ML and thorough bootstrap analysis were performed, using 100 parsimony starting trees and 1000 bootstrap replicates, using the PROTCAT model with estimated amino acid frequencies. The amino acid substitution matrix used for each family was taken from the output of the mixed model analysis from MrBayes.
LTR and ITR insertions for each TE family were identified using S. rosetta 5′ query sequences. Terminal sequences were downloaded from NCBI Trace Archive against ‘Proterospongia’ with a threshold of e-05. LTR sequences were used as queries for the retrotransposon families; for DNA transposons, 300 bp of the ITR and UTR sequences were used as query sequences. TSDs were identified to distinguish individual insertions; if identical 5′ and 3′ TSDs from the Trace Archive were present, the two termini of the same element could be identified. Partially sequenced LTRs were excluded from further analyses, as their category of element could not be determined. LTRs adjacent to internal TE DNA were labelled as being from full-length elements and LTRs flanked only by genomic DNA were labelled as solo LTRs.
TE family ML trees were generated using the raxmlGUI from 100 starting parsimony trees, using the GTRCAT model and supported with 1000 bootstrap replicates. MrBayes analysis was employed using the protocol stated for the amino acid datasets, although the GTR + I + Γ nucleotide substitution model was used.
Levels of nucleotide diversity for each TE family were calculated, using values of Nei’s π , with DnaSP version 5. 10. 01 . Values of π were calculated DNA transposon families by the analysis of 5′ ITR/UTR alignments, with a maximum length of 300 bp aligned in MAFFT. Values of π for LTR retrotransposon families were determined with individual alignments, generated with MAFFT, containing solo elements, individual FLEs and combined FLE and solo elements to produce All, FLE and Solo Phylip files.
Determining TE family expression levels
Raw Illumina RNA-Seq transcriptome reads (NCBI SRA files SRX042046-SRX042054 and SRX3432761-SRX3432762–74.8 million reads) were downloaded from NCBI and mapped to the TE gene sequences through SMALT v. 0.2.6 (https://www.sanger.ac.uk/science/tools/smalt-0). The families Srosgyp1, Sroscv2, Sroscv5 and SrosS contained repetitive regions in their full-length CDS, which were over-represented in comparison to other regions of the CDS in the SMALT output. As it is unclear if the repetitive regions are also present in non-TE regions of the S. rosetta genome they were excluded from the final estimation of TE gene expression (the edited sequences are shown in Additional file 12). The total number of reads for each family was calculated from the SMALT output SAM files in Tablet v.1.17.08.06 .
Analysis of codon usage bias in transposable element families
For each TE family in the S. rosetta genome values of Nc, Fop and GC3s were calculated for all coding sequences using CodonW 1.4.4 . The fop.coa file for S. rosetta was taken from Southworth et al. . Codons overlapping multiple regions of the TE, e.g. with LTR sequences as well as the overlapping gag pol regions of Sroscv4 and Sroscv5, were excluded from analyses. The GC content of non-coding DNA from each TE family was also determined in CodonW.
In order to assess any contribution of selection on translational accuracy, the codons which encode amino acids in functional domains were separated from those that encode non-domain amino acids into individual FASTA sequences. Domain regions were identified by analysing each Pol and Tnpase protein sequence in BLASTp. Fop values were then determined for the concatenated domain and concatenated non-domain codons in each family.
The authors wish to thank Jarosław Bryk for advice on the bioinformatics analyses undertaken in the research. We also thank two anonymous reviewers for comments on the initial submission of the manuscript.
MC, JS, CAG and AOM devised the research. MC, JS, CAG, AOM and NF undertook the analyses. MC and JS wrote the manuscript. All authors read and approved the final manuscript.
This work was supported by a Leverhulme Trust Postdoctoral Training grant for JS.
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The authors declare that they have no competing interests.
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