Copyright 0 1997 by the Genetics Society of America Evolution of Repeated Sequence Arrays in the D-Loop Region of Bat Mitochondrial DNA Gerald S. Wilkinson,* Frieder Mayer; Gerald Ked*and Barbara Petri5 *Department of Zoology, University of Maryland, College Park, Maryland, tInstitut fur Zoologie II, Universitat Erlangen-Nurnberg, Erlangen, Germany, Theodor-Bovmi-Institut, UniversitatWurzburg, Wurzburg, Germany and Zoologisches Institut, Universitat Munich, Munich, Germany Manuscript received September 1, 1996 Accepted for publication March 17, 1997 ABSTRACT Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near thetRNA-Pro gene are present allin vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from15% to 63%. Fewer repeats among heteroplasmic than homoplasmic individuals in a species with up to nine repeats indicates selection may act against long arrays. A lower limit of two repeats and morerepeats among heteroplasmic than homoplasmic individuals in two species with few repeats suggests length mutations are biased. Significant regressions of heteroplasmy, 0 and n, on repeat number furthersuggest that repeatduplication rate increases with repeat number.Comparison of vespertilionine bat consensus repeats to mammal control region sequences revealed that tandem repeats of similar size, sequence and numberalso occur inshrews, cats and bighorn sheep. Thepresence of two conserved protein-binding sequences in all repeat units indicates that convergent evolution has occurred by duplication of functional units.We speculate that D-loop region tandem repeats may provide signal redundancy anda primitive repair mechanism in the event of somatic mutations to these binding sites. NIMAL mitochondria contain circulara l6kb DNA two sites in the central,conserved portion of the control A molecule, encoding 13 protein, 22 transfer RNA region (CHANGet al. 1985; KING and LOW 1987; CLAY- (tRNA) and two ribosomal RNA genes (ANDERSON et TON 1992). Each strand of the mtDNA molecule, re- al. 1981). The small size and compact organization of ferred to as heavy (H) and light (L) based on differ- the mitochondrial DNA (mtDNA) genome has been ences in base composition, has different promoter re- suggested to be the result of selection for rapid organ- gions that bind nuclearcoded proteins (GHMZZANIet elle replication (HARRISON 1989; RAND 1993).However, al. 1993b; NASS 1995) and that differ in nucleotide se- recent discovery of length variation in the noncoding quence between species (KING and LOW1987). Replica- control region, which lies between the tRNA-Pro and tion of the H-strand is primed by RNA transcribed be- tRNA-Phe genes, in a varietyof vertebrate species tween the L-strand promoter (LSP) and the H-strand (DENSMOREet al. 1985; MORITZand BROWN1987; BURO- origin of replication (OH,CHANG and CLAWON 1985). KER et al. 1990; HAYASAKA et al. 1991; WILKINSONand H-strand replication is usually terminated shortly there- CHAPMAN1991; ARNMON and RAND 1992; BROWNet al. after at termination-associated sequences (TAS) re- 1992, 1996; HOELZELet al. 1993,1994; STEWART and sulting in short 7s DNA strands (DODAet al. 1981; CLAY- BAKER 1994; xu and hMON1994; YANG et al. 1994; TON 1991). 7s DNA strands remain associated with the CECCONIet al. 1995; PETRIet al. 1996; FUMAGALLIet al. L-strand and displace the original H-strand to create a 1996) is not consistent with this hypothesis and has not three-stranded structure known as the displacement or yet been adequately explained (WOLSTENHOLME1992; D-loop. In mice, only 5% of replication events continue RAND 1993). Because the proteins encoded by mtDNA beyond the control repon (BOGENHAGENand CLAWON genes play critical roles in oxidative metabolism and 1978). Sequence-specific DNA binding proteins inter- control region length might influencethe rate of act with TAS elements (MADSEN et al. 1993b) between mtDNA transcription or replication (ANNEX and WIL two conserved regions, mt 5 (OHNOet al. 1991), which LJMS 1990), the metabolic rate of the organism and is also referred to as region J (KING and LOW 1987), possibly its survival could be affected by length varia- and mt 6 (KUMAR et al. 1995). Initiation of replication tion. of the L-strand occurs only when H-strand replication Transcription of mitochondrial genes is initiated at is two-thirds complete and the conserved 012sequence, which in mammals lies between tRNA-Cy s and tRNA- Corresponding author: Gerald S. Wilkinson, Department of Zoology, University of Maryland, College Park, MD 20742. Asn, is exposed (CLAYTON1982). E-mail: [email protected] While the function of the D-loop is not well under- Genetics 146: 1035-1048 (July, 1997) 1036 G. S. Wilkinson et al. stood (WOLSTENHOLME1992), its structure and size are plasmy among seven species of vespertilionine bats in likely to influence mtDNA replication. A high propor- order to identify evolutionary processes that influence tion of triplex to duplex forms correlates with mtDNA repeat array size. If the mutational process that gives copy number, mtRNA abundance and therate of oxida- rise to heteroplasmy is unbiased, we would expect ho- tive metabolism in different tissues (ANNEX and WIL- moplasmic and heteroplasmic individuals to have equal LIAMS 1990). The length of the 7s DNA strand, and numbers of R1 repeats (BROWNet al. 1996). Deviations therefore the size of the D-loop, varies depending on from this expectation indicate mutational bias or selec- which TAS site is used for termination (DODA et al. tion. Similarly, the proportion of heteroplasmic individ- 1981). Consequently, tandem repeats containing TAS uals is expected tobe determined by a balance between elements should alter D-loop size. TAS elements occur mutation and organelle segregation (CWUC1988; BIRKY within tandem repeats of evening bats (WILKINSONand et al. 1989) since paternal transmission is rare (HAR- CHAPMAN1991), shrews (STEWARTand BAKER1994; Fu- MSON 1989; SKIBINSKIet al. 1994). Thus, variation in MAGALLI et al. 1996),bighorn sheep (ZARDOYA et al. heteroplasmy should reflect variation in repeat muta- 1995), treefrogs FANGet al. 1994), minnows (BROUCH- tion rate if the number of organelles per cell does not TON and DOWLING1994), sturgeon(BROWN et al. 1996), vary. Finally, we compare consensus sequences from cod (ARNASON and RAND 1992; LEE et al. 1995), and vespertilionine bats with repeats to control region se- seabass (CECCONIet al. 1995). Despite the distant taxo- quences of other mammals with and without repeats to nomic affiliations among these species, in most cases determine if R1 repeated arrays have evolved multiple these R1 repeats (FUMAGALLIet al. 1996), Figure 1) are times in mammals and might influence organism func- -80 bp in length. In some fish and frogs the 80-bp tion. repeat contains two or more smaller units. In several species, R1 repeats have been predicted to form ther- MATERIALSAND METHODS modynamically stable secondary structures (BUROKERet al. 1990; WILKINSONand CHAPMAN1991; STEWARTand Sampling locations: Bats were captured by netting atroost- ing and foraging sites in Europe, Malaysia, United States, BAKER1994; FUMAGALLIet al. 1996). R1 repeat duplica- Central America, South America, and Africa. Nycticeius humer- tions and deletions are thoughtto occur by competitive alis were captured at six attic nursery colonies in Missouri strand displacement among the three strands of the D- and one in North Carolina (WILKINSONand CHAPMAN1991). loop (BUROKERet al. 1990) resulting in a unidirectional Eptesicus fuscus and Myotis lucifugus were captured in a single mutational process (WILKINSONand CHAPMAN1991). barn near the town of Princeton, Missouri. Leptonycteris cura- soae and L. nivalis were captured in day roosts in Mexico; Short, tandem repeats on the opposite side of the Glossophaga soricinawas netted in Guanacaste, Costa Rica (WIL- central conserved portion of the control region have KINSON and FLEMING1996). Four species were captured in also been reported in a variety of mammals including the Transvaal, South Africa: Epomophorus cvpturus and N. several carnivores (HOELZELet al. 1994), pinnipeds(AR- schle@nii were netted over streams near the town of Skukuzu in Kruger National Park while Nycteris thebaica and Rhinolophus NASON et al. 1993; HOELZELet al. 1993), pigs (GHMZ- clivosus were captured in a mine tunnel just southof Kruger ZANI et al. 1993a), horses (ISHIDAet al. 1994; XU and National Park. Four species were also captured from a cave ARNASON 1994), rabbits (MIGNOTTE et al. 1990; BIJU- on Tamana Hill in Trinidad, West Indies: Pteronotus parnelli, DWAL et al. 1991),shrews (FUMAGALLIet al. 1996), mar- Momoops megalophylla, Natalustumidirostris and Phyllostomus supials UANKE et al. 1994) and bats (PETRIet al. 1996; hastatus. Saccoptqx bilineata were captured at La Selva, Costa E. PETIT,personal communication). These R2 repeats Rica. Sixspecies were captured in peninsular Malaysia: Hippos- ideros diadema, R afJinis, R. sedulus, Murina suilla, Nyctophilus (FUMAGALLIet al. 1996) typically involve variable num- gouldii and Keriwoula papillosa. Seven species were collected in bers of short 6 to 30-bp units, which often contain the Germany: Nyctalus noctula in Brandenburg and Bavaria, and 4bp motif GTAC, and exhibit length variation similar E. nilssoni, M. myotis, M. bechsteini, Pipistrellus Pipistrellus, P. to that described for nuclear microsatellite