CR1 clade of non-LTR retrotransposons from Maculinea butterflies (Lepidoptera: Lycaenidae): evidence for recent horizontal transmission
© Novikova et al; licensee BioMed Central Ltd. 2007
Received: 24 November 2006
Accepted: 25 June 2007
Published: 25 June 2007
Non-long terminal repeat (non-LTR) retrotransposons are mobile genetic elements that propagate themselves by reverse transcription of an RNA intermediate. Non-LTR retrotransposons are known to evolve mainly via vertical transmission and random loss. Horizontal transmission is believed to be a very rare event in non-LTR retrotransposons. Our knowledge of distribution and diversity of insect non-LTR retrotransposons is limited to a few species – mainly model organisms such as dipteran genera Drosophila, Anopheles, and Aedes. However, diversity of non-LTR retroelements in arthropods seems to be much richer. The present study extends the analysis of non-LTR retroelements to CR1 clade from four butterfly species of genus Maculinea (Lepidoptera: Lycaenidae).
The lycaenid genus Maculinea, the object of interest for evolutionary biologists and also a model group for European biodiversity studies, possesses a unique, specialized myrmecophilous lifestyle at larval stage. Their caterpillars, after three weeks of phytophagous life on specific food plants drop to the ground where they are adopted to the ant nest by Myrmica foraging workers.
We found that the genome of Maculinea butterflies contains multiple CR1 lineages of non-LTR retrotransposons, including those from MacCR1A, MacCR1B and T1Q families. A comparative analysis of RT nucleotide sequences demonstrated an extremely high similarity among elements both in interspecific and intraspecific comparisons. CR1A-like elements were found only in family Lycaenidae. In contrast, MacCR1B lineage clones were extremely similar to CR1B non-LTR retrotransposons from Bombycidae moths: silkworm Bombyx mori and Oberthueria caeca.
The degree of coding sequence similarity of the studied elements, their discontinuous distribution, and results of divergence-versus-age analysis make it highly unlikely that these sequences diverged at the same time as their host taxa. The only reasonable alternative explanation is horizontal transfer. In addition, phylogenetic markers for population analysis of Maculinea could be developed based on the described non-LTR retrotransposons.
Non-long terminal repeat (non-LTR) retrotransposons are mobile genetic elements that propagate by reverse transcription of an RNA intermediate. These elements lack terminal repeats and utilize a simplified target-primed reverse transcription (TPRT) mechanism for their retrotransposition. During TPRT, the element-encoded endonuclease cleaves the genomic DNA, and reverse transcriptase (RT) uses this break to prime reverse transcription from the element's RNA. Resulting cDNA copy is then integrated into the target site [1, 2].
Based on their structure, non-LTR retrotransposons can be classified into two groups. The first group has a single open reading frame (ORF) that encodes RT in the middle and a restriction enzyme-like endonuclease (RLE) near its C-terminus. The second group of non-LTR retrotransposons has two ORFs: ORF1 and ORF2; the latter encodes two domains responsible for retrotransposition: apurinic/apyrimidinic endonuclease (APE)-like endonuclease domain at the N-terminus and RT domain in the middle.
The RT domain has been used to classify non-LTR retrotransposons into phylogenetic groups, or clades . Originally, 11 clades were distinguished among non-LTR retrotransposons. Later, the total number of clades increased to 16, with the addition of Genie , NeSL-1 , Ingi, Rex1 , and L2 clades . This number is likely to increase further since almost every detailed study of non-LTR retrotransposons brings additional information about their phylogeny and diversity [8–10]. For example, a new family encoding both RLE and APE endonucleases was described recently .
Non-LTR retrotransposons represent a large fraction of known retroelements in insects. At the same time, most of our knowledge on distribution and diversity of non-LTR retrotransposons from insects is limited to model organisms such as dipteran genera Drosophila, Anopheles, and Aedes [8, 11, 12]. Almost nothing is known about many other insect groups. Several studies attempted to test a wide range of arthropods for the presence of particular clades, e.g. distribution of R1 and R2 clades has been explored in detail in different arthropods .
Our recent study of scorpions (Arachnida) revealed an unexpected diversity of non-LTR retrotransposons within several clades in this ancient arthropod group, with at least three distant clusters in CR1 clade . Previously, only one family, T1Q-like elements, was described inside this clade from arthropods [3, 8, 11]. Studies of non-LTR retrotransposon diversity can provide a valuable contribution into understanding of evolution and spreading of mobile elements in arthropods.
The mode of retrotransposition and phylogenetic studies suggested that evolution of non-LTR retrotransposons proceeds mainly via vertical transmission and random loss of elements from a population . Horizontal transmission is believed to be a very rare event for this class of retrotransposons. Nevertheless, at least one well-documented case of horizontal transmission is known. Horizontal transmission of Bov-B elements from an ancestral snake lineage (Boidae) to the ancestor of ruminants has been confirmed on the basis of discontinuous distribution, extreme nucleotide sequence conservation, and phylogenetic analysis .
In the present study, we examined the diversity of CR1 clade of non-LTR retrotransposons in lycaenid butterflies of the genus Maculinea. Molecular systematic studies confirmed the existence of seven Maculinea species, most of them with several subspecies . Maculinea arion L., M. alcon (Denis et Schiffermüller), and M. teleius (Bergsträsser) are widely distributed from western Europe to East Asia, while M. nausithous (Bergsträsser) ranges from West Europe to Central Asia. M. cyanecula (Eversmann) is restricted to Central Asia, while M. arionides (Staudinger) and M. kurenzovi (Sibatani, Saigusa et Hirowatari) are found in East Asia [16–18]. In Europe, Maculinea are an object of interest of evolutionary biologists and model species for biodiversity studies since they are considered vulnerable or threatened [19–21] and also because of their fascinating biology. All Maculinea species are characterized by a specialized myrmecophilous lifestyle at larval stage. Their caterpillars, after three weeks of phytophagous life on specific food plants drop to the ground where they are adopted to the ant nest by Myrmica foraging workers. Larvae remain within the nest for a period of approximately 10 or 22 months, during which time they increase their body mass almost 100 times without moulting [22–24]. Three Maculinea species, M. teleius, M. nausithous and M. arion, are obligatory predators of the ant larvae. However, M. alcon, so-called "cuckoo" species, is a fully integrated social parasite and is fed by workers by trophic eggs and regurgitation .
Their unusual life history traits, such as parasitic life style, differentiated feeding strategies, exceptions to normal insect growth rules as an adaptation to long starvation period  and one or two-year mode of caterpillar's growth, make genus Maculinea a very interesting model for evolutionary studies, including studies of non-LTR retrotransposons diversity and evolution. Moreover, non-LTR retrotransposons could be very useful as molecular markers in intraspecific phylogeography of Maculinea.
We implemented broad analysis for CR1 group in an attempt to cover the diversity of CR1-like elements from Maculinea butterflies and evaluate the possibilities for development of phylogenetic markers. Distinct evolutionary lineages of the CR1-like non-LTR retrotransposons were identified, showing significant variation among CR1 clade elements.
Surprisingly, retroelements highly similar to one of the detected CR1 families have been recently identified in silkworm Bombyx mori. We reconstructed the complete element BmCR1B from B. mori using the genomic fragments available in databases. Extremely high similarity of B. mori and Maculinea elements was intriguing since these lepidopteran species are not closely related. We performed additional PCR screening of butterflies from distinct groups (suborder Ditrysia) to evaluate distribution of newly isolated families from CR1 clade. CR1A-like elements were found only in family Lycaenidae. In contrast, CR1B-like non-LTR retrotransposons were detected in Bombycidae moths: silkworm Bombyx mori and Oberthueria caeca. The degree of coding sequence similarity of the studied elements, their discontinuous distribution and results of divergence-versus-age analysis make it highly unlikely that these sequences diverged at the same time as their host taxa. Thus, new evidence was obtained for horizontal transmission of non-LTR retrotransposons.
Results and discussion
Multiple lineages of CR1-like retroelements represent in Maculinea
Maculinea species used in this study, lineages detected in CR1 clade, and GenBank accession numbers of isolated clones.
No. of clones with RT
In total, 52 clones were obtained for CR1 clade and sequenced (Table 1). After preliminary identification of RT fragments by comparison with sequences in the GenBank database, we found that 49 clones showed clear similarity with RT domain of non-LTR retrotransposons, while three sequences revealed no presence of RT. The total number of clones with RT for each species is listed in Table 1.
The BLAST search showed a strong similarity of translation products of those clones that contained RT to the elements described from CR1 clade. In general, the designed PCR primers for CR1 proved to be suitable for isolation of an RT domain from Maculinea. However, two sequences unexpectedly showed clear similarity to the elements belonging not to CR1 but to R1 clade of non-LTR retrotransposons. These sequences were named MnaR1 (M aculinea na usithous R1 element) [GenBank:DQ836391] and MalR1 (M aculinea al con R1 element) [GenBank:DQ836392]. Representatives of R1 clade appear to play a very important role in telomere maintenance in insects [27, 28]. For example, TRAS1 and SART1 elements from Bombyx mori are site-specific for telomeric repeats . Two isolated Maculinea elements shared more than 57 % homology in amino acid sequence and were highly similar to R1-like elements from other insects. Similarity between amino acid sequences of MnaR1 and RT1 element from Anopheles gambiae  was 51 %. Amplification of R1 elements using CR1 pair of primers could possibly be a result of primer degeneracy.
The majority of detected CR1-like elements revealed defective translated products (29 clones). In total, 18 elements represented putatively intact RT sequences. Of 29 defective clones, 15 has a single stop mutation and no frameshifts, while 14 clones contained variable-length indels and resulted in frameshifts.
Lineage A of CR1-like elements from Maculinea – MacCR1A family
In total, 29 clones from all four studied species formed this lineage in NJ analysis based on nucleotide sequences of RT domain (Figure 1). Comparative analysis of RT nucleotide sequences showed an extremely high similarity among elements both within and between species. Average intraspecific sequence similarity ranged from 98.6 % in M. alcon to 99.5 % in M. teleius. Less than 2 % of intraspecific divergence was detected in M. nausithous. Three of eight clones isolated from M. alcon appeared to be absolutely identical (clones MalCR1-4, 5, and 7 - M aculinea al con CR1 clones 4, 5, and 7).
The potential translation products of the sequences were determined. Eleven clones contained intact RT sequences and could represent the master sequences of elements with RT activity. Eight of isolated elements had stop mutations but no frameshifts. The remaining ten clones contained indels in comparison with the consensus sequence. Indels resulted in frame-shifts in five elements. All indels seems to be unique for a specific element copy since single copies were detected for each case of insertion or deletion.
Kimura-corrected distances were calculated within and between species using 14 selected copies (only one copy was used for the identical MalCR1-4, 5, and 7). The average distance was 3.1% within M. teleius, 2.7% within M. nauthitous, and 3.6% within M. alcon. Interspecific distances ranged from 3.1% between M. teleius and M. nausithous to 3.6% between M. teleius and M. alcon. These low Kimura-corrected distances indicate relatively recent retrotransposition events for Lineage A elements.
Phylogeny of Maculinea based on nucleotide sequences of CR-1-like elements was not congruent with the known phylogeny of host species (Figure 1). Moreover, no relationships could be reconstructed within Lineage A elements. Altogether, 100 variable sites per 553 bp were detected among aligned nucleotide sequences, and 49 variable amino acid residues per 184 sites of amino acid sequence alignment. However, only six variable sites were parsimoniously informative at DNA level, and only two, at amino acid level. Other variable sites were represented by singletons and were non-informative for phylogenetic analysis.
Lineage B of CR1-like elements from Maculinea – MacCR1B family
Lineage B of CR1-like elements from Maculinea butterflies was represented by 14 clones. The phylogenetic tree based on NJ analysis of nucleotide sequences showed a strong support for separation of this lineage from elements of the Lineage A (bootstrap 100 %). However, the topology within Lineage B was not supported.
A comparative analysis of nucleotide and amino acid sequences within the Lineage B demonstrated high similarity both within and among species. Two clones from M. teleius (MteCR1-12 and MteCR1-16) appeared to be identical; both carried 11 bp insertion and 40 bp deletion. One more clone MteCR1-7 contained 17 bp insertion and 32 bp deletion. The remaining sequences had no indels; however, two of them (MteCR1-17 and MnaCR1-11) represented interrupted translated products. Six sequences seemed to be the copies of putatively intact retrotransposons, with the average genetic distance less than 2 %. The most distant copy (MteCR1-6) showed nucleotide sequence divergence 4.1 %.
For phylogenetic analysis, we selected eight copies without compensatory frameshift mutations. Nucleotide and amino acid sequences were aligned. Variable and parsimoniously informative sites were determined for both alignments. As in MacCR1A lineage, number of parsimoniously informative sites was very low. Among 66 variable sites per 547 bp of sequence only eight were informative for phylogenetic analysis while 58 substitutions were copy-specific. Number of sites with variable amino acid residues was less than in the Lineage A, only 36 sites per 182 amino acid residues. Four parsimoniously informative sites were detected, compared to two in lineage A; such low number of informative sites does not allow meaningful phylogeny reconstruction among lineage B. Low Kimura-corrected distances within and among species (average 3.3% in lineage B) suggest a relatively recent retrotransposon activity.
MacCR1A and MacCR1B elements were only 73.5 % similar at DNA level but 88.5 % similar at amino acid level.
Lineage C – T1Q-like non-LTR retroelements from Maculinea
Only four retroelements from lineage C were isolated from two species: M. teleius and M. nausithous (Figure 1). MnaCR1-6 and MnaCR1-7 showed 100 % similarity, whereas MteCR1-1 and MteCR1-2 differ in length and contain two substitutions per 411 bp of common sequence. MteCR1-1 sequence is 546 bp long; MteCR1-2 is truncated at 5' portion and 411 bp long. Translated products of MteCR1-2 and MteCR1-1 contain no stop mutations or frameshifts and seem to be putatively intact. MnaCR1-6 and MnaCR1-7 clones contained not only stop codons but also frameshifts. We reconstructed the reading frame for MnaCR1-6/7 element. Comparison of amino acid sequences showed 66.3 % similarity between MteCR1-1 and reconstructed MnaCR1-6/7, but only 55.9 % similarity was found at the DNA level.
Elements from MacCR1C lineage share high similarity with T1 and Q elements and seem to belong to the T1Q lineage [3, 31, 32]. The amino acid sequence similarity ranged from 53.3 % between MnaCR1-6/7 and Q element to 54.7 % between MteCR1-1 and Q element; the isolated elements were less than 50% similar to the CR1 retrotransposon from chicken Gallus gallus . The T1Q lineage was detected from genomes of two out of four studied species of Maculinea. One element was isolated from M. nausithous, and two closely related copies, from M. teleius. Small fraction of T1Q-like elements among isolated clones could be due to a very low copy numbers of this lineage in the studied species. It could be also a consequence of high divergence among the elements in the sequences annealing to PCR primers.
BmCR1B element from B. mori
The nucleotide and amino acid sequences of MacCR1A and MacCR1B elements were used as queries in BLAST search against databases for arthropods. No species among those available in databases gave the positive match except of silkworm Bombyx mori. MacCR1B was found to be present in B. mori genome. Surprisingly, CR1B elements from Maculinea and B. mori showed extremely high sequence similarity.
BmCR1B element could be almost completely reconstructed by fusion of two contigs: Ctg011054 [GenBank:AADK01011054] and Ctg006777 [GenBank:AADK01006777]. The fragment Ctg011054 lacks approximately 400 bp of element at the 3' end. The second contig, Ctg00677, represents almost entire element BmCR1B but lacks 400 bp at 5' end. Other contigs contain incomplete sequences of BmCR1B retroelement to a greater extent, either due to their position at a contig border (Ctg034636; Ctg022133; Ctg011753; Ctg028411; Ctg016714) or to insertion of sequences of an unknown origin (Ctg010781 and Ctg004626).
Distribution of CR1B and CR1A families in other butterflies
We designed specific PCR primers for both discovered families (MacCR1A and MacCR1B) and screened a selection of species from other related groups of Lepidoptera. Primers were based on sequences isolated from Maculinea spp. and BmCR1B element (Figure 4) and tested by PCR and sequencing in all four species of Maculinea. The primers annealed specifically either to MacCR1A or to MacCR1B template.
List of Lepidoptera species used in present study in addition to Maculinea spp. Results of PCR screening with degenerate primers for CR1 clade, and specific primers for CR1A and CR1B families.
In total, 17 species were used in the further PCR screening in addition to Maculinea spp. (Table 2). We also performed PCR with original degenerate CR1 primers (CR1-S and CR1-A) to demonstrate presence of CR1 clade in all studied species. All specimens gave us the positive signals with the original CR1 primers (Figure 5). The products with the expected size, 550 bp in length, were detected by electrophoresis in all reactions. Thus, we can assume that all studied species contain CR1 clade elements in their genomes.
PCR screening with specific primers demonstrated presence of the CR1A family in all Lycaenidae but its lack in all other studied species. PCR products of the expected size were detected in Scolitantides orion, Shijimaeoides divina, and Plebejus argus. The genera Scolitantides and Shijimaeoides are the closest relatives of Maculinea. It appears that CR1A family is found in Lycaenidae, but is absent from other butterflies even in closely related families Pieridae (Pieris napi), Nymphalidae (Araschnia levana and Melitaea phoebe), and Satyridae (Erebia theano). On the other hand, CR1A-like elements could further diverge in other species to the extent that designed primers would be not appropriate for their detection.
Amino acid divergences of genes and CR1 non-LTR retrotransposons from B. mori and M. teleius.
Amino Acid Divergence (%)
Bombyx versus Maculinea
Bombyx versus Maculinea
Bombyx versus Maculinea
CR1 RT non-LTR retrotransposons
BmCR1B Bombyx versus CR1B Maculinea
BmCR1B Bombyx versus CR1A Maculinea
BmCR1B Bombyx versus OcaCR1B Oberthueria
OcaCR1B Oberthueria versus CR1B Maculinea
OcaCR1B Oberthueria versus CR1A Maculinea
PargCR1A Plebejus versus CR1A Maculinea
SoriCR1A Scolitantides versus CR1A Maculinea
ShdivCR1A Shijimaeoides versus CR1A Maculinea
Positive results in PCR reaction with CR1B primers were observed for Maculinea and representatives of the family Bombycidae: O. caeca and B. mori (Figure 5). The CR1B products were cloned and sequenced. Surprisingly, the CR1B elements from closely related O. caeca (OcaCR1B) and B. mori (BmCR1B) showed higher level of amino acid sequence divergence than CR1B retrotransposons from Maculinea and B. mori (Table 3). Taxa which are most closely related to Maculinea, S. orion and Sh. divina, did not demonstrate a presence of this family in PCR screening (Figure 5). The discontinuous distribution, high sequence similarity between MacCR1B and BmCR1B elements from divergent species and lower similarity between BmCR1B and OcaCR1B elements from closely related species could be the evidences for horizontal transmission for non-LTR retrotransposons from CR1B family.
Evidence of horizontal transmission
Most examples of horizontal gene transfer in eukaryotes involve transposable elements [35, 36]. Such transfers are usually recognized by the presence of very closely related elements in distantly related host taxa. Evolutionary dynamics of transposable elements could sufficiently differ even in closely related taxa. As a result, phylogenetic studies of mobile elements often show incongruence with host species phylogenies. First, transposable elements are multicopy components of genomes. Thus, comparisons of paralogous copies of these elements instead of orthologs along with varying rates of sequence evolution of mobile elements copies are the main sources for phylogenetic incongruence, which could be misidentified as horizontal transmission. Second, random loss of transposable elements from a few taxa, ancestral polymorphism and independent assortment of copies into descendant species, and unequal DNA substitution rates in different species all can lead to incongruence in phylogenetic reconstruction.
Traditionally, horizontal transfer has been implied when highly similar transposable elements have been found in distantly related taxa accompanied by their discontinuous distribution, and such phenomenon could not be explained in terms of vertical inheritance [37–39].
By comparing the amino acid sequence divergence for host genes and transposable elements evolving under presumably similar selective pressure the case of horizontal transmission could be detected if divergence among mobile elements is significantly lower than that observed for the proteins encoded by host genes. We compared the partial amino acid and nucleotide sequences of the elongation factor 1 alpha (EF-1α) gene from Maculinea spp. and B. mori since only this nuclear gene from Maculinea was available in databases. It is necessary to note that EF-1α gene is highly conserved in amino acid sequence and is usually used for reconstruction of high-level phylogenies, especially in insects [40–42].
The EF-1α gene comparisons demonstrated almost the same level of amino acid divergence as the mobile elements from CR1B family, 2.8% for EF-1a genes and 2.7% for CR1B elements (Table 3). The nucleotide sequences of EF-1α gene had much lower similarity to each other. Only 83% similarity was detected for EF-1α gene whereas more than 96% similarity showed BmCR1B and MacCR1B non-LTR retrotransposons at DNA level. It seems unlikely that 96% nucleotide and 98.3% amino acid sequence similarity could be selectively or otherwise maintained in retrotransposons that diverged from a common ancestor.
The amino acid sequences of mitochondrial cytochrome oxidase subunit I (COI) and subunit II (COII) genes also were compared among studied species. It seems that RT of CR1B elements is considerably more conserved than compared mitochondrial proteins. We found 14.4% and 15.3% divergence at the amino acid level in COI and COII genes, respectively and only 2.7% average divergence between RT BmCR1B and MacCR1B elements (Table 3).
Amino acid divergences and evolutionary rates in the CR1 and Jockey clades.
LCA (divergence time in MYA)
Amino Acid Divergence (%)
Evolutionary rate (10-9)
Q Anopheles versus worf Drosophila
T1 Anopheles versus worf Drosophila
BmCR1B Bombyx versus CR1B Maculinea
SoriCR1A Scolitantides versus CR1A Maculinea
BmCR1B Bombyx versus OcaCR1B Oberthueria
MteQ M. teleius versus Q Anopheles
Jockey D. melanogaster versus Jockey D. funebris
AMY B. mori versus Helena D. mauritiana
JuanA Aedes versus Jockey D. melanogaster
NLR1Cth Chironomus versus Jockey D. melanogaster
NLR1Cth Chironomus versus AMY B. mori
Overall, our data clearly indicate that the high degree of similarity we observed between the Bombyx mori BmCR1B and Maculinea CR1B is not due to selection. The only reasonable alternative explanation is horizontal transfer.
Horizontal transmission in non-LTR retrotransposons
Horizontal transfer can be defined as the process by which genes can move between reproductively isolated species. It is not surprising that many examples of horizontal transfer of transposons have been identified in eukaryotes. There are several features of transposon behavior that make them particularly prone to horizontal transfer. Transposable elements have the capacity to insert themselves into the chromosomes of possible vectors and, subsequently, into host chromosomes. Subsequent to transfer, they can spread rapidly throughout a given species, as is evidenced by the rapid spread of P elements in D. melanogaster . Most studied cases of horizontal transmission involve DNA transposons [38, 39].
Among retrotransposons, several examples of horizontal transfer have been documented between closely related species (mostly among Drosophila) for LTR retrotransposons such as copia [44, 45] and gypsy . The possible mechanism of horizontal transfer of LTR retrotransposons could be their ability to ride along on cross-species viral infections. The presence of envelope-like coding sequences makes some of the LTR retrotransposons capable of virus-like particle formation.
In contrast, non-LTR retrotransposons are believed to be inherited exclusively vertically. Extensive phylogenetic and comparative studies dismissed majority of putative horizontal transfer reports for non-LTR retrotransposons . Nevertheless, strong evidence of horizontal transmission that cannot be neglected, were provided by Zupunski et al. (2001)  for Bov-B retroelements which have been relatively recently transferred from the ancestral snake lineage (Boidae) to the ancestor of ruminant mammals [15, 47]. Thus, one should not exclude a possibility of occasional horizontal transmission events in non-LTR retrotransposons.
The compared Bov-B elements from Squamata and Ruminantia (LCA 310 Mya) showed the same divergence at the amino acid level as that between Mus and Rattus L1 elements (LCA 15 Mya). The divergence rate in Squamata versus Ruminantia Bov-B was found to be very low in comparison with other elements from the same clade .
The slowdown effect on evolutionary rates was also observed in our study. We estimated sequence divergence rates for non-LTR retrotransposons from insects, including those for CR1 and Jockey clades (Table 4). The divergence rates appeared to be almost the same in all comparisons with exception of drosophilid Jockey elements and CR1B elements from butterflies. However, while in the case of Jockey elements from Drosophila melanogaster and D. funebris the rate was much higher in comparison with other pairs, CR1B retrotransposons from B. mori and Maculinea spp. showed significantly low rate of divergence. It was much lower than for the elements from closely related species: SoriCR1A from Scolitantides and CR1A from Maculinea; BmCR1B from Bombyx and OcaCR1B from Oberthueria (Table 4).
The actual mechanisms of horizontal transfer are still unknown for mobile elements from eukaryotes since it is not possible experimentally to show how the horizontal transmission can occur. Parasites, symbionts, bacteria, or viruses all could be suggested as potential vectors for horizontal transmission. However, only those putative vectors could be under consideration, which have wide host range and access to germ line cells . Since horizontal transfer detected in present study occurred within butterflies and moths, all viruses and parasites common for Lepidoptera could be suspect as possible vectors.
The direction of CR1B retroelements horizontal transfer is also under question. Since it is unlikely that horizontal transfer could occur simultaneously and independently to two distantly related taxa, those elements could hardly be transmitted recently to B. mori and genus Maculinea from an unknown species. On the other hand CR1B elements were detected not only in B. mori, but also in other representative of Bombycidae family – O. caeca. It is highly possible that one of the moths from family Bombycidae was a source of the CR1B elements for a common ancestor of Maculinea species.
CR1A and CR1B as potential molecular markers
Insertion events of retrotransposable elements have been recently demonstrated to be powerful tools in phylogenetics and population genetics studies in various organisms [49–51]. Integration of a non-LTR retrotransposon to a new place is an irreversible event. Non-LTR retrotransposons, once inserted in chromosomal DNA, appear to be fixed. Since retrotransposition is thought to be more or less random with respect to the region of insertion, insertions at exactly the same location appear to be unlikely. The most popular transposon-based marker method is the Sequence-Specific Amplification Polymorphism approach (S-SAP), also called "transposon display" . The S-SAP markers were developed for wide range of taxa, in particular in plants [52, 53], insects , and fungi .
CR1A and CR1B non-LTR retrotransposons from Maculinea species showed very high homogeneity among studied species, and thus cannot be used for inferring interspecific phylogenies; however, these elements could be used as markers in population genetics studies.
Sequences with extremely high similarity to Maculinea were identified in genome of silkworm Bombyx mori. A non-LTR retrotransposon BmCR1B belonging to CR1B lineage was reconstructed based on short fragments available in databases. The distribution of CR1A and CR1B families was studied among 17 species of Lepidoptera from nine superfamilies. Only representatives of the family Lycaenidae gave positive signal in PCR amplification for the CR1A lineage. None of the studied species showed presence of CR1B family, except Maculinea and two moths from family Bombycidae: Oberthueria caeca and Bombyx mori.
The studied MacCR1B and BmCR1B elements are considerably more conserved than the mitochondrial proteins and have the same level of conservation as EF-1α protein, one of the most conserved proteins in eukaryotes. In summary, the data are consistent with horizontal transfer of CR1B elements between species of Lepidoptera at some point well after the divergence of Bombycoidea and Papilionoidea. The degree of similarity of coding sequences in these elements, their discontinuous distribution, and the results of divergence-versus-age analysis, make it highly unlikely that these sequences diverged at the same time as their host taxa. A presence of the CR1B family in two closely related species from family Bombycidea (Oberthueria caeca and Bombyx mori) allowed us to suppose that horizontal transmission occurred from Bombycidae to the common ancestor of Maculinea species. This study presents a new example of horizontal transmission of non-LTR retrotransposons from CR1 clade.
Species collection and total DNA isolation
Table 1 lists Maculinea species used in the present study. M. teleius, M. nausithous, and M. arion specimens originated from southern Poland. M. alcon specimens originated from western Siberia (Novosibirsk, Russia). Taxonomy is given after Als et al. (2004)  and Bereczki et al. (2005) . Live Maculinea larvae were collected in nature and preserved in 96 % ethanol. Species identification of larval stages was performed using the morphology key by Śliwińska et al. (2006) . Total DNA was isolated from several larvae.
Table 2 lists Lepidoptera species from suborder Ditrysia used in present study and their taxonomy. All material was kindly provided by Dr. Oleg Kosterin (Institute of Cytology and Genetics, Novosibirsk) and Dr. Vladimir Dubatolov (Institute of Animal Systematics and Ecology, Novosibirsk). All listed Lepidoptera, except Maculinea spp. and Bombyx mori, were collected in nature and stored at -40°C. Detailed label data are available from the authors. Genomic DNA was isolated from the thorax and head of two to four individuals for all species except Plebejus argus, Calidula sp. and Oberthueria caeca. Total DNA extraction and PCR amplification have been done according to standard techniques . For B. mori, the DNA of strain Dazao was used in present study.
CR1 clade PCR amplification and sequencing
Degenerate PCR primers for CR1 clade of non-LTR retrotransposons were designed by inspection of conserved amino acid sequences in the reverse transcriptase (RT) domains of different published non-LTR retroelements. Primer sequences were: CR1-S= 5'-TATCTTCTTCTCCnggnccngaygg-3' and CR1-A= 5'-CAAAAACACTGCCytgnggnacncc-3', where Y = C + T, and N = A + G + C + T. The designed pair of primers flanked the sequence between conserved regions 0 and 4 of RT domain [3, 58]. The length of expected PCR products was about 550 bp.
PCR amplification with degenerate primers was performed using 0.1 μg of genomic DNA in 10-μl volume of 10 mM Tris-HCl (pH 8.9), 1 mM (NH4)2SO4, 4 mM MgCl2, 200 μM each of four dNTPs, 0.5 μM primers, and 2.5 units of Taq polymerase. After an initial denaturation step for 3 min at 94°C, the PCR reactions were subjected to 30 cycles of amplification consisting of 30 sec denaturation at 94°C, 42 sec annealing at 52°C, and 1 min extension at 72°C. PCR results were assayed by agarose gel electrophoresis.
The resulting PCR products were directly ligated into a pGEM vector using a pGEM-T-easy cloning kit (Promega) for sequence determination. Clones were amplified by PCR with M13 primers, and 40 ng of the product was used in a 10 μl cycle sequencing reaction with the ABI BigDye Terminator Kit on an ABI 377 DNA sequencer. Sequences were deposited to GenBank under accession numbers [GenBank:DQ822995-DQ823039], [GenBank:DQ836362 - DQ836391] and [GenBank:DQ994657 - DQ994665] (Table 1).
BmCR1B retrotransposon reconstruction
Nucleotide and protein sequence searches of the GenBank databases with MacCR1A and MacCR1B were performed with BLAST  search programs of the NCBI . The following sequence databases were searched at NCBI: nonredundant (NR) and Bombyx mori WGS database for Dazao  and p50T strains . BmCR1B element sequence was reconstructed by assembling of partial sequences from different contigs (Figure 2). The sequence of the entire BmCR1B element could be found in Supplementary Material (Additional file 2), as well as the list of contigs used for reconstruction (Additional file 3).
Based on the sequence of reconstructed BmCR1B element, we designed PCR primers with the following sequences: BmCR1B-S1 5'-CCCTTTCCTTCCCCACCCC; BmCR1B-S2 5'-TAGCACCTGCGCTGACTCGG; BmCR1B-S3 5'-GGTTCATTGGCATATTCCGC; BmCR1B-S4 5'-GCGGAACGGAGAGTTAAAGTCG; BmCR1B-A1 5'-TCTTCCACATCCGGCACACG; BmCR1B-A2 5'-GTCCAGAGTCGAGTTTCCCGC; BmCR1B-A3 5'-CGCAGTGCCAAAGGATCTAGC; BmCR1B-A4 5'-GAGGCAAACGAGCAAGACGGG.
PCR amplification with designed pairs of primers was performed using 0.1 μg of M. teleius genomic DNA in 10-μl volume of 10 mM Tris-HCl (pH 8.9), 1 mM (NH4)2SO4, 2 mM MgCl2, 200 μM each of four dNTPs, 0.5 μM primers, and 2.5 units of Taq polymerase. After an initial denaturation step for 3 min at 94°C, the PCR reactions were subjected to 30 cycles of amplification consisting of 30 sec denaturation at 94°C, 42 sec annealing at 54°C, and 1 min extension at 72°C. PCR results were assayed by agarose gel electrophoresis (Figure 6).
CR1A and CR1B families PCR screening
Based on newly isolated MacCR1A, MacCR1B and BmCR1B elements, specific primers for PCR were designed for each family. The location of primers is shown on Figure 4. Sequences of primers were: for MacCR1A CR1A-S 5'-TCTTTTCGGTTCTCCTACTC and CR1A-A 5'-GCATCAACAGACTGAGTTTCCGAG; for MacCR1B CR1B-S 5'-CTTATTCAGACACTCTTATGTCC and CR1B-A 5'-GAGCATGAGCCGTCTACGA PCR amplification with CR1A and CR1B pairs of primers was performed using 0.1 μg of genomic DNA in 10-μl volume of 10 mM Tris-HCl (pH 8.9), 1 mM (NH4)2SO4, 1.5 mM MgCl2, 200 μM each of four dNTPs, 0.5 μM primers, and 2.5 units of Taq polymerase. After an initial denaturation step for 3 min at 94°C, the PCR reactions were subjected to 30 cycles of amplification consisting of 30 sec denaturation at 94°C, 42 sec annealing at 52°C, and 1 min extension at 72°C. PCR results were assayed by agarose gel electrophoresis.
The resulting PCR products were directly ligated into a pGEM vector using a pGEM-T-easy cloning kit (Promega) for sequence determination. Clones were amplified by PCR with M13 primers, and 40 ng of the product was used in a 10 μl cycle sequencing reaction with the ABI BigDye Terminator Kit on an ABI 377 DNA sequencer. Sequences were deposited to GenBank under accession numbers [GenBank:EF591634], [GenBank:EF592105], [GenBank:EF592106], [GenBank:EF592107 ], [GenBank:EF592108].
Sequence and phylogenetic analysis
Multiple DNA sequence alignment was performed by ClustalW  and used for phylogenetic tree construction based on the neighbor-joining (NJ) method, implemented in MEGA 3.0 . Pairwise distances were estimated under Kimura's two parameter model by MEGA 3.0. The amino acid sequences of newly identified RT and RT from GenBank were aligned using ClustalW  and edited manually. Phylogenetic analysis was carried out using the NJ method in MEGA 3.0 program  and Maximum Likelihood (ML) optimality criteria in PHYML algorithm . For the ML analysis, the appropriate substitution model was calculated using ProTest program, version 1.3 . The best evolutionary models for the analyzed amino-acids data set were rtREV based on the Akaike Information Criterion (AIC)  and Blosum62 based on Bayesian Information Criterion (BIC) . Statistical support for the trees was evaluated by bootstrapping: 1000 replications for NJ and 100 replications for the ML .
Evolutionary rates were estimated by standard methods . Poisson correction distances (d) were estimated from the equation d = -ln(1 - p), where p represents the proportion of different amino acids. The rate of amino acid substitution (r) was estimated by the standard equation r = d/2T, where T is the divergence time of the last common ancestor (LCA) of the compared species. Amino acid distances used in divergence-versus-age analysis were calculated from sequences of the partial RT domain (~300 aa) using MEGA 3.0 .
long terminal repeat
target-primed reverse transcription
open reading frame
restriction enzyme-like endonuclease
untranslated terminal region
elongation factor 1 alpha
oxidase subunit I
oxidase subunit II
last common ancestor
million years ago
maximum parsimony method
method of maximum likelihood
Sequence-Specific Amplification Polymorphism
We thank Dr. Oleg Kosterin and Dr. Vladimir Dubatolov for providing the material of butterflies. This study was financed by EC within its RTD project EVK2-CT-2001-00126 and EVK2-CT-2002-80009 as well as by Polish Ministry of Science within its project SPUB-3024. We declare that all the experiments in this study were performed in Poland and Russia and comply with current Polish and Russian law.
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