Evolution of the Transposable Element Mariner in Drosophila

Evolution of the Transposable Elementmariner in Drosophila Species

Kyoko Maruyama and Daniel L. Hart1 Department of Genetics, Washington University School of Medicine, St. Louis, Missouri 631 10-1095 Manuscript received September 2 1, 1990 Accepted for publication February16, 199 1

ABSTRACT The distribution of the transposable elementmariner was examined in the genusDrosophila. Among the eight species comprisingthe melanogaster species subgroup,the element is present in D.mauritiana, D. simulans, D. sechellia, D.yakuba and D. teissieri, but it is absent in D. melanogaster, D. erecta and D. orena. Multiple copies ofmariner were sequenced from each species in whichthe element occurs.The inferred phylogenyof the elements andthe pattern of divergence were examined inorder to evaluate whether horizontal transfer among speciesor stochastic loss couldbetter account for the discontinuous distribution of the element amongthe species. The data suggest that the element was present in the ancestral species beforethe melanogaster subgroup diverged andwas lost in the lineage leading to D. melanogaster and the lineage leading to D. erecta and D. orena. This inference is consistent with the finding that mariner also occurs in members of several other species subgroups within the overall melanogaster species group, Within the melanogaster species subgroup, the average divergence of mariner copies between species was lower than the coding region of the alcohol dehydrogenase (Adh) gene. However, the divergence of mariner elements within species was as great as that observed for Adh. We conclude that the relative sequence homogeneityof mariner elements within species is more likely a result of rapid amplification of a few ancestral elements than of concerted evolution. The mariner element may also have had unequal mutation rates in different lineages.

RANSPOSABLE elements comprise a class of ment in Drosophila. The P element appears to have T multigene families whose members are capable spread in Drosophila melanogaster only in the past few of moving from one chromosomallocation to another. decades, since it is prevalent in contemporary natural Although subject to extensive genetic and molecular populations but completely absent in old laboratory study,transposable elements are littleunderstood strains andin species closely related to D. melanogaster from the standpoint of evolutionary history. Trans- (KIDWELL1983). However, homologues of the P ele- posable elements can rapidly increase or decrease in ment are prevalent in more distantly related species copy numberwithout apparent phenotypic effects, comprising the willistoni and sultans species groups which distinguishes them from conventional single- (DANIELSet al. 1984; DANIELS and STRAUSBAUCH copy nuclear genes. A seconddistinction is that at 1986; LANSMANet al. 1987) [see ENGELS(1989) for least some transposable elements can undergo hori- review]. The discovery of a functional P element in zontal transfer between different species. For these D.willistoni that is virtually identical in nucleotide reasons and others, tranposable elements are subject sequence with the D. melanogaster element strongly to somewhat different evolutionary forces than con- argues that the elementwas transferred recently into ventional single-copy genes. the D. melanogaster genome from oneof these species Phylogenetic studies indicate that many transposa- (DANIELSet al. 1990). On the other hand,many trans- ble elements are distributed in discontinuous fashion posable elements reveal a more complex pattern in amongrelated taxa (DOWSETTand YOUNG 1982; their species distribution. For example, the transpos- MARTIN,WIERNASZ and SCHEDL1983; STACEYet al. ableelement designated I is also thoughtto have 1986). When a particular species lacks a transposable spread recently in populations ofD. melanogaster, sim- element that is found in closely related species, the ilar to thesituation with P. However, inactive elements finding is generally attributed to stochastic loss of the homologous to I are found in sibling species of D. element in the lineage of the particular species. Con- melanogaster, and it is not clear whether rapid spread versely, when a species contains a transposable ele- of the I element was initiated by interspecific transfer ment not foundin near relatives, the presence of the or whether some process allowed the activationof element is generally attributed to novel acquisition of previously inactive elements already in the genome theelement by interspecifictransfer from a more (BUCHETONet al. 1986) [see FINNEGAN(1989) for distantly related taxon. The occurrence of interspe- review]. cific transfer is illustrated by the P transposable ele- Inaddition to issues concerningthe distribution

Genetics 128: 319-329 (June, 1991) 320 K. Maruyama and D. L. Hart1 and abundance of transposable elements, much re- A mains to be learned about forces governing the extent orena of nucleotidesequence variation between different crccrn copies of transposableelements within andamong teissieri species. Although it has been suggested that dispersed, Y- moderately repetitive sequences mightundergo rapid concerted evolution as do tandemlyrepeated se- nulanogaster quences (ARNHEIM 1983),there is no strong evidence -sechellia for this view [see DOVER(1988) for review]. For the Simulnnr rDNA gene family in humans, it has been shown that -mnuriIia3u concerted evolution of rDNA sequences within the same chromosome is much more rapid than among B orena rDNA sequences in nonhomologouschromosomes erecta (SEPARACK,SLATKIN and ARNHEIM1988). In thecase of transposable elements, if sequences within a family teissieri are virtually identical, then this could result either - from concerted evolutionby specific genetic exchange melonogacrer mechanisms, such as gene conversion, or from recent amplification of a few ancestral copies of the element. -sechellia We report here a systematic study of the evolution Simulons of the transposable element mariner in the melano- -moluitiaM gaster species subgroup of Drosophila. The autono- FIGURE1.-Two alternative phylogenetic trees of the melano- mous mariner element is 1286 nucleotides in length, gaster species subgroup. Unequivocal relationships are indicated by thick lines and equivocal relationships by thin lines (redrawn from includes a single, long open reading frame encoding LACHAISEet al. 1988). a putative protein of 345 amino acids (presumed to be a transposase necessary for transposition), and has naturallyfound in D. melanogaster, D. erecta or D. terminal inverted repeats of 28 base pairs (JACOBSON, orena. MEDHORAand HARTL 1986). Eight species are as- Two hypotheses may be proposed to explain the signed to the melanogaster species subgroup, which discontinuous distribution of mariner in the melano- radiated in the Afro-tropical region. Two alternative gaster species subgroup. One model is that mariner trees have been proposed for the species phylogeny, was present in the common ancestor before the eight with three distinct species complexes recognized contemporary species divergedbut was lostin the within thesubgroup (Figure 1, modified from LA- lineages leading to D. orena, D. erecta and D. melanogaster. If this model is correct, the gene phylogeny of CHAISE et al. 1988). Based on genetic distance data derived from allozymes and nucleotide sequences of mariner sequences among the species is expected to the Adh genes, the three complexes are thought to be congruent with the phylogeny of the species, and sequences homologous to mariner might be found in have split 6-15 million years ago (BODMERand ASH- other species related tothe melanogaster species BURNER 1984; LACHAISEet al. 1988). The species D. subgroup. The other model for the distribution of erecta and D. orena are restricted to a small part of mariner assumes that one or more recent horizontal Africa, whereas D. teissieri and D. yakuba are wide- transfers introduced the element into thefive species spread on the African mainland. These four species possessing it some time after the divergence of the are reproductively completely isolated from one an- subgroup began. Inthis case the phylogeny of mariner other, and they do not hybridize even in the labora- sequences would have no necessary correlation with tory. In the melanogaster species complex, D. sechellia the phylogeny of the species, andthe mariner se- and D. mauritiana are insular species (restricted to the quencesshould all be very similar to each other. Seychelles and Mauritius, respectively), while D. si- Although the models are not necessarily mutually mulans and D. melanogaster have spread worldwide. exclusive, the weight of the evidence may be deter- The three most closely related species are D. simulans, mined by comparing the phylogeny of mariner se- D. sechellia and D. mauritiana,and they produce fertile quences with the phylogeny of the species that contain female offspring when mated with each other (LA- them. These comparisons require knowledge of mar- CHAISE et al. 1988). Althoughthe mariner element was iner sequence variation, both within and between the first identified in D.mauritiana, homologous se- species in the subgroup. quences are also present in D. simulans, D. sechellia, We have carried out Southern blot hybridizations D. yakuba and D. teissieri. However, the species distri- on representative species of the major Drosophila spe- bution of mariner is discontinuous in that it is not cies groups in the genus Drosophila in order to detect mariner Evolution in Drosophila 32 1 the presence of mariner homologues. The genus Dro- TABLE 1 sophila consists of twomajor subgenera. The subgenus Distribution of sequences hybridizing with mariner Sophophora contains sevenspecies groups, one of whichis the melanogaster species group. Although Subgenus Sophophora Group willistani further subdivisionof the species groups into Group melanogaster - capricorni subgroups is difficult, the melanogaster species group Subgroup melanogaster - nebulosa is conventionally divided into 10 to 1 1 subgroups, - erecta - paulistorum ++ mauritiana Group saltans including the melanogaster subgroup (ASHBURNER - melanogaster - neocordata 1989; THROCKMORTON1975; LEMEUNIER et al. 1986). - orena - saltans We have found that sequences homologous to mariner ++ sechellia - sturtmanti are prevalent within the melanogaster species group, ++ simulans Subgenus Drosophila and we have cloned and sequenced multiple copies of ++ tkssieri Group virilis ++ yakuba - littoralis the element from various members of the melanogaster Subgroup tahhashii - lummei species subgroup withinthis species group. Judged - lutescens - mmtana from these sequences, the phylogeny of the mariner - paralutea - Uirilis elements is congruent with the phylogeny of the spe- - prostipennis Group robusta cies within the subgroup. The rate of sequence evo- - pseudotukahashii - lacertosa - takahashii - mclanica lution of mariner appears to besomewhat slowerthan Subgroup eugracilis - robusta that of the single-copy nuclear gene, alcohol dehydro- - eugracilis - sordidula genase (Adh). Furthermore, mariner elements from Subgroup suxukii Group repleta closely related species can share nucleotide polymor- + lucipennis - arkonensis phisms,implying that concerted evolution hashad + mimetica - buzzatii + pulchrella - eohydei little effect on mariner evolution over the time scale + rajasehri - mojavensis of evolution of these species. Subgroup montium - mercatorum - auraria - mulleri MATERIALS AND METHODS + barbarae - neohydei + bicornutu - paranaensis Fly stocks: Isofemale lines of species in the melanogaster - birchii - repleta species subgroup were obtained from the following individ- + diplacantha Group immigrans uals: D. simulans, courtesy J. R. DAVIDand R. S. SINGH;D. - jambulina - albomicans melanogaster and D. sechellia, courtesy of J. R. DAVID;and + kikkawai - immigrans D. yakuba, D. teissieri, D.erecta and D. orena, courtesy of M. + nikananu - nasuta SOLIGNAC. DNAfrom various Hawaiian Drosophila species + pennae Groupfunebris was kindly provided by R. DESALLE.All other species were + quadraria -funebris obtained from the National Drosophila Species Resource ++ seguyi Hawaiian Drosophila Center, Bowling Green State University, Bowling Green, + serrata - adiastola Ohio. All species examined for thepresence of the sequences ++ tsacasi - grimshawi homologous to the mariner element are listed in Table 1. - vulcana - heteroneura DNA probes: Four overlapping regions covering mariner Subgroup ananassae - mimica were subcloned into pUC18 to use as probes, as shown in -+ ananassae - neutralis Figure 2 (see also JACOFSON,MEDHORA and HARTL1986). + malerkotliana - punalua Plasmid DNA was prepared by CsCl density gradient cen- Group obscura Subgenus Dorsilopha trifugation, and probe DNA was isolated by a modification - aflnis - busckii of the glass-powder method of VOCELSTEINand GILLESPIE - algonquin (1979). - bfasciata Genus Zaprionus DNA blotting experiments:Genomic DNA for Southern - miranda - inermis blot hybridization experiments was prepared as described - persirnilis ++ tuberadatus in LIS,SIMON and SUTTON(1 983). Restriction digestion, gel - Pseudoobsczcra electrophoresis, and transfer to filters were performed ac- Species examined for the presence of sequences that hybridize cording to MANIATIS, FRITSCHand SAMBROOK(1982). with mariner. The ++ and + signs indicate the relative abundance Southern blots were carried out essentially as in SOUTHERN of hybridizing sequences; - indicates no detectable hybridization (1975). Probes were labeled with '*P to high specific activity signal. by the method of FEINBERCand VWEISTEIN (1983, 1984). Filter hybridizations were performed at 65" for 12-16 hr isolated from the gel and ligated into the BamHI site of the in a solution of 1 % Sarcosyl, 100 mg/mlsalmon sperm lambda vector EMBL3. Recombinant lambda phage were DNA, 0.5 M NaCI, 0.1 M Na2HP04,5 mM Na2EDTA, pH packaged in vitro with Gigapack (Stratagene, La Jolla, Cali- 7.0. Filters were washed at room temperature,first with 1% fornia), and the resulting libraries were screened with the Sarcosyl, 1 mM Tris (pH 7.0), and then three times with 1 &PI-NheI fragment of mariner (Figure 2). To avoid sequenc- mM Tris (pH 8.0). ing the same mariner element twice, the genomic position Cloning and sequencing:Genomic DNA from each spe- of each clone was examined by preparing DNA from each cies was prepared according to KUNERet al. (1985). DNA positive lambda clone (HELMSet al. 1985), digesting the was partially digested with Sau3A and size-fractionated by DNA with one or more endonucleases having no restriction gel electrophoresis. DNA fragments of 13-22 kilobases were sites within the mariner element, and separating the frag- 322 K. Maruyama and D. L. Hart1

56 34 9987 647 1183

Probe 5 -"Probe 4 FIGURE2.-Restriction map of mariner, showing the - regions covered by different probes. 4 Pro& 3 b -Protm 2 - ments on agarose gels alongside genomic DNA digested with the same restriction enzymes. After transfer to filters andhybridization with mariner as probe, lambda clones representing independent mariner elements were selected by identifying those distinct fragments that comigrated with genomic DNA fragments also hybridizing with the probe. The DNA fragment containing mariner from each lambda clone was subcloned into M13mp18 and M13mp19 and sequenced by the dideoxy chaintermination method of SANCER, NICKLENand CoULsoN (1977) usingSequenase 6.5 kb- ' (United States Biochemical). Sequences of Adh from D. teissieri and D. yakuba were kindly provided by P. JEFFSand M. ASHBURNER. 3.5 kb-

RESULTS Distribution of mariner in the D. melanogaster species subgroup: In order to estimate the number of copies of mariner in the genome, DNA digested with various combinations of restriction enzymes that - do not cleave within the element was transferred to FIGURE3.-Southern blot hybridization of species in the mcla- nylon membranes and probed with an internal SspI- nogaster species subgroup.Each lane contains DNA from about 10 NheI mariner fragment that includes most of the ele- flies digested with Hind111 and BamHI. The filter was hybridized ment (probe 3 in Figure 2). To detect the presence of with the SalISphl fragment (probe2). Hybridization withthe S@I- deleted elements or restriction-site polymorphisms, NheI fragment (probe 3) revealed no additional hybridizing bands. the same filters were also hybridized with two distinct mariner in the D. simulans linesis generallysmall internal fragments, an SspI-XhoII fragment that in- (occasionally none), but lines from Afro-tropical re- cludes approximatelythe 5' half ofthe element (probe gions show the highest number of bands. However, 5), and an XhoII-NheI fragment that includes approx- the variation in genomic position is not very great, imately the 3' half of the element (probe 4). and a few hybridizingbands of the samesize are Figures 3 and 4 show that the mariner element is present in the majority of the strains representing distributed discontinuously among species in the mel- regions as far apart asAfrica and North America anogaster species subgroup. For example, sequences hybridizing with mariner are found in the threeclosely (Figure 4). For example, a 6.6 kb EcoRI-BamHI band related species, D. mauritiana, D. simulans and D. is observed in strains from Capetown, Congo,and the sechellia, but not in their sibling species, D. melano- United States. Thus, it appears that mariner has a low gaster. Hybridizing sequences are also found in D. copy number in most strains of D. simulans, and some yakuba and D. teissieri, but no detectable hybridization genomic siteswith insertions appear to be nearly fixed. is observed in D. erecta or D. orena. Among the species Intraspecific variationin the distribution of mariner containing the element, a striking difference is found was also studied by Southern blot experiments using in the copy number and the pattern of intraspecific multiple isofemale linesfrom each species: 12 lines of variation. D. mauritiana has the largest number of D. mauritiana, five lines of D. sechellia, six lines of D. bands hybridizing with mariner, but its sibling species yakuba, seven lines of D. teissieri and two lines of D. D. simulans and D. sechellia have the lowest number erecta. For D. orena only one strain was used because of bands. Furthermore, D. sechellia does not show any all the extant lines are derived from one collection in evidence for intraspecific variation in the copy num- 1975 from Cameroon (LACHAISEet al. 1988). For the ber or genomic positions ofthe elements. cosmopolitan speciesD. simulans and D. melanogaster, Figure 4 also illustrates intraspecific variationin the more than 15 isofemale lines representing different copy number and genomic positions of mariner ele- regions of the world were screened for the presence ments in strains of D. simulans from different regions of mariner. Southern blot hybridization with distinct of the world. The number of bands hybridizing with internal fragments of mariner does not revealany mariner Evolution in Drosophila 323

FIGURE4.-Southern blot hybridization of D. simulans lines. The leftmost lane containsD. mauriliana asa control. The y w strainis a laboratory strain maintained for many years at the California Institute of Technology. The w strain is a laboratory strain obtained from the Indiana University Stock Center. All other strains were isolated recently from natural populations. Each lane contains DNA from about 10 flies digested with EcoRI and BamHI. The filter was hybridized with theSspI-NheI fragment(probe 3). The Morro Bay strain showstwo hybridizing bands inother blots (data not shown). Blots using DNA from single flies show that the heavy bands are shared by all the individuals in the strain, whereas the weak bands are present only in some individuals (data not shown).

detectable difference in hybridization pattern in D. ofAfrica show from 4-8 hybridizingbands. Two mauritiana, indicating that there are no major dele- examples are illustrated in Figure 5. Unlike D. sechel- tions or restriction site polymorphisms among the Zia, hybridization with distinct internal fragments of elements (Figure 5). Each of the D. mauritiana strains mariner does not detect any large deletions in the examined also shows a unique pattern of hybridiza- elements (compare Figure 5, A with B). The presence tion, suggesting that at least some of the elements in of a moderate number of distinct bands suggests the the genome are active (data not shown). The present presence of at least some active mariner elements in observations confirm and extend the results OfJACOB- the D. yakuba genome. SON, MEDHORAand HARTL(1986), who reported that In D. teissieri, hybridization with two distinct inter- D. mauritiana has 20-30 copies of mariner per ge- nal mariner fragments shows a strikingly different nome, great variation in the genomic positions ofthe pattern. In all strains examined, the SspI-XhoII frag- elements, and few deleted copies. ment from the 5’ end hybridizes to over 15 genomic All five isolates of D. sechellia examined show the bands (Figure 5A),while the XhoII-NheI fragment same pattern ofhybridization as thethree strains from the 3’ end hybridizes onlyto a few bands (Figure shown in Figure 3. When the entire mariner element 5B). This result indicates that the majority of mariner is used as probe, two bands are observed, but only copies in this species havea deletion of most ofthe 3’ one hybridizes with the XhoII-NheI fragment from the half ofthe gene. The sequence ofthe deleted elements 3‘ end (compare Figure 5, A with B). This result is shown below. suggests that there aretwo copies ofmariner at fixed Distribution in other Drosophila species: Species locations in the genome, and that one copyhas a from other Drosophila species groups were alsoprobed deletion of most of the 3’ half of the element. with mariner. Some representative examples are The sibling species D. yakuba and D. teissieri are shown in Figure 6, and the results concerning pres- widespread in Africa (LACHAISEet al. 1988). Variation ence (+ or ++, according to relative abundance) or in the distribution of mariner within each species was absence (-) of hybridizingfragments are summarized studied using strains collected from areas spanning in Table 1. In addition to the melanogaster species the continent. Strains of D. yakuba from different parts subgroup, mariner homologues are found in several 324 K.and Maruyama D. L. Hart1

FIGURE5.”Southern blot hybridization showing large deletions in mariner elements in some species of the melano- gasterspecies subgroup. DNA was digested with EcoRI and BamHI, except for D. sechellia DNA, which was digested with BamHI and HindIII. (A) Hybridi- zation with the Sspl-XhoII fragment (probe 5). (B) Hybridization with the Xholl-NheI fragment (probe 4).

FIGURE6.”Southern blot hybridization of species of representative species groups. See also Table 1. DNA was digested withEcoRI and BamHI, and the filter was probed with the XhoII- NheI fragment(probe 4). In the ananassae subgroup, D. ananassae and D. malerkotliana do not show any hybridization when probed with the SspI-XhoII fragment (probe 5). Some species of the montium subgroup (D.Cikhawai, D. diplacantha, D. nikananu, D. seguyi, D. tsacasi and D. vulcana) show an abundanceof restriction fragments that hybridize with the mariner probe. Sequences homologous to mariner have not been found in the takahashii (D.lutescens, D. takahashii) or eugracilis subgroups, which are generally considered to be most closely related to the melanogaster subgroup (LEMEUNIER et al. 1986). In species outside the melanogaster species group (Table 1). we have observed no sequences that hybridize with mariner. However, notable exceptions are species within the genus Zaprionw. When the hybridization is carried out at low stringency, homologous sequences are detected in some species of the immigransspecies group (data not shown). In the blot, lane 6 is intentionally left blank.

other subgroups in the melanogaster species group, the melanogaster species group (Table l), we have namely, the montium, surukii and ananassae observed no mariner homologues, with the notable subgroups. However, presence of homologousele- exception of species withinthe genus Zaprionus. When ments has not been detected in the takahashii and the hybridization is carried out at lower stringency, eugracilis subgroups, which are generally considered however, homologous sequencesare detected in Some to be more closely related tothe melanogaster species of the immigrans species group, which the subgroup (LEMEUNIERet al. 1986). In species outside genus Zaprionus is related to (data not shown). mariner Evolution in Drosophila 325

4 Yak2

6 181832

5 Yak3

5 Pch

4 Me 1 Ma351 0 ' r2Mor1 FIGURE8.-Maximum parsimony tree of eight mariner se- TCAGACGAAACGACCAAGAGCCCTAAGTGTTTTAT-TGG quences, using PAUP (SWOFFORD1985). Deletions at the ends of TCAGACACARAAACCACCAATGTGCTTTAT-TGG ME4 and Teis32 weretreated asunique singlecharacters. The TCAGACGAAGCGATCAAGGGCCCAATGTGCTTTAT-TGG ** ** * * *** **** * compensating insertion/deletion at positions 1248 and 1254 was FIGURE7.-Alignment of eight mariner sequences. Onlysites treatedas a singlecharacter. The number of mutationalsteps differing among the sequences are shown. The numbers at the top between the nodes are indicatedalong the branches. indicate the positions of the variable sites in the mariner sequence. Deletions are indicatedby minus signs. Phylogenetically informative the two distinct internal fragments. Among 14 clones sites are indicated by asterisks. The best alignment is obtained by a that showedhybridization with theentire mariner deletion of T at position 1248, and addition of A at position 1254, fragment, only three hybridizedwith both internal in the sequences of pch and MEI. Thus, the numbering of nucleotides is through 1287 instead of 1286. The open reading frame fragments, whereas 11 hybridized with the SspI-XhoII runs from 172 through 1209. fragment but not with the XhoII-NheI fragment. Thus, the ratio of intact copies tothe total number of DNA sequence analysis: The aligned nucleotide elements is 3: 14, which is roughly consistent with the sequences from fourspecies ofthe melanogaster species results of the Southern blot experiments. One of the subgroup are presented in Figure 7. For D. mauri- three apparently complete mariner copies (Teis32) was tiana, sequences of three mariner copies are shown. chosen for further study. As seen in Figure 7,while The copyof the element denoted peach (pch) was the element possesses an intact open reading frame, it previouslycloned from the white locus UACOBSON, is missing a portion of the 5' terminal inverted repeat. MEDHORAand HARTL1986), and the Mosl sequence We also studied the sequences of three elements ran- is a highly active mariner element that causes a high domly chosen from the 11 clones containing mariner rate of excision ofthe peach element from its insertion elements with a deletion in the 3' half of the gene. point in the white locus (MEDHORA, MACPEEKand All three share an identical internal deletion that spans HARTL 1988; MEDHORA, MARUYAMAand HARTL nucleotide positions 544-1 260, which includes the 1991). The sequence denoted Ma351 was obtained putative stop codon of the open reading frame and from a clone chosen at random from a u3" library the putative polyadenylationsite (JACOBSON, MED- (JACOBSON, MEDHORAand HARTL1986). Figure 7 HORA and HARTL1986). indicates that all three elements from D. mauritiana Phylogeny of mariner elements A phylogenetic are 1286 nucleotides in length. An additional five tree was constructed from the eight mariner sequences copies of mariner isolated from D. mauritiana also in Figure 7. All of the phylogenetically informative show no heterogeneity in length (data not shown). sites were consistent with each other, and therefore For D. simulans and D.yakuba, two copies ofmariner maximum parsimony analysis yields onlyone treewith from each species were sequenced. In the Morro Bay no inconsistencies (Figure 8). In Figure 8, the numbers strain of D. simulans, one copy (designated MBI) was represent mutational steps separating any two nodes. full-length (1286 base pairs), while the other (desig- Although these numbers are small, it is clear that the nated MB4) had a few base pairs deleted at the ends mariner sequences from D.yakuba and D.teissieri form of the terminal inverted repeats. The two D. yakuba one phylogenetic group andthose from D.mauritiana copies (Yak2 and Yak3) were both full-length, and and D. simulans form another. Within D. mauritiana there were no chain-terminating base substitutions and D. simulans, however, the mariner sequences do observed in the open reading frame of the sequence, not form clusters according to the species classifica- consistent with the Southern blot experiments sug- tion. That is, MBl from D. simulans is grouped with gesting that these elements are, at least potentially, the mariner sequences from D.mauritiana, while MB4 active. is off by itself. The simplest explanation of these For D.teissieri, which was found to possess a number shared polymorphisms is that they arose prior to spe- of deleted copies, the genomic library was screened ciation, which would indicate that mariner elements first using the entire mariner element as a probe, and have not undergone rapid concerted evolution within positive clones were then hybridized separately with these genomes. These species are closely related, and 326 K. Maruyama and D. L. Hart1 Db.1 1111351 pcb IO1 I54 Yak2 Yak3 To1.32 In themelanogaster species group, mariner homologs m.1 0.002 0.007 0.003 0.008 0.01s 0.019 0.019 are found in some other subgroups as well as the -3s1 0.006 0.002 0.007 0.019 0.0190.019 melanogaster species subgroup, namely the montium,

pcb 0.004 0.0230.0240.0230.012 suxukii, and ananassae subgroups, although the element is not observed in the subgroups takahashii and Jm 0.0190.0200.0190.008 eugracilis. Furthermore, the montium, suzuki and an- I54 0.0180.0180.017 anassae subgroups are mainly Oriental in distribution, 0.0070.007 whereas the melanogaster species subgroup is Afro- rak3 0.008 tropical (LEMEUNIERet al. 1986). Thus, the distribu- lbi.32 tion of mariner is not limited to one subgroup or to FIGURE9.-Pairwise divergence among the eight mariner se- one geographic area, as any simple form of the recent quences. The divergence was calculated as the number of nucleo- invasion hypothesis would predict. tides differing between anytwo sequences divided by thetotal Maximum parsimony analysis of the mariner se- number of nucleotides in mariner. quences from the melanogaster species subgroup gives they are known to share polymorphisms in the Adh only one phylogenetic tree with no inconsistencies gene that arose prior speciationto (COYNEand KREIT- (Figure 8). When superimposed on thetwo alternative MAN 1986; STEPHENSand NEI 1985). species trees (shown in Figure l), the mariner phylo- Figure 9 summarizes the pairwise divergences (per- geny is consistent with either 1A or 1B. (The absence cent of nucleotidesthat differ) among mariner se- of mariner in both D. erecta and D.orena prevents quences from the four species. There is an unexpect- discrimination.) Assuming that mariner was present edly low rate of divergence between D. mauritiana- before the diversification of the melanogaster species D-simulans and D.yakuba-D. teissieri lineages. The subgroup, both trees require that mariner was lost at table indicatesthat theaverage divergence of mariner least twice in lineages leading to D. melanogaster and between these two species clusters is less than 2%. A D. orena-D. erecta. The possibility that a species could similar comparison for the coding region of the Adh lose a transposable element is supported by the distri- gene gave 5% divergence (P. JEFFS and M. ASHBUR- bution of mariner in D. simulans,in which some strains NER, personal communication). On the other hand, lack the element entirely. the average divergence of mariner elements within Assuming that mariner was present in the ancestral species is about 1% and is roughly compatible with group beforespeciation, we might expect thatmariner the intraspecific variation in the Adh gene of D. mela- sequences would be as divergent as any single-copy nogaster (KREITMAN1983). Including additional mar- gene between the species. However, the mariner se- iner sequences from D. mauritiana indicates that ran- quences among the species have not diverged to the dom mariner sequences from this species may differ same extent as the Adh gene from the same species. by as much as 1.1% (data not shown), but this does We compared the rates of divergence between the not affect the comparison between species. coding regionof the Adh gene and the entiresequence of the mariner gene, since it is believed that most of DISCUSSION the 1286 nucleotide sequence of mariner encodes a Two hypotheses may be proposed to account for transposase protein (JACOBSON, MEDHORAand HARTL the discontinuous distribution of the transposable ele- 1986). If the sequences were under similar selective ment mariner in the melanogaster species subgroup of pressure, we should see a somewhat greater rate of the genus Drosophila. According to the stochastic loss divergence in mariner, since a small amount of non- hypothesis, mariner was present in the ancestral group coding region is included in the mariner sequence. and was subsequently lost from some of the species The divergence in the 771-nucleotide coding region during their evolution. On the other hand,according of Adh is 5% between D. mauritiana and D. yakuba, to the recentinvasion hypothesis, mariner was recently and this is roughly consistent with other data on the transferred from an outside source into some species genetic distance between the two species. However, of the melanogaster species subgroup subsequent to its the average divergence of mariner sequences is ap- divergence. Several features of our results donot proximately 2%, roughly half that of the Adh gene. support the recent invasion hypothesis. First, mariner Comparison between D. mauritiana and D. teissieri is not limited to the melanogaster species subgroup. gives a similar result. Thus, it seems that mariner has Second, the mariner gene phylogeny is congruent with diverged more slowly than the coding region of the the species subgroup phylogeny. Third, adetailed Adh gene, which itself has diverged at a relatively slow sequence analysis of mariner copies shows that the rate (SHARPand LI 1989). pattern of divergence between species is not compat- Although theother evidenceargues against the ible with the pattern expected based on recent inter- recent invasion hypothesis, the slow rate of mariner specific transfer. These findingsare elaborated below. evolution requires reconsideration of the possibility marinerDrosophila Evolution in 327 two random copies of mariner from this species may cOreMerecta differ by as much as 1.1%. The relatively high rate of mariner polymorphism within D. mauritiana, as contrasted with the relatively low rate of divergence among species, suggests the melanogaster possibility that mariner elements may have different sechellia mutation rates in different lineages. Another example sirnulams of possible differential mutation rates was reported in mauritiana a region near the glue gene cluster in D. melanogaster (MARTIN and MEYEROWITZ1986), in which noncod- FIGURE10,”Two scenarios of mariner invasion after the divergence of the melanogaster species subgroup. In scenario X, mariner ing sequences in salivary region 68C adjacent to the invaded before D.teissieri and D. yakuba split, whereas in scenario gene clusterare significantly more conserved than the Y, the invasion occurred after the species had split. nearby coding regions. The hypothesis of differential mutation rates might be particularly apt for transpos- that mariner may have entered themelanogaster species able elements. Some quiescent transposable elements subgroup after the species diverged. This would give in Drosophila are located in heterochromatic regions the mariner elements a more recent common ancestor where gene expression appears to be low (BUCHETON than the common ancestorof the Adh gene, andcould et al. 1986; DANIELSet al. 1984; MIKLOS et al. 1988; explain the discrepancy in the rates of sequence evo- SIMONELIGet al. 1988). In species where the elements lution. One possibility is that mariner independently undergo active transposition they are usually found in entered each of the ancestralspecies of the yakuba and euchromatic regions. In D. mauritiana, where mariner melanogaster species complexes before the divergence reaches the highest copy number among the sibling of D. yakuba and D. teissieri (scenario X in Figure 10). species, the elements are located primarily in euchro- This model would predict that mariner and Adh should matic regions (data not shown), and some copies have be equally divergent betweenD. yakuba and D. teissieri, been shown to be highly active (BRYAN and HARTL but this is not the case. In these species mariner se- 1988; MEDHORA,MARUYAMA and HARTL 1991). In quences are 0.8% divergent but Adh sequences are simulans, however, mariner is located in both het- more than 2% divergent. An alternative possibility is D. erochomatic and euchromaticregions (data not that mariner invaded the melanogaster species shown). If genes in heterochromatin have reduced subgroup several times after the speciation of D. yak- mutation rates, then it is possible that mariner could uba and D. teissieri (scenario Y in Figure 10). Scenario Y requires at least three independent invasions, into have long resided in heterochromatin and only re- D. yakuba, D. teissieri and the species ancestral to D. cently become active in D. mauritiana, resulting in a simulans, D. mauritianaand D. sechellia. Although this higher mutation rate in this species. model might explain why mariner is less divergent The slow rate of evolution and shared polymor- between species than the Adh gene, it creates another phisms betwen closely related species are often ex- difficulty. For example, if we accept that interspecific plained by introgression between species. Although transfer of mariner was frequent enough toallow three the introgression of mtDNA is speculated to have independent invasion events in the melanogaster occurred from the population of D. simulans in Mad- subgroup, then we have to explain why mariner has agascar to that of D. mauritiana endemic to Mauritius not invaded other species of Drosophila and why the (SOLIGNACand MONNEROT1986), thereis no evidence phylogeny of mariner is congruent with the species for introgression of any nuclear genes in natural pop- subgroup phylogeny. ulations. In the caseof the Adh gene, the simulans We thereforeprefer the hypothesis that mariner alleles most closely related to mauritiana alleles are was present in the ancestral species prior to the radia- not limited to populations in the Afro-tropical region tion of the melanogaster species subgroup, and that the where the two species coexist, suggesting thatthe element was lost in the lineages leading to D. melano- shared polymorphisms are due to retention of poly- gaster and D. orena-D. erecta.Acceptance of this model morphisms that predated speciation and not due to implies that mariner has evolved rather slowly in the introgression (COYNE andKREITMAN 1986; STEPHENS melanogaster species subgroup. Although genes under and NEI 1985). Two observations support the same different selective pressures are known to have differ- conclusion for mariner. First, theMorro Bay strain ent rates of nucleotidesubstitution, the paucity of that we studied was collected in California, geograph- substitutions, even atthe presumed silent sites in icallyvery distant from Mauritius, where D.mauri- mariner, remains difficult to explain. On the other tiana is endemic.Second, active copies of mariner, hand, the data onintraspecific variation have revealed which constitute a subset of all mariner sequences (K. many nucleotide polymorphisms within mariner ele- MARUYAMA,unpublished data), are foundin D. simu- ments in D. mauritiana, as evidenced by the fact that lans populations worldwide as well as in D. mauritiana, 328 K. Maruyama and D. L. Hart1 indicating that active mariner elements existed in the equilibrium state. However, given the wide range of common ancestor of the two species (CAPYet al. 1990). variation, it is possible that none of the species are at A similar observation holds forthe pair of sibling equilibrium, and that the different states merely rep- species D. yakuba and D. teissieri (unpublished data, resent different stages in the evolution of a transpos- but see Figure 8). Although their mtDNA shows strik- able element. We have provided evidence suggesting ing similarity, these two species show almost complete that mariner was lost at least twice in the melanogaster reproductive isolation, and there is no evidence for subgroup. The absence of mariner in the takahashii nuclearintrogression (MONNEROT, SOLIGNACand and eugracilis subgroups, considered to be the closest WOLSTENHOLME1990). Furthermore, species of the relatives of the melanogaster subgroup, also suggests melanogaster species complex (D.mauritiana and D. that the element was lost completely in the lineages simulans) and the yakuba complex (D.yalzuba and D. leading to these subgroups. Theoretical work suggests teissieri) do not hybridize either under laboratory con- that, if the transposition process depends on copy ditions or in nature. Thus, the relatively slow rate of number, then the transposable element system will go sequence evolution of mariner in the melanogaster extinct in the genome unless autonomous copies have subgroup and the shared polymorphisms between D. some advantage in terms of frequency of transposition mauritiana and D. simulans are not likely to result over nonautonomous copies (KAPLAN, DARDEN and from introgression. LANGLEY1985). This is a rather pessimistic model The retention of ancestral polymorphisms suggests from the standpoint of a transposable element, and if that the mariner family ofelements has not undergone it is accepted, then rare instances of horizontal trans- rapid sequence homogenization. Althoughit has been fer to new species would provide a necessary mecha- suggested that transposable elements might undergo nism for an element to remain extant over long pe- rapidconcerted evolution or homogenization (HEY riods of evolutionary time. Indeed, it seems very likely 1989), the concept is difficult to apply to dispersed that some kind of horizontal transfer of mariner be- genes that can change rapidly in copy number. The tween species must be invoked toaccount for the shared polymorphisms and lack of species-specific nu- presence of the element in the genus Zaprionus. cleotide changes between closely related species sug- We would like to thank J. CARULLI, J.DAVID, R. DESALLE,L, gests the absence of concerted evolution of mariner PARK,R. SINGH,M. SOLIGNACand the National Drosophila Species transposable elements. Indeed, the relative homoge- Stock Center for providing Drosophila stocks, and M. ASHBURNER neity of mariner sequences within species may simply and P. JEFFS for sharing theAdh sequences. We thank G. BRYAN,J. reflect rapid amplification of the elements from afew DAVID,R. DUBOSE,D. DYKHUIZEN,D. GARZA,L. GREEN, JACOB-J. SON, M. MEDHORAand I. MORI for helpful discussions, J. AJIOKA, ancestral sequences andthe coalescence process M. ASHBURNER andR. DUBOSEfor critical comments on the man- (KINGMAN1980), without any involvement of partic- uscript, and A. MACPEEK, K. SHAWand K. SCHOORfor technical ular homogenizing mechanism relevant to multigene assistance. This work was supported by National Institutes of Health families. grant GM33741. 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