Vertical Evolution and Horizontal Transfer of CR1 Non-LTR
Retrotransposons and Tc1/mariner DNA Transposons in
Lepidoptera Species
Irina Sormacheva,*,1 Georgiy Smyshlyaev,1 Vladimir Mayorov,2 Alexander Blinov,1 Anton Novikov,1 and
Olga Novikovaz,1
1
Department of Molecular Genetic Systems, Institute of Cytology and Genetics, Novosibirsk, Russia
Department of Basic Medical Sciences, Mercer University School of Medicine
z
Present address: Department of Biological Sciences, University at Albany
*Corresponding author: E-mail: [email protected].
Associate Editor: Norihiro Okada
The sequences of CR1 non-LTR retrotransposons; MLEs; and full-length Macmar1, Macmar3, MacmarY, and BhmarY elements
from Maculinea and Bombyx, as well as chimeric CR1B/MLE elements from Maculinea, were deposited in GenBank (accession nos.
JQ580972–JQ580974). Sequences of CR1 non-LTR retrotransposons were deposited in GenBank under accession numbers
HQ284259–HQ284570. The sequences of these MLEs were deposited in GenBank under accession numbers HQ606331–HQ606343,
HQ606345–HQ606368, HQ606371–HQ606381, HQ606402, HQ606410, and HQ606415–HQ606462. The sequences of full-length
Macmar1, Macmar3, and MacmarY elements from Maculinea were deposited in GenBank under accession numbers HQ606330,
HQ606344, HQ606387–HQ606390, HQ606392–HQ606396, HQ606399, HQ606401, HQ606405–HQ606409, and HQ606411–
HQ606414. The sequences of chimeric CR1B/MLE elements from Maculinea were deposited in GenBank under accession numbers
JQ580972–JQ580974.
2
Horizontal transfer (HT) is a complex phenomenon usually used as an explanation of phylogenetic inconsistence, which cannot
be interpreted in terms of vertical evolution. Most examples of HT of eukaryotic genes involve transposable elements. An
intriguing feature of HT is that its frequency differs among transposable elements classes. Although HT is well known for DNA
transposons and long terminal repeat (LTR) retrotransposons, non-LTR retrotransposons rarely undergo HT, and their phylogenies are largely congruent to those of their hosts. Previously, we described HT of CR1-like non-LTR retrotransposons between
butterflies (Maculinea) and moths (Bombyx), which occurred less than 5 million years ago (Novikova O, Sliwinska E, Fet V,
Settele J, Blinov A, Woyciechowski M. 2007. CR1 clade of non-LTR retrotransposons from Maculinea butterflies (Lepidoptera:
Lycaenidae): evidence for recent horizontal transmission. BMC Evol Biol. 7:93). In this study, we continued to explore the diversity
of CR1 non-LTR retrotransposons among lepidopterans providing additional evidences to support HT hypothesis. We also
hypothesized that DNA transposons could be involved in HT of non-LTR retrotransposons. Thus, we performed analysis of one of
the groups of DNA transposons, mariner-like DNA elements, as potential vectors for HT of non-LTR retrotransposons. Our
results demonstrate multiple HTs between Maculinea and Bombyx genera. Although we did not find strong evidence for our
hypothesis of the involvement of DNA transposons in HT of non-LTR retrotransposons, we demonstrated that recurrent and/or
simultaneous flow of TEs took place between distantly related moths and butterflies.
Key words: Tc1/mariner DNA transposons, Lepidoptera, phylogeny, horizontal transfer.
Introduction
Horizontal transfer (HT) is a complex phenomenon generally
used to explain the inconsistencies of phylogeny of the same
species, reconstructed on the basis of different markers
(Shields and Sharp 1989; Capy et al. 1994; Powell and
Gleason 1996; Clark and Kidwell 1997). HT is considered an
important part of the evolutionary process of prokaryotes;
however, it can also occur between eukaryotic species (Wenzl
et al. 2005; Slot and Hibbett 2007; Khaldi et al. 2008; Alsmark
et al. 2009; Andersson 2009). Although the majority of known
cases of HT of eukaryotic genes are due to the activity of
transposable elements (Kidwell 1992; Hamada et al. 1997;
Hartl et al. 1997; Kordis and Gubensek 1998; Malik et al.
1999; Volff et al. 2000; Sanchez-Gracia et al. 2005; Novikova
et al. 2007; Novikova et al. 2009), HT of functional genes in
eukaryotes is also possible (Khaldi et al. 2008; Richards et al.
2009; Slot and Rokas 2011).
One of the main properties of the transposable elements
(TEs) is their ability to transpose within their host’s genome.
This property allows TEs to overcome the barrier between
species and to participate in the process of HT. To date, many
cases of HT of TEs have been reported in a variety of taxonomic groups of eukaryotes, including plants, animals,
fungi, and protozoa (Hamada et al. 1997; Volff et al. 2000;
ß The Author 2012. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
Mol. Biol. Evol. 29(12):3685–3702 doi:10.1093/molbev/mss181 Advance Access publication July 23, 2012
3685
Research article
Abstract
MBE
Sormacheva et al. . doi:10.1093/molbev/mss181
Sanchez-Gracia et al. 2005; Novikova et al. 2007; Novikova
et al. 2009).
The mechanism of HT is still unknown. This is primarily
due to the lack of opportunities for experimental studies of
this phenomenon. Viruses, symbiotic microorganisms (especially intracellular), and other organisms can act as potential
vectors of HT (Silva and Kidwell 2000; de Almeida and
Carareto 2005). Apparently, for each described case of the
proposed HT, its own model of transfer could be implemented. The different groups of TEs show substantial variety
in the potency of HT (Kordis and Gubensek 1999; Volff et al.
2000; Zupunski et al. 2001; Novikova et al. 2007; Novikova
et al. 2009; Kojima et al. 2011). Thus, for Drosophila, approximately 96% of all possible cases of HT were associated with
long terminal repeat (LTR) retrotransposons and DNA transposons, compared with only 4% with non-LTR retrotransposons (Loreto et al. 2008).
Differences in the mechanisms of formation of a new copy
between groups of TEs most probably affect the abovementioned ratio. In contrast to both DNA transposons and
LTR retrotransposons, non-LTR retrotransposons do not have
extrachromosomal copies, and the reverse transcription of
these elements with the formation of new copies is carried
out in the nucleus, directly at their integration sites. Therefore,
their transfer outside of the cell nucleus is significantly decreased (Malik et al. 1999; Malik and Eickbush 2001).
Previously, it was assumed that non-LTR retrotransposons
could not be horizontally transferred because of restrictions
imposed by the mechanism of their transfer. Nevertheless,
several cases of proposed HT are well documented today:
1) HT of Bov-B elements between ruminant animals and
snakes (Kordis and Gubensek 1998; Zupunski et al. 2001);
2) multiple events of HT of various non-LTR retrotransposons
between closely related species of the genus Drosophila have
been described with the use of bioinformatic methods
(Sanchez-Gracia et al. 2005); 3) Tad elements probably have
been transferred between fungi of the groups Eurotiomycetes
and Sordariomycetes (Novikova et al. 2009); and 4) HT of
CR1B elements between Maculinea butterflies and Bombyx
moths (or between common ancestors), which occurred less
than 5 million years ago (Novikova et al. 2007).
As mentioned earlier, DNA transposons seem to be routinely horizontally transferred. Many events of DNA transposon HT have been proposed between organisms from
different phyla (Robertson 1993; Robertson and Lamp 1995;
Jehle et al. 1998; Yoshiyama et al. 2001; Silva and Kidwell
2004). Moreover, transposons are even active in organisms
that do not belong to the kingdom as their natural host. It is
now widely accepted that HT is a necessary part of a DNA
transposon’s life cycle. DNA transposons move to another
host to escape elimination that may be caused by the vertical
inactivation mechanism of the host (Silva et al. 2004;
Munoz-Lopez and Garcia-Perez 2010). In these cases, HT
might allow the TE to colonize a new genome in which
host suppression mechanisms are inefficient, possibly because
they have not had time to co-evolve (Schaack et al. 2010) or
because of other reasons. These remarkable features have
made DNA transposons as attractive molecular tools for
3686
the manipulation of the genomes of their natural hosts
and, more importantly, the genomes of species that are not
closely related. There are quite a few DNA transposons that
can be used for gene transfer among many different species
(Kikuta and Kawakami 2009; Zhu et al. 2010; Clark et al. 2011;
Grabundzija et al. 2011; Kim and Pyykko 2011). Tol2 system
has been used in zebrafish, Xenopus, chicken embryos, and
cultured vertebrate cells. This system is based on the transposon from hAT superfamily derived from the medaka fish
Oryzias latipes (for review, see Munoz-Lopez and Garcia-Perez
2010). Transposable element Minos isolated from Drosophila
hydei was shown to be exceptionally useful for invertebrate
transformations including various insects and even germline
cells of the ciliate Ciona intestinalis (Sasakura et al. 2003).
We hypothesized that DNA transposons can be the natural vectors for HT of non-LTR retrotransposons between species. To test this hypothesis, we used as a starting point the
previously described HT of CR1B non-LTR retrotransposons
between butterflies (Maculinea) and moths (Bombyx) and
performed an in-depth analysis of CR1-like non-LTR retrotransposons and mariner-like elements (MLEs) from a
number of Lepidoptera species. Our data suggest that recurrent HT of several MLEs and CR1B retrotransposons recently
took place between Maculinea and Bombyx. A few chimeric
CR1B/MLE sequences were discovered in the genomes of
Bombyx and Maculinea, which could represent vestiges of
those transposon-based vectors involved in HT of the CR1B
retrotransposon. Alternatively, full-length CR1B non-LTR retrotransposons and MLEs located in close proximity to each
other could be transferred between butterflies and moths as a
part of a single large DNA fragment.
Materials and Methods
Species Collection and Total DNA Isolation
Butterfly and moth species used in this study and their taxonomy are listed in table 1. All entomological material was
kindly provided by the ATOLep collection (as part of the
LepTree project—http://www.leptree.net/, last accessed
August 2, 2012; contact person Dr Charles Mitter,
University of Maryland, MD), Dr Oleg Kosterin (Institute of
Cytology and Genetics, Novosibirsk, Russia), Dr Vladimir
Dubatolov (Institute of Animal Systematics and Ecology,
Novosibirsk, Russia), and Dr Eva Sliwinska (Jagiellonian
University, Poland). Total DNA was isolated from the
thorax and head of one to four individuals using the
DNeasy Blood & Tissue Kit (QIAGEN, Valencia, CA) according
to the manufacturer’s protocol.
PCR Amplification, Cloning, and Sequencing
Sets of degenerate primers were used to amplify partial sequences of the CR1 non-LTR retrotransposon reverse transcriptase (RT) gene (Novikova et al. 2007) and partial
sequences of the MLE transposase gene (Robertson 1993).
Primer sequences were CR1-S= 50 -TATCTTCTTCTCCNG
GNCCNGAYGG-30 and CR1-A= 50 -CAAAAACACTGCCYT
GNGGNACNCC-30 for CR1 RT and MAR-124F=50 -TGGGT
NCCNCAYGARYT-30 and MAR-276R=50 -GGNGCNNARRT
MBE
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
Table 1. List of Macrolepidoptera Species Used in This study.
Superfamily
Papilionoidea
Family
Lycaenidae
Riodinidae
Nymphalidae
Hesperioidea
Geometroidea
Papilionidae
Pieridae
Hesperiidae
Geometridae
Drepanoidea
Calliduloidea
Noctuoidea
Drepanidae
Callidulidae
Lymantriidae
Noctuidae
Bombycoidea
Sphinigidae
Saturniidae
Bombycidae
Species
Plebejus argus
Scolitantides orion
Shijimaeoides divina
Pseudozizeeria mahab
Maculinea teleius
Maculinea arion
Maculinea nausithous
Maculinea alcon
Pseudolucia collinab
Thecla betulae
Narathura japonicab
Brangas neorab
Eumaeus godartib
Curetis regula
Anteros formosusb
Emesis lucindab
Euselasia chrysippe
Catocyclotis adelinab
Theope virgiliusb
Symmachia xypeteb
Mesosemia lamachusb
Caria rhacotisb
Araschnia levana
Melitaea phoebe
Erebia theano
Coenonympha glycerion
Oeneis magna dubia
Oeneis sculda
Parnassius stubbendortii
Colias hyale
Heteropterus morphaeus
Scopula ornata
Semiothisa clathrata
Calospilos sylvata
Brephos parthenius
Drepana sp.
Callidula sp.
Lymantria dispar
Polia nebulosa
Mythimna sp.
Agrotis exclamationis
Erinnyis ellob
Perigonia ilusb
Agrius cingulatab
Dovania poecilab
Paonias myopsb
Aglia tau
Janiodes laverna nigropunktab
Asthenidia transversariab
Oxytenis modestiab
Bombyx huttonib
Bombyx mori
Bombyx mandarinab
Colla glaucescens
CR1 PCR
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+c
+
+
+
+
+
+c
+c
+
+
+
+
+
+c
+
+c
+
+
+
+
+
+
+c
+
+c
+c
+
+
+
+
+
MLEs PCRa
+
+
+
+ (1)
+ (2)
+
+
+
+
+
+ (3)
+ (4)
+
+
+
+
+
(5)
+
+ (6)
+ (7)
+ (8)
+ (9)
(10)
(11)
+ (12)
+
+ (13)
+ (14)
+ (15)
+
(16)
(17)
(18)
(19)
+
+ (20)
+ (21)
(22)
(continued)
3687
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Sormacheva et al. . doi:10.1093/molbev/mss181
Table 1. Continued
Superfamily
Lasiocampoidea
Family
Species
CR1 PCR
MLEs PCRa
Lasiocampidae
Oberthueria caeca
Ernolatia mooreib
Quentalia chromanab
Trilocha sp.b
Truncina brunneab
Lasiocampa quercus
+
+c
+c
+c
+
+
+
+ (23)
+ (24)
+ (25)
a
Numbers indicate corresponding species used in dot-blot hybridization (data presented on fig. 5).
Specimen was kindly provided by the ATOLep collection (as part of the LepTree project – http://www.leptree.net/, last accessed August 2, 2012; contact person Dr. Charles
Mitter, University of Maryland, MD).
c
No CR1-like non-LTR retrotransposons were detected after cloning and sequencing (for more details: supplementary table S1, Supplementary Material online).
b
CNGG-30 for transposase. Polymerase chain reaction (PCR)
amplification with degenerate primers was performed using
0.1 g of genomic DNA in a 20 l volume of 10 mM Tris–HCl
(pH 8.9), 1 mM (NH4)2SO4, 4 mM MgCl2, 200 M each of four
dNTPs, 0.5 M primers, and 2.5 units of Taq DNA polymerase. After an initial denaturation step for 3 min at 94 C, the
PCR samples were subjected to 30 cycles of amplification
consisting of 30 s denaturation at 94 C, 42 s annealing at
52 C, and 1 min extension at 72 C, followed by a final extension at 72 C for 10 min.
Full-length Bmmar1, Bmmar3, Bmmar6, and BmmarY
MLEs were amplified using primers that were designed
based on the sequences of their inverted terminal repeats
(ITRs). The primers were Bmmar1-ITR= 50 -CTTAGTCTGGC
CATAAATACTGTTACAAAA-30 (Robertson and Asplund
1996), Bmmar3-ITR= 50 -CCTTACATATGAAATTAGCG-30
(Kumaresan and Mathavan 2004), Bmmar6-ITR= 50 -MCTA
GTCAGGTCATAARTWYTGTCAC-30
(Robertson
and
Walden 2003), and BmmarY-ITR= 50 -GGCTGCACTAAAAGT
ATCGGGA-30 (this study), where Y = C + T, M = A + C,
R = A + G, W = A + T and N = A + G + C + T. PCR amplification was performed using 0.1 g of genomic DNA in a 20 -l
volume of 10 mM Tris–HCl (pH 8.9), 1 mM (NH4)2SO4,
1.5 mM MgCl2, 200 M each of four dNTPs, 0.5 M primers,
and 2.5 units of Taq DNA polymerase. After an initial denaturation step for 3 min at 94 C, the PCR samples were subjected to 30 cycles of amplification consisting of 30 s
denaturation at 94 C, 30 sec annealing at 52 C, and 1.5 min
extension at 72 C, with a final extension at 72 C for 10 min.
DNA hybridization probe templates (Bmmar1, BmmarY,
Bmmar3, and CR1B) were prepared by amplification with
primer Bmmar1-ITR, Bmmar3-ITR, or BmmarY-ITR; the set
of primers for CR1B amplification was B. mori CR1B
(BmCR1B)-S1
5’-CCCTTTCCTTCCCCACCCC-3’
and
BmCR1B-A1 5’-TCTTCCACATCCGGCACACG-3’ (Novikova
et al. 2007). Bombyx mori total DNA was used as a template
in all amplifications.
PCR products were separated by agarose gel electrophoresis. The resulting PCR products were purified using a
QIAquick Gel Extraction Kit (QIAGEN, USA; http://www
.qiagen.com/, last accessed August 2, 2012) and directly
ligated into a pGEM vector using a pGEM-T cloning kit
(Promega; http://www.promega.com/, last accessed August
2, 2012) for sequence determination. Clones were amplified
by PCR with M13 primers, and 40 ng of the product was used
3688
in a 10 -l cycle sequencing reaction with the ABI BigDye
Terminator Kit on an ABI 310 Genetic Analyzer (Applied
Biosystems). Alternatively, sequencing reactions were performed with the Dye Terminator Cycle Sequencing Kit
(Beckman Coulter) and analyzed on a CEQ 8000 Genetic
Analysis System.
The sequences of CR1 non-LTR retrotransposons; MLEs;
and full-length Macmar1, Macmar3, MacmarY, and BhmarY
elements from Maculinea and Bombyx, as well as chimeric
CR1B/MLE elements from Maculinea, were deposited in
GenBank (accession nos. JQ580972–JQ580974).
Genomic Sequence Screening, Sequencing, and
Phylogenetic Analysis
The genome sequence of the silkworm B. mori is available at
SilkDB (http://www.silkdb.org/, last accessed August 2, 2012).
BmCR1B non-LTR retrotransposons were localized using
BlastN (Altschul et al. 1990). To test the presence of the
transposase gene in the surrounding sequences, we performed BlastN and BlastP analyses in 20-kb windows centered
on identified CR1B RT domains. Basic Local Alignment Search
Tool was essentially performed using sequence databases accessible from the National Center of Biotechnology
Information (NCBI) server (Altschul et al. 1990; http://blast.
ncbi.nlm.nih.gov/, last accessed August 2, 2012) and SilkDB
(http://www.silkdb.org/). The novel mariner-like DNA transposon search was performed by BlastN and BlastP using the
sequences of known Bmmar elements as queries and the
silkworm genome assembly database at SilkDB (http://
silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi, last accessed
August 2, 2012; Mita et al. 2004).
Multiple DNA alignments were performed by ClustalW
(Thompson et al. 1994) and edited manually. Phylogenetic
analyses were performed using the neighbor-joining (NJ)
method in MEGA5 software (Tamura et al. 2011). Statistical
support for the NJ tree was evaluated by bootstrapping (1,000
replications) (Felsenstein 1985). Amino acid distances used in
a divergence-versus-age analysis were calculated from sequences of the transposase and RT domain using MEGA5
software (Tamura et al. 2011).
A codon alignment for Bmmar1, BmmarY, and Bmmar3
sequences was used to estimate the mean dS (the number of
synonymous substitutions per site) values for Maculinea
versus B. mori and the level of codon usage bias (CUB) for
each of the analyzed genes. To include all sequences in this
analysis, we slightly edited some sequences to repair
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
insertions. Sequences of three nuclear genes, wingless (wg),
elongation factor 1 alpha (EF1a) and histone H3 (H3), were
used in comparison with transposons. Their dS and CUB were
also estimated to test the HT hypothesis. Sequences obtained
in GenBank and their accession numbers were as follows:
wg—Maculinea alcon (HQ918074), Maculinea arion
(HQ918080), Maculinea nausithous (HQ918085), Maculinea
teleius (HQ918090), and B. mori (EU033069); EF1a—
M. alcon (HQ918097), M. arion (HQ918114), M. nausithous
(HQ918116), M. teleius (HQ918117), and B. mori (EU136667);
and H3—M. alcon (HQ917973), M. arion (GQ128823), M.
nausithous (HQ917976), M. teleius (HQ917978), and B. mori
(DQ443228).
The dS was estimated using the Nei and Gojobori (1986)
method in MEGA5 (Tamura et al. 2011). Fisher’s exact test
(one-tailed) was used to verify if transposon dS values were
statistically lower than those presented by the host genes.
Fisher’s exact test was conducted using 2 2 tables:
Bmmar1, BmmarY, or Bmmar3 and the nuclear gene versus
the number of synonymous variable sites (dS number of
synonymous sites) and the number of synonymous
non-variable sites {[1 – dS] number of synonymous sites}.
The one-tailed P value was considered because we supposed
one direction of HT (the dS for transposons is lower than for
the gene). The CUB level of transposons and host genes was
checked by the effective number of codons (Wright 1990)
and the codon bias index (Morton 1993), which were computed using DNASP 5.0 software (Librado and Rozas 2009).
Dot-Blot Hybridization
Dot-blots were prepared by applying genomic DNA (500 and
100 ng) to Nytran SuPer Charge nylon membranes, using the
alkali blotting protocol as described in the user’s manual and
then crosslinked by UV light using a Bio-Rad GS Genelinker
(Bio-Rad, USA; http://www.bio-rad.com/). The blots were hybridized for 1 h at 65 C in a solution containing QuikHyb
Hybridization Solution (Stratagene, http://www.stratagene
.com, last accessed August 2, 2012), 100 g/ml fragmented
and denatured herring sperm DNA, and a DNA probe labeled
with 32P by random priming using a DNA Polymerase I Large
(Klenow) Fragment Mini Kit (Promega). The membrane was
washed twice in a wash solution (0.1 sodium chloridesodium citrate [SSC], 0.1% sodium dodecyl sulfate [SDS])
for 45 min at 65 C and then exposed to a phosphor screen
(GE Healthcare, USA; http://www.gelifesciences.com/, last
accessed August 2, 2012). The following protocol was used
for stripping: incubation in freshly prepared 0.4 N NaOH for
60 min at 65 C, followed by two washes in a wash solution
(0.1 SSC, 0.1% SDS) for 60 min at 55 C.
MtemarY1 from M. teleius was used as a probe in
BmmarY-like MLEs hybridization, and Mtemar1.2 from
M. arion was used as a probe for Bmmar1-like elements. To
prepare CR1B probe, we amplified 50 -UTR of the CR1B
non-LTR retrotransposon from M. teleius using primers:
MteCR1B_50 UTR-S=50 -GTAGTAGTAGATCCAAGTCTGCAG
TTCG-30 and MteCR1B_ORF-A=50 -TTATTGGGCTCAGTTTG
MBE
CACCC-30 . The same hybridization and stripping protocols
were used for PCR mini-library screening.
PCR Mini-Library Preparation and Screening
PCR mini-libraries were prepared for four species: M.
teleius, M. arion, B. mori, and Bombyx mandarina. Primers
used to amplify putative chimeric sequences were
BmCR1B-A1=50 -TCTTCCACATCCGGCACACG-30 (Novikova
et al. 2007); MacmarY-S1=50 -CTTGTCAAACTTGTTTAGTCG
TTGA-30 ; BmmarY-A1=50 -AAGGAAGTGACACAACATTAC30 ; and Bmmar1-S1=50 -TGTGTTTTCTCATTTTGGCGCCA-30 ;
and Bmmar1-ITR=50 -CTTAGTCTGGCCATAAATACTGTTAC
AAAA-30 (Robertson and Asplund 1996). Combinations of
the above-mentioned primers for MLE and CR1B were used
to amplify the fused CR1B/MLE. The amplified fragments
were visualized by agarose gel electrophoresis, purified using
a QIAquick Gel Extraction Kit (QIAGEN), and ligated into a
pGEM vector using a pGEM-T cloning kit (Promega).
A colony lift assay followed by colony hybridization was performed as described by Sambrook et al. (1989). A 32P-labeled
DNA probe specific for the MLE was used in the first step
(either Bmmar1 or BmmarY). After stripping, the same membranes were used for hybridization with the 32P-labeled 50 UTR
CR1B-specific probe.
Results
Two Lineages of the CR1-Like Non-LTR
Retrotransposons from Lepidopterans
Both Bombyx and Maculinea belong to the group
Macrolepidoptera within the order Lepidoptera. To expand
our understanding of the evolutionary tendencies that led to
the current diversity of CR1 non-LTR retrotransposons in
lepidopterans, we chose a number of species from
Macrolepidoptera for further experiments (table 1; fig. 1A).
In total, 60 species from 15 families were screened using
PCR with previously designed, clade-specific degenerate CR1
primers (table 1; Novikova et al. 2007). We covered all six
superfamilies within the Macrolepidoptera and included
more species related to Bombyx and Maculinea (families
Bombycidae and Lycaenidae). After cloning PCR products,
all obtained clones were sequenced and analyzed for the
presence of RT fragments using BlastP against the
non-redundant GenBank database (Altschul et al. 1990).
Altogether, 318 clones showed similarity to the RT domains
of non-LTR retrotransposons. Only a few clones did not reveal
any similarity to RT. This indicated that the primers we used
were efficient for amplifying non-LTR retrotransposons from
diverse moths and butterflies. Sequences of CR1 non-LTR
retrotransposons were deposited in GenBank under accession
numbers HQ284259–HQ284570.
Surprisingly, further comparative and phylogenetic analyses demonstrated that not all clones derived from CR1-like
non-LTR retrotransposons. Some RT fragments that belonged
to the T1Q, Jockey, and R1 clades were detected among the
cloned fragments, in addition to CR1-like retrotransposons
(see supplementary fig. S1 and table S1, Supplementary
Material online for details). In total, 156 clones obtained
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Sormacheva et al. . doi:10.1093/molbev/mss181
A
B
FIG. 1. (A) Phylogenetic relationships among the investigated Macrolepidoptera families, represented according to the Tree of Life Web Project (http://
tolweb.org/, last accessed August 2, 2012) with minor modifications. The number of analyzed species is shown for each family. True butterflies (light-grey
area; superfamily Papilionoidea) and moths (dark-gray area; superfamily Bombycoidea) are shown. (B) Neighbor-joining (NJ) analysis implemented
based on partial nucleotide sequences of RT domains (approximately 500 bp in length) from newly isolated CR1-like non-LTR retroelements. Estimated
divergence times are indicated for some nodes (see text). Statistical support for NJ phylogeny was evaluated by bootstrapping (1,000 replications); nodes
with bootstrap values over 50% are indicated. CR1-like non-LTR retrotransposons from true butterflies (light-gray area; superfamily Papilionoidea) and
moths (dark-gray area; superfamily Bombycoidea) are highlighted within the Fabre clade. The number of sequences for each compressed subtree is
shown in the corresponding area. Putative horizontal transfer (HT) is indicated. The detailed phylogenetic tree is available in supplementary figure S1,
Supplementary Material online.
from 37 species contained the RT sequences of CR1 retroelements (table 1). It is possible that those 23 species, which
did not yield PCR products or CR1 fragment clones, had lost
this type of retrotransposon. However, most likely, their CR1
non-LTR retrotransposons had highly diverged at the primer
sites and could not be detected using the designed pair of
3690
primers. Another possibility that cannot be ruled out at this
point is the presence of only heavily 50 -truncated copies of
these elements in genomes of these particular specimens
(DNA from a single imago was used in many cases).
Further NJ analysis was performed using amino acid sequences of newly obtained RT fragments and known CR1-like
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
non-LTR retrotransposons: BmCR1B (Novikova et al. 2007)
and Kendo (GenBank: AB126052) from B. mori; CR1A and
CR1B from Maculinea species (Novikova et al. 2007); and two
retrotransposons from the fall armyworm Spodoptera frugiperda (Noctuidae; GenBank: FP340404 and FP340409). At
least two distinct lineages can be found in the resultant phylogenetic tree (fig. 1B). The first lineage, which was named
Fabre, was widely distributed and included retrotransposons
from 20 species that belonged to 11 families: “true” butterflies
from families Lycaenidae, Riodinidae, Nymphalidae, and
Papilionidae; representatives of Drepanidae, Hesperiidae,
Geometridae, Sphingidae, and Noctuidae; and moths from
Bombycidae. CR1-like non-LTR retrotransposons from the
Fabre lineage exhibited a high level of intraspecific similarity.
For example, the average divergence at the DNA level was
3.7% for elements from Maculinea and 2.3% for retrotransposons from Bombyx. The second lineage, named Aurivillius, had
a more narrow distribution and was found in 14 species from
six diverse families: Lasiocampidae, Saturniidae, Nymphalidae,
Geometridae, Sphingidae, and Noctuidae.
The evolutionary history and dynamics of non-LTR retrotransposons from any given eukaryotic group may include
bursts of retrotransposition, balanced retrotransposition
and loss, or loss of the retrotransposons by some of the taxonomic groups but maintenance by closely related ones. All
these processes lead to the incongruence between phylogenetic trees of host species and those reconstructed based on
the sequences of non-LTR retrotransposons. However, the
topology of the CR1-like non-LTR retrotransposon phylogenetic tree in general showed high congruence with the known
phylogeny of butterflies and moths (fig. 1). This indicates that
the vertical inheritance and maintenance of the low diversity
of the sequences of CR1-like non-LTR retrotransposons has
prevailed in lepidopteran genomes. In addition, it is possible
that these retrotransposons are represented by a low copy
number per genome for the majority of the investigated
butterflies and moths. The results of our PCR cloning experiment indirectly confirm this suggestion. As only a few CR1
copies were available for amplification, PCR amplification
with degenerate primers yielded not only products from
CR1-like retrotransposons but also fragments from other
non-LTR retrotransposons that belonged to different clades.
Evolutionary Rates and HT of CR1-Like Non-LTR
Retrotransposons
Although the congruence between phylogenies is remarkable,
especially in the Fabre lineage of CR1-like non-LTR retrotransposons, several serious discrepancies can be found.
Previously, we described HT of CR1B non-LTR retrotransposons between ancestors of the Maculinea (Lycaenidae) and
Bombyx (Bombycidae) genera, which took place approximately 5 million years ago (Ma; Novikova et al. 2007). To
detect other examples of putative HT in our current dataset,
a divergence-versus-age analysis was performed (Kordis and
Gubensek 1998; Malik et al. 1999; Novikova et al. 2007;
Novikova et al. 2009). Divergence-versus-age analysis allows
for a comparison of evolutionary rates among
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retrotransposons in a convenient visual form. In this analysis,
the divergence rates between the RT domains of the non-LTR
retrotransposons were plotted against the host divergence
time estimates. It is necessary to note that the divergenceversus-age analysis should include comparisons of those retrotransposons believed to be exclusively vertically inherited,
such as representatives of R1, R2, and/or Jockey clades (Malik
et al. 1999).
Nearly all modern orders of insects, including butterflies
and moths, appear in the fossil record by the Triassic period
(245 Ma). As estimated based on the molecular data, Diptera
and Lepidoptera share their last common ancestor approximately 340–380 Ma (Douzery et al. 2004). The time since the
divergence of the major Macrolepidoptera superfamilies (e.g.,
Bombycoidea and Papilionoidea) is approximately 140 Ma
(Gaunt and Miles 2002). Other useful time divergence estimates include 43 My between Lycaenidae and Nymphalidae
and 100 My between Lycaenidae and Papilionidae (Nazari
et al. 2007). Amino acid sequence distances between the
RT domains of the non-LTR retrotransposons from lepidopterans along with other known elements from arthropods
(arthropod curve), mostly from Diptera, and vertebrates (vertebrate curve) were plotted against estimates of host divergence times (fig. 2; Malik et al. 1999). Our current extended
dataset contained non-LTR retrotransposons that belonged
to R1, Jockey, T1Q, and CR1 clades from diverse families of
Macrolepidoptera. All R1, Jockey, and T1Q comparisons fell
very close to the previously reported arthropod curve (Malik
et al. 1999), suggesting that there are no cases of HT of R1,
Jockey, or T1Q between butterflies and moths (comparisons
shown between 10 and 140 Ma in fig. 2) or lepidopterans and
dipterans (comparisons at 380 Ma in fig. 2). The observed
distribution of estimates from Lepidoptera lineages also indicated that evolutionary rates within the R1, Jockey, and T1Q
clades from butterflies and moths are not different from
those estimated for other arthropods.
On the basis of the results of a previous divergenceversus-age analysis, Malik et al. (1999) have proposed that
non-LTR retrotransposon sequence evolution is slower in vertebrates (Malik et al. 1999). Surprisingly, the estimates for
CR1-like non-LTR retrotransposons from lepidopterans fall
near or even lower than the vertebrate curve, indicating
that slower evolutionary rates are a feature of a particular
clade(s) of non-LTR retrotransposons rather than of a
whole pool of non-LTR sequences from a given taxonomical
group (vertebrates in this present case). Several examples in
which the estimates fell markedly below all curves were
BomHutCR1B-1 from Bombyx huttoni (Bombycidae) versus
MteCR1B from M. teleius (Lycaenidae); ObecaeCR1B-8 from
Oberthueria caeca (Bombycidae) versus MteCR1B; and
BmCR1 from silkworm B. mori (Bombycidae) versus
MteCR1B (all points at 140 Ma). All indicated estimates represented the potential cases of HT and included representatives of the CR1B family of the Fabre lineage within the CR1
clade of non-LTR retrotransposons. Comparative analysis of
RT nucleotide sequences showed an extremely high similarity
among elements from the CR1B family within and among
species. The average intraspecies divergence varied from 1.8%
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Sormacheva et al. . doi:10.1093/molbev/mss181
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FIG. 2. Divergence-versus-age analysis of the non-LTR retrotransposons including the CR1 clade of Macrolepidoptera. Amino acid sequence distances
were calculated from the partial sequences of the reverse transcriptase (RT) domains. The curves for arthropods (solid line) and vertebrates (dotted line)
are reproduced from Malik et al. (1999). For each host divergence time estimate, the elements used are as follows: for R1 clade: BraNeoR1-32 versus
OenSculR1-28, and StryMelR1-2 versus EreTheR1-29 or CoeGlyR1-7 compared at 43 My; BraNeoR1-32 versus TrichspR1-6, and StryMelR1-2 versus
AglTauR1-8 at 140 My; R1Dm (GenBank: X51968) from Drosophila melanogaster versus R1Bm (GenBank: M19755) from Bombyx mori at 380 My; for
Jockey clade: BmJockey from B. mori (GenBank: AAA17752) versus TrichSpJockey-16 compared at 10 My; NarJapJockey-2 versus ParStubJockey-17 at 100
My; TheBetJockey-17 versus BmJockey at 140 My; BmJockey versus DmJockey (GenBank: M22874) from D. melanogaster at 380 My; for CR1 clade:
AraLevCR1-29 versus MteCR1A (GenBank: DQ823008) from Maculinea teleius compared at 43 My; ParStubCR1-18 versus ShiDivCR1-15 at 100 My;
BomHutCR1B-1 versus MacCR1A at 140 My; BomHutCR1B-1 versus MteCR1B (GenBank: DQ823029) from M. teleius; ObecaeCR1B-8 from Oberthueria
caeca versus MteCR1B; BmCR1B (Novikova et al. 2007) from B. mori versus MteCR1B compared at 140 My; for T1Q clade: EreTheT1Q-25 versus
ScoOriT1Q-2 at 43 Myr; LasQueT1Q-1 versus ScoOriT1Q-38 at 140 Myr; DmT1Q (Repbase; http://www.girinst.org/, last accessed August 2, 2012:
DMCR1A) from D. melanogaster versus MteT1Q (DQ836363) from M. teleius at 380 My. Species divergence times are based on estimates by Nazari et al.
(2007) for Lycaenidae versus Nymphalidae (43 Ma) and Lycaenidae versus Papilionidae (100 Ma) comparisons; by Gaunt and Miles (2002) for
comparisons between Papilionoidea and Bombycoidea (140 Ma); and by Douzery et al. (2004) for comparisons between Lepidoptera and Diptera
(380 Ma).
to 4%. The interspecies DNA divergence among CR1B-like
non-LTR retrotransposons varied from 3.6% to 4.8%. No
other examples of non-LTR retrotransposon HT were found
in this study.
The lower rates of divergence and the slowdown effect on
evolutionary rates do not necessarily indicate HT events
(Kordis and Gubensek 1998; Malik et al. 1999). The alternative
hypothesis, which includes strict evolutionary constraints, is
especially attractive in the case of CR1 non-LTR retrotransposons from lepidopterans because the slower-than-usual evolutionary rates were observed in all estimates (fig. 2). However,
the observed evolutionary rates for CR1B retrotransposons
can be explained only by extremely strict selective pressures
comparable with those acting on the most conservative proteins, such as EF1a (Novikova et al. 2007). The hypothesis of
strict constraints could be implemented only in the case of
the functional importance of the CR1B non-LTR retrotransposons in a few evolutionarily scattered lepidopteran species,
which is highly unlikely. The internal regulatory elements of
retrotransposons can alter gene expression and be selectively
advantageous. However, typically, only these regulatory
3692
components evolve under selective pressure (Medstrand
et al. 2001; Ono et al. 2001).
The mechanism(s) of HT of non-LTR retrotransposons is
not clear. However, the use of DNA-based vectors is the most
probable candidate. We hypothesized that DNA transposons
played the role of such a vector at the initial steps of HT. The
HT of various DNA transposons between various taxonomic
groups is well documented (e.g., Robertson 1993; Robertson
and Lamp 1995; Jehle et al. 1998; Yoshiyama et al. 2001; Silva
and Kidwell 2004), and insertion of non-LTR retrotransposons
into DNA transposons has been described previously (e.g.,
Kumaresan and Mathavan 2004; Xia et al. 2004; Abe et al.
2005).
Mariner-Like Elements from Butterflies and Moths
To test our hypothesis concerning the possible role of DNA
transposons in the HT of non-LTR retrotransposons, first,
we mined MLEs using bioinformatic and experimental
approaches. Whole-genome sequence (WGS) data and/or
expressed sequence tag (EST) data are limited for butterflies
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
and moths. Nevertheless, ESTs for several model species can
be accessed via ButterflyBase (Papanicolaou et al. 2008;
http://butterflybase.ice.mpg.de/, last accessed August 2,
2012) and SPODOBASE (EST database for Spodoptera frugiperda; Negre et al. 2006), in addition to the WGS data for B.
mori (http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi, last
accessed August 2, 2012; Mita et al. 2004) and the nucleotide
sequence data available at NCBI GenBank (Benson et al.
2011).
MLEs have been previously classified into three distinct
families: mariner, ludens, and mori (Rouault et al. 2009). The
mariner family contains 15 subfamilies, the ludens family contains 2 subfamilies, and the mori family contains only 1 subfamily, correspondingly. Six MLEs from mariner and mori
families have been described in silkworm B. mori and designated as Bmmar1-6 (Robertson and Asplund 1996; Robertson
and Walden 2003; Kumaresan and Mathavan 2004). Our
mining of MLEs from WGS data of B. mori yielded one
more mariner-like DNA transposon, which was named
BmmarY. The search was performed by BlastN and BlastP
using the sequences of known Bmmar elements as queries
and the silkworm genome assembly database at SilkDB
(http://silkbase.ab.a.u-tokyo.ac.jp/cgi-bin/index.cgi, last accessed August 2, 2012; Mita et al. 2004). The consensus BmmarY
element was a typical mariner-like DNA transposon. BmmarY
was 1,287 bp in length and carried rather short ITRs, which
were 26 bp in length and shared 84.6% DNA similarity.
Consensus BmmarY showed the presence of a single open
reading frame, which was 1,050 bp in length and encoded a
transposase, the essential enzyme for transposition of
mariner-like DNA elements. BmmarY transposase had a characteristic D,D(35)D cation-binding domain, and nuclear localization signals GRPR and NLS were also identified (fig. 3; Doak
et al. 1994; Hartl et al. 1997; Kosugi et al. 2009). A search of
the B. mori EST database, which is also available at SilkDB,
with the BmmarY consensus sequence using BlastN did not
show any significant matches, suggesting that BmmarY is not
expressed. The rough estimate for its copy number suggested
approximately 100 copies of full-length BmmarY per haploid
genome plus multiple short vestiges.
In addition to the search of MLEs in the B. mori genome,
we gathered sequences of mariners from other databases. In
total, 52 MLEs were identified in GenBank (using BlastN;
Altschul et al. 1990) from diverse insect species, including
27 MLEs from Lepidoptera (full list is available in supplementary table S2 and fig. S2, Supplementary Material online). No
mariner-like sequences were detected in the EST data at
ButterflyBase (Papanicolaou et al. 2008; http://butterflybase
.ice.mpg.de/, last accessed August 2, 2012).
For further in-depth analysis of MLE diversity in butterflies
and moths, we used PCR with the degenerate primers
MAR-124F and MAR-276R (Robertson 1993), which were
specific to the transposase gene of the MLEs, to investigate
the presence of MLEs in the same set of Macrolepidoptera
species that we used to investigate the CR1-like non-LTR
retrotransposons. The results of this PCR amplification are
summarized in table 1. Thirty-six of 60 investigated species
showed positive PCR results. Twenty-five species that
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belonged to the families Lycaenidae, Riodinidae, and
Bombycidae were chosen for further cloning of the obtained
putative MLE fragments. In total, 190 clones were collected
and sequenced; among them, 145 clones from 18 species
(including representatives of the Maculinea and Bombyx
genera) showed high similarity with known MLE transposases.
The sequences of these MLEs were deposited in
GenBank under accession numbers HQ606331–HQ606343,
HQ606345–HQ606368, HQ606371–HQ606381, HQ606402,
HQ606410, and HQ606415–HQ606462. These sequences,
together with sequences obtained in our in silico mining
and other MLEs from insects and invertebrates (supplementary table S2 and fig. S2, Supplementary Material online),
were used for subsequent phylogenetic tree reconstruction.
The NJ phylogenetic analysis demonstrated the presence
of both the mori and ludens families and 10 subfamilies of the
mariner family in the insect genomes (supplementary figs. S2
and S3, Supplementary Material online). MLEs from the
Lepidoptera species analyzed in silico have been found in
six subfamilies of the mariner family (cecropia, mellifera, vertumana, mauritiana, irritans, and CRI) and in the mori family
(mori subfamily). The mariner element from the tobacco
budworm Heliothis virescens (Noctuidae) was originally described as the representative of the irritans subfamily (Ren
et al. 2006). Our phylogenetic analysis clearly showed that this
element belongs to the vertumana subfamily (supplementary
figs. S2 and S3, Supplementary Material online). The sequences obtained in our PCR analysis belonged to four distinct MLE subfamilies: cecropia, mellifera, mauritiana, and
vertumana (only one species: nine clones from Pseudolucia
collina, Lycaenidae). Cecropia and mellifera seemed to be
widely distributed among the tested species. The representatives of the mauritiana subfamily were found in the
Bombycidae species only (fig. 4A). To summarize, at least
seven subfamilies of MLEs are distributed in the
Macrolepidoptera species: cecropia, mellifera, mauritiana, irritans, CRI, vertumana, and mori.
The degenerate primer pair MAR-124F and MAR-276R was
useful for the amplification of MLEs from butterflies and
moths. However, on the basis of the results for B. mori, we
suggest that these primers did not permit amplification of the
full diversity of MLEs. Most probably, using MAR-124F and
MAR-276R yielded amplification of the most prevalent subfamilies of MLEs, that is, those that had a higher copy number
(such as cecropia and mellifera). To explore the diversity of
MLEs in Maculinea and some other Lepidoptera species further, we used another approach: amplification with primers
specific for ITRs.
Recent Horizontal Transfer of Mariner-Like DNA
Transposons between Maculinea and Bombyx
The primers selected for the ITR sequences of Bmmar1 and
Bmmar6 (both from the mori family) and Bmmar3 (cecropia
subfamily) were previously published (Robertson and
Asplund 1996; Robertson and Walden 2003; Kumaresan
and Mathavan 2004). The primers for BmmarY (vertumana
subfamily) were designed in this study. No Bmmar2
3693
Sormacheva et al. . doi:10.1093/molbev/mss181
MBE
A
B
FIG. 3. (A) Structure of BmmarY mariner-like element (MLE) from Bombyx mori and pairwise alignment of its inverted terminal repeats. 50 and 30 ITRs,
50 and 30 inverted terminal repeats; ORF, open reading frame; GRPR and NLS, nuclear localization signals, identified using NLS Mapper (Kosugi et al.
2009). The location of the conservative D160, D252, and D287 from a characteristic transposase D,D(35)D cation-binding domain is also demonstrated.
(B) Multiple alignment of transposase gene from diverse mariner-like DNA transposons from insects. Conservative D160, D252, and D287 from a
characteristic transposase D,D(35)D cation-binding domain as well as the highly conserved D282 are indicated by arrows; *stop-codon. The GenBank
accession numbers of MLEs used for multiple alignment are as follows: BmmarY B. mori DQ443409; BhmarY.1 Bombyx huttoni HQ606344; MalmarY.1
Maculinea alcon HQ606387; MnamarY.3 Maculinea nausithous HQ606407; Bmmar1 B. mori AAS47739; Mnamar1.1 M. nausithous HQ606403;
Mtemar1.3 Maculinea teleius HQ606411; Lqmar1.1 Lasiocampa quercus HQ606384; Bmmar3 B. mori BAA23532; Bmamar3 Bombyx mandarina
AB237543; Mtemar3.4 M. teleius HQ606412; Marmar3.1 Maculinea arion HQ606394; Attacus atlas AB006464; Papilio xuthus AB055185; Hamar2.3
Helicoverpa armigera HM807611; Bmmar6 B. mori AF461149; Bmamar6 B. mandarina BAH20555; Apis mellifera XM_001121351; Alvcmar1.14 Alvinella
caudata AJ496133; Adoxophyes honmai AB020617; Agrilus planipennis GQ398105; Chrysoperla plorabunda U11654; and Ceratitis rosa AY034623.
ITR-specific primer was designed due to the lack of complete
copies of this element in databases. All 28 investigated species
showed positive PCR results with the ITR-specific primer for
Bmmar3. These data agree with the results of PCR amplification with the degenerate primers and confirm the presence of
the cecropia subfamily in all investigated Macrolepidoptera
species (supplementary table S3, Supplementary Material
online).
Positive PCR results were also obtained with primer
BmmarY-ITR for Bombycidae (B. mori and B. huttoni) and
Lycaenidae (all Maculinea species) as well as in reactions
with Bmmar1-ITR and Bmmar6-ITR for Bombycidae
3694
(B. mori and B. mandarina) and Lycaenidae (all Maculinea
species). No positive PCR results with these primers were
observed for the remaining investigated species. The resulting
PCR products from the selected species were isolated from
agarose gels, cloned into plasmid vectors, and sequenced. In
total, 5 Bmmar3-like MLEs, 3 Bmmar1-like MLEs, and 13
BmmarY-like MLEs were obtained from 4 Maculinea species
(Lycaenidae). In addition, a single sequence of the Bombyx
huttoni mariner Y (BhmarY) element was isolated from
B. huttoni (Bombycidae) (fig. 3B). The sequences of full-length
Macmar1, Macmar3, and MacmarY elements from Maculinea
were deposited in GenBank under accession numbers
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
A
MBE
B
C
FIG. 4. (A) Schematic representation of the phylogenetic tree reconstructed based on sequences of mariner-like elements (MLEs). Only clades (indicated
on the right) containing novel MLEs from Lepidoptera are shown. The number of sequences obtained for each species is indicated after the species
name. Bombyx mori MLEs derived from whole-genome shotgun sequences. The detailed phylogenetic tree is available as supplementary figure S3,
Supplementary Material online. (B) Detailed mori family subtree of MLEs from Bombyx and Maculinea species. (C) Detailed vertumana subfamily
subtree of MLEs isolated from Lepidoptera species, including Bombyx and Maculinea. Statistical support for NJ phylogeny was evaluated by bootstrapping (1,000 replications); nodes with bootstrap values over 50% are indicated. Putative horizontal transfers (HTs) are indicated.
HQ606330, HQ606344, HQ606387–HQ606390, HQ606392–
HQ606396, HQ606399, HQ606401, HQ606405–HQ606409,
and HQ606411–HQ606414. All sequences were used in further phylogenetic analysis together with other available MLEs
(figs.4B and 4C; supplementary fig. S3, Supplementary
Material online).
To confirm the results of PCR, which is sensitive and prone
to contamination, we used dot-blot hybridization. Totally, 25
species from 11 families were analyzed including butterflies
M. teleius and M. arion and moths B. mori and B. mandarina
(table 1 and fig. 5). The distinct strong signals in hybridization
with Bmmar1 and BmmarY as probes were detected only for
Maculinea and Bombyx. The much weaker signals were also
detected in some other investigated species. However, most
probably, these signals are results of cross-hybridization between diverse MLEs (data not shown). In addition, the
dot-blot hybridization for CR1B was performed. Distinct, signals in the hybridizations were detected only for Maculinea
and Bombyx (fig. 5).
Together, our PCR and dot-blot data suggest that the distribution of Bmmar1/Macmar1 (Bmmar1-like MLEs from
Maculinea) and BmmarY/MacmarY (BmmarY-like MLEs
3695
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Sormacheva et al. . doi:10.1093/molbev/mss181
DNA
BmmarY
500 ng
100 ng
1
2
3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
19
20
21
22 23
24
25
13
500 ng
100 ng
Bmmar1
500 ng
100 ng
1
2
3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
19
20
21
22 23
24
25
13
500 ng
100 ng
CR1B
500 ng
100 ng
1
2
3
4
5
6
7
8
9
10
11
12
14
15
16
17
18
19
20
21
22 23
24
25
13
500 ng
100 ng
FIG. 5. Dot-blot hybridization of 25 butterfly and moth species with BmmarY and Bmmar1 DNA transposons and CR1B non-LTR retrotransposon.
Numbers indicate species and correspond to the numbers in table 1.
from Maculinea) is patchy and limited to two genera:
Maculinea and Bombyx. To investigate whether the high similarity presented among transposon sequences from diverse
species is a consequence of HT, we chose to analyze the rate
of substitutions at the synonymous sites (dS). This approach
offers a measure of neutral evolution in the absence of strong
CUB. If a transposon was acquired via vertical transfer, its dS
values should be similar to the dS values of its host’s genes.
Alternatively, if the TE was horizontally transmitted to the
host genome, the TE dS should be significantly lower than
those of its host’s genes (Silva and Kidwell 2000; Ludwig et al.
2008; Bartolome et al. 2009; Mota et al. 2010). Sequences of
the nuclear genes wg, EF1a, and H3 were obtained from
GenBank and used as examples of vertical evolution in the
comparison with Bmmar1/Macmar1 and BmmarY/
MacmarY, for which HT was assumed. In addition, Bmmar3
and Macmar3 were also employed as examples of presumably
vertical transmission of transposons. The transposon sequences of each species were grouped, and the mean dS
values were obtained for each pair of species for each
3696
particular transposon. The differences between transposon
and host gene dS values were examined using Fisher’s exact
test. In all comparisons, the transposon dS values were significantly lower than those of the host’s genes, except for
Bmmar3/Macmar3 (fig. 6). These data support the
Bmmar1/Macmar1 and BmmarY/MacmarY HT hypothesis.
Although Bmmar3 showed an unexpectedly low dS, it was
not significantly lower than the wg gene dS for the B. mori and
M. arion pair, thus suggesting vertical evolution. For two other
pairs of species (B. mori and M. nausithous, B. mori, and M.
teleius), this difference was significant at the 0.05 level, thus
questioning the vertical evolution of this transposon.
Chimeric CR1B/MLE Sequences Exist in Genomes of
Bombyx and Maculinea
On one hand, the HT of CR1B non-LTR retrotransposons,
Bmmar1/Macmar1 and BmmarY/MacmarY, between species
of the different genera (or their ancestors) of the order
Lepidoptera—Maculinea and Bombyx—can be coincidence.
On the other hand, considering the nature and mechanism of
MBE
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
***
Bmmar1/Macmar1
***
NS
B. mori vs. M. arion
BmmarY/MacmarY
Bmmar3/Macmar3
EF1a
wg
***
***
H3
*
B. mori vs. M. nausithous
***
***
*
B. mori vs. M. teleius
***
B. mori vs. M. alcon
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
dS
FIG. 6. Mean dS values for mariner-like elements Bmmar1, BmmarY, and Bmmar3, and the elongation factor 1 alpha (EF1a), wingless (wg), and histone
H3 (H3) genes in species pairwise comparisons for Bombyx mori, Maculinea arion, Maculinea nausithous, Maculinea teleius, and Maculinea alcon. Results
of Fisher’s exact test: ***P < 0.001; **P < 0.01; *P < 0.05; NS, non-significant.
transposition of non-LTR retrotransposons, such a coincidence is highly improbable. The described cases of HTs are
unique. They are all recent and occurred not more than 5–10
Ma. Thus, if MLEs played roles in the HT of CR1B non-LTR
retrotransposons, we still could be able to identify chimeric
CR1B/MLE sequences or their vestiges in the Maculinea and/
or Bombyx genomes.
We performed in silico analysis of the B. mori genome to
detect chimeric sequences that represented Bmmar MLEs
with the BmCR1B non-LTR retrotransposon inserted. As a
result of this search, we found several loci that contained
Bmmar and BmCR1B sequences. All sequences were too
short, except two, in loci nscaf2823 and nscaf2901 (numbers
are given according to SilkDB; http://www.silkdb.org/, last
accessed August 2, 2012). Unfortunately, the nucleotide
sequences of these contigs were not completely sequenced.
Nevertheless, we were able to reconstruct the structures of
the chimeric sequences (fig. 7A). The chimeric element
BmCR1B/Bmmar1 represented a full-length Bmmar1 DNA
transposon that carried a heavily truncated BmCR1B
non-LTR retrotransposon, just 237 bp in length, flanked by
target sequence duplications (TSDs; ctctga. . . ctctga).
Chimeric BmCR1B/Bmmar6 was a full-length Bmmar6 MLE
with a 50 -truncated BmCR1B inserted in the orientation opposite to Bmmar6. Both elements showed the presence of
TSDs, suggesting recent activity of the Bmmar6 MLE and the
BmCR1B non-LTR retrotransposon.
These findings prompted us to investigate the chimeric
sequences in the Maculinea and Bombyx species. For this
purpose, several pairs of primers corresponding to both
Bmmar/Macmar and CR1B elements were used in amplification reactions with total DNA from four species: M. teleius,
M. arion, B. mori, and B. mandarina. The PCR fragments were
used to prepare PCR-based mini-libraries. The mini-libraries
were further screened for the presence of clones containing
both Bmmar/Macmar-like and CR1B sequences using a
colony lift assay (see Materials and Methods for details).
Although numerous colonies were positive for Bmmar, only
a few gave a hybridization signal with the CR1B probe. In total,
two clones containing both a CR1B and an MLE were isolated
from M. teleius; one from M. arion; and none from B. mori and
B. mandarina. All isolated clones differed among species, from
each other and from the chimeric elements detected in the
B. mori WGS. Their structures are shown in figure 7B. The
sequences of chimeric CR1B/MLE elements from Maculinea
were deposited in GenBank under accession numbers
JQ580972–JQ580974.
One of the clones from M. teleius (MacCR1B/MtemarY-1)
showed the presence of a CR1B non-LTR retrotransposon
inserted into MacmarY without interrupting the MacmarY
transposase coding regions; theoretically, it might represent a
HT cassette (GenBank: JQ580972).
Discussion
In this study, we investigated the evolutionary history of the
CR1 clade of non-LTR retrotransposons and MLEs in a fairly
large set of species from the Macrolepidoptera group.
Although the distribution and evolution of the investigated
3697
MBE
Sormacheva et al. . doi:10.1093/molbev/mss181
A
5’truncated
ITR
polyN
BmCR1B/Bmmar1
ma
Bm
1273 bp
Bombyx mori nscaf2823
r1
352
482
1
964
TSD [tattatataa]
BmCR1B/Bmmar6
Bm
201
1
polyA
TSD [ggttaca]3537
polyN
2691 bp
Bombyx mori nscaf2901
BmCR1B
TSD [ctctga] 3310
3538 TSD [ctctga]
ITR
1178
1314
3045
polyA
ma
BmCR1B5’truncated TSD [tattatataa]
1929
r6
polyN
1234
965
B
2374
TSD [ggttaca]
17
1242
1307
100 bp
1113
MacCR1B
MacCR1B/MtemarY-1
MtemarY
2070 bp
Maculinea teleius
976
142
4
1113
MacCR1B
MacCR1B/MtemarY-2
1620 bp
Maculinea teleius
MtemarY
142
(463-486) 703
(933-957)
484
MacCR1B
MacCR1B/MarmarY
5’truncated
1113
MarmarY
1070 bp
Maculinea arion
100 bp
142
581
FIG. 7. (A) Structure of chimeric BmCR1B/MLE transposable elements detected in Bombyx mori genomic sequences (http://www.silkdb.org/, last
accessed August 2, 2012). Numbers below indicate positions corresponding to the consensus MLE; numbers on the top correspond to the position in
the consensus sequence of the BmCR1B non-LTR retrotransposon. ITR, inverted terminal repeat; TSD, target site duplication; polyA, poly-adenine
sequence; polyN, track of unknown sequence. (B) Structural organization of the fragments of putative chimeric MacCR1B/MLE transposable elements
isolated from Maculinea species. Numbers below indicate positions corresponding to the consensus MLE; numbers on the top correspond to the
position in the consensus sequence of the MacCR1B non-LTR retrotransposon. MacCR1B/MtemarY-2 has two short deletions: from position 463–486
in comparison with the consensus MtemarY transposon and from position 933–957 in comparison with the MacCR1B non-LTR retrotransposon. The
orientation of CR1B insertion is indicated by arrows.
TEs were generally in accord with vertical evolution, a few
striking discrepancies were found. Unexpectedly high similarities, discontinuous distribution, and slowdown effects on
evolutionary rates were detected for some transposable elements. It appears that the HT of the non-LTR retrotransposon
CR1B (Novikova et al. 2007) as well as the MLEs Bmmar1 and
BmmarY took place between Maculinea (true butterflies;
Papilionoidea) and Bombyx (moths; Bombycoidea; fig. 8).
The HT of different TEs (non-LTR retrotransposons and
DNA transposons) between the same distantly related genera
(Maculinea and Bombyx) has never been demonstrated
before. Recurrent HTs of different TEs were previously reported only for closely related Drosophila species (for
review: Loreto et al. 2008). We infer that HT of CR1B retrotransposons and HT of MLEs between butterflies (Maculinea)
and moths (Bombyx) could be linked. Unfortunately, the direction of the described HTs remains unknown, which complicates further analyses of the event. The circumstantial
evidence indicates that an ancestral moth of the
Bombycidae family might be the source of the HT. For example, the distribution of CR1B non-LTR retrotransposons and
MLEs is wider among Bombycidae (covering at least two different genera in the case of CR1B elements; Novikova et al.
2007) than Lycaenidae species, but this observation could be
3698
biased because only the modern groups of Bombycidae and
Lycaenidae were included in the analysis.
HT of non-LTR retrotransposons is believed to be a very
rare event. Nevertheless, several cases of non-LTR retrotransposon HTs have been well documented. Unfortunately, the
mechanisms of the HTs remain unknown (Takasaki et al.
1996; Kordis and Gubensek 1998; Zupunski et al. 2001;
Sanchez-Gracia et al. 2005; Novikova et al. 2007; Novikova
et al. 2009). Lack of extrachromosomal copies of non-LTR
retrotransposons and the formation of new copies in the
nucleus directly at their integration sites might be sufficient
for the rarity of non-LTR retrotransposon HT (Malik et al.
1999; Silva et al. 2004). There are two possible pathways for
HT of non-LTR retrotransposons: 1) via an RNA intermediate
and 2) via a DNA intermediate.
RNA-mediated transfer of non-LTR retrotransposons
could involve different types of viruses, including retroviruses,
as vectors for the HT via packaging of RNA transcript into a
virion. Through their capacities to enter and infect eukaryotic
cells, viruses are potential vehicles of HT (Miller and Miller
1982; Fraser et al. 1985; Jehle et al. 1998; Malik et al. 2000;
Piskurek and Okada 2007; Schaack et al. 2010; Thomas et al.
2010). It was shown recently that flock house virus virions
package a variety of host RNAs, including TEs. The packaging
of these RNAs elicits the possibility of horizontal gene transfer
MBE
~140 MYA
CM
AR
Y
/M
A
MA
RY
BM
MA
R1
BM
1B
CR
1
CR
~40 MYA
~100 MYA
LYCAENIDAE
RIODINIDAE
NYMPHALIDAE
PAPILIONOIDAE
HESPERIOIDEA
GEOMETROIDEA
DREPANOIDEA
CALLIDULOIDEA
SATURNIIDAE
BOMBYCIDAE
SPHINIGIDAE
LASIOCAMPOIDEA
LYMANTRIIDAE
NOCTUIDAE
PYRALOIDEA
CE
CR
OP
IA
/M
A
(B
M
MA
R3
CM
AR
1
)
Horizontal Transfer of TEs between Lepidopterans . doi:10.1093/molbev/mss181
Maculinea
Bombyx
FIG. 8. Distribution of CR1-like non-LTR retrotransposons, including CR1B, and MLEs (from subfamily cecropia; Bmmar1- and BmmarY-like) among the
investigated families of Lepidoptera. Phylogenetic relationships among the investigated Macrolepidoptera families are represented according to the Tree
of Life Web Project (http://tolweb.org/) with minor modifications.
between eukaryotic hosts that share a viral pathogen (Routh
et al. 2012). The simultaneous HT of CR1B non-LTR retrotransposon and MLEs detected in this study could occur as a
result of the co-packaging of their RNA (CR1B) and DNA
(Bmmar1 and BmmarY) into a virion.
Another possible means of HT could occur if a non-LTR
retrotransposon inserts into a DNA transposon, which are
abundant in genomic DNA, such a construct (chimeric element) has a chance to be horizontally transferred; especially if
the coding sequences of the DNA transposon were not disrupted. The possible involvement of DNA transposons in
non-LTR retrotransposon HT was proposed earlier by
Takasaki et al. (1996). The identification of a chimeric element in which a unit of the HpaI non-LTR retrotransposon,
found in salmonid, was integrated within the Tc1-like DNA
transposon led the authors to suggest a DNA transposonmediated HT pathway (Takasaki et al. 1996).
Our search for chimeric CR1B/MLEs revealed the presence
of a few constructs in the genomes of Maculinea species
(M. teleius and M. arion) and B. mori. Although we did not
find identical chimeric sequences in the investigated genomes
and it is hard to tell whether the detected chimeric CR1B/
MLEs are actually the result of the post-HT activity of TEs, we
suggest that the detected chimeric elements might represent
the vestiges of the vector(s) for CR1B HT. Taken together, our
results suggest that DNA transposon-mediated HT of CR1B
non-LTR retrotransposons is the most parsimonious explanation. First, it explains the simultaneous transfer of a few
diverse TEs. Second, because the transmitted molecule is
DNA and no RNA intermediate is necessary, non-LTR retrotransposons easily overcome the limitations imposed by their
mechanism of retrotransposition (Malik et al. 1999; Silva et al.
2004). Finally, the process of HT for such chimeric elements
should be identical to the HT of a single DNA transposon. In
cases when insertion of CR1B elements within MLEs disrupts
a transposase gene, such chimeric elements could be
cross-mobilized by closely related autonomous MLEs, similar
to the cross-mobilization of most other non-autonomous
transposons (Lampe et al. 2001; Hua-Van et al. 2002;
Feschotte et al. 2005).
Both viruses and intracellular parasites could present possible secondary vectors for MLE HTs and DNA transposonmediated HT of non-LTR retrotransposons (chimeric
elements). Bracoviruses and polydnaviruses may accidentally
serve as viral intermediates for DNA transposon delivery to
lepidopteran cells (Gundersen-Rindal and Lynn 2003; Doucet
et al. 2007; Dupuy et al. 2011). Intracellular symbiotic bacteria, such as Wolbachia and Spiroplasma, have also been considered as possible vectors (Montenegro et al. 2005; Dunning
Hotopp et al. 2007; Loreto et al. 2008).
In both RNA-mediated and DNA transposon-mediated
pathways, contact between species (Maculinea and
Bombyx) is essential for the HT event to occur. Butterflies
of the genus Maculinea spend most of their life as social
parasites inside Myrmica ant colonies (Thomas 1984; Elmes
et al. 1991; Witek et al. 2006), which are the ideal places for
3699
Sormacheva et al. . doi:10.1093/molbev/mss181
recurrent and lengthy contact between Maculinea and
Bombyx. Contact between a common ancestor of the
Maculinea butterflies and a common ancestor of the
Bombycidae moths in ant nests could have led to HT of
these elements and their spread throughout all Maculinea
species.
Conclusion
To summarize, the main issue in HT investigations is the
ability to analyze events only post-factum. The comprehensive analysis of related species in this study proved helpful,
and similar methodologies may aid other HT research studies.
Our results demonstrate multiple HTs between two genera,
which could be overlooked if only a few species and/or only
one group of TEs were included in the analysis. Although we
did not find strong evidence for our hypothesis of the involvement of DNA transposons in HT of non-LTR retrotransposons, we demonstrated that recurrent and/or simultaneous
flow of TEs took place between distantly related moths and
butterflies. The investigated HTs are unique. They occurred
recently (approximately 5 Ma) and affected a group of species
that diverged more than 140 Ma (Gaunt and Miles 2002). The
search for possible vectors, such as viruses (or virions) and
intracellular parasites, seems warranted.
Supplementary Material
Supplementary tables S1–S3 and figures S1–S3 are available at
Molecular Biology and Evolution online (http://mbe.oxford
journals.org/).
Acknowledgments
We thank ATOLep collection (as part of the LepTree project,
http://www.leptree.net/, last accessed August 2, 2012) and
personally Dr Charles Mitter (University of Maryland, MD);
Dr Oleg Kosterin (Institute of Cytology and Genetics,
Novosibirsk, Russia); Dr Vladimir Dubatolov (Institute of
Animal Systematics and Ecology, Novosibirsk, Russia); and
Dr Eva Sliwinska (Jagiellonian University, Poland) for their
help in sample collection and species identification. This
work was supported by the Ministry of Education and
Science of the Russian Federation (State Contract no. P1044).
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