Evolutionary Dynamics of Mitochondrial DNA Duplications in Parthenogenetic Geckos, Heteronotia Binoei

Evolutionary Dynamics of Mitochondrial DNA Duplications in Parthenogenetic Geckos, Heteronotia binoei

Craig Moritz

Department of Zoology, University of Queensland, Brisbane, Queensland, Australia 4072 Manuscript received August 22, 1990 Accepted for publication May 1 1, 199 1

ABSTRACT Mitochondrial DNA (mtDNA) fromtriploid parthenogenetic geckos of the Heteronotia binoei complex varies in size from 17.2 to 27.6 kilobases (kb). Comparisons of long us. short genomes using restriction endonucleases revealed a series of tandem direct duplications rangingin size from 1.2 to 10.4 kb. This interpretationwas supported by transfer-hybridization experimentswhich also demon- strated that coding sequenceswere involved. Someof the duplications have been modifiedby deletion and restriction site changes, butno other rearrangements were detected. Analysis of the phylogenetic and geographic distribution of length variation suggests that duplications have arisen repeatedly within the parthenogenetic form of H. binoei. The parthenogens, and thus the duplications, areof recent origin; modificationsof the duplicated sequences, particularly by deletion, has therefore been rapid. The absence of duplications from the mtDNA of the diploid sexual populations of H. binoei reinforces the correlation between nuclear polyploidy and duplication of mtDNA sequences reported for other lizards. In comparison to the genomes of sexual H. binoei and of most other animals, the mtDNA of these parthenogenetic geckosis extraordinarily variablein length and organization.

ONTRARY to previous assertions of extreme acting against thestrong geneticdrift of oocyte C economy and conservative organization (WAL- mtDNA, to maintainheteroplasmy (DENSMORE, LACE 1982; SEDEROEF1984; ATTARDI1985), there WRIGHT and BROWN1985; RAND and HARRISON have been several recent reports ofanimal mitochon- 1986; BUROKERet al. 1990). Analysis of segregation drial DNAs (mtDNAs) with large-scale size variation among the progenyof heteroplasmic females suggests attributableto repetitive sequences (Table 1). To- that smaller molecules may be at an advantage (RAND gether with numerous reports of intra-individual size and HARRISON1986), althoughin Drosophila the bias polymorphism (heteroplasmy, Table 1 and BIRMING- varies with the age of the female (SOLIGNACet al. HAM, LAMBand AVISE 1986) and variation in gene 1987). Comparisons among related species of lizards order (WOLSTENHOLMEet al. 1985; DUBIN,HSU-CHEN (DENSMORE,WRIGHT and BROWN 1985;DENSMORE et and TILLOTSEN1986; HAUCKEand GELLISSEN1988; al. 1989; MORITZ,WRIGHT and BROWN 1989),crick- GAREY andWOLSTENHOLME 1989; DESJARDINSand ets(RAND and HARRISON1989) and bark weevils MORAIS1990) these studies reveal a genome more (BOYCE,ZWICK and AQUADRO1989) revealed that plastic than previously supposed. It now seems that these noncoding tandem repeatsare often maintained the duplication and transposition of sequences is an through speciation events. Thus, any selection against importantmode of mtDNAevolution (MORITZ, large molecules must be balanced by recurrent muta- DOWLINGand BROWN1987; JACOBS et al. 1989; CAN- tion. TATORE et al. 1987). Most of the length variation that has been charac- A different form of length variation, involving the terized is due tovariation in the number of copies of tandemduplication of coding sequences, has been tandemlyrepeated noncoding sequences fromthe reported in mtDNAsfrom newts, nematodes, and control region (Table 1). Repeatunits of this type lizards (Table 1). These duplications vary in size from vary in size from 64 base pairs (bp) in mtDNA from 1.1 to 8.0 kb and typically span or flank the control Cnemidophorus lizards (DENSMORE,WRIGHT and region. All known duplications of animal mtDNA BROWN 1985)to 2.0 kb in bark weevils (BOYCE,ZWICK coding sequences are tandem and direct,except in the and AQUADRO1989). In two groups with exception- nematode Romanomermis (HYMAN,BECK and WEISS ally large mtDNA, scallops (SNYDERet al. 1987; LA 1988) where there appears to be a partial, inverted ROCHEet al. 1990) and bark weevils, the additional copy disjunct from three tandem direct repeats. In DNA is largely due to these tandemly repeated se- contrastto the noncodingrepeats, duplications of quences. Several studies have suggested that copy coding sequencesare rarely heteroplasmicand appear number of the noncoding repeats changes rapidly, to beephemeral in thatnot one is sharedamong

(knetics 129: 221-230 (September, 1991) 222 C. Moritz

TABLE 1 Characteristics of repeated sequences (>20 bp) in animal mtDNA

Shared between Location or Species Size species? Copy No. content Heteroplasmy Reference 1. Noncoding sequences Cnemidophorus spp. 64 bp Yes 3-9 Control region 15/92 DENSMORE,WRIGHK and BROWN(1 985) Acipens transmontanus 82 bp ? 1-4 Control region 521128 BUROKERet al. (1990) Gryllus spp. 206 bp Yes 2-7 Control region 1471319 RANDand HARRISON(1989) Drosophila spp. 470 pb Yes 2-6 Control region 17/92 SOLIGNACet al. (1986); HALEand SINGH(1 986) Pissodes spp. 0.8-2.0 kb Yes Control region 2191219 BOYCE,ZWICK and AQUADRO(1 989) Alosa sapidissma 1.5 kb ? 2-3 Control region 301244 BENTZEN,LEGGETT and BROWN(1 988) Placopecten magellias 1.4 kb 2-7 ? 181250 LA ROCHEet al. (1990) 2. Coding sequences Cnemidophurus spp. 1.5-8.0 kb No 2 Variable 1/43 MORITZand BROWN(1 987) Triturus 1.1-8.5 kb NO 2-3 Variable 213 WALLIS(1 987) Romanomermis 3.0 kb ? 3-5 ? None HYMAN,BECK and WEISS(1988) closely related species (Table 1) (MORITZ, DOWLING SEXUAL lcAsl S M6 and BROWN 1987). DIPLOID (0153) (0150) Previously reported duplications of mtDNA coding 9 sequences have been taxonomically or geographically isolated, offering little scope for analysis of their evo- CABISM6 PARTHENOGENETIC lutionary dynamics. In particular, thereis no evidence DIPLOID 7 (7) on the form and rate of sequence evolution within repeated mtDNAgenes. Is there selection for a return to small genome size? Doesthe presence of redundant sequences permitforms of sequence evolution (re- viewedin BROWN1985) not usually seen in animal mtDNA? J PARTHENOGENETIC ‘A’ ‘B + C’ This report concerns variation in the size and dis- TRIPLOIDS tribution of large tandem duplications of coding se- mlCA61SMB mlSM6lSM6 quences in mtDNA from parthenogenetic(all-female) (25154) (32133) FIGURE1 .-Evolutionary history of parthenogenetic H. binoei geckos of the Heteronotia binoei complex. These par- and their mtDNA (boxed). Analyses of chromosome and allozyme thenogens are triploid and arose via multiple inde- variants suggest thatthe parthenogens arose through multiple pendent hybridization events involving two chromo- hybridization events between the “CA6” and ”SM6” sexual races some races (“CA6” and “SM6”) of sexual H. binoei (MORITZ1984; MORITZet al. 1989). The predicted diploid-hybrid (Figure 1) (MORITZ 1983). The parthenogenetic line- intermediate has never been found despite intensive collecting and may no longer exist. The mtDNA of the parthenogens analyzed in ages are now distributed throughout most of the this reportare most similar to those from western Australian central and western deserts of Australia and, for a populations of the CA6 type (MORITZ 1991). The “A” and “B + C” parthenogenetic vertebrate, have extraordinarily high are designations for the two major chromosome classes of triploids genetic (allozymic and chromosomal) diversity within that are derived from different types of backcross. The frequency and between lineages (MORITZet al. 1989). There are of duplications in each type of lizard is given in parentheses. two major chromosome lineages that differ in the dosage of the parental genes and which are thought hybrid genome of the parthenogens, and the diploid to represent the result of different hybridizations (Fig- recombining nonhybrid backgroundof their maternal ure 1). In contrast to the variability of the nuclear sexual parents.Comparisons of mtDNAs in sexual genome, mtDNAs from the parthenogens have few and parthenogenetic lizards, and in different types of restriction site differences other than those due to parthenogenetic lineages may illuminate the processes length changes. These data suggest that the parthen- involved. In particular, thepresence in the partheno- ogens arose recently,ie., within the last few thousand gens of mtDNAs that differ only by length mutations years, from a small geographic area, probably in the provides a simple system for mapping and character- west (MORITZ 1991). izing the mutations. This paper presents an analysis The studies on the parthenogens provide ahistori- of the physical properties and the geographic and cal framework for interpreting changes in their mt- phylogenetic distributions of mtDNA duplications. DNAs. Similar mtDNAs occur in two very different This revealed a highly dynamic system of duplication nuclearbackgrounds; the triploidnon-recombining and deletion,which is in stark contrast to the stability Evolution of mtDNA Duplications 223

TABLE 2 kb s L1 L2L3L5 L7L7 02 Ls L11LS Localities sampled andresults obtained

Sample Length Locality size variants" Aileron Stn., NT 1 Alice Springs, NT 9 70km W Alice Springs, NT 1 Bullabulling Stn., WA 4 3' Bullardoo Stn., WA 4 i Coondambo Stn., SA 3 Cunyu Stn.. WA 1 De Rose Hill Stn., SA 2 1 Faraheedy Stn., WA 2 Glenayle Stn., WA 1 Granite Downs Stn., SA 5 Granite Peak Stn., WA 1 Kathleen Valley, WA 2 Kirkalocka Stn., WA 1 FIGURE2.-Autoradiogram ofBg/lI-digested mtDNAs from par- Lake Violet Stn., WA 6 thenogenetic H. binoei showing the effects of different size dupli- Laverton Downs Stn., WA 13 cations on fragment patterns. The smallest duplication, Lt, results Leinster Downs Stn., WA 2 in a type 1 change; in others the duplicated sequence is present as Mt Willoughby Stn., SA 5 an additional fragment (type 11 modification). The sample labeled Millbillillie Stn., WA 1 Ls has a 6.1-kb duplication but, because of a site loss, the original Mt Ebenezer Stn., NT 1 1.9- and 6.1-kb fragments have combined to produce an 8.0-kb Munarra Stn., WA 1 fragment. The L7 samples had a 7.2-kb duplication, but have lost Neds Creek Stn., WA 4 0.4 kb from the 6.0-kb fragment, resulting in a net gain of 6.8 kb New Springs Stn., WA 3 (Ds, Table 4). Similarly, the LS sample has an additional 8.8-kb Oakden Hills Stn., SA 2 fragment (comigrating with the largest fragment) which in D2 has Tea Tree, NT 1 been reduced by a 0.38-kb deletion. The designation of length Wirraminna Stn., SA 3 mutations follows Tables 3 and 4. Arrows indicate novel or altered Yellowdine, WA 1 fragments relative to S mtDNA. Yundamindra, WA 2 Yunndaga Stn., WA 5 RESULTS The designation of length variants is described in Tables 3 Size and natureof insertions: Analysis of the frag- and 4. ment patterns producedby digestion with a variety of of mtDNA structure in sexual If. binoei and most other restrictionendonucleases revealed that mtDNA of animals. Heteronotia typically varies between 17.0 and 17.4 kb and that heteroplasmy for minor length variation MATERIALS ANDMETHODS is common (MORITZ 1991; unpublished data). How- Lizards were captured from homestead rubbish tips in ever, someparthenogenetic individuals hadlarger the central and western deserts of Australia (Table 2). Each genomes, with the size of insertions ranging from 1.2 individual was karyotyped from short-term leucocyte cul- to 10.4 kb (Figure2). The total genome size therefore tures (MORITZ 1984) and the preserved specimens were lodged with the University of Michigan Museum of Zoology varies from 17 to 27 kb. and the Queensland Museum. The nature of these insertions was investigated by mtDNA was purified by ultracentrifugation of CsCI-pro- comparing fragment patterns of normal length (S) pidium iodide gradients using a scaleddown method appro- and long (L)genomes using restriction endonucleases priate to the smaller volumes of theBeckman TLS-55 swing for which the cleavage sites in the S genome had been bucket rotor in theTL-100 benchtopultracentrifuge (DOWLING,MORITZ and PALMER1990). mtDNAs were di- mapped. Three types of modification were observed gested with restriction endonucleases following the sup- (Table 3) as in previous studies (MORITZand BROWN plier's instructions, end-labeled with ["PIdNTPs, and elec- 1986, 1987). For some enzymes, one fragment was trophoresed through 1.2% agarose and 3.5% polyacryl- larger in the L genome (type I). For others, there was amide gels (BROWN1980; DOWLINC,MORITZ and PALMER one additional fragment in the L genome (type 11). 1990). Southern transfer-hybridization experiments were done Finally, digestion with some enzymes produced mul- using acid depurination followed by alkali transfer (REED tiple additional fragments in the L genome, all but and MANN 1985) from agarose gels to charged nylon mem- one of which comigrated with fragments also seen in branes (GeneScreen Plus, Du Pont). The filter-immobilized the S genome (type 111). The total size increment for DNA was hybridized sequentially with nick-translated each enzyme was the same within experimental error; mtDNA that had no duplication and with a cloned 1.4-kb fragment of the 12s rRNA from gorilla mtDNA (HIXSON i.e., the length increase for type I changes was the and BROWN1986) at 2 X SSC, 65" for 18 hr with 10% same size as the single novel fragment for type I1 dextran sulfate. changes and as the sum of the additional fragments 224 C. Moritz

TABLE 3 Characteristicsof large mtDNA genomes in parthenogenetic H. binoei

Duplication LI L2 Ls L. Ls h L7 LS L L,o LI I 1.2 2.8 5.8 6.0 6.1 6.0 5.8Size (kb) 2.8 1.2 6.6 6.8" 7.6 8.8 10.4 9.4 Frequency "A" 10 3 04 0 4 0 11 0 2 "B+C"3 0 6 10 7 2 10 0 3 0 mtDNA hap D D I, A M. L J K AN N 0 lotype

Am1 (a) 11.0 I 9.0 5.9 8.8I1 7.7 7.2 Bcll (c) 2.8 1.2 1.54.3, 4.3, 1.5 4.3, 1.5 3.9.2.15 4.3,2.4 4.3.3.7 4.3.4.3 4.3,3.9 0.78 0.78 0.78 0.78 0.78 0.78 0.78 2.8 5.8 5.9 6.1 6.6 7.2 7.6 7.2 6.6 6.1 BdII 5.9 (9) 5.8 I 2.8 8.8 10.5 9.0 EcoO 109 (e) 1.2 I 5.1,1.04.7, 1.0 4.3,1.95 3.9,1.3' 4.3, 2.3 4.3, 4.3,2.0 2.3 4.3,2.0 0.78,0.22 1 .O 1 .o 1.0, 0.822.0, 1.0 1.0.0.82 0.78 0.48 0.78 EcoRV (v)6.1 5.8 7.2 5.1.3.8 5.1.4.3 5.6, 5.1 Hind111 (h) 6.1 I I I I

MluI (m) I1 6.1 6.8 8.8 Ncol (0) 119.4 8.86.1 6.8 NheI (n) 1 I I 1 pvUII (P) I I 6.1 8.8I 7.5 9.5 8.4, 1.15 SPeI(d) 1.53.4,1.5 3.0, 1.51.25 2.6, 1.5, 1.2 4.4.2.5 1.54.4,5.9,1.5 6.1, 1.5 5.6, 1.5 1.3,0.3 1.3, 0.3 1.3, 0.3 1.3,0.3 1.3, 0.3 1.3,3.1, 0.3 1.25, 0.3 2.8 4.6, 1.7 6.8 7.1, 1.7 7.1, 6.8 1.7 SStII4.6, (s) 2.8 XbaI (x) 1.2 I 5.86.1 6.1 6.6 9.0 6.8 8.8 7.6 8.6, 1.2 The number observed for each of the major chromosome types("A" or "B + C")is given. mtDNA haplotypes are defined by the gain and loss of cleavage sites(MORITZ 1991). The modificationsin fragment pattern relative to digests of S genomes are shown against each enzyme. "I" indicates a type I modification. Numbers are the size in kh of additional fragments-those in bold are novel fragments, others comigrate with fragments also present in the S digests. These samples have superimposeda 400-bp deletion on a 7.2-kb duplication (seeD2. Table 4). 'Site gain 1.0 + 0.78 + 0.22. for type 111 modifications. Forexample, the total amount of additional DNA in the LS variant ranged from 5.7 to 5.9 kb (Table 3). However, for the two samples with the LIIvariant, the sum of additional fragments in digests with BcZI and EcoO109 (type I11 changes) were lower than expected (Table 3). Further analysis of these genomes is necessary to characterize and map the length mutations. Someother exceptions are discussed below. These three types of modification are consistent FIGURE3.-Autoradiograms of filter-hybridhation experiments with the presence of direct tandem duplications and where short (S, lane 1) and long (D4,lane 2; Ls, lane 3) mtDNAs were hybridized with S mtDNA (A) and with 12s rDNA sequences are inconsistent with inversions or inverted repeats from primate mtDNA (B) after digestion with four restriction (see DOWLING,MORITZ and PALMER1990). Under endonucleases. The arrowheads indicate the position of duplication the duplication model, type I modifications are pre- bearing fragments. dicted for enzymes that have no site in the duplicated exogenous mtDNA sequencewould mimic the regular sequence, type I for enzymes with one site duplicated, fragment changes expected for a duplication. and type I11 for enzymes with multiple sites within the Location and gene content: The location of the duplication. duplications was determined by comparing the frag- The possibility that the additional DNA is exoge- ment changesfor each enzyme to thosepredicted nous was tested by probing digests of S and L genomes from the location of cleavage sites in the S genome with S mtDNA (Figure 3). The additional fragments map (Figure 4A). For example, L2 (Table 3) is a 2.8- present in type I1 and I11 digests of L genomes hy- kb duplication that spans the 1.5-kb SpeI fragment bridized with the S probe,confirming their origin (type I11 modification) and includes single sites for from mtDNA. It is also extremely unlikely that an BgZII, BcZl and SstII (type I1 changes). This duplication Evolution of mtDNA Duplications 225

A. DUPLICATIONS

L1 1.2kb "* L2 2.8kb ...-... FIGURE4.-(A) Size and location La 5.8kb ...... " ...... of mtDNA duplications in relation to Le &lkb ...... the cleavage map for S mtDNA from Le &6kb ...... the parthenogens and to part of the L, 7.2kb genetic map for vertebrate mtDNA. The cross-hatched bar indicates the La 7.6kb ...... region definitely included within the L, 6.0kb duplication and the adjacent dots in- Lm OAkb dicate the maximum extension in Lo 8.8kb either direction. The sawtooth in the map indicates the position of minor high frequency length variation. The predicted gene content of the region covered by the duplications is shown below the cleavage map, ND = GENE CONTENT NO2 ND1 res 12s CR Cyt bND6 NDS ND4 1-2 ..... -4 NADH dehydrogenase, CR = control region, Cytb = cytochrome b, 16s and 12s are the large and small ribosomal RNAs; respectively. Enzyme abbrevi- ations and duplication designations follow Table 3. (B) Location of deletions in relation to the restriction map of the region duplicated in the L.9 ..E...... -.. variant. Da OAkb D~ o.3w 0, 2;5kb .....a ...... D~ 0.2~ excludes sites for Ec00109 and NcoI (type I changes). included in the various duplications can be inferred These data localize the L2 duplication to the region on the assumption that the size and order of mtDNA shown in Figure 4A. The survey of all mtDNAs re- genes is the same in the geckos as in frogs, mammals vealed 11 different duplications that varied in sizeand (reviewed by BROWN1985), whiptail lizards (D.STAN- location (Table 3, Figure 4A). Only one type of du- TON, C. MORITZand W. M. BROWN, unpublished data) plication was observed in anyone individual and there and fish (JOHANSEN, GUDDALand JOHANSEN 1990). was no evidence for multiple overlapping duplications. All but the smallest of the duplications (LI)border on Comparison of the ten fully characterized duplica- or span the control region. Downstream from the tions (Figure 4A) revealsthree notable features. First, control region, most duplications appear to include the duplications are clustered in a single region of the both ribosomal genes and all or most of NDI. The genome. Second, most of the left-hand boundaries of gene content upstream is less certain because of the the duplications are located in a small region (0.3 kb) unknown size of the control region, but the larger between a PvuII and a BcZl site, whereas the right duplications may extend as far as the 3' end of ND5 hand boundaries appear tobe more widely dispersed. (Figure 4A). Third, the region included in each of the smaller Modifications of duplicate sequences: The frag- duplications is encompassed by the location of larger ment comparisons provided evidence for both dele- duplications. Only the two smallest duplications, L1 tions and changes in restriction sites within the dupli- and Lp, do not overlap in content. cated sequences. The presence of a deletion within These duplications span between 7 and 61% of the one copy of the duplication was suggested by (1) a S mtDNA. Giventhe tight packing of genes in animal consistent reduction in size of novel fragments or of mtDNA, it seems probable that eachmust include fragments also present in the S genome, and (2) by sequences for one or more genes. The presence of the the elimination of some cleavage sites witha concom- highly conserved SstII sites within several of the du- itant reduction in the size of the new combined frag- plications (Table 3) suggests that these include rRNA ment. Four deletions ranging in size from 0.38 to 2.5 gene sequences. This was confirmed for the L9 dupli- kb were characterized in detail (Table 4). Three of cation by the hybridization of a mitochondrial 12s these, Dl, 02and Dq, occurred in genomes with the rDNA probe to novel fragments in the L genome LS duplication. The Dsdeletion appears to have oc- (Figure 3B). On the basis of their location, it appears curred in one copy of a 7.2-kb duplication, resulting that all ofthe duplications include at least somerDNA in a net gain of 6.8 kb (6L7 in Table 3). sequences (Figure 4A). The identity of the othergenes The locationof these deletions was determined 226 C. Moritz

TABLE 4 Alterations in fragment patterns used todefine deletions within duplicated sequences

Length variant

Duplication Ls Deletions

Enzyme +8.8 D,-0.19 De -0.38 D, -0.40 04-2.5

AvaI 8.8 8.8 + 8.7 8.87.4 + 8.5 9.5+ 7.2 + 7.2 BglII 8.8 8.8 + 8.6 8.86.1 --f 8.4 9.2+ 5.6 + 6.8 Bcll 0.783.7 4.3 3.7 + 2.4 4.3 ”-* 3.9 7.5 + 5.3 EcoO 109 0.782.02.0 4.3 + 1.81 2.0 + 1.624.3 + 3.9 1.0 0.82 EcoRV 3.8 5.1 5.1 + 4.7 8.4 + 8.0 (3.4 + 5.1) + 6.1 NcoI 8.8 9.6 + 7.3 PvulI 8.8 8.8 + 8.2 + 14.8 SstII 7.1 1.7 (1.7 + 5.5) + 6.811.0 + 8.5 Spe I 5.6 1.6 1.3 5.6 + 5.1(1.6 + 1.3)-+2.5 0.3 Xba I 8.8 13.0 + 10.5 The additional fragments for the LS duplication are listed for comparison. Numbers in bold are novel fragments altered by deletion. from patterns of overlap in the affected fragmentsor mosome types of parthenogens (Figure l), but were restriction sites (Figure 4B). The 0.4-kb deletion in- significantly more common in the “B + C” group than cludes the SstII site located in the 16s rRNA gene. in the “A” group (x2= 29.7, P < 0.001). In contrast, The location and gene contentof the 0.19-, 0.38- and mtDNA duplications were absent among53 surveyed 2.5-kb deletions is less well defined, but they appear representatives of the sexual race that provided the to be involve sequences in the ND5-ND6 region. The mtDNA in the parthenogens, i.e., the CA6 race (Fig- location of the 2.5-kb D4 deletion in the right hand ure 1) (see MORITZ199 1). This difference in incidence copy of the Lg duplication was confirmed by the hy- is highly significant (x2= 58.6, d.f. = 1, P < 0.001). bridization experiments (Figure 3). Had the deleted A more detailed study of the phylogenetic distri- sites for EcoRV and PuuII beenin the left hand copy, bution of length variants was done using the results then the largerof the AuaI, BgZII and NcoI fragments of a survey of the same mtDNAs with a suite of 4-bp- that hybridize with the rDNA probewould have been recognizing restriction endonucleases(MORITZ 1991). reduced in size. These were unaffected (Figure 3B). This complementarystudy, which specifically ex- In contrast tothe deletions, gains and lossesof cluded fragment changesdue to the lengthmutations, restriction sites were enzyme specific (Table 3). One revealed 15 different haplotypes among the parthen- of the four samples with the D2 variant had gainedan ogens. These haplotypes differed by between one and EcoO109 site in one copy of the duplication (4.3 kb ninerestriction sites andthree, a, d and n, were 44.1 + 0.16). Similarly, both L6 samples had an extra common and present in both major chromosometypes EcoO109 site in one copy (1.0 kb + 0.78 + 0.22). of triploid. The relationships among the various hap- The LI1samples had modifications for SpeI and XbaI lotypes was estimated by arranging them into a mini- suggestive of site gains (Table 3), but this interpreta- mum length network (Figure 5). Placing the length tion is tentative until other ambiguities for these sam- variants onto this network reveals that samples with ples are resolved. and without duplicationsare scattered throughout the A notable feature of both types of modification is network. Among the individuals with two of the com- that the changes were restricted to one or other copy mon mtDNA haplotypes, a and d, some lacked dupli- of theduplicated sequence. In any one individual cations, whereas others had a range of duplication deletions were restricted to one copy of the duplica- sizes. The othercommon haplotype, n, was character- tion, but across all the individuals assayed, deletions ized by the Lg variant and deletions derived from it were observed in both the upstream and downstream (Dl, D2 and D4). Each specific length variant is local- copies. Restriction site changes werealso only present ized within the mtDNA phylogeny, suggesting that in one or other copy. Changes in both copies would each has a separate origin, either by deletion from a have been obvious in comparison to theS genome but larger duplication, or by de novo duplication from an were not seen. S genome. Phylogenetic and geographic distribution:Of the Heteroplasmy for the presence of duplications (ie., 87 parthenogens surveyed, 57 had duplications. Du- S and L genomes in the same individual) was rare. plications occurred in mtDNA from both major chro- Only four of the 87 samples appeared to be hetero- Evolution of mtDNA Duplications 227

S f-6 S 02 I i 0 S -0.38

0 # Lll S L7

FIGURE5.-Minimum length network of mtDNA from parthenogenetic H. binoei based on the gain and loss of cleavage sites for 4-bp recognizing endonucleases independent of length changes (details in MORITZ 1991). Each node is identified by haplotype (lower case letter) and length mutation(s) (as in Tables 3 and 4). The L, variant was not analyzed in sufflcient detail to be included here. Boxed changes on the branches indicate the inferred location of some duplication or deletion events. Narrow bars indicate changes in cleavage sites. plasmic for S and L genomes. Also, aside from the GARDINER1989). In contrast, the mtDNA duplica- ubiquitous minor length variation, only one size of tions in Cnemidophorus (MORITZ and BROWN 1987) duplication was seen in any one individual. and Heteronotia have no obvious phenotypic effect. Parthenogens from both central and western Aus- The localization of duplications to this part of the tralia had mtDNA duplications (Figure 6). However, genome may reflect a bias in the mutation process. there is a striking geographic pattern in that individ- MORITZ and BROWN(1987) noted that this region uals from westernAustralia tended to havesmall remains single stranded for a lengthy period during duplications or to lack them altogether, whereas the the highlyasymmetric replication process and that central Australian parthenogens all had large dupli- most ofthe rearrangementboundaries coincided with cations. Each variant had a well defined geographic the predicted location oftRNA genes. They suggested distribution. At one extreme, theLS variant was wide- that the secondary structure of the tRNAs may act as spread in central Australia (Table 2, Figure 6). At the a signal for the duplication process, perhaps through other, several variants(e.g., L1 and L4) were onlyfound illegitimate initiation of replication (see alsoJACOBS et in one place. Three of the deletion variantswere al. 1989). In Heteronotia, the left boundary of many found in central Australia and the fourth was from duplications occurs in the same small region, but the the southwestern limit of the distribution (Figure 6). gene content of the boundaries is not certain. Further information on theduplication process in Heteronotia DISCUSSION mtDNA will be gained through sequencing studies now in progress. The presence in Heteronotia of mtDNAs with a The origin duplications: The mtDNA duplica- range of duplication sizes and of identical genomes of without duplications offers an unusual opportunity to tions are present in some, but not all, parthenogens investigate the evolutionary origins and consequences and are absent from their sexual relatives (Figure 1). of excess, presumablyredundant, coding sequences in This provides strong evidence that the duplications an otherwise economical genome. A novel feature of arose in the parthenogenetic lineages, rather than in this study is that previous investigations on the evo- their sexual ancestors. Further, the dispersion of the lution of the parthenogenetic form provide a historical L and S genomes throughout therestriction-site based framework for interpreting thechanges in mtDNA. mtDNA network and the presence of both S and L Gene content and location of the duplications: genomes within particular haplotypessuggests that Except for the smallest variant (L1),all of the dupli- duplications have arisen repeatedly within the par- cations border on or include the control region. The thenogens. general location and content of the duplications is It is not clear whether these events occurred in the therefore similar to that reported forcoding sequence triploid lineages or in their hypotheticaldiploid- duplications in other animal mtDNAs (Table l), es- hybrid parthenogenetic precursors (Figure 1). The pecially those in Cnemidophorus lizards (MORITZ and presence ofsome variants (e.g., L,) in both major BROWN1987). In all, these duplications span an ap- chromosome types of triploid suggests that they arose proximately 1 1-kb region bounded on one side by the in the common diploid ancestor. However, the differ- origin of light strand replication and on the otherby ence in the incidence of duplications in the “A” vs. the ND4 sequence. The only duplication so far re- “B + C” chromosome types and the stronggeographic ported toinclude the origin of light strand replication patterns suggest that at least some of the duplications is heteroplasmic and is associated with mitochondrial arose in the triploid lineages. dysfunction in humans (POULTON,DEADMAN and The association between parthenogenesis and du- 228 C. Moritz

FIGURE6.-Map of southwestern Australia showing the geographic distribution of mtDNA length variants. mtDNAs without duplications are in- dicated as open boxes, duplications by filled boxes and the numbers 1 to 11 (L1 to LII) and deletions by circled numbers 1 to 4 (Dl to D4).

plication in H. binoei may be related to the fundamen- Evolutionary dynamics of duplicated sequences: tal changes in nucleargenes that accompany the Parthenogenetic Heteronotia have very little restric- change from sexual to parthenogenetic reproduction. tion site variation other than that due to the length In particular, the parthenogens are polyploid and mutations, suggesting that they arose recently, prob- have non-recombining hybrid genomes. In Cnemido- ablywithin the pastfew thousand years (MORITZ phorus, mtDNA duplications werefound in both sex- 1991). This means that the duplications, which are ual and parthenogenetic lizards, but were significantly only found in the parthenogens, must also be recent. more common in polyploids than in diploids (MORITZ It follows that the modificationof the duplicated and BROWN1987). In that study, there was no signif- sequences by deletion and restriction site change has icant difference in the incidence of duplications in been rapid. Similarly, HYMANand SLATER(1990) hybrid us. nonhybrid nuclear backgrounds. The ap- reported a deletion within one copy of a duplicated parent predisposition of parthenogenetic lizards to sequence that had arisen within 170 generations of a mtDNA duplication is reinforced by the presence of nematode lineage. These observations contrast a duplication in one population of another triploid strongly with the usual structural stability of animal parthenogenetic species, Hemidactylusgarnotii (C. mtDNA. MORITZ, unpublished data). The small size of animal mtDNAis often attributed The mechanism of duplication of mtDNA coding to strong selection against the accumulation of DNA sequences is not known. The association between fun- (WALLACE1982; SEDEROFF1984; ATTARDI 1985; damental changes in the nuclear genome of lizards CLARK-WALKER1985), perhaps because smaller mol- and duplications suggests that the nuclear control of ecules can replicate more efficiently (WALLACE1982; mtDNA replication has been perturbed. The replica- RAND andHARRISON 1986; WALLIS1987). The evi- tion and transcription of mtDNA depends onthe dence for Heteronotia is consistent with a strong bias import of nuclearly encoded enzymes, transcription toward smaller genome size. Although genome size factors and RNA moeties (CLAYTON1982, 1984; can be increased by 6 1% without obvious phenotypic CHANGand CLAYTON1987). That the nuclear geno- effects (but see POULTON,DEADMAN and GARDINER type can have a direct effect on mtDNA structure is 1989), thephylogenetic analysis suggeststhat the du- evident from the observation that in humans, mito- plicated sequences are prone to rapid deletion. This chondrial myopathy, which involveslarge deletions of was mostobvious forthe L9 variant where three mtDNA, is inherited as an autosomal dominant trait different deletions were found in one or other copy (ZEVIANI et al. 1989). Itis conceivable that themtDNA of the 8.8-kb duplication. It is also likely that some of duplications in these lizards result from changes in the smaller duplications are derived from larger ones cell cycle time or gene expression that may be associ- by deletion of sequences from the internal junction; ated with the transition from diploidy to triploidy or such changes are indistinguishable from de novo du- from some effect of hybridity.This hypothesis can, in plication using restriction fragment analysis. The ob- principle, be tested by manipulating cell lines. servation thatthe additional sequences are rapidly EvolutionDuplications of mtDNA 229 reduced by deletion is consistent with their restricted and restriction site heteroplasmy in the mitochondrial DNA of phylogenetic distribution (MORITZand BROWN1987; American shad (Alosa sapidissima).Genetics 118: 509-5 18. BIRMINGHAM,E., T. LAMBand J. C. AVISE,1986 Size polymor- MORITZ, DOWLINGand BROWN1987) and provides phism and heteroplasmy in the mitochondrial DNA of lower further evidence that large coding sequence duplica- vertebrates. Heredity 77: 249-252. tions are ephemeral. BOYCE, T. M., M. E. ZWICK and C. F. AQUADRO, Eachof the modificationsof the duplicated se- 1989 Mitochondrial DNA in the bark weevils: size, structure quences encountered in this survey was restricted to and heteroplasmy. Genetics 123: 825-836. BROWN,W. M., 1980 Polymorphism in mitochondrial DNAof one copy of the duplication. The evolutionary inde- humans as revealed by restriction endonuclease analysis. Proc. pendence of each copy of a duplicated sequence was Natl. Acad. Sci. USA 77: 3605-3609. also observed for Cnemidophorus mtDNAs (MORITZ BROWN,W. M., 1985 The mitochondrial genome of animals, pp. and BROWN1987). This differs from the situation for 95-130 in MolecularEvotutionary Genetics, edited byR. J. the phylogenetically conserved inverted repeat of MACINTYRE.Plenum Press, New York. BUROKER,N. E., J. R. BROWN,T. A. GILBERT,P. J. O’HARA,A. T. land-plant chloroplast DNA where copy-correction is BECKENBACH,W. K. THOMASand M. J. SMITH,1990 Length virtually instantaneous (reviewed by PALMER1985). heteroplasmy of sturgeon mitochondrial DNA: an illegitimate It also differs from the situation for non-coding re- elongation model. Genetics 124: 157-163. peats in animal mtDNA which appear to show con- CANTATORE, P.,M. N. GADALETA,M. ROBERTI,C. SACCONEand certed evolution (SOLIGNAC,MONNEROT and MOUN- A. C. WILSON,1987 Duplication and remoulding of tRNA genes during theevolutionary rearrangement of mitochondrial OLOU 1986; BUROKERet al. 1990). The apparent genomes. Nature 329 853-855. homogenization of these smaller noncoding repeats CHANG,D. D., and D. A. CLAYTON,1987 A mitochondrial RNA may be due to their high frequency of deletion and processing activity contains nuclearly encoded RNA. Science reduplication. This process would not operate in the 235: 1178-1 184. large coding sequence duplications because they are CLARK-WALKER,G. D., 1985 Basis of diversity in mitochondrial DNAs, pp. 277-297 in The Evolution of Genome Size, edited by generated less often and because only one additional T. CAVALIERSMITH. John Wiley & Sons, New York. copy is present. CLARK-WALKER,G. D., 1989 In vivo rearrangement of mitochon- The tight packing ofgenes in most animals mtDNAs drial DNAin Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. may usually preclude structural rearrangements, ef- USA 86 8847-8851. fectively freezing the genome structure (WALLACE CLAYTON,D. A,, 1982 Replication of animal mitochondrial DNA. Cell 28: 693-705. 1982; SEDEROFF1984; ATTARDI 1985; CLARK- CLAYTON,D. A,,1984 Transcription of the mammalian mito- WALKER1985; BROWN1985). The presence of redun- chondrial genome. Annu. Rev. Biochem. 53: 573-594. dant sequences in Heteronotia mtDNA should relax DENSMORE,L. D., J. WRIGHTand W. M. BROWN,1985 Length selection againstrearrangements and, with the appro- variation and heteroplasmy are frequent in mitochondrial DNA priate deletions, might evenchange gene order (MOR- from parthenogenetic and bisexual lizards (genus Cnemidopho- rus). Genetics 110 689-707. ITZ, DOWLINCand BROWN 1987; CLARK-WALKER DENSMORE,L. D., C. MORITZ,J. WRIGHTand W. M. BROWN, 1989; DFSJARDINSand MORAIS 1990). In the absence 1989 Mitochondrial DNA analysesand theorigin and relative of redundant sequences, deletions of coding sequences age of parthenogenetic lizards (genus Cnemidophorus). IV. Nine are only tolerated in the heteroplasmic state and even sexlineatus group unisexuals. Evolution 43: 969-983. then can be deleterious (reviewed by WALLACE1989). DESJARDINS,P., and R.MORAIS, 1990 Sequence and gene organization of the chicken mitochondrial genome. J. Mol. Biol. Several Heteronotia with duplicate sequenceswere 212: 599-634. homoplasmic for deletions in one or other copy (but DOWLING,T. E., C. MORITZ and J. PALMER,1990 Nucleic acids never both), confirming that there is redundancy of 11: restriction site analysis, pp. 251-317, in Molecular System- coding sequences. However, the survey of 87 mtDNAs atics, edited by D. M. HILLISand C. MORITZ. Sinauer, Sunder- did not reveal any inversions or transpositions. This land, Mass. DUBIN, D. T., C. HSU-CHEN and L. E. TILLOTSEN, suggests that even in the absence of constraints im- 1986 Mosquito mitochondrial transfer tRNAs for valine, gly- posed by tightly packed genes, large inversions and cine and glutamate: RNA and gene sequences and vicinal transpositions occur less often than deletions and the genome organization. Curr. Genet. 10 701-707. gain or loss of cleavage sites. CAREY,J. R., and D.R. WOLSTENHOLME, 1989 Platyhelminth mitochondrial DNA: evidence for early evolutionary origin of Thanks to S. LAVERY,A. HEIDEMANand E. ZEVERING for assist- a tRNA”‘ AGN that contains dihydrouridine arm replacement ance in the laboratory, to s. BALDAUF,D. CLARK-WALKER,R. SLADE loop, and of serine-specifying AGA and AGG codons. J. Mol. and C. E. ZEVERINCfor comments on the manuscript, and to the Evol. 28: 374-387. Australian Research Council, the National Science Foundation, the HALE,L. R., and R. S. SINGH,1986 Extensive variation and National Geographic Society (United States), 2nd the University of heteroplasmy in size of mitochondrial DNA among geographic Queensland for support. populations of Drosophila melanoguster. Proc. Natl. Acad. Sci. USA 83: 8813-8817. LITERATURECITED HAUCKE,H.-R., and G. GELLISSEN,1988 Diffferent mitochondrial gene orders among insects: exchanged tRNA gene positions in ATTARDI,G., 1985 Animal mitochondrial DNA: an extreme of the COlI/COIIl region between an orthopteran anda dipteran genetic economy. Int. Rev. Cytol. 93: 93-145. species. Curr. Genet. 14 471-476. BENTZEN,P., W.C. LEGCETT and G. G. BROWN,1988 Length HIXSON,J. E., and W. M. BROWN,1986 A comparison of the 230 C. Moritz

small ribosomal RNA genes from the mitochondrial DNA of DNA in plants and algae, pp. 131-240 in Molecular Evolution- great apes and humans: sequence, structure, and phylogenetic ary Genetics, edited by R. MACINTYRE.Plenum Press, New implications. Mol. Biol. Evol. 3: 1-18. York. HYMAN,B. C., and T. M. SLATER,1990 Recent appearance and POULTON,J., M. E. DEADMANand R. M. GARDINER,1989 Tandem molecular characterization of mitochondrial DNA deletions direct duplication of mitochondrial DNAin mitochondrial within a defined nematode pedigree. Genetics 124: 845-853. myopathy: analysis of nucleotide sequence and tissue distribu- HYMAN,B. C., J. L. BECK and K. C. WEISS, 1988 Sequence tion. Nucleic Acids Res. 17: 10223-10229. amplification and gene rearrangement in parasitic nematode RAND,D. M., and R. G. HARRISON,1986 Mitochondrial DNA mitochondrial DNA. Genetics 120 707-712. transmission genetics in crickets. Genetics 114 955-970. .JACOBS,H. T., S. ASAKAWA,T. ARAKI,K. MIURA,M. J. SMITHand RAND,D. M., and R. G. HARRISON,1989 Molecular population K. WATANABE,1989 Conserved tRNA cluster in starfish mi- genetics of mtDNA size variation in crickets. Genetics 121: tochondrial DNA. Curr. Genet. 15: 193-206. 551-569. JOHANSEN, S., P. H. GUDDAL and T. JOHANSEN, REED,K. C., and D. A. MANN,1985 Rapid transfer of DNA from 1990 Organization of the mitochondrial genome of Atlantic agarose to nylon membranes. NucleicAcids Res. 13: 7207- cod, Gadus morhua. Nucleic Acids Res. 18: 41 1-419. 7221. LA ROCHE,J., M. SNYDER,D. I. COOK,K. FULLERand E. ZOUROS, SEDEROFF,R. R., 1984 Structural variation in mitochondrial 1990 Molecular characterization of a repeat element causing DNA. Adv. Genet. 22: 1-108. large-scale size variation in the mitochondrial DNA of the sea SNYDER,M., A. R. FRASER, J. LA ROCHE,K. E. GARTNER-KEPKAY scallop Placopecten mugellanicus. Mol. Biol. Evol. 7: 45-64. and E. ZOUR~S,1987 Atypical mitochondrial DNA from MORITZ, C., 1983 Parthenogenesis in the endemic Australian deep-sea scallopPlacopecten mugellanicus. Proc. Natl. Acad. Sci. lizard Heteronotia binoei (Gekkonidae). Science 220: 735-737. USA 84: 7595-7599. MORITZ,C., 1984 The origin and evolution of parthenogenesis SOLIGNAC, M., M. MONNEROT and J.-C. MOUNOLOU, 1986 Concerted evolution of sequence repeats in Drosophila in Heteronotia binoei (Gekkonidae). Chromosoma 89 15 1-162. mitochondrial DNA. J. Mol. Evol. 24: 53-60. MORITZ, C., 1991 The origin and evolution of parthenogenesis SOLIGNAC,M., J. GENERMONT,M. MONNEROTand J.-C. MOUNOLOU, in Heteronotia binoei (Gekkonidae): evidence for recentand 1987 Drosophila mitochondrial genetics: evolution of heter- restricted origins of widespread clones. Genetics 129 2 1 1-2 19. oplasmy through germ line cell divisions. Genetics 117: 687- MORITZ, C., and W. BROWN,1986 Tandem duplication of D- M. 696. loop and ribosomal RNA sequences in lizard mitochondrial WALLACE,D. C., 1982 Structure and evolution of organelle ge- DNA. Science 233: 1425-1427. nomes. Microbiol. Rev. 46 208-240. MORITZ,C., and W.M. BROWN,1987 Tandem duplications in WALLACE,D. C., 1989 Mitochondrial DNA mutations and neu- animal mitochondrial DNAs: variation in incidence and gene romuscular disease. Trends Genet. 5: 9-13. content among lizards. Proc. Natl. Acad. Sci. USA 84: 7183- WALLIS,G. P., 1987 Mitochondrial DNA insertion polymorphism 7187. and germ line heteroplasmy in the Triturus cristatus complex. MORITZ,C., T. E. DOWLINGand W. M. BROWN,1987 Evolution Heredity 58229-238. of animal mitochondrial DNA: relevance for population biol- WOLSTENHOLME,D. R., D. 0. CLARY,J. L. MACFARLANE,J. A. ogy and systematics. Annu. Rev. Ecol. Syst. 18: 269-292. WAHLEITHNERand L. WILCOX,1985 Organization and evo- MORITZ, C., J. W. WRIGHT and W. M. BROWN, lution of invertebrate mitochondrial genomes, pp 61-69 in 1989 Mitochondrial DNA and the origin and relative age of Achievements and Perspective in Mitochondriat Research, Vol. 11: parthenogenetic lizards (genus Cnemidophorus). 111. C. uelox Biogenesis, edited by E. QUAGLIARELLIO,E. C. SLATER,F. and C. exsunpis. Evolution 43: 958-968. PALMIERI,C. SACCONE,et al. Elsevier, New York. MORITZ,C., S. DONNELLAN,M. ADAMSand P. R. BAVERSTOCK, ZEVIANI,M., S. SERVIDEI,C. GELLERA,E. BERTINI,S. DIMAUROand 1989 The origin and evolution of parthenogenesis in Heter- S. DIDONATO,1989 An autosomal dominantdisorder with onotia binoei (Gekkonidae): extensive genotypic diversity among multiple deletions of mitochondrial DNA starting at theD-loop parthenogens. Evolution 43: 994-1 003. region. Nature 339: 309-31 1. PALMER,J. D., 1985 Evolution of chloroplast and mitochondrial Communicating editor: M. J. SIMMONS