Characterization of mariner-like transposons of the mauritianaSubfamily in seven tree aphid species

Imen Kharrat • Maha Mezghani • Nathalie Casse • Francoise Denis •

Aurore Caruso • Hanem Makni • Pierre Capy • Jacques-Deric Rouault •

Benoıt Chenais • Mohamed Makni

Received: 4 September 2014 / Accepted: 26 December 2014 / Published online: 3 January 2015! Springer International Publishing Switzerland 2015

Abstract Mariner-like elements (MLEs) are Class IItransposons present in all eukaryotic genomes in which

MLEs have been searched for. This article reports the

detection of MLEs in seven of the main fruit tree aphidspecies out of eight species studied. Deleted MLE

sequences of 916–919 bp were characterized, using the

terminal-inverted repeats (TIRs) of mariner elementsbelonging to the mauritiana Subfamily as primers. All the

sequences detected were deleted copies of full-length

elements that included the 30- and 50-TIRs but displayedinternal deletions affecting Mos1 activity. Networks based

on the mtDNA cytochrome oxidase subunit-I (CO-I) and

MLE sequences were incongruent, suggesting that muta-tions in transposon sequences had accumulated before

speciation of tree aphid species occurred, and that they

have been maintained in this species via vertical trans-missions. This is the first evidence of the widespread

occurrence of MLEs in aphids.

Keywords MLEs ! Transposable elements ! Aphididae !Internal deletion ! CO-I

Introduction

Eukaryotic genomes contain a diverse array of transposableelements (TEs), which are DNA sequences that are able to

move from one chromosomal site to another. TEs typically

fall into two main groups on the basis of their mechanism oftransposition (Finnegan 1989; Wicker et al. 2007; Chenais

et al. 2012). Class I elements, or retro-elements, transposevia an RNA intermediate, whereas Class II elements trans-

pose from one chromosomal site to another by an excision/

insertion mechanism (cut and paste) using a transposase, anenzyme encoded by the element itself (Plasterk et al. 1999;

Hua-Van et al. 2005). Mariner-like elements (MLEs) are

Class II transposons belonging to the Tc1-mariner-IS630SuperFamily, which is one of the most diverse and wide-

spread families of TEs (Wicker et al. 2007). MLEs are

characterized by having a very simple structure, i.e. a singlegene boarded by untranslated sequences (in 50 and 30), the

complete element being flanked by two short terminal

inverted repeats (TIRs). The total length of the MLE isabout 1,300 bp, and its unique gene encodes a transposase

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10709-014-9814-1) contains supplementarymaterial, which is available to authorized users.

I. Kharrat ! M. Mezghani ! H. Makni ! M. Makni (&)Faculte des Sciences de Tunis, Universite de Tunis El Manar,UR11ES10 Genomique des insectes ravageurs, 2092 Manar II,Tunisiae-mail: Md.Makni@fst.rnu.tn

N. Casse ! F. Denis ! A. Caruso ! B. Chenais (&)Laboratoire Mer, Molecules, Sante, Universite du Maine, EA2160 Avenue Olivier Messiaen, 72085 Le Mans Cedex 9, Francee-mail: bchenais@univ-lemans.fr

F. DenisUMR 7208 BOREA, Biologie des Organismes et EcosystemesAquatiques, Museum National d’Histoire Naturelle (MNHN),CP 26, 43 rue Cuvier, 75231 Paris Cedex 05, France

H. MakniInstitut Superieur de l’Animation pour la Jeunesse et la Culturede Bir-El-Bey, Universite de Tunis, 2055 Tunis, Tunisia

P. Capy ! J.-D. RouaultLaboratoire Evolution, Genomes et Speciation, UPR9034,CNRS, 91198 Gif-sur-Yvette, France

P. Capy ! J.-D. RouaultUniversite Paris-Sud, 91405 Orsay, France

123

Genetica (2015) 143:63–72

DOI 10.1007/s10709-014-9814-1

of about 340–360 amino acid residues (Robertson 2002;

Claudianos et al. 2002; Feschotte et al. 2005). The firstmariner element was discovered in the fruit fly Drosophila

mauritiana as the result of an analysis of an unstable

mutation in the white gene. This 1,286-bp element, initiallydesignated pch, includes a single open reading frame

encoding a protein of 345 amino acids with TIRs of 28 bp at

each extremity (Jacobson et al. 1986). Since then, numerousmariner elements have been characterized in nematodes

(Leroy et al. 2003), crustaceans (Casse et al. 2006; Bui et al.2007, 2008), fish (Ivics et al. 1997), human beings (Auge-

Gouillou et al. 1995; Robertson and Zumpano 1997; Rob-

ertson and Martos 1997), plants (Casacuberta et al. 1998;Feschotte and Wessler 2002; Zhou et al. 2010), protozoans

(Silva et al. 2005), and insects (Robertson 1993; Robertson

and McLeod 1993; Rouleux-Bonnin et al. 2005; Wang et al.2011; Rezende-Teixeira et al. 2012).

Five main MLE subfamilies have been defined on the basis

of phylogenetic studies and sequence similarities, i.e. mau-ritiana, cecropia, mellifera, irritans, and elegans (Robertson

and McLeod 1993). So far, only three of the mariner elements

characterized have been shown to be naturally active, i.e. to beable to be mobilized by their own transposase, which must

therefore be a catalytically active enzyme: (1) Mos1, found in

D. mauritiana (Medhora et al. 1991), (2) Famar1, a functionalcoding sequences of the earwig Forficula auricularia (Barry

et al. 2004), and (3) Mboumar-9 discovered in the satellite

DNA of the ant Messor bouvieri (Munoz-Lopez et al. 2008).Molecular analysis has shown that most MLE sequences have

accumulated mutations such as deletions, insertions, stop

codons, or frameshifts that have led to the inactivation of theelements. Deleted elements with intact TIRs and internal

deletions are often observed. Some studies of Class II ele-

ments, such as Brunet et al. (2002) on MLEs or, more recently,Negoua et al. (2013) on Lemi elements, have shown that

internal deletions do not occur randomly, but involve very

small direct repeats, known as microhomologies, that can belocalized at or close to the breakpoints (BPs) of the deletion.

To date, with regard to aphid species, only internal partial

sequences of mariner belonging to the irritans and melliferasubfamilies had been identified in the Soybean Aphid Aphis

glycine (Mittapalli et al. 2011). In the study reported here,

deleted copies of full-length mauritiana MLEs were char-acterized in seven fruit tree aphid species, and their classi-

fication and haplotype network relationships were inferred.

Materials and methods

Collection and identification of the aphid species

Eight species belonging to the Aphididae family, i.e. Aphisgossypii, Aphis pomi, Aphis punicae, Aphis spiraecola,

Brachycaudus amygdalinus, Hyalopterus pruni, Pterochloro-

ides persicae, and Toxoptera aurantii were collected from dif-ferent locations in northern Tunisia (Supplementary Data S1).

Species identification of the specimens was performed based on

the identification keys of Leclant (2000) and Blackman andEastop (2007). Samples were preserved in 96 % ethanol at

-20 "C before the genomic DNA (gDNA) was extracted.

gDNA extraction, PCR amplifications of MLE and CO-

I sequences

Genomic DNA was extracted using the hexadecyltrimethyl

ammonium bromide method (Doyle and Doyle 1987). Toamplify MLE from an aphid, a degenerated primer Mos1

(50-TAY CAG GTG TAC AAG TAK GRA A-30) was

designed on the basis of eleven TIR alignments from themauritiana Subfamily (Bigot et al. 2005). The CO-I mito-

chondrial DNA region was amplified for all the samples

using the universal primers designed by Folmer et al.(1994), i.e. forward LCO1490 (50-GGTCAACAAATCA-

TAAAGATATTGG-30) and reverse HCO2198 (50-TAAAC

TTCAGGGTGACCAAAAAATCA-30). PCR amplificationswere carried out using 50–100 ng of genomic DNA in a

25-ll reaction mixture comprising 0.1 U of GoTaq poly-

merase (Promega), 1X PCR buffer, 2 mM MgCl2, 0.1 mMof each primer, and 0.2 mM dNTPs. Amplification was

performed in a 2,720 thermal cycler (Applied Biosystems),

programmed as follows: an initial denaturing step at 94 "Cfor 5 min was followed by either 40 cycles (94 "C, 60 s;

48–50 "C, 60 s; 72 "C, 90 s) for MLEs, or 35 cycles (94 "C,

60 s; 48 "C, 60 s; 72 "C, 60 s) for CO-I, and ending with afinal extension at 72 "C for 5 min.

Cloning of PCR products in plasmid vectorsand sequencing

PCR products of the expected size were excised fromagarose gel and purified using Wizard SV Gel and PCR

Clean up System kits (Promega). Eluted DNA was then

cloned in pGEM-T Vector System (Promega), according tothe Manufacturer’s protocol. E. coli DH5a cells (New

England Biolabs) were transformed and selected as

described by Bui et al. (2007). Positive clones werescreened during a subsequent PCR using T7 and SP6

primers. Plasmids from positive colonies were isolated and

purified using a Wizard Plus Minipreps DNA Purificationsystem (Promega), and sequenced on both strands by the

Cogenics-Genome express company.

Sequence analyses

Similarity searches for nucleotide and amino-acid sequen-ces were carried out through BLAST programs with default

64 Genetica (2015) 143:63–72

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parameters (Altschul et al. 1990) using the GenBank

database. Nucleic and translated sequence multiple align-ments were performed using ClustalW with default settings

(Thompson et al. 1994) and distances were calculated using

the MEGA5 software (Tamura et al. 2011). The conceptualtranslation products of the MLE sequences were manually

constructed by means of the ‘‘judicious’’ introduction of

frameshifts and gaps after translation using the Embosstools (Rice et al. 2000). Motif signatures of MLEs were

identified by sequence comparison, whereas Helix-Turn-Helix (HTH) motif and putative nuclear localization signal

(NLS) were searched for using the GYM2.0 (Narasimhan

et al. 2002) and cNLS Mapper (Kosugi et al. 2009) tools,respectively.

Classification of MLE elements

MLE copies were classified as belonging to the Tc1-mar-

iner–IS630 Superfamily on the basis of a previouslydescribed automated method based on pairwise distances

(Rouault et al. 2009). The nucleotide sequences were

aligned pairwise, and the distances between them werecomputed. An ascending aggregative process was then

performed in order to draw a tree that summarized the

classification. There are two main differences between thismethod and the UPGMA (Unweighted Pair Group Method

with Arithmetic mean) method: (1) there is no consensus,

and the distance between two groups is the mean of thedistances between the elements; (2) in the computation of

the distance, the weight of the gaps is progressively

reduced from 1 to 0, in order to group complete and deletedsequences separately. This Variation of Metric process

allowed us to include sequences of very different lengths.

The UPGM-VM method is applied to the whole nucleotidesequences of TEs.

Haplotype network comparison

Mitochondrial CO-I sequences from the tree aphid species

were generated from two individuals per species: onecorresponded to the same individual as had been used for

MLE detection, and the other was derived from GenBank.

The total length of the CO-I product was 710 bp. In orderto avoid any accidental base changes due to artifacts during

experimental procedures, and to compare identified CO-I

sequences with sequences from GenBank, we considered asingle 658-bp fragment for all sequences. CO-I sequences

were deposited in Genbank under accession numbers

KF114022–KF114028. Parsimonious haplotype networkswere drawn using the Fluxus Network 4.6 software (Polzin

and Daneschmand 2003) available at http://www.fluxus-

engineering.com. Genetic distance between CO-I

sequences was calculated with the MEGA5 software

(Tamura et al. 2011) using the Kimura 2-parameter model(Kimura 1980).

Results

Characterization of aphid MLEs

For seven out of eight aphid species studied, PCR productsof about 900 bp were obtained with the mauritiana specific

primers. A posteriori, the absence of amplification in the A.

punicae specimens may serve as a negative control, indi-cating the absence of contamination with MLE-positive

DNA. After cloning, five clones per species were

sequenced, and BLAST results revealed MLE sequences inall seven aphids species. A total of 28 deleted copies of

full-length MLEs were identified. For A. gossypii, A. pomi,

A. spiraecola, and H. pruni, all five cloned sequences wereidentified as MLEs, whereas for T. aurantii, B. amygdali,

and P. persicae, only 1–4 clones were MLEs (Table 1).

Pairwise comparison showed a high degree of similarity(98–99 %) between the MLEs isolated, which made it

possible to constitute a single consensus sequence, desig-

nated Aphidmarcons, for Aphid mariner consensus, andavailable as Supplementary Data S2. Comparison of Ap-

hidmarcons to the reference sequence Mos1 from D.

mauritiana highlights the presence of internal deletions inthe Aphid sequence (Fig. 1).

Microhomologies were manually searched for by

exploration of the flanking regions of the deletion breakingpoints (BPs), and only deletions of more than 5 bp were

taken into consideration. A total of 16 deletions were

detected, with deletion sizes ranging from 6 to 72 bp. Thepart of the element susceptible to being deleted seemed to

be similar for the 50 and 30 ends. Indeed, 67 % of micro-

homologies were near both BPs (BPNN: breaking pointnear near) considering ‘‘near’’ as comprised between 1 and

10 bp, 20 % with one exactly at the BPs, and the other near

the BPs (BPEN: breaking point exact near), and 13 % wereexactly at both BPs (BPEE: breaking point exact exact).

Microhomologies sizes ranged from 3 to 5 bp, 62 % of

which were 3 bp long. Moreover, the frequency of shortdirect repeats (SDRs) was 56 %, whereas the frequency of

short inverted repeats (SIRs) was 44 %. Microhomology

sizes were always 3 bp long for SDRs, but more than 3 bplong for 57 % of SIRs. The high frequency of SDRs and

SIRs at or near to the breakpoints of the deletions strongly

suggests that most deletions do not occur randomly. Sinceall 28 sequences were very similar, we can conclude that

deletions may derive from an ancestral event that occurred

before the aphid tribes diverged.

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Analysis of the translated protein sequencesof Aphidmarcons

In-silico translation of Aphidmarcons gave rise to a deletedtransposase of only 242 amino-acids. Alignment of Ap-

hidmarcons and Mos1 transposases showed seven deletions

located along the transposase (Fig. 2). Several canonicalmotifs of MLEs, such as helix turn helix (HTH) and

nuclear localization sequence (NLS), were highly con-

served in Aphidmarcons transposase, whereas some othermotifs were slightly modified, such as WVPHEL (Rob-

ertson 1993; Bui et al. 2007) replaced by WV(NL)EL.

Moreover, the motifs surrounding the first two Ds of thecatalytic core (DDD) had been changed from TGDEKWI

and FLHDNARPH (Leroy et al. 2000; Auge-Gouillou et al.

2001) to T(I)D(K)K(R)I and LHD(S)A(PS)H, respectively.

Table 1 Number and size of MLE sequences identified per species

Tribe Tested species Number ofclonedsequences

Number ofsequencescorrespondingto MLEs

MLE denomination Size (bp) Accession numbers

Aphidini Aphis gossypii 5 5 Agosmar 1.1–1.5 917–919 AB858399–AB858403

Aphis pomi 5 5 Apommar 1.1–1.5 917 AB858404–AB858408

Aphis spiraecola 5 5 Aspimar 1.1–1.5 917 AB858409–AB858413

Hyalopterus pruni 5 5 Hprumar 1.1–1.5 916–919 AB858417–AB858421

Toxoptera aurantii 5 1 Taurmar 1.1 917 AB858430

Macrosiphini Brachycaudus amygdalinus 5 3 Bamymar 1.1–1.5 917 AB858414–AB858416

Pterochloroides persicae 5 4 Ppermar 1.1–1.4 917 AB858422–AB858425

a Aphid sampling data are available in Supplementary Data S1

Fig. 1 Nucleic acid sequence alignment of Aphidmarcons and Mos-1from D. mauritiana. Both ‘‘short direct repeat’’ (SDR) and ‘‘shortinverted repeat’’ (SIR) microhomologies are indicated by whiteboxes. The different kind of microhomologies observed at or close tothe breaking points (BPs) of the deletions are indicated as follows:

BPNN breaking point near near, BPEN breaking point exact near andBPEN breaking point exact exact. The start codon, the terminationand polyadenylation sites are indicated in black boxes, the 50-TIR and30-TIR are in gray boxes

66 Genetica (2015) 143:63–72

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The third conserved motif, YSPDLAP (Robertson 1993;

Bui et al. 2007), was missing and included in a large

deletion of the C-terminal domain.

Classification of aphid MLEs within the Tc1-mariner-

IS630 SuperFamily

To classify the 28 aphid MLEs within the Tc1-mariner-IS630

SuperFamily, all sequences were compared using the UPGM-VM method to 285 known, full-length, mariner elements

belonging to various different mariner SubFamilies available

from Genbank in order to specify the exact position of theAphid sequences in this Subfamily (Fig. 3a). The classifica-

tion of the 28 aphid MLEs, i.e. Aphidmar copies, amongst all

the MLEs in our databank showed that they belonged to themauritiana Subfamily (Fig. 3a, b). This Subfamily consists of

a set of Tribes: Dipteris (including Mos1), Hymenopteris, and

other Tribes that are less precisely defined, because of theabsence of complete sequences. The 28 Aphidmar sequences

are grouped to form a new Tribe, known as Puceronis, which

is clearly distinct from the other Tribes of the mauritianaSubfamily (Fig. 3b). In addition, branches between Puceronis

pairwise sequences are very short, due to the high similaritybetween aphid MLE sequences.

This classification strongly suggests that the inventory of

MLEs is not yet complete, and that many other Tribes remainto be discovered in other groups of insects or animals.

Molecular network analyses based on CO-I and MLEsequences

The mitochondrial CO-I barcoding marker has proved to bean effective tool for characterizing aphid species (Park

et al. 2011); therefore the genetic distance between species

were calculated in order to evaluate the divergences

between the molecular markers of studied species (Sup-plementary Data S3). This result showed low interspecific

distances that allowed performing a molecular network on

the CO-I sequences in order to compare the mtDNA rela-tionships with the MLE relationships. So, the CO-I net-

work, based on one sequence per species obtained from the

same individuals as MLEs and one GenBank entry for eachspecies, revealed seven haplotypes corresponding to the

seven aphids tested, which are encoded according to a

single change per base position (Fig. 4a). This CO-I-basednetwork supports the existence of two tribes according to

Blackman and Eastop (2000) and Remaudiere and Rem-

audiere (1997) i.e. the Macrosiphini, including P. persicaeand B. amygdalinus, and the Aphidini, which includes A.

gossypii, A. pomi, A. spiraecola, H. pruni, and T. aurantii.

Analysis of MLE relationships for the same seven treeaphid species was used to construct an MLE network

(Fig. 4b), indicating that species that do or do not belong to

the same tribe may share closely-related MLEs. Thus,phylogenic relationships constructed from MLE and CO-I

sequences were incongruent for the seven species tested inthis work. Aphid MLE sequences showed evidence of an

erratic distribution. The incongruence of the two networks

may suggest that MLEs have evolved independently fromthe speciation events during the evolution of aphid species.

Discussion

Elements of the mauritiana Subfamily were detected inseven tree aphid species (i.e. A. gossypii, A. pomi, A.

Fig. 2 Sequence alignment of the Mos-1 transposase (accessionnumber: X78906) and Aphidmarcons in silico translation. Black boxesindicate highly conserved amino acids residues. Green boxescorrespond to conserved motifs of the catalytic triad, each D of thecatalytic core is positioned below and indicated by an arrow. Red

boxes correspond to the predicted bipartite and monopartite NLS, theblue dashed box corresponds to the Helix-Turn-Helix motif of theTIR binding domain, and the yellow box to a motif conserved inmariner elements (Robertson 1993; Bui et al. 2007)

Genetica (2015) 143:63–72 67

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spiraecola, B. amygdalinus, H. pruni, P. persicae, and T.

aurantii) genomes using the inverted terminal repeat of theMos1 element as primers, whereas the eighth species (A.

punicae) was negative. This finding is not surprising, and is

in agreement with previous reports showing that MLEs arewidespread and diverse in insects (Robertson 1993; Rou-

leux-Bonnin et al. 2005; Wang et al. 2011). Other mariner

elements belonging to other Subfamilies may exist in theseaphid species as reported by Mittapalli et al. (2011), which

have shown the presence of irritans (Agmar1) and melli-

fera (Agmar2) elements in the genome of the soybeanaphid A. glycine. By contrast the in silico genome mining

of the model aphid Acyrthosiphon pisum retrieved no sig-

nificant hit for MLEs belonging to the mauritiana andmellifera subfamilies, whereas some irritans elements have

been found (data not shown). The absence of mauritiana

and mellifera MLEs in the sequenced genome of A. pisummay be related to the fact that only 50 % of the genome is

currently annotated. This may also indicate that all aphid

genomes do not contain the same MLEs.

Using an absolute quantification protocol as well as acomparative estimation with the single copy gene RPL7

(Mittapalli et al. 2011), quantitative PCR results indicate

that MLEs are present in aphids in a low copy number (i.e.1 or 2 copies), below the detection limit of quantitative

PCR (data not shown). This is in agreement with the results

of Mittapalli et al. (2011) obtained for MLEs in A. glycine.Low copy MLEs have also been reported in Drosophila

ananassae (Robertson and Lampe 1995) and D. sechellia

(Capy et al. 1992).In our study, a total of 28 full-length aphid elements

were obtained displaying 68 % homology with D. simulans

and D. teissieri mariner elements. Sequences analysisrevealed defective elements containing several kinds of

mutations generating stop codons, frameshifts, and non-

functional transposase, suggesting that transposase genesmay now be evolving as pseudogenes, accumulating

mutations neutrally by vertical inactivation as are several

elements belonging to the Tc1-mariner-IS630 SuperFamily(Hartl et al. 1997; Brunet et al. 2002). Pairwise comparison

of the MLEs isolated showed a high degree of similarity,

indicating that they shared a common origin. It is possiblethat the deletion occurred in an ancestral population before

the Tribes diverged in the Eocene (Kim et al. 2011). If so,

all these deletions can be expected to have resulted fromthe same ancestral event.

Alignment of aphid MLEs with Mos1 showed that the

internal deletion is flanked by short direct repeats of

b Fig. 3 Classification of the Aphid MLEs in the mauritiana Subfamilyof the mariner Family. a A set of 313 sequences was classified by theUPGM-VM method on the basis of the available whole nucleotidesequences. The main Families of the Tc1-mariner-IS630 Superfamilyof transposable elements are located at the lower right part of therosette: Chl (Chlorophyllis = Plant mariner = DD39D in plants),Gam (Gambol = DD34E in animals), Pog (Pogo in animals andLemis in plants), Tco (Tc1 = DD34E in animals), Lud (Luden-sis = maT pp. = Rosa in animals), Tct (Tc3 in animals), Msq(Mosquitis = DD37E in animals), Fot (Fotis in fungi), Mel (Melilotisin bacteria with ISRm10), Jap (Japonis in bacteria with IS870), Son(Sonneis in bacteria with ID630), Mat (Matelotis = maT pp. = Mor-i = DD37D in animals). The mariner Family (DD34D in animals)splits into two subgroups: Atlantis (Atl including the Irritans) andMareNostrum (Mar). In this second group, the main SubFamilies arerepresented: Mellifera (Mel), Elegans (Ele), Cecropia (Cem and Cec),Chitwoodis (Chi), Indianus (Ind), Briggsae (Bri) and Cemar2 (Ce2).The mauritiana Subfamily appears to be structured into a largenumber of Tribes. Hymenopteris (Hym), Dipteris (Dip, with Mos1)are well characterized. The other Tribes, e.g. Nikananis (Nik), are stillspeculative, because of the small number of sequences (most of whichwere only partial sequences) and the small number of host species.The last Tribe is Puceronis (Puc), which includes all the newsequences of fruit tree aphid elements presented in this paper. Thisclassification clearly shows that the Puceronis Tribe belongs to theMauritiana Subfamily. The complete list of the 313 sequences, withaccession numbers, lengths, and host species names is available inSupplementary Data S4. b Zoom on the Mauritiana Subfamilyshowing the puceronis Tribe

68 Genetica (2015) 143:63–72

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3–5 bp. Such repeats were found at the extremities of

internal deletions of some MLEs (Brunet et al. 2002).Indeed, it seems that the part of MLE element likely to be

deleted is similar at the 50 and 30 ends. Analogous results

were obtained for Lemi elements, in which deletions werescattered throughout the sequences in the Ogris and Po-

ucetis Subfamilies, and three categories of microhomolo-

gies were detected (Negoua et al. 2013). However for themauritiana Subfamily within Drosophilidae species,

internal deletions occur mainly in the 50 part of elements

(Brunet et al. 2002). MLE Aphid sequences also revealedmicrohomologies, implying that homologous recombina-

tion between small direct repeats may be the cause of

deleted elements among MLEs. Various mechanisms couldexplain the origin of deletions in conjunction with the

presence of SDRs, such as ectopic recombination and

abortive gap repair (Negoua et al. 2013). It should thereforebe possible to find independent deletions with similar

breakpoints.

The predicted consensus transposase Aphidmarconsdisplays the MLE signature sequence DD(34)D, as well as

important deletions in some key regions. These deletions

span the NLS motif in the N terminal part of the protein,and also the third ‘‘D’’ of the catalytic domain in the

C-terminal region (Doak et al. 1994). The main conserved

motif of the mariner transposase WVPHEL was slightlymodified by WV(N)(L)EL, however the second conserved

motif, YSPDLAP, was deleted in all species. This deletion

is specific to aphids and may be a selective mutation sharedby all aphid species.

The comparison of nucleotide sequences has shown high

degree of similarity among tree aphid MLE copies indi-cating either (1) an earlier invasion of Mos1 copies into the

host genome dating from the Eocene followed by a phase

of senescence in the common aphid ancestor and by somesubstitutions or (2) an horizontal transfer of deleted copies

through transposase from endogenous active elements.

Like all genes, TEs are transmitted vertically from acommon ancestor in the process of speciation. In this case,

the relationships between sequences of elements must

reflect the phylogenetic relationships between their hosts.Therefore, a comparative CO-I based network showing the

relationships between the seven aphid species studied was

constructed, and was consistent with classical phylogeneticanalyses based on morphological and molecular charac-

teristics (Foottit et al. 2008; Cœur D’Acier et al. 2008; Kim

et al. 2011; Blackman and Eastop 2007; Leclant 2000). Themitochondrial CO-I barcoding marker has proved an

effective tool for characterising aphid species (Park et al.

2011). Rigorous statistical proof of horizontal transmissionrequires the demonstration of a statistically-significant

inconsistency in the molecular phylogeny (or haplotype

network) of the transposable element as compared to thoseof single-copy, non-transposable sequences from the same

A. gossypii

A. pomi

A. spiraecola

H. pruni

B. amygdalinus

P. persicae

T. aurantii

10 mutations 1 mutation

Ap

Ap Ap

Ap Ap

Pp

Pp

Pp

Pp

Pp

Ta

Hp

Hp

Hp

Hp Hp

As

As

As As

Ag

Ag

Ag

Ag Ag

Ba

Ba

Ba

As

(A) (B)

Fig. 4 Parsimonious haplotype network generated from CO-Isequences (a) and MLE sequences (b) within seven aphids species.a The ellipse size corresponds to the haplotype frequency. Red circlesindicate mutation steps corresponding to hypothetical haplotypes,which were not detected among the specimens analyzed. For eachspecies, one sequence was sequenced from the same individual usedfor TIR detection, the other one being based on the followingGenBank entries: Aphis gossypii (EU701419.1), Aphis pomi(EU701479.1), Aphis spiraecola (EU701501.1), Toxoptera aurantii

(EU701931.1), Hyalopterus pruni (GU457791.1), Pterochloroidespersicae (JN644646.1), Myzus persicae (EU701803.1). b Red circlesrepresent forms inferred from, but not detected amongst the haplotypesamples; Ag, Agosmar 1–5 from Aphis gossypii; Ap, Apommar 1–5from Aphis pomi, As, Aspimar 1–5 from Aphis spiraecola; Ba,Bamymar 1–3 from Brachycaudus amygdalinus; Hp, Hprumar 1–5from Hyalopterus pruni, Pp, Ppermar 1–4; from Pterochloroidespersicae, and Ta, Taurmar1 from Toxoptera aurantii

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genomes (Lawrence and Hartl 1992; Clark et al. 1994;

Robertson and Lampe 1995; Capy et al. 1994a, b). Here,the topologies of haplotype networks are highly incon-

gruent, showing that MLEs have evolved independently of

the speciation events. A similar observation was also beenreported for the Bambusoideae Subfamily (Zhou et al.

2011). These results imply that horizontal transfer events

might occur between species originating within a relativelysmall geographic range. This is reflected in particular by

the presence of nearly identical MLEs in distantly-relatedspecies, and the presence of very diverse MLEs within the

same species. This may be due to the occurrence of hori-

zontal transfer events between phylogenetically-distantspecies during aphid evolution or the existence of an

ancestral MLEs polymorphism followed by divergent

evolution and stochastic loss (Hartl et al. 1997). In thiswork, we found that all copies of MLEs were deleted, and

that the deletions were similar for both the Macrosiphini

tribe, including Pterochloroides persicae and Brachycau-dus amygdalinus, and the Aphidini tribe, which includes

Aphis gossypii, Aphis pomi, Aphis spiraecola, Hyalopterus

pruni, and Toxoptera aurantii.In conclusion, there are two possible explanations for

such a topology. Either, amplification must have occurred

within tree aphid tribes after the inactivation of MLEcopies and before ancestral divergence occurred between

the groups. Or, alternatively, MLEs copies are long-term

inactivated copies, but they were recently amplified byanother copy encoding an active transposase. However, no

active copy has so far been detected in these species. These

results highlight the difficulties of explaining the evolutionof the Mos1-like elements in tree aphid species. While

mariner elements may have been present in the ancestor of

tree aphid species, this does not exclude the possibility ofseveral losses and horizontal transfer (Brunet et al. 1999).

This observation is unexpected, because it is highly

improbable that long forms (916–919 bp) of a single MLEelement could have been transferred between species sev-

eral times during evolution. Concerning the way the

putative horizontal transfer occurs, several scenarios can beproposed, but as mentioned by Loreto et al. (2008) none of

them have been demonstrated. The aphids species studied

here infect each a different plant from two plant Families(i.e. Rutacae and Rosacae) but some aphids are polypha-

gous (e.g. Aphis gossypii or A. spiraecola) and virus

present in the aphid (e.g. Tristeza virus in A. spiraecola)may be transmitted to the host plant and then represent a

potential vector of horizontal transfer. Moreover, whatever

aphids are fed on the same plants, many vectors can be alsoconsidered like common virus, bacteria or other parasites.

Apart from horizontal transfer, another hypothesis could

explain the incongruence between networks based on CO-Iand TEs. Ancestral duplication and/or polymorphism could

have created paralogous copies of TEs that have evolved

distinctly in different genomes (Capy et al. 1994a, b).A better understanding of insect TEs will enable us to

make better-informed use of the currently available TE-

based tools. This means that fundamental studies havesignificant implications for the applications of TE-based

molecular tools. As in so many areas of biology, the

availability of genome sequences from related species aswell as individuals within populations will greatly facilitate

the investigation and application of insect TEs.

Acknowledgments This work received funding for the UR11 ES10by the Tunisian Ministry of Higher Education and ScientificResearch. The authors are grateful to Monika Ghosh for revising theEnglish text.

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