The evolutionary history of mariner elements in stalk-eyed flies reveals the horizontal transfer of transposons from insects into the genome of the cnidarian Hydra vulgaris

Abstract

The stalk-eyed flies (Diopsidae, Diptera) are a family of approximately 100 species of calypterate dipterans, characterised by extended head capsules. Species within the family have previously been shown to possess six subfamilies of mariner transposons, with nucleotide substitution patterns suggesting that at least two subfamilies are currently active. The vertumnana subfamily has been shown to have been involved in a horizontal transfer event involving Diopsidae and a second dipteran family in the Tephritidae. Presented here are cloned and sequenced mariner elements from three further diopsid species, in addition to a bioinformatic analysis of mariner elements identified in transcriptomic and genomic data from the genus Teleopsis. The newly identified mariner elements predominantly fall into previously recognised subfamilies, however the publicly available Teleopsis data also revealed a novel subfamily. Three of the seven identified subfamilies are shown to have undergone horizontal transfer, two of which appear to involve diopsid donor species. One recipient group of a diopsid mariner is the Bactrocera genus of tephritid flies, the transfer of which was previously proposed in an earlier study of diopsid mariner elements. The second horizontal transfer, of the mauritiana subfamily, can be traced from the Teleopsis genus to the cnidarian Hydra vulgaris. The mauritiana elements are shown to be active in the recipient H. vulgaris and transposase expression is observed in all body tissues examined in both species. The increased diversity of diopsid mariner elements points to a minimum of four subfamilies being present in the ancestral genome. Both vertical inheritance and stochastic loss of TEs have subsequently occurred within the diopsid radiation. The TE complement of H. vulgaris contains at least two mariner subfamilies of insect origin. Despite the phylogenetic distance between donor and recipient species, both subfamilies are shown to be active and proliferating within H. vulgaris.

Full text

RESEARCH ARTICLE
The evolutionary history of mariner elements
in stalk-eyed flies reveals the horizontal
transfer of transposons from insects into the
genome of the cnidarian Hydra vulgaris
C. Alastair Grace
1,2
, Martin CarrID
2
*
1Department of Biology, University of York, Heslington, York, United Kingdom, 2Department of Biological &
Geographical Sciences, School of Applied Sciences University of Huddersfield, Huddersfield, United
Kingdom
*m.carr@hud.ac.uk
Abstract
The stalk-eyed flies (Diopsidae, Diptera) are a family of approximately 100 species of calyp-
terate dipterans, characterised by extended head capsules. Species within the family have
previously been shown to possess six subfamilies of mariner transposons, with nucleotide
substitution patterns suggesting that at least two subfamilies are currently active. The ver-
tumnana subfamily has been shown to have been involved in a horizontal transfer event
involving Diopsidae and a second dipteran family in the Tephritidae. Presented here are
cloned and sequenced mariner elements from three further diopsid species, in addition to a
bioinformatic analysis of mariner elements identified in transcriptomic and genomic data
from the genus Teleopsis. The newly identified mariner elements predominantly fall into pre-
viously recognised subfamilies, however the publicly available Teleopsis data also revealed
a novel subfamily. Three of the seven identified subfamilies are shown to have undergone
horizontal transfer, two of which appear to involve diopsid donor species. One recipient
group of a diopsid mariner is the Bactrocera genus of tephritid flies, the transfer of which
was previously proposed in an earlier study of diopsid mariner elements. The second hori-
zontal transfer, of the mauritiana subfamily, can be traced from the Teleopsis genus to the
cnidarian Hydra vulgaris. The mauritiana elements are shown to be active in the recipient H.
vulgaris and transposase expression is observed in all body tissues examined in both spe-
cies. The increased diversity of diopsid mariner elements points to a minimum of four sub-
families being present in the ancestral genome. Both vertical inheritance and stochastic loss
of TEs have subsequently occurred within the diopsid radiation. The TE complement of H.
vulgaris contains at least two mariner subfamilies of insect origin. Despite the phylogenetic
distance between donor and recipient species, both subfamilies are shown to be active and
proliferating within H.vulgaris.
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OPEN ACCESS
Citation: Grace CA, Carr M (2020) The evolutionary
history of mariner elements in stalk-eyed flies
reveals the horizontal transfer of transposons from
insects into the genome of the cnidarian Hydra
vulgaris. PLoS ONE 15(7): e0235984. https://doi.
org/10.1371/journal.pone.0235984
Editor: Ruslan Kalendar, University of Helsinki,
FINLAND
Received: April 20, 2020
Accepted: June 25, 2020
Published: July 13, 2020
Copyright: ©2020 Grace, Carr. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All newly generated
sequences have been deposited in GenBank and
the accession numbers stated in the manuscript.
(accession numbers MN719915-MN719940).
Newly assembled transposon sequences are
provided in the Supporting Information file S1
Dataset. Alignments used in the phylogenetics
analyses are provided in the Supporting
Information file S2 Dataset.
Funding: The author(s) received no specific
funding for this work.

Introduction
Transposable elements (TEs) are almost universal components of eukaryotic genomes [1], that
are capable of driving their own replication and movement within their host genome. They are
divided into two classes, based upon their transposition mechanism. The Class I elements are
retrotransposons, which transpose via an RNA intermediate; in contrast, Class II elements are
transposons that move as DNA molecules. Transposition of many DNA transposons is facili-
tated by a Transposase (Tnpase) enzyme. The Tnpase binds to the inverted terminal repeats
(ITRs) that flank the transposon and generates double stranded DNA breaks in order to excise
the parental element and then integrate the element into a new genomic location. Daughter
elements may be generated during transposition if the double stranded break created to excise
the parental element is repaired using a copy of the transposon as a template (reviewed in [2]).
DNA transposons can be subdivided through the phylogenetic analysis of their Tnpase
sequences. Within Metazoa, one of the most widespread forms of DNA transposon is the Tc1/
mariner superfamily, named after elements originally discovered in Caenorhabditis elegans [3]
and Drosophila mauritiana [4].
TEs are recognised as a major source of mutation within their hosts’ genomes. Mutations
may be deleterious due to a variety of different mechanisms. Insertion mutations result from
TEs integrating within or adjacent to host genes and thereby either altering their expression or
mRNA sequence [5,6], whilst recombination between similar TEs in different genomic loca-
tions can produce gross chromosomal rearrangements and result in selection against ectopic
exchange [7]. The presence of TEs within their hosts’ genomes results in a metabolic burden
due to RNA production, protein synthesis and the repair of double stranded DNA breaks, so
the transposition process itself can be deleterious to the host organism [8]. It can be seen that
for each of these processes individuals harbouring higher TE copy numbers will be at a greater
selective disadvantage than those with lower copy numbers. TEs can be considered to be in a
state of genomic conflict with their hosts, as their ability to proliferate may be opposed by nat-
ural selection through their hosts. Furthermore, active elements are constantly acquiring
mutations and it has been proposed that TEs have a natural life cycle within their hosts, with
elements entering a naïve genome and proliferating, before deactivating mutations, as well as
host repression mechanisms, result in the loss of all active copies [9].
TE families may maintain on-going transposition through their horizontal transfer into a
new host population. The new host is unlikely to have defences, such as RIP, RNAi or protein
targeting [10–12], against the invading TE, which will only evolve once the host adapts to the
new TE family [13]. Horizontal transfer has been shown to be a common feature of TE evolu-
tion [14–17] and has been shown to occur between closely related species [18], as well as spe-
cies from different eukaryotic supergroups [19]. The different mechanisms which underpin
horizontal transfer are currently unknown, although it has been speculated that shared para-
sites, viruses and introgression between closely related taxa may facilitate the transfer of TEs
from one species to another [12,20,21]. Horizontal transfer events may be identified through
incongruencies between phylogenetic trees, where TE phylogenies show strongly supported
differences to host species phylogenies. As phylogenies frequently have poor support, due to
the rapid rate of TE evolution [22,23], phylogenetic trees may often be consistent with both
horizontal transfer and vertical inheritance. The direction of a horizontal transfer event may
however be established if TEs from one taxon are nested, with strong support, within the TEs
of the second taxon. In such circumstances the nested taxon is likely to be the recipient that
has acquired the TE from the donor in which its TEs are nested.
The Tc1/mariner superfamily has been extensively studied with regard to both horizontal
transfer and vertical inheritance [24–26]. Carr [16] showed that 14 species of diopsid stalk-
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Competing interests: The authors have declared
that no competing interests exist.

eyed fly possess a minimum of six subfamilies of the mariner elements, with evidence for on-
going transposition uncovered in two families. Diopsids form a family of acalyptrate dipterans
(Fig 1) that exhibit hypercephaly, with head capsules laterally extended into eyestalks [27].
One subfamily of the Diopsidae, Centrioncinae, is non-hypercephalic, whilst the second sub-
family, Diopsinae, is only made up from hypercephalic species [28]. The Diopsinae are further
divided into two tribes, the Sphyracephalini and the Diopsini [27].
One of the two identified putatively active mariner subfamilies within the diopsids, the ver-
tumnana subfamily, presented a phylogeny consistent with the vertical inheritance of the
transposon through the Diopsini tribe [16]. In addition, a vertumnana mariner tnpase from
the dipteran Bactrocera neohumeralis (Tephritidae) was recovered as nested, with strong sup-
port, within the diopsid vertumnana sequences, consistent with the horizontal transfer of the
vertumnana subfamily from the diopsids into Bactrocera [16]. The geographically close habitat
ranges of Bactrocera and the potential donor genus Teleopsis led to the proposal that the hori-
zontal transfer may have occurred in New Guinea. The species composition of Teleopsis is
under debate [29–31], however here it is used within its broadest sense to include the puta-
tively nested or synonymous genera Cyrtodiopsis and Megalobops.
Experimental aims
The presented work expands upon the original diopsid mariner survey of Carr [16] through a
PCR screen of two additional species from the Diopsinae, Diasemopsis aethiopica and Diopsis
apicalis, and the first investigated species from the Centrioncinae in Teloglabrus entabensis.
Furthermore whole genome bioinformatic surveys of Teleopsis dalmanni and Sphyracephala
Fig 1. Representative phylogeny of diopsid species. Cladogram highlighting the relationships between the diopsid species involved in this study, based
upon Kotrba and Balke [28] and Kotrba et al [29].
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brevicornis were performed in order to overcome the limitations of PCR screening to investi-
gate diopsid mariner diversity. Additional bioinformatic analyses were performed using
RNA-Seq reads to identify novel mariner sequences in Teleopsis species and determine relative
gene expression levels. The diopsid-Bactrocera horizontal transfer, proposed in Carr [16], is
re-evaluated and two further horizontal transfers, between insects and the cnidiarian Hydra
vulgaris (Hydridae), are analysed in depth.
Methods
DNA extraction and PCR
Whole flies of D.aethiopica,Di.apicalis and Teloglabrus entabensis were provided by Andrew
Pomiankowski of University College, London. DNA was extracted from individual specimens
using the Proteinase K/NaCl protocol of Carr et al. [32]. PCR was carried out in 50μl volumes
(5U Abgene Red Hot DNA Polymerase, 2.5 mM MgCl2, 0.4mM dNTP), using the MAR124F
and MAR276R primers of Robertson [24], which amplify multiple mariner subfamilies. The
annealing temperature was 52˚C, with an extension step of 72 ˚C of 1 minute per cycle other
than the final cycle which utilised a 10 minute extension step. PCR was undertaken over 25
cycles.
PCR products were ligated into the pGEM-T Easy Vector (Promega) and transformed into
Subcloning Efficiency DH5αChemically Competent Cells (Invitrogen). Plasmid DNA was
extracted using the Qiagen Spin Miniprep kit and mariner DNA sequenced using T7 and SP6
primers (Macrogen Inc, Seoul, Korea). All sequences have been deposited in GenBank (acces-
sion numbers MN719915-MN719940).
Bioinformatic identification of novel mariner sequences
Genomes of T.dalmanni (assemblies NLCU01 and JXPO01), S.brevicornis (JXPL01), H.vulga-
ris (ACZU01 and ABRM01), H.oligactis (PJUT01) and H.viridissima (PJUU01) were down-
loaded from NCBI. Each was screened with RepeatMasker [33] specifying a library of
Drosophila sequences available in RepBase [34] with the following options: -species drosophila
-pa 4-nolow -no_is -inv -a. Custom mariner RepeatMasker libraries were compiled and
genomes re-screened to obtain all hits not collected using Drosophila sequences using the
same options as above with the exception of -lib custom_library.
Putative miniature inverted-repeat transposable element (MITE) families were identified in
the genomes of T.dalmanni,H.vulgaris and S.brevicornis using MITE Tracker [35]. The
resulting families_nr.fasta file was BLASTed using Tdmar,Hvmar and Sbmar nucleotide que-
ries to ascertain the regions where MITEs may have been derived from the full-length mariner
sequences.
Full-length mariner elements were identified in the T.dalmanni genome by undertaking
BLASTn similarity searches of the whole genome shotgun contigs using the partial, putatively
autonomous, tnpase sequences identified through PCR and RepeatMasker screening. Contigs
with tnpase hits for individual subfamilies were aligned with MAFFT v7.309 [36] using the
L-INS-I strategy and default parameters; this strategy resulted in the mariner elements in the
contigs being aligned with each other. Diagnostic TA target site duplications were identified to
confirm that the 5’ and 3’ termini had been identified. Illumina RNA-Seq sequencing reads
from T.quinqueguttata (SRX1490590, SRX1490591) and T.whitei (SRX485305) were mapped
onto the complete tnpase (coding sequence) cds from each T.dalmanni subfamily in order to
generate reconstructed species-specific sequences (S1 Dataset). Reads were mapped onto
tnpase cds with SMALT v. 0.2.6 (https://www.sanger.ac.uk/science/tools/smalt-0). The number
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of reads mapped to each tnpase was calculated in Tablet v.1.19.09.03 [37] from the SMALT
output SAM files.
A full-length mauritiana subfamily sequence from H.vulgaris was produced using 180bp
query sequences from Tdmar2 (NLCU01006112, 92267–90983). Reads were identified using
the Trace Archive Nucleotide BLAST on the Hydra magnipapillata–wgs database. A H.vulga-
ris mosellana partial tnpase was uncovered (accession number ABRM01005801) in the
BLASTn screening for the subfamily phylogeny, using Tdmar2 as a query sequence. The
ABRM01005801 hit was then used as a query sequences for the H.vulgaris whole genome shot-
gun contigs, using BLASTn, in order to identify contigs containing mosellana subfamily ele-
ments. Full-length sequences were then uncovered by aligning contigs in MAFFT. Contig
ABRM01012171 was assembled with an intact mosellana element that possessed a premature
stop codon at positions 431–433. Screening of the Trace Archive for the Hydra magnipapil-
lata–wgs database revealed the majority of reads showed guanosine at position 431 rather than
the thymine present in ABRM01012171. Replacing the thymine with guanosine results in a
glutamate residue in the Tnpase, as opposed to the stop codon represented in contig
ABRM01012171 (S1 Dataset).
Phylogenetic analyses
The sequenced diopsid mariner clones were aligned against homologous regions of tnpase
from publicly available diopsid sequences, as well as the uncovered tnpase sequences identified
in the whole genome contigs of S.brevicornis and T.dalmanni and the sequence read archive
(SRA) RNA-Seq files of T.whitei in MAFFT using the L-INS-I strategy and default parameters.
The alignment was manually edited by eye in order to minimise indel regions. The resulting
alignment was then subjected to maximum likelihood analysis with the raxmlGUI [38], using
the thorough bootstrapping methodology and 1,000 bootstrap replicates. The ML tree was gen-
erated from 100 starting parsimony trees and created with the GTRCAT model, following the
RAxML author’s recommendation. Bayesian inference phylogenies were created with MrBayes
3.2.6 [39] on the Cipres Science Gateway server [40]. The analyses were run with the GTR+I
+Γmodel and a four category gamma distribution to correct for among site rate variation. The
MCMC analyses consisted of 5,000,000 generations with two parallel chain sets run at default
temperatures and a sampling frequency of 1000, with a burnin value of 1250.
The diopsid irritans subfamily dataset was constructed only from putatively autonomous
sequences generated through PCR and bioinformatic screening. Nucleotide sequences were
acquired for the mosellana subfamily phylogeny using BLASTn, with the tnpase cds of Tdmar4
used as a query sequence. The nr/nt database was screened without an organism limitation,
whilst the wgs database was limited to screening Metazoa (taxid: 33208). Bactrocera sequences
in NCBI were screened for the vertumnana subfamily phylogeny using the T.quinqueguttata
sequence Tqmar1.1(DQ197023) with BLASTn with screened organisms limited to Bactrocera
(taxid: 27456) in both the nr/nt and wgs databases. Extracted sequences were aligned to diop-
sid sequences in MAFFT. Maximum likelihood and Bayesian inference phylogenies were cre-
ated for the irritans,mauritiana and mosellana subfamilies using the same protocols as for the
diopsid mariner sequences.
Subfamily maximum likelihood and Bayesian inference phylogenies, created with individ-
ual insertion sequences, were generated using the same protocols as the diopsid mariner phy-
logeny. For Hvmar1 and Hvmar2 the Trace Archive for the Hydra magnipapillata–wgs
database was screened through NCBI using the 5’ ITR and untranslated region (UTR) as query
sequences. 5’ITR/UTR query sequences were also used for Tdmar2-4 insertions phylogenies.
BLASTn searches were conducted using the SRA database on the file SRX2950777, which
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contained 9,283,997 reads of male T.dalmanni genomic DNA. For both the T.dalmanni and
H.vulgaris TEs, individual insertions were identified using the 5’ flanking DNA in the
sequencing read, with reads that contained less than 12bp of flanking DNA discarded.
Sequences were additionally discarded if the 5’ and 3’ termini of the ITR/UTR regions were
not intact.
All alignment used in the phylogenetic analyses are presented in the Nexus format in the S2
Dataset.
Gene expression analysis of mariner tnpase
Raw Illumina RNA-Seq transcriptome read files were downloaded from NCBI (see S3 Table
for accession numbers, as well as the tissue type and number of reads for each file). Reads were
mapped onto tnpase cds with SMALT. The number of reads mapped to each tnpase was calcu-
lated in Tablet v.1.19.09.03 [37] from the SMALT output SAM files. Normalised gene expres-
sion levels were calculated as transcripts per million (TPM) [41].
Results
A revised phylogeny of mariner within Diopsidae
Cloned PCR products amplified from genomic DNA extractions of Diasemopsis aethiopica,
Diopsis apicalis and Teloglabrus entabensis were subject to BLASTn similarity searching, which
resulted in 26 partial mariner sequences being identified (S1 Table). The PCR screen only
identified the vertumnana subfamily in Di.apicalis. One clone did not show any obvious null
mutations and may have been amplified from an autonomous element, whilst the other two
clones contained premature stop codons in the amplified region. The irritans,mauritiana and
mellifera subfamilies were amplified from the genomic DNA of D.aethiopica. Only one
sequence, from the irritans subfamily, did not contain premature stop codons. The genome of
the centrioncid Te.entabensis harbours a minimum of four subfamilies, with the capitata,irri-
tans,mellifera and vertumnana subfamilies all amplified in the PCR screen. Possible autono-
mous elements, lacking premature stop codons, were amplified from the irritans,mellifera and
vertumnana subfamilies, whilst the single copy amplified from the capitata subfamily con-
tained multiple internal stop codons.
The newly generated sequences were combined with the diopsid mariner sequences from
Carr [16] to produce a customised RepeatMasker [33] library, in order to screen the whole
genome contigs of T.dalmanni and S.brevicornis. The S.brevicornis screen revealed the pres-
ence of two mariner elements with putative, albeit imperfect, ITRs (accession numbers:
JXPL01000092 and JXPL01000142, S1 Dataset). Neither copy was assembled as encoding a
functional tnpase, indicating that mariner is no longer active in S.brevicornis. The screen of T.
dalmanni identified 93 mariner-like sequences within the assembled whole genome shotgun
(wgs) contigs. Phylogenetic analyses of the T.dalmanni sequences resulted in four distinct
clades (97–100% maximum likelihood bootstrap percentage (mlBP); 1.00 bayesian inference
posterior probability (biPP)), which correspond to four different mariner subfamilies (S1 Fig).
Confirming the results of the PCR screen in Carr [16], the mauritiana and vertumnana sub-
families were uncovered with RepeatMasker; furthermore the irritans subfamily and a previ-
ously unidentified subfamily were also present. The mariner complement of T.dalmanni
recovered in the RepeatMasker screen was dominated by elements from the mauritiana sub-
family, with 68 of 93 identified insertions belonging to this subfamily. Full length, putatively
autonomous, elements were uncovered for three of the subfamilies in the T.dalmanni whole
genome contigs (S1 Dataset) through the presence of inverted terminal repeats and diagnostic
TA target site duplications generated by mariner elements [42]. No functional copy of the
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vertumnana subfamily was identified, with only degraded pseudogenes present in the assem-
bled genome.
Further bioinformatic searching for diopsid mariner sequences was performed by mapping
SRA sequencing reads onto the mariner clones sequenced here and in Carr [16], as well as the
full-length mariner tnpase open reading frames (ORFs) identified in the contigs of T.dalmanni.
RNA-Seq SRA files were screened for T.quinqueguttata and T.whitei. The vertumnana,cecro-
pia,mauritiana and mellifera subfamilies, previously identified in T.quinqueguttata [16], were
all expressed at relatively low levels (S2 Table), however no additional subfamilies were identi-
fied in the T.quinqueguttata RNA-Seq reads. From the RNA-Seq reads of T.whitei complete
tnpase ORFs could be reconstructed for the irritans,mauritiana subfamilies as well as the novel
subfamily uncovered in T.dalmanni (S1 Dataset).
The combined diopsid dataset for all identified mariner sequences comprised 217
sequences and was analysed using both ML and BI methodologies (Fig 2). The six mariner sub-
families identified in diopsids by Carr [16] were recovered, however the capitata subfamily
was not recovered as monophyletic, albeit with only moderate phylogenetic support (55%
mlBP, 0.81 biPP). A seventh subfamily was identified, with strong support (100% mlBP, 1.00
biPP), which was made up only from mariner elements from T.dalmanni and T.whitei. The
two non-functional S.brevicornis mariner elements were recovered with strong support (79%
mlBP, 1.00 biPP) as members of the irritans subfamily.
The presence and identity of subfamilies in the screen undertaken here and Carr [16], as
well as the absence of families in the whole genome contigs of S.brevicornis and T.dalmanni
are shown in Table 1.
Due to low numbers of putatively autonomous copies, individual phylogenies were not cre-
ated for the diopsid representatives of the capitata,cecropia,mellifera subfamilies and the
novel subfamily uncovered in Teleopsis. The capitata and mellifera subfamilies were both iden-
tified in the centrioncid Te.entabensis, as well as the Sphyracephalini and Diopsini tribes of
Diopsinae (Fig 2). The cecropia subfamily elements are currently limited to two species, D.
dubia and T.quinqueguttata, with no additional members identified in either the PCR or bio-
informatics screens conducted here.
Four subfamilies were each analysed phylogenetically. The irritans subfamily was identi-
fied in six species, of which five harboured potentially autonomous elements. A phylogeny
of the putatively autonomous diopsid elements of the irritans subfamily, rooted with
sequences from the centrioncid Te.entabensis, is broadly congruent with the host species
phylogeny; the only element in an unexpected position is a long-branched sequence from
D.aethiopica which clusters with moderate to strong support (65% mlBP; 0.98 biPP) with an
irritans tnpase from S.europaea (S2 Fig). The phylogenies of the mauritiana and vertum-
nana subfamilies, as well as the novel subfamily uncovered in T.dalmani are presented indi-
vidually below.
Phylogenetic analysis of the mosellana subfamily
The seventh mariner subfamily, identified within the whole genome shotgun contigs of T.dal-
manni and RNA-Seq transcriptome reads of T.whitei, was not uncovered in the diopsid PCR
screen of Carr [16]. In order to investigate the evolutionary origins of the subfamily in the
diopsids BLASTn similarity searching, through NCBI, was undertaken with the nucleotide
sequence of a putatively autonomous tnpase from T.dalmanni. Only the top hit uncovered for
each species was extracted for phylogenetic analysis. The screen uncovered the presence of 41
tnpase sequences that clustered with the novel Teleopsis sequences (95% mlBP; 1.00 biPP, Fig
3). The earliest published sequence from the subfamily was identified in the genome of the
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Fig 2. Maximum likelihood phylogeny of diopsid mariner sequences. The phylogeny was constructed from 480 aligned
nucleotide positions using the GTRCAT model, and estimated nucleotide frequencies. Values for mlBP and biPP are shown
above and below the branches respectively. 100% mlBP and 1.00 biPP are both denoted by “”. Values <70% mlBP and <0.97
biPP are denoted by “-”. The scale bar represents the number of substitutions per site. Individual mariner subfamilies are
bracketed and colour-coded.
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dipteran Sitodiplosis mosellana and accordingly the subfamily is named here as the mosellana
subfamily.
The Teleopsis sequences fell within two distinct groups in the mosellana subfamily, however
there were no strongly supported branches (70%mlBP and 0.97biPP) separating the two
clusters (Fig 3). Of the two groups, one consisted of three non-autonomous T.dalmanni
sequences which all harboured premature stop codons. The second group possessed sequences
from both T.dalmanni and T.whitei, with both species harbouring putatively functional
tnpase sequences. The potentially active Teleopsis elements formed a monophyletic group with
tnpase sequences from three Bactrocera species and S.mosellana, however this clade of dip-
teran elements had no phylogenetic support.
Thirty nine of the mosellana sequences were uncovered from pancrustacean species, with
hosts falling within the insect orders Archaeognatha, Coleoptera, Diptera, Hymenoptera and
Lepidoptera, as well as the crustacean Copepoda order. The two remaining sequences were
identified in the whole genome shotgun contigs of the hydrozoan cnidarian Hydra vulgaris
(synonymised with H.magnipapillata,H.littoralis and H.attenuata) and the ray-finned fish
Cyprinodon variegatus. The sequence from the C.variegatus contig showed a single nucleotide
difference (99.9% nucleotide identity) from a mosellana subfamily element uncovered in the
copepod Caligus rogercresseyi. This is consistent with a horizontal transfer between the two
species, with C.variegatus being a marine fish and C.rogercresseyi a sea louse parasite of fish.
The more biologically plausible direction of transfer would be from C.rogercresseyi to
Table 1. The presence of mariner subfamilies within screened diopsid species.
Species capitata cecropia irritans mauritiana mellifera mosellana vertumnana
Centrioncinae
Te.entabensis Temar4 -Temar2 -Temar3 -Temar1
Sphyracephalini
C.seyrigi - - - - Csmar1-2 - -
S.beccarii - - - - - - -
S.brevicornis Absence
a
Absence
a
Sbmar1 Absence
a
Absence
a
Absence
a
Absence
a
S.europaea Semar3,Semar4 -Semar1 Semar2 Semar5 - -
Diopsini
D.aethiopica - - Damar3 Damar1 Damar2 - -
D.comoroensis - - - - Dcmar2 -Dcmar1
D.dubia Ddmar2 Ddmar3 - - Ddmar1 - -
D.meigenii - - - - - - Dmmar1
D.signata - - - - Dsmar2 -Dsmar1
Di.apicalis - - - - - - Dioamar1
T.breviscopium - - - Tbmar2 Tbmar3 -Tbmar1
T.dalmanni Absence
a
Absence
a
Tdmar3 Tdmar2 Absence
a
Tdmar4 Tdmar1
T.quadriguttata - - - - - - Tqdmar1
T.quinqueguttata -Tqmar4 -Tqmar2 Tqmar3 -Tqmar1
T.rubicunda - - - - - - Trmar1
T.thaii - - - - - - Ttmar1
T.whitei - - Twmar3
b
Twmar2
b
-Twmar4
b
Twmar1
Dash represents the absence of the subfamily in PCR and transcriptome screens.
a
: The subfamily is absent from the assembled whole genome contigs.
b
: Sequences identified in RNA-Seq reads.
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Fig 3. Maximum likelihood phylogeny of the mosellana subfamily. The phylogeny was constructed from 1032 aligned nucleotide positions
using the GTRCAT model, and estimated nucleotide frequencies. The mosellana elements are bracketed and rooted with tnpase sequences
from the irritans and mellifera subfamilies. Values for mlBP and biPP are shown above and below the branches respectively. Diopsid
sequences are shown in purple, other dipteran sequences are shown in light blue. Dark blue sequences are from hymenopteran hosts, pink
sequences from lepidopteran hosts, the orange sequence is from an archaeognath host and light green sequences are from copepod hosts. The
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C.variegatus, as the fish mariner sequence is nested within those of the pancrustaceans by
three strongly supported branches (all 96% mlBP and 1.00 biPP). An alternative explanation
is that the sequenced genomic DNA of C.variegatus was contaminated by DNA from C.roger-
cresseyi. Consistent with this hypothesis only a single copy of the transposon was uncovered in
the whole genome contigs of C.varietgatus, present in a contig (JPKM01108314) which only
contained 53bp of flanking DNA. Two copies, possessing different flanking DNA, of the mosel-
lana element were identified in C.rogercresseyi contigs (LBBV01023767 and LBBU01012271).
Mapping RNA-Seq reads of C.rogercresseyi (54.6 million reads, SRX864101-2 and
SRX1481244) to the mosellana subfamily sequence (accession number LBBV01023767)
revealed 468 reads that spanned the entire coding region. The subfamily however could not be
identified in C.variegatus RNA-Seq reads (124.7 million reads, SRX3140005-6, SRX3140009-
11, SRX5103143, SRX5103155 and SRX5103167). The lack of expression in C.variegatus sug-
gests either an unsuccessful horizontal transfer or contamination of genomic DNA during
whole genome sequencing.
A full-length consensus sequence, labelled Hvmar1, was generated for the mosellana sub-
family element in the genome of H.vulgaris (S1 Dataset). The H.vulgaris tnpase clustered with
mariner elements from the dipteran Bactrocera tryoni and the hymenopteran ants Camponotus
floridanus and Pseudomyrmex gracilis (100% mlBP, 1.00biPP, Fig 3). Hvmar1 exhibited 83.7%
and 87.0% nucleotide identity with the C.floridanus (accession number XM_011264844) and
P.gracilis (accession number XM_020442418) mariner elements respectively, despite cnidari-
ans and insects last sharing a common ancestor approximately between 600–700 million years
ago [43–45]. Hvmar1 appears to be a genuine component of the H.vulgaris genome, as simi-
larity searching of the NCBI Hydra magnipapillata wgs Trace Archive with BLASTn revealed
148 distinct 5’ termini. Furthermore, mapping RNA-Seq reads to the tnpase also showed
Hvmar1 to be expressed in whole polyps, as well as head, foot, tentacle and body tissue (S2
Table).
The high nucleotide identity between Hvmar1 and the hymenopteran mariners, as well as
the nested position of Hvmar1 within the insect mosellana sequences is consistent with a hori-
zontal transfer event. The donor species would appear to be an insect, although the donor
insect order cannot be confirmed, as Hvmar1 clusters with mariner elements from dipteran
and hymenopteran species but is not nested within either group. Similarity searching of whole
genome contigs, with BLASTn, using Hvmar1 as a query sequence failed to uncover mosellana
sequences in either H.viridis or H.oligactis, as well as the genomes of other cnidarians. The
absence of the subfamily within other Hydra species is consistent with H.vulgaris being the
recipient species of the horizontal transfer event in Cnidaria.
A reassessment of the diopsid-Bactrocera vertumnana horizontal transfer
Carr [16] proposed a putative horizontal transfer event between Diopsini stalk-eyed flies and
the tephritid Bactrocera neohumeralis. Due to the B.neohumeralis sequence (clone Bnmar29,
accession number AF348438.1) being nested within the Teleopsis sequences, it was suggested
that the direction of transfer was from Teleopsis to Bactrocera, although the actual donor and
recipient species could not be confirmed. The identification of vertumnana sequences from
two additional diopsid species (Te.entabensis and Di.apicalis), as well as the additional
brown sequence is from a hydrozoan host and the mustard sequence from a fish host. The outgroup sequences are from the irritans
subfamily (Semar1 and Temar2.2) and the mellifera subfamily (Temar3.1). 100% mlBP and 1.00 biPP are both denoted by “”. Values <50%
mlBP and <0.70 biPP are denoted by “-”. The scale bar represents the number of substitutions per site.
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sequencing of whole genomes from Bactrocera species, allowed a review of the phylogenetic
relationships between vertumnana elements from diopsid and Bactrocera hosts.
BLASTn similarity searching of Bactrocera wgs contigs and the nr/nt database, using
Tqmar1.1(Accession Number DQ197023), uncovered vertumnana sequences from five spe-
cies in addition to clone Bnmar29 from B.neohumeralis. The vertumnana subfamily therefore
has a greater distribution within Bactrocera than recognised in Carr [16]. Diopsid vertumnana
sequences, taken from all twelve species in which the subfamily has been identified, were
aligned with seven Bactrocera elements. A single sequence was used for each diopsid species in
which the subfamily has been identified with the exception of T.dalmanni; due to internal
deletions two sequences were used for T.dalmanni. The resulting alignment was subjected to
maximum likelihood and Bayesian inference analyses (Fig 4). The Bactrocera elements formed
a strongly supported monophyletic group (96% mlBP, 1.00 biPP), as did the vertumnana
sequences from Teleopsis (94% mlBP, 1.00 biPP) and Diasemopsis (88% mlBP, 0.99 biPP). The
expanded vertumnana phylogeny therefore rejects the placement of the B.neohumeralis trans-
poson within the grouping of Teleopsis elements recovered in the Carr [16] ML phylogeny.
Rooting the phylogeny with the mariner sequenced from the earliest branching diopsid genus,
Teloglabrus, recovers the expected relationships between elements from the host diopsid gen-
era under the model of vertical inheritance. The Bactrocera elements are recovered as nested
within the Diopsini vertumnana elements (100% mlBP, 1.00 biPP), as the sister-group to the
tnpases sequenced from Teleopsis species (99% mlBP, 1.00 biPP). Re-rooting the phylogeny
Fig 4. Maximum likelihood phylogeny of the vertumnana subfamily. The phylogeny was constructed from 510 aligned nucleotide positions using the
GTRCAT model and estimated nucleotide frequencies. Support values are shown in the same format as Fig 3.
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with the Bactrocera sequences fails to recover any of the expected genera relationships based
upon the host species phylogeny. All further potential rooting of the phylogeny also fail to
recover the expected genera relationships based upon the host species phylogeny. The most
biologically plausible rooting for the phylogeny is therefore between the Teloglabrus tnpase
and the remaining transposons, with the tephritid vertumnana being recovered as the sister-
group to the Teleopsis transposons. The increased dataset presented here therefore provides
further evidence for a horizontal transfer event of the vertumnana subfamily from the Diopsini
to Bactrocera.
Phylogenetic evidence for a cross-phyla horizontal transfer event from
Teleopsis to Hydra
BLAST screening of the newly sequenced mariner elements from D.aethiopica unexpectedly
recovered a high scoring hit (E value: 5e-175) from H.vulgaris (accession number U51187,
clone Hydra.vulgaris.6) when Damar1.1of the mauritiana subfamily was used as a query
sequence. The only other mariner sequences uncovered with similarly high BLAST scores
were from other diopsid species. A BLASTn screen of the H.vulgaris whole-genome shotgun
contigs, using sequence Hydra.vulgaris.6 as a query sequence, confirmed the presence of 218
similar sequences recovered with E values of 0.0 indicating that the original Hydra.vulgaris.6
sequence was a genuine component of the H.vulgaris genome and not a PCR contaminant.
Robertson [46] highlighted the relationship of clone Hydra.vulgaris.6 with insect mariner
sequences, but did not propose whether a horizontal transfer donor was a cnidarian or an
insect.
The finding raised the possibility of the identification of a second horizontal transfer event
of a mariner subfamily from diopsids, as well as a second insect to Hydra horizontal transfer. A
reciprocal BLASTn of Hydra.vulgaris.6 against diopsid sequences in the nr/nt database
resulted in high scoring hits (E value: 0.0, query coverage: 100%, nucleotide identity: >95%)
for mariner sequences deposited from T.breviscopium,T.dalmanni and T.quinqueguttata. A
BLASTn screen of the whole-genome shotgun contigs of T.dalmanni uncovered a complete,
intact mauritiana mariner element (Accession Number NLCU01006112, position 92,267–
90,983), designated as Tdmar2 on the basis of identity with the sequenced clones generated by
Carr [16]. Tdmar2 possessed 27bp inverted terminal repeats (ITRs) and a putative tnpase ORF
of 1,038bp in length (S1 Dataset).
Screening the NCBI Sequencing Trace Archive of the H.vulgaris genome with 180bp query
sequences of Tdmar2 uncovered hits across the entire element. The concatenated hits pro-
duced a putative mauritiana subfamily element, designated as Hvmar2, which exhibited 31
nucleotide differences from Tdmar2 out of 1,287 sites (97.6% nucleotide identity). The coding
regions of Tdmar2 and Hvmar2 showed 19 nucleotide differences (98.2% nucleotide identity),
which resulted in 14 amino acid differences between the putative Tnpases.
Similarity screening with BLASTn of the wgs contigs of H.oligactis and H.viridis, as well as
all available cnidarian whole-genome contigs in NCBI failed to uncover orthologous mauriti-
ana subfamily sequences. Phylogenetic analyses of Hvmar2 and the diopsid mauritiana ele-
ments clustered the Hvmar2 with the Teleopsis elements in a group with robust support (84%
mlBP, 0.99 biPP, Fig 5) and furthermore nested Hvmar2 within the Teleopsis sequences (97%
mlBP, 1.00 biPP). Rooting the mauritiana sequences with those from the Sphyracephalini S.
europaea recovered the expected relationships between the diopsid TEs based upon their host
species, consistent with their vertical inheritance since the origin of the Diopsinae. An alterna-
tive rooting, between Hvmar2 and the diopsid elements, failed to recover the expected species
relationships, indicating that this is not the correct root for the phylogenetic tree. The
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mauritiana subfamily Hvmar2 subfamily therefore appears to have had a recent origin from
within the diopsids and specifically from the Teleopsis genus. T.dalmanni does not appear to
be the donor species for Hvmar2, as Tdmar2 is recovered as being more closely related to
mauritiana elements from both T.whitei and T.breviscopium in Fig 5. Based upon the phylog-
eny the donor species would appear to be a closer relative of T.breviscopium,T.dalmanni and
T.whitei than T.quinqueguttata, as the T.quinqueguttata elements are recovered with strong
support at the base of the Teleopsis mauritiana elements. The basal position of the T.quinque-
guttata elements mirrors the basal position of T.quinqueguttata in the host species genus [28,
29], further highlighting the reliability of the phylogenetic signal in the mariner sequences.
Mauritiana tnpase is expressed in the genomes of Teleopsis species and H.
vulgaris
The phylogenetic analyses of tnpase from the mauritiana subfamily indicated that an active
mariner element had undergone horizontal transfer from an unknown Teleopsis species into
the cnidarian H.vulgaris. In order to investigate this proposed transfer the activity of mariner
Fig 5. Maximum likelihood phylogeny of the mauritiana subfamily. The phylogeny was constructed from 492 aligned nucleotide positions using the
GTRCAT model and estimated nucleotide frequencies. Support values are shown in the same format as Fig 3.
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was analysed in both Teleopsis, as the putative donor genus, and H.vulgaris, from the putative
recipient genus.
RNA-Seq reads were mapped onto the mariner subfamily tnpase sequences of T.dalmanni,
T.quinqueguttata and T.whitei (S2 Table). Within T.dalmanni it was possible to investigate
gene expression within the male and female germ cells, as well as whole larvae and adult head
cells. Consistent with the lack of identified autonomous vertumana elements in T.dalmanni,
the subfamily exhibited the lowest mapping coverage (<1.5 TPM, 0.001% of RNA-Seq reads
in all five tissue types), albeit based upon a shorter CDS (S2 Table). The irritans,mauritiana
and mellifera subfamilies were all expressed in both ovary and testes cells. Expression was also
observed in male and female heads, raising the possibility that transposition occurs in both
somatic and germline cells. Across the examined five tissue types in T.dalmanni,tnpase TPM
and the absolute number of mapped reads was higher for the mosellana subfamily (Tdmar4)
than the mauritiana subfamily (Tdmar2). T.whitei testes expression patterns were similar to
those of T.dalmanni, with the vertumnana TPM being an order of magnitude lower than val-
ues of the irritans,mauritiana and mosellana subfamilies. As in T.dalmanni testes, the mosel-
lana subfamily TPM was more than sevenfold than higher than the value of the mauritiana
subfamily. In contrast to T.dalmanni and T.whitei, the vertumnana subfamily showed the
highest TPM in T.quinqueguttata testes. However only 39 out of 84,490,068 sequencing reads
mapped to the cecropia,mauritiana and mellifera subfamilies, indicating that the other sub-
families may either be non-functional or silenced in T.quinqueguttata testes.
Unlike the diopsids, H.vulgaris lacks a sequestered germline [47], as gametes develop from
interstitial cells which also produce neurons, cnidocytes and secretory cells [48,49]. As with
Hvmar1 of the mosellana subfamily, Hvmar2 was shown to be expressed in all cell types (S2
Table), consistent with its on-going transposition. Mapping coverage for the mosellana sub-
family was fourfold to eightfold higher compared to the mauritiana subfamily for all examined
tissues, mirroring the expression patterns observed in T.dalmanni and T.whitei.
Phylogenetic analyses of individual mariner insertions in the genomes of H.
vulgaris and T.dalmanni
The expression of tnpase is required for the transposition of mariner in both species. However,
the observed expression does not confirm that the mauritiana elements are transposing, since
the expression of tnpase mRNA could reflect the presence of a repressor isoform, such as the
KP protein that supresses transposition of the Pelement in Drosophila melanogaster [8,50].
The mariner elements in both H.vulgaris and T.dalmanni were therefore subjected to phylo-
genetic analysis in order to uncover evidence of on-going transposition. Upon transposition
daughter elements should possess identical sequences to their parental elements in phyloge-
netic trees, despite being present at different genomic locations. Nucleotide differences will
accumulate over time, as the parental and daughter elements begin to diverge following
transposition.
A phylogeny of the Hvmar1 5’ ITR/UTR region revealed that insertions were mainly pres-
ent on short terminal branches (S3 Fig), with 62 identical copies at different genomic locations.
The Hvmar2 phylogeny of 103 insertions was also generated from 5’ ITR/UTR sequences. The
phylogeny was similar to that of Hvmar1, with a large number of short branched sequences, as
well as 26 identical paralogous sequences. At the base of the phylogeny was a weakly supported
grouping of long branched sequences, which appear to be older elements that are no longer
transposing. The long branch sequences contained unique indels, consistent with their greater
antiquity in the H.vulgaris genome, whilst the presence of identical paralogous sequences
indicate that there is on-going transposition of both Hvmar1 and Hvmar2 in H.vulgaris.
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The 5’ termini sequences identified in the sequencing reads of T.dalmanni also uncovered
identical paralogous copies for the subfamilies Tdmar2-4 (S4 Fig). Within the 86 sequences of
Tdmar2, four insertions at different genomic locations showed identical sequences; for
Tdmar3 there were 9 identical paralogous insertions across 134 sequences. In contrast to the
RepeatMasker tnpase screen of the whole genome contigs, which only uncovered 11 Tdmar4
sequences, the BLAST screen of the 5’ ITR/UTR of sequencing reads identified 2,422 distinct
insertions. Of those, 1,790 copies were identical to other copies in different genomic locations,
indicating a high level of recent transposition of the mosellana subfamily in T.dalmanni.
Mariner-derived MITEs are abundant in the genome of T.dalmanni
Due to the discrepancy between the results of the T.dalmanni RepeatMasker tnpase screen,
where Tdmar2 appeared to have the highest copy number, and the ITR/UTR wgs Trace
Archive BLAST screen, which was dominated by Tdmar4 insertions, the genomes of T.dal-
manni,S.brevicornis and H.vulgaris were screened for MITEs. MITEs are non-autonomous
transposons that possess ITR sequences, but lack internal coding tnpase sequences [51]. MITE
families are derived from autonomous transposons and are mobilised via the Tnpase enzymes
of autonomous element copies [52]. The T.dalmanni screen uncovered the presence of 342
putative MITE families, of which 55 appeared to have originated from mariner elements
(Table 2). Within the mariner-derived MITEs 25 families had an origin within Tdmar4 of the
mosellana subfamily and Tdmar4-derived MITEs contributed 2017 out of 2489 MITE inser-
tions in the whole genome contigs (Table 3). Consistent with the lack of autonomous Tdmar1
copies, the vertumnana subfamily possessed the fewest MITEs and only contributed 73 MITE
insertions, split across eight families.
The S.brevicornis genome possessed 36 MITE families, none of which appeared to have a
mariner-derived origin (Table 2), further highlighting the paucity of mariner-like elements
within the species. In contrast to S.brevicornis the genome of H.vulgaris was rich in MITE ele-
ments. A total of 126 MITE families were identified, however none of the families were found
to have an origin from mariner elements (Table 2).
Table 2. MITE sequences BLASTed against full-length sequences Hvmar1-2,Sbmar1 and Tdmar1-4.
Species Total number of MITE families Number potentially mariner -derived
Diopsidae
S.brevicornis 36 0
T.dalmanni 342 55
Hydridae
H.vulgaris 126 0
https://doi.org/10.1371/journal.pone.0235984.t002
Table 3. Characterisation of MITE families for each mariner subfamily in T.dalmanni.
Subfamily No. of MITE families Total copy number
Tdmar1 8 73
Tdmar2 5 98
Tdmar3 17 301
Tdmar4 25 2017
MITEs sharing identity with multiple subfamilies were assigned to that with the most significant e-value.
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Discussion
Multiple mariner subfamilies were present in the ancestral diopsid
The original diopsid mariner study of Carr [16] identified six subfamilies of mariner transpo-
sons within 14 species of diopsid, however, with the exception of the vertumnana subfamily,
no attempt was made to determine the evolutionary origins of the subfamilies. The sequencing
of thirteen mariner clones from the centrioncid Te.entabensis, allows greater insight into the
subfamilies present in the last common ancestor (LCA) of diopsids.
Through the use of subfamily phylogenies, as well as the distribution of subfamilies across
species, it is possible to estimate the latest evolutionary points of origin of the subfamilies
within the diopsids (S5 Fig). The capitata,irritans,mellifera and vertumnana subfamilies are
all present in the genome of the centrioncid Te.entabensis as well as multiple Diopsinae spe-
cies, consistent with their presence in the diopsid LCA. Subfamily phylogenies of both irritans
and vertumnana also indicate their vertical inheritance since the origin of the Diopsidae.
The mauritiana subfamily was amplified in species across the Diopsinae, but not from the
centrioncid Te.entabensis. Only 13 clones were sequenced from the genomic DNA of Te.enta-
bensis, so it is possible that the absence of the mauritiana subfamily was due to the limited sam-
ple size. The distribution of mauritiana elements, as well as the phylogenetic relationships
presented in Fig 5 indicate that the subfamily was present in the genome of the Diopsinae
LCA, but an earlier origin in the ancestor of all diopsids cannot be excluded on the basis of the
current dataset.
The cecropia subfamily has only been identified within Diopsini species (Table 1), but its
absence in both the Sphyracephalini and Centrioncinae is currently mainly posited on small-
scale PCR screens which may not uncover low copy number, or non-functional degenerate,
subfamilies. Finally, only two Teleopsis species have been shown to harbour mosellana ele-
ments, consistent with a late origin of the subfamily in the diopsids. The subfamily has not
been amplified through PCR in any diopsid species or the earlier screen of H.vulgaris genomic
DNA [46]. The WVPHEL amino acid motif on which the Robertson [24] forward degenerate
mariner primer was designed is not present in the reconstructed mosellana Tnpase sequences
in T.dalmanni,T.whitei and H.vulgaris (S1 Dataset). Conceptual translations of the mosellana
tnpase sequences used to generate the subfamily phylogeny in Fig 3 also lack the WVPHEL
motif. The homologous region of the Tnpase could be translated in 33 species and 19 encoded
the amino acids LVPKEL. The Robertson primers appear to lack specificity to mosellana ele-
ments in a broad range of species; therefore the absence of amplified PCR product may be the
result of failed primer binding rather than the absence of mosellana elements in a species’
genome. The loss of the highly conserved WVPHEL motif will require the design of alternative
degenerate primers to amplify mosellana elements from genomic DNA. The lack of the mosel-
lana subfamily from the transcriptome of T.quinqueguttata is consistent with an origin of the
subfamily in Teleopsis after the lineage leading to T dalmanni and T.whitei split from the T.
quinqueguttata lineage. It remains however possible that the mosellana subfamily has greater
antiquity in Teleopsis, and perhaps other diopsid taxa, and has undergone stochastic loss in T.
quinqueguttata.
The mariner subfamilies present in the T.dalmanni genome have persisted for sufficient
time in order to generate non-autonomous MITE families. The MITE complement is domi-
nated by transposons generated from Tdmar4 of the mosellana subfamily, however all four
subfamilies have produced multiple MITE families. The available data suggest that the ances-
tral diopsid possessed a diverse complement of mariner elements, with a minimum of four
subfamilies residing in the genome. Due to the limitations of small-scale PCR screens and lim-
ited whole genome availability across the Diopsidae, the origins of the cecropia,mauritiana
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and mosellana subfamilies are unresolved and they may be either ancestral or more recent
acquisitions into diopsid genomes. Stochastic loss of mariner subfamilies has occurred within
the diopsids, as can been seen by the absence of any active copies in the sequenced genome of
S.brevicornis, as well as the absence of mariner elements in PCR screen of the genomic DNA
of S.beccarii reported in Carr [16]. S brevicornis is a closer relative of S.europaea, a species
which possess multiple mariner subfamilies, than S.beccarrii (Fig 1), indicating the absence of
mariner elements in S.beccarrii and S.brevicornis is due to independent losses.
Within T.dalmanni the irritans,mauritiana and mosellana subfamilies are all expressed in
both the male and female germline. This finding is consistent with mariner transposition
occurring in both sexes, unlike the sex-restricted transposition of some TE families, such as
copia and Doc, observed in D.melanogaster [53].
The vertumnana subfamily diopsid-Bactrocera horizontal transfer event
The phylogeny presented here recovers a nested position of the Bactrocera elements within
those of the Diopsini in the vertumana subfamily (Fig 4). Carr [16] proposed a horizontal
transfer event within New Guinea from either a Teleopsis species, or related diopsid, to Bactro-
cera, based primarily upon the phylogeny of the mariner elements, but also the then recognised
distribution of Teleopsis and Bactrocera species. More recently, Feijen and Feijen [54] stated
that the direction of horizontal transfer should be re-evaluated, as they considered Teleopsis
species to be absent from New Guinea. The enlarged vertumnana phylogeny presented here,
with additional diopsid and Bactrocera mariner sequences, provides an ideal opportunity to
reassess the inheritance of the subfamily. The increased diversity of Bactrocera mariner ele-
ments from the vertumnana subfamily highlights that the proposed horizontal transfer event
did not occur into B.neohumeralis, but within an ancestor of at least five Bactrocera species.
The monophyly of the Bactrocera elements, which are nested within the paraphyletic Diopsini
vertumnana elements with strong support, points toward the diopsids being the donor group
and Bactrocera being the recipients. Feijen and Feijen’s [54] alternative argument failed to take
into account the required horizontal transfer of a vertumnana mariner from the Australasian
B.neohumeralis into African Diasemopsis species if the hypothesis of Bactrocera being the
donor group was correct. The revised phylogeny presented here provides additional evidence
for the Diopsidae being the donor to Bactrocera. The putatively recipient Bactrocera species
are present across South Asia [55] and not confined to Australasia, thereby expunging the
argument that the direction of transfer could not have been from diopsids to Bactrocera due to
the absence of Teleopsis from New Guinea. The presence of the vertumana subfamily in addi-
tional African diopsid genera, in Diopsis and Teloglabrus, would require a further two inde-
pendent, intercontinental horizontal transfer events, under the Bactrocera to Diopsidae
horizontal transfer route. The phylogeny presented here requires a single horizontal transfer
event from Diopsini to Bactrocera within South East Asia, as was the case in the original Carr
[16] phylogenetic tree. However the alternative route from Bactrocera to Diopsidae requires a
minimum of four independent horizontal transfer events, into the genera Diasemopsis,Diopsis,
Teleopsis and Teloglabrus.
Horizontal transfer events of mariner from insects to H.vulgaris
The genome of H.vulgaris is rich in TEs, with approximately 57% of the genome being made
up from over 500 TE families [49]. The H.vulgaris genome is considerably larger than that of
its distant congener H.viridis [56] and the difference has been speculated to be the result of
bursts of transposition by multiple TE families [49,56,57]. DNA transposons contribute to
21% of the H.vulgaris genome, with mariner elements alone making up 4% of the sequenced
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genome [49]. PCR screens of the genomes of H.vulgaris, including the North American sub-
species/sister-species H.littoralis, have identified mariner elements from the capitata,cecropia,
irritans and mauritiana subfamilies [46].
Two of the mariner subfamilies identified here in diopsid species are also present in the
genome of H.vulgaris, these being the mauritiana and mosellana subfamilies. Orthologous
mariner elements appear to be absent from the sequenced genomes of both H.oligactis and H.
viridis, consistent with their invasion of Hydra occurring after the H.vulgaris lineage split
from other Hydra approximately 21–28 million years ago [58]. The presence of the subfamilies
in the ancestors of either all three Hydra species or only H.vulgaris and H.oligactis is a less
parsimonious explanation, which would require multiple stochastic loss events in addition to
the gains of the two subfamilies through horizontal transfer. The mosellana subfamily, desig-
nated Hvmar1, was not identified in a previously published PCR screen in either H.vulgaris or
H.littoralis genomic DNA [46], however this may be due to the lack of subfamily primer speci-
ficity due to the loss of the WVPHEL amino acid motif. The phylogeny of the mosellana sub-
family presented here indicates a horizontal transfer event from an unknown insect donor into
H.vulgaris (Fig 3). The lack of a clear donor species, or even donor insect order, has resulted
in Hvmar1 being placed on a relatively long branch within the subfamily phylogeny; it is there-
fore unclear as to whether the horizontal transfer was an ancient or more recent event. The
transfer of Hvmar1 appears to have been a successful one. The tnpase is expressed across multi-
ple cell types and, based upon the ITR/UTR phylogeny which showed 62 identical paralogous
copies, Hvmar1 is currently transposing within the H.vulgaris genome.
In contrast to the mosellana subfamily, the mauritiana subfamily, designated here as
Hvmar2, was amplified by Robertson with clone Hydra.vulgaris.6 [46]. The relationship of
Hydra.vulgaris.6 to other mariner elements was not robustly resolved in the Robertson phylog-
eny, but it was nested within a clade of insect mariner elements. No diopsid mariner elements
were included in the phylogeny, with Hydra.vulgaris.6 clustering with mauritiana elements
from D.mauritiana and the hymenopteran Myrmecia occidentalis. The mauritiana phylogeny
presented here robustly nests Hvmar2 within the diopsid elements and highlights a putative
horizontal transfer between an unknown Teleopsis species and H.vulgaris. The Tnpases of the
mauritiana subfamily were shown by Carr [16] to be evolving under purifying selection on
their amino acid sequences, indicating the elements are active and therefore potentially viable
donors. The mauritiana horizontal transfer into H.vulgaris has also been successful, with
Hvmar2 tnpase expression observed across body tissues and multiple identical paralogous
insertions identified in the whole genome sequencing reads.
The mechanism, or mechanisms, that have facilitated the horizontal transfer events from
insects into H.vulgaris are difficult to envisage. Aquatic cnidarians and terrestrial insects shar-
ing mutual parasites or viruses appears to be unlikely, given their approximately 600 million
year divergence time and different habitats. Terrestrial insect larvae or imagos which fall into
the water column may be preyed upon by Hydra, which are known to feed upon dipteran lar-
vae and can engulf prey items in excess of 30mm in length [59]. As Teleopsis species, as well as
members of other diopsid genera, often live over bodies of water [29,30,60] opportunistic pre-
dation may potentially allow diopsid mariner DNA to be taken up by Hydra cells resulting in
horizontal transfer.
The lack of mariner-derived MITEs is consistent with both Hvmar1 and Hvmar2 being
recent invaders in the H.vulgaris genome and contrasts with the proliferation of MITE fami-
lies in T.dalmanni. The absence of orthologous families of Hvmar1 and Hvmar2 in the whole
genome contigs of both H.viridis and H.oligactis suggests that the horizontal transfer events
from insect donors occurred within the H.vulgaris species complex. The lack of available
sequence data means that the approximate age of the mosellana transfer cannot be gauged,
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Cross-phyla horizontal transfers of mariner elements
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however the very low nucleotide divergence (~2.5%) between Tdmar2 and Hvmar2 suggest
that the mauritiana transfer occurred very recently in the evolutionary history of Hydra. A
donor Teleopsis species has not been identified, with the Hvmar2 tnpase showing 98.2% and
98.5% nucleotide identity to the tnpase sequences of Tdmar2 and Twmar2 respectively. Teleop-
sis species are restricted to eastern Asia, with many species present in south east Asia [30,54]
therefore a broader screen of Teleopsis species will be required in order to determine if the
genus harbours the donor species. The subspecies, or strain, of H.vulgaris which has been
shown to harbour Hvmar2 in its genome, H.magnipapillata, was isolated from Japan and
closely related populations are present in south east Asia [58], overlapping with Teleopsis spe-
cies and indicating a possible east Asian location for the mauritiana subfamily horizontal
transfer event. The H.vulgaris AEP strain is a North American laboratory-produced line, gen-
erated through a cross of strains from California and Pennsylvania [58]. The presence of
Hvmar2 in the transcriptome reads from the AEP strain highlights the transcontinental move-
ment of this mauritiana element in the H.vulgaris global population.
Conclusions
Sequencing of mariner elements from the basal centrioncid Te.entabensis points to a mini-
mum of four subfamilies being present in the ancestral diopsid. A total of seven subfamilies
have now been identified within Diopsidae genomes. The identification of the mosellana sub-
family in whole genome sequence data highlights the inherent dangers of relying upon degen-
erate primers in PCR screening for mariner elements, as the widely used primers designed by
Robertson [37] do not amplify this subfamily. Two diopsid mariner subfamilies appear to have
undergone horizontal transfer to species outside of the family. One of the putative recipient
species, H.vulgaris, has also acquired a mariner element from a second, unidentified, insect
donor. Despite the great evolutionary distance between insects and cnidarians, both trans-
ferred mariner elements have successfully proliferated in Hydra contributing to the diverse TE
complement of this species.
Supporting information
S1 Dataset. Annotated sequences of the mariner subfamilies characterized in S.brevicornis,
Teleopsis species and Hydra vulgaris.The full-length sequences for each identified subfamily
are presented, along with putative open-reading frames, untranslated regions and flanking
repeats. Conceptual translations of encoded proteins are provided.
(TXT)
S2 Dataset. Alignments used in the phylogenetic analyses. Alignments are provided in the
Newick format. Columns present within square brackets were excluded from the phylogenetic
analyses.
(TXT)
S1 Fig. Maximum likelihood phylogeny of the T.dalmanni mariner sequences uncovered
in whole genome shotgun contigs using the customized mariner RepeatMasker library.
The phylogeny was constructed from 1099 aligned nucleotide positions using the GTRCAT
model, and estimated nucleotide frequencies. Values for mlBP and biPP are shown above and
below the branches respectively. 100% mlBP and 1.00 biPP are both denoted by “”. Values
<50% mlBP and <0.70 biPP are denoted by “-”. The scale bar represents the number of substi-
tutions per site. Individual mariner subfamilies are bracketed and colour-coded.
(PDF)
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Cross-phyla horizontal transfers of mariner elements
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S2 Fig. Maximum likelihood phylogeny of diopsid irritans tnpase sequences. The phylogeny
was constructed from 500 aligned nucleotide positions using the GTRCAT model, and esti-
mated nucleotide frequencies. The phylogeny layout is the same as in S1 Fig.
(PDF)
S3 Fig. Maximum likelihood phylogenies of mariner 5’ ITR/UTR sequences within the H.
vulgaris genome. Phylogenies were generated using the GTRCAT model with empirical base
frequencies. A) Hvmar1 created from 246 aligned nucleotide positions, B) Hvmar2 created
from 291 aligned nucleotide positions. OTU labels are the 5’ flanking DNA of the ITR. The
phylogeny layouts are otherwise the same as in S1 Fig.
(PDF)
S4 Fig. Maximum likelihood phylogenies of mariner 5’ ITR/UTR sequences within the T.
dalmanni genome. Phylogenies were generated using the GTRCAT model with empirical
base frequencies. A) Tdmar2 created from 187 aligned nucleotide positions, B) Tdmar3 created
from 204 aligned nucleotide positions, C) Tdmar4 created from 197 aligned nucleotide posi-
tions. OTU labels are the 5’ flanking DNA of the ITR for A and B. C is presented as a radial
tree and both the support values and OTU labels are omitted due to the large number of
sequences. The phylogeny layouts are otherwise the same as in S1 Fig.
(PDF)
S5 Fig. Representative diopsid phylogeny showing the latest possible points of origins of
mariner subfamilies. Circles represent the putative origin points of the subfamilies. The tree
layout is the same as Fig 1.
(PDF)
S1 Table. mariner sequences generated in this study.
(DOCX)
S2 Table. Number of RNA-Seq reads mapped to mariner tnpase sequences.
(DOCX)
S3 Table. SRA RNA-Seq files used in gene expression analyses.
(DOCX)
Acknowledgments
The authors are grateful to Andrew Pomiankowski for providing whole flies of D.aethiopica,
Di.apicalis and Te.entabensis.
Author Contributions
Conceptualization: Martin Carr.
Data curation: Martin Carr.
Formal analysis: C. Alastair Grace, Martin Carr.
Investigation: C. Alastair Grace, Martin Carr.
Methodology: Martin Carr.
Project administration: Martin Carr.
Writing – original draft: C. Alastair Grace, Martin Carr.
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Cross-phyla horizontal transfers of mariner elements
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References
1. Pritham EJ. Transposable elements and factors influencing their success in eukaryotes. J Hered. 2009;
100: 648–655. https://doi.org/10.1093/jhered/esp065 PMID: 19666747
2. Hartl DL. Discovery of the transposable element Mariner. Genetics 2001; 157: 471–476. PMID:
11156971
3. Emmons SW, Yesner L, Ruan K-s, Katzenberg D. Evidence for a transposon in Caenorhabditis ele-
gans. Cell 1983; 32: 55–65. https://doi.org/10.1016/0092-8674(83)90496-8 PMID: 6297788
4. Jacobson JW, Medhora MM, Hartl DL. Molecular structure of a somatically instable transposable ele-
ment in Drosophila. Proc Natl Acad Sci USA 1986; 83: 8684–8688. https://doi.org/10.1073/pnas.83.22.
8684 PMID: 3022302
5. Charlesworth B, Charlesworth D. The population dynamics of transposable elements. Genet Res 1983;
42: 1–27.
6. Montgomery EA, Charlesworth B, Langley CH. A test for the role of natural selection in the stabilization
of transposable element copy number in a population of Drosophila melanogaster. Genet Res 1987; 49:
31–41. https://doi.org/10.1017/s0016672300026707 PMID: 3032743
7. Langley CH, Montgomery EA, Hudson R, Kaplan N, Charlesworth B. On the role of unequal exchange
in the containment of transposable element copy number. Genet Res 1988; 52: 223–235. https://doi.
org/10.1017/s0016672300027695 PMID: 2854088
8. Brookfield JFY. Models of transposition repression in P-M hybrid dysgenesis and by zygotically
encoded repressor proteins. Genetics 1991; 128: 471–486. PMID: 1649073
9. Brookfield JFY. The ecology of the genome–mobile DNA elements and their hosts. Nat Rev Genet
2005; 6: 128–36. https://doi.org/10.1038/nrg1524 PMID: 15640810
10. Tabara H, Sarkissian M, Kelly WG, Fleenor J, Grishok A, Timmons L, et al. Therde-1 gene, RNA inter-
ference and transposon silencing in C.elegans. Cell 1999; 99: 123–132. https://doi.org/10.1016/s0092-
8674(00)81644-x PMID: 10535731
11. Hood ME, Katawczik M, Giraud T. Repeat-induced point mutation and the population structure of trans-
posable elements in Microbotryum violaceum. Genetics 2005; 170: 1081–1089. https://doi.org/10.
1534/genetics.105.042564 PMID: 15911572
12. deHaro D, Kines KJ, Sokolowski M, Dauchy RT, Streva VA, Hill SM, et al. Regulation of L1 expression
and retrotransposition by melatonin and its receptor: implications for cancer risk associated with light
exposure at night. Nucleic Acids Res 2014; 42: 7694–7707. https://doi.org/10.1093/nar/gku503 PMID:
24914052
13. Kofler R. Dynamics of transposable element invasions with piRNA clusters. Mol Biol Evol 2019; 36:
1457–1472. https://doi.org/10.1093/molbev/msz079 PMID: 30968135
14. Daniels SB, Peterson KR, Strausbaugh LD, Kidwell MG, Chovnick A. Evidence for horizontal transmis-
sion of the Ptransposable element between Drosophila species. Genetics 1990; 124: 339–355. PMID:
2155157
15. Sa
´nchez-Gracia A, Maside X, Charlesworth B. High rate of horizontal transfer of transposable elements
in Drosophila. Trends Genet 2005; 21: 200–203. https://doi.org/10.1016/j.tig.2005.02.001 PMID:
15797612
16. Carr M. Multiple subfamilies of mariner transposable elements are present in stalk-eyed flies (Diptera:
Diopsidae). Genetica 2008; 132: 113–122. https://doi.org/10.1007/s10709-007-9157-2 PMID:
17562187
17. Kuraku S, Qiu H, Meyer A. Horizontal transfers of Tc1 elements between teleost fishes and their verte-
brate parasites, lampreys. Genome Biol Evol 2012; 4: 929–936. https://doi.org/10.1093/gbe/evs069
PMID: 22887124
18. Carr M, Bensasson D, Bergman CM. Evolutionary genomics of transposable elements in Saccharomy-
ces cerevisiae. PLoS One. 2012; 7: e50978. https://doi.org/10.1371/journal.pone.0050978 PMID:
23226439
19. Southworth J, Grace CA, Marron AO, Fatima N, Carr M. A genomic survey of transposable elements in
the choanoflagellate Salpingoeca rosetta reveals selection on codon usage. Mobile DNA-UK 2019; 10:
44.
20. Yoshiyama M, Tu Z, Kainoh Y, Honda H, Shono T, Kimura K. Possible horizontal transfer of a transpos-
able element from host to parasitoid. Mol Biol Evol 2001; 18: 1952–1958. https://doi.org/10.1093/
oxfordjournals.molbev.a003735 PMID: 11557800
21. Gilbert C, Chateigner A, Ernenwein L, Barbe V, Be
´zier A, Herniou EA, et al. Population genomic sup-
ports baculoviruses as vectors of horizontal transfer of insect transposons. Nat Comm 2014; 5: 3348.
PLOS ONE
Cross-phyla horizontal transfers of mariner elements
PLOS ONE | https://doi.org/10.1371/journal.pone.0235984 July 13, 2020 22 / 24

22. Peterson-Burch BD, Voytas DF. Genes of the Pseudoviridae (Ty1/copia Retrotransposons). Mol Biol
Evol 2002; 19: 1832–1845. https://doi.org/10.1093/oxfordjournals.molbev.a004008 PMID: 12411593
23. Carr M, Nelson M, Leadbeater BSC, Baldauf SL. Three families of LTR retrotransposon are present in
the genome of the choanoflagellate Monosiga brevicollis. Protist 2008; 159: 579–590. https://doi.org/
10.1016/j.protis.2008.05.001 PMID: 18621583
24. Robertson HM. The mariner element is widespread in insects. Nature 1993; 362: 241–245. https://doi.
org/10.1038/362241a0 PMID: 8384700
25. Robertson HM, Lampe DJ. Recent horizontal transfer of a mariner transposable element among and
between Diptera and Neuroptera. Mol Biol Evol 1995; 12: 850–862. https://doi.org/10.1093/
oxfordjournals.molbev.a040262 PMID: 7476131
26. Casse N, Bui QT, Nicolas V, Renault S, Bigot Y, Laulier M. Species sympatry and horizontal transfers of
mariner transposons in marine crustacean genomes. Mol Phylogenet Evol 2006; 40: 609–619. https://
doi.org/10.1016/j.ympev.2006.02.005 PMID: 16690328
27. Shillito JF. The genera of Diopsidae (Insecta: Diptera) Zool J Linn Soc 1971; 50: 287–295.
28. Kotrba M, Balke M. The systematic position of Cladodiopsis Se
´guy, 1949 and the origin of sexual dimor-
phism in stalk-eyed flies (Diptera: Diopsidae) inferred from DNA sequence data. Mol Phyl Evol 2006;
38: 843–847.
29. Kotrba M, Carr M, Balke M. The systematic position of Diopsina Curran, 1928 (Diptera: Diopsidae)
inferred from DNA sequence data. Insect Syst Evol 2010; 41: 295–302.
30. Fo
¨ldva
´ri M, Pomiankowski A, Cotton S, Carr M. A comprehensive morphological and molecular descrip-
tion of a new Teleopsis species (Diptera, Diopsidae) from Thailand. Zootaxa 2007; 1620: 37–51
31. Feijen HR, Feijen C. An annotated catalogue of the stalk-eyed flies (Diopsidae: Diptera) of India with
description of new species in Megalabops Frey and Teleopsis Rondani. Israel J Entom 2019; 49: 35–
72.
32. Carr M, Cotton S, Fo
¨ldva
´ri M, Kotrba M. A description of a new species of Diasemopsis (Diptera, Diopsi-
dae) from the Comoro Islands with morphological, molecular and allometric data. Zootaxa 2006; 1211:
1–19.
33. Smit, AFA, Hubley, Green P (2013–2015) RepeatMasker Open-4.0.
34. Bao W, Kojima KK, Kohany O. Repbase Update, a database of repetitive elements in eukaryotic
genomes. Mobile DNA 2015; 6: 11. https://doi.org/10.1186/s13100-015-0041-9 PMID: 26045719
35. Crescente JM, Zavallo D, Helguera M, Vanzetti LS. MITE Tracker: an accurate approach to identify min-
iature inverted-repeat transposable elements in large genomes. BMC Bioinformatics 2018; 19: 348.
https://doi.org/10.1186/s12859-018-2376-y PMID: 30285604
36. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in per-
formance and usability. Mol Biol Evol 2013; 30: 772–780. https://doi.org/10.1093/molbev/mst010 PMID:
23329690
37. Milne I, Stephen G, Bayer M, Cock PJA, Pritchard L, Cardle L, et al. Using Tablet for visual exploration
of second-generation sequencing data. Brief Bioinform 2013; 14: 193–202. https://doi.org/10.1093/bib/
bbs012 PMID: 22445902
38. Silvestro D, Michalak I. raxmlGUI: a graphical front-end for RAxML. Org Divers Evol. 2011; 12: 335–337
39. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, et al. MrBayes 3.2: efficient
Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61:
539–42. https://doi.org/10.1093/sysbio/sys029 PMID: 22357727
40. Miller MA, Pfeiffer W, Schwartz T. Creating the CIPRES Science Gateway for inference of large phylo-
genetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), 14 Nov.
2010, New Orleans, LA, pp. 1–8.
41. Wagner GP, Kin K, Lynch VJ. Measurement of mRNA abundance using RNA-seq data: RPKM mea-
sure is inconsistent among samples. Theory Biosci 2012; 131: 281–285. https://doi.org/10.1007/
s12064-012-0162-3 PMID: 22872506
42. Plasterk RHA, Izsva
´k Z, Ivics Z. Resident aliens: The Tc1/mariner superfamily of transposable ele-
ments. Trends Genet 1999; 15: 326–332. https://doi.org/10.1016/s0168-9525(99)01777-1 PMID:
10431195
43. dos Reis M, Thawornwattana Y, Angelis K, Telford MJ, Donoghue PC, Yang Z. Uncertainty in the timing
of origin of animals and the limits of precision in molecular timescales. Curr Biol 2015; 25: 2939–2950.
https://doi.org/10.1016/j.cub.2015.09.066 PMID: 26603774
44. Miyazawa H, Ueda C, Yahata K, Su ZH. Molecular phylogeny of Myriapoda provides insights into evolu-
tionary patterns of the mode in post-embryonic development. Sci Rep 2014; 4: 4127 https://doi.org/10.
1038/srep04127 PMID: 24535281
PLOS ONE
Cross-phyla horizontal transfers of mariner elements
PLOS ONE | https://doi.org/10.1371/journal.pone.0235984 July 13, 2020 23 / 24

45. Peterson KJ, Cotton JA, Gehling JG, Pisani D. The Ediacaran emergence of bilaterians: congruence
between the genetic and the geological fossil records. Philos Trans R Soc Lond B Biol Sci 2008; 363:
1435–1443. https://doi.org/10.1098/rstb.2007.2233 PMID: 18192191
46. Robertson HM. Multiple mariner transposons in flatworms and Hydras are related to those of insects. J
Hered 1997; 88: 195–201. https://doi.org/10.1093/oxfordjournals.jhered.a023088 PMID: 9183847
47. Bosch TCG, David CN. Stem cells of Hydra magnigpapillata can differentiate into somatic and germ line
cells. Dev Biol 1987; 121: 182–191.
48. Martı
´nez DE. Mortality patterns suggest lack of senescence in Hydra. Exp Gerontology 1998;33: 217–
225.
49. Chapman JA, Kirkness EF, Simakov O, Hampson SE, Mitros T et al. The dynamic genome of Hydra.
Nature 2010; 464: 592–596. https://doi.org/10.1038/nature08830 PMID: 20228792
50. Lee CC, Mul YM, Rio DC. The Drosophila P-element KP repressor protein dimerizes and interacts with
multiple sites on P-element DNA. Mo Cell Biol 1996; 16: 5616–5622.
51. Wessler SR, Bureau TE, White SE. LTR-retrotransposons and MITEs: important players in the evolu-
tion of plant genomes. Curr Opin Genet Dev 1995; 5: 814–821. https://doi.org/10.1016/0959-437x(95)
80016-x PMID: 8745082
52. Feschotte C, Osterlund MT, Peeler R, Wessler SR. DNA-binding specificity of rice mariner-like transpo-
sases and interactions with Stowaway MITEs. Nucleic Acids Res 2005; 44: 2153–2165.
53. Pasyukova EG, Nuzhdin SV, Filatov DA. The relationship between the rate of transposition and trans-
posable element copy number for copia and Doc retrotransposons of Drosophila melanogaster. Genet
Res 1998; 72: 1–11. https://doi.org/10.1017/s0016672398003358 PMID: 9802257
54. Feijen HR, Feijen C. On the biogeographic range of the genus Teleopsis Rondani (Diptera: Diopsidae),
with redescription of Teleopsis sykesii from India and description of a new species from Borneo. Zool
Med Leident 2011; 85: 141–159.
55. Qin Y, Paini DR, Wang C, Fang Y, Li Z. Global establishment risk of economically important fruitfly spe-
cies (Tephritidae). PLoS ONE 2015; 10: e0116424. https://doi.org/10.1371/journal.pone.0116424
PMID: 25588025
56. Zacharias H, Anokhin B, Khalturin K, Bosch TCG. Genome sizes and chromosomes in the basal meta-
zoan Hydra. Zoology 2004; 107: 219–227. https://doi.org/10.1016/j.zool.2004.04.005 PMID: 16351940
57. Wong WY, Simakov O, Bridge DM, Cartwright P, Bellantuono AJ, Kuhn A, et al. Expansion of a single
transposable element family is associated with genome-size increase and radiation in the genus Hydra.
Proc Natl Acad Sci USA 2019; 116: 22915–22917. https://doi.org/10.1073/pnas.1910106116 PMID:
31659034
58. Martı
´nez DE, Iñiguez AR, Percell KM, Willner JB, Signorovitch J, Campbell RD. Phylogeny and bioge-
ography of Hydra (Cnidaria: Hydridae) using mitochondrial and nuclear DNA sequences. Mol Phylo-
genet Evol 2010; 57: 403–410. https://doi.org/10.1016/j.ympev.2010.06.016 PMID: 20601008
59. Deserti MI, Esquius KS, Escalante AH, Acuña FH. Trophic ecology and diet of Hydra vulgaris (Cnidaria;
Hydrozoa). Anim Biol 2017; 67: 287–300.
60. Feijen HR, Feijen C. Diopsis (Diopsidae, Diptera) with unusual wing spots: two new species from
Malawi with a longer eye span in females than in males. Zool Med Leiden 2009; 83: 701–722.
PLOS ONE
Cross-phyla horizontal transfers of mariner elements
PLOS ONE | https://doi.org/10.1371/journal.pone.0235984 July 13, 2020 24 / 24