Evolution of Repeated Sequence Arrays in the D - Loop Region of Bat Mitochondrial DNA Gerald

Analysis of mitochondrial DNA control region sequences from 41 species of bats representing 11 families revealed that repeated sequence arrays near the tRNA-Pro gene are present in all vespertilionine bats. Across 18 species tandem repeats varied in size from 78 to 85 bp and contained two to nine repeats. Heteroplasmy ranged from 15% 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 more repeats 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 further suggest that repeat duplication 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 number also occur in shrews, cats and bighorn sheep. The presence 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 and a primitive repair mechanism in the event of somatic mutations to these binding sites. A NIMAL mitochondria contain a circular l 6 k b DNA molecule, encoding 13 protein, 22 transfer RNA (tRNA) and two ribosomal RNA genes (ANDERSON et al. 1981). The small size and compact organization of the mitochondrial DNA (mtDNA) genome has been suggested to be the result of selection for rapid organelle replication (HARRISON 1989; RAND 1993). However, recent discovery of length variation in the noncoding control region, which lies between the tRNA-Pro and tRNA-Phe genes, in a variety of vertebrate species (DENSMORE et al. 1985; MORITZ and BROWN 1987; BUROKER et al. 1990; HAYASAKA et al. 1991; WILKINSON and CHAPMAN 1991; ARNMON and RAND 1992; BROWN et al. 1992, 1996; HOELZEL et al. 1993, 1994; STEWART and BAKER 1994; xu and h M O N 1994; YANG et al. 1994; CECCONI et al. 1995; PETRI et al. 1996; FUMAGALLI et al. 1996) is not consistent with this hypothesis and has not yet been adequately explained (WOLSTENHOLME 1992; RAND 1993). Because the proteins encoded by mtDNA genes play critical roles in oxidative metabolism and control region length might influence the rate of mtDNA transcription or replication (ANNEX and WIL LJMS 1990), the metabolic rate of the organism and possibly its survival could be affected by length variation. Transcription of mitochondrial genes is initiated at Corresponding author: Gerald S. Wilkinson, Department of Zoology, University of Maryland, College Park, MD 20742. E-mail: [email protected] two sites in the central, conserved portion of the control region (CHANG et al. 1985; KING and LOW 1987; CLAYTON 1992). Each strand of the mtDNA molecule, referred to as heavy (H) and light (L) based on differences in base composition, has different promoter regions that bind nuclearcoded proteins (GHMZZANI et al. 1993b; NASS 1995) and that differ in nucleotide sequence between species (KING and LOW 1987). Replication of the H-strand is primed by RNA transcribed between the L-strand promoter (LSP) and the H-strand origin of replication (OH, CHANG and CLAWON 1985). H-strand replication is usually terminated shortly thereafter at termination-associated sequences (TAS) resulting in short 7s DNA strands (DODA et al. 1981; CLAYTON 1991). 7s DNA strands remain associated with the L-strand and displace the original H-strand to create a three-stranded structure known as the displacement or D-loop. In mice, only 5% of replication events continue beyond the control repon (BOGENHAGEN and CLAWON 1978). Sequence-specific DNA binding proteins interact with TAS elements (MADSEN et al. 1993b) between two conserved regions, mt 5 (OHNO et al. 1991), which is also referred to as region J (KING and LOW 1987), and mt 6 (KUMAR et al. 1995). Initiation of replication of the L-strand occurs only when H-strand replication is two-thirds complete and the conserved 012 sequence, which in mammals lies between tRNA-Cy s and tRNAAsn, is exposed (CLAYTON 1982). While the function of the D-loop is not well underGenetics 146: 1035-1048 (July, 1997) 1036 G. S. Wilkinson et al. stood (WOLSTENHOLME 1992), its structure and size are likely to influence mtDNA replication. A high proportion of triplex to duplex forms correlates with mtDNA copy number, mtRNA abundance and the rate of oxidative metabolism in different tissues (ANNEX and WILLIAMS 1990). The length of the 7s DNA strand, and therefore the size of the D-loop, varies depending on which TAS site is used for termination (DODA et al. 1981). Consequently, tandem repeats containing TAS elements should alter D-loop size. TAS elements occur within tandem repeats of evening bats (WILKINSON and CHAPMAN 1991), shrews (STEWART and BAKER 1994; FuMAGALLI et al. 1996), bighorn sheep (ZARDOYA et al. 1995), treefrogs FANG et al. 1994), minnows (BROUCHTON and DOWLING 1994), sturgeon (BROWN et al. 1996), cod (ARNASON and RAND 1992; LEE et al. 1995), and seabass (CECCONI et al. 1995). Despite the distant taxonomic affiliations among these species, in most cases these R1 repeats (FUMAGALLI et al. 1996), Figure 1) are -80 bp in length. In some fish and frogs the 80-bp repeat contains two or more smaller units. In several species, R1 repeats have been predicted to form thermodynamically stable secondary structures (BUROKER et al. 1990; WILKINSON and CHAPMAN 1991; STEWART and BAKER 1994; FUMAGALLI et al. 1996). R1 repeat duplications and deletions are thought to occur by competitive strand displacement among the three strands of the Dloop (BUROKER et al. 1990) resulting in a unidirectional mutational process (WILKINSON and CHAPMAN 1991). Short, tandem repeats on the opposite side of the central conserved portion of the control region have also been reported in a variety of mammals including several carnivores (HOELZEL et al. 1994), pinnipeds ( A R NASON et al. 1993; HOELZEL et al. 1993), pigs (GHMZZANI et al. 1993a), horses (ISHIDA et al. 1994; XU and ARNASON 1994), rabbits (MIGNOTTE et al. 1990; BIJUDWAL et al. 1991), shrews (FUMAGALLI et al. 1996), marsupials UANKE et al. 1994) and bats (PETRI et al. 1996; E. PETIT, personal communication). These R2 repeats (FUMAGALLI et al. 1996) typically involve variable numbers of short 6 to 30-bp units, which often contain the 4bp motif GTAC, and exhibit length variation similar to that described for nuclear microsatellite loci (CHARLESWORTH et al. 1994). Because these short repeats occur upstream from the origin of H-strand replication, they probably do not influence D-loop size. Consequently, their formation is more likely to be caused by replication slippage (LEVINSON and GUTMANN 1987; MADSEN et al. 1993a) than competitive strand displacement. In this paper we present data on the presence and number of tandem R1 repeats among 41 species of bats representing most families in the order Chiroptera. By comparing sequence similarity between species with and without repeats we provide evidence for the evolutionary origin of R1 repeats in vespertilionine bats. We then compare the number of R1 repeats and heteroplasmy among seven species of vespertilionine bats in order to identify evolutionary processes that influence repeat array size. If the mutational process that gives rise to heteroplasmy is unbiased, we would expect homoplasmic and heteroplasmic individuals to have equal numbers of R1 repeats (BROWN et al. 1996). Deviations from this expectation indicate mutational bias or selection. Similarly, the proportion of heteroplasmic individuals is expected to be determined by a balance between mutation and organelle segregation (CWUC 1988; BIRKY et al. 1989) since paternal transmission is rare (HARMSON 1989; SKIBINSKI et al. 1994). Thus, variation in heteroplasmy should reflect variation in repeat mutation rate if the number of organelles per cell does not vary. Finally, we compare consensus sequences from vespertilionine bats with repeats to control region sequences of other mammals with and without repeats to determine if R1 repeated arrays have evolved multiple times in mammals and might influence organism function. MATERIALS AND METHODS Sampling locations: Bats were captured by netting at roosting and foraging sites in Europe, Malaysia, United States, Central America, South America, and Africa. Nycticeius humeralis were captured at six attic nursery colonies in Missouri and one in North Carolina (WILKINSON and CHAPMAN 1991). Eptesicus fuscus and Myotis lucifugus were captured in a single barn near the town of Princeton, Missouri. Leptonycteris curasoae and L. nivalis were captured in day roosts in Mexico; Glossophaga soricinawas netted in Guanacaste, Costa Rica (WILKINSON and FLEMING 1996). Four species were captured in the Transvaal, South Africa: Epomophorus cvpturus and N. schle@nii were netted over streams near the town of Skukuzu in Kruger National Park while Nycteris thebaica and Rhinolophus clivosus were captured in a mine tunnel just south of Kruger National Park. Four species were also captured from a cave on Tamana Hill in Trinidad, West Indies: Pteronotus parnelli, Momoops megalophylla, Natalus tumidirostris and Phyllostomus hastatus. Saccoptqx bilineata were captured at La Selva, Costa Rica. Six species were captured in peninsular Malaysia: Hipposideros diadema, R afJinis, R. sedulus, Murina suilla, Nyctophilus gouldii and Keriwoula papillosa. Seven species were collected in Germany: Nyctalus noctula in Brandenburg and Bavaria, and E. nilssoni, M. myotis, M. bechsteini, Pipistrellus Pipistrellus, P. nathusii and Vespertilio murinus in Bavaria. Seven species were netted in Greece: E. serotinus, N. leisleri, N. lasiopterus, Minioptems schreibersi, P. kuhli, Tadarida teniotis and R. f m m e q u i n u m . Samples from R. f m m e q u i n u m were obtained from Switzerland and Luxemburg. DNA extraction, amplification and sequencing: A small piece of patagia1 membrane, -10 mm‘, was excised from each individual with biopsy punches and stored either in a concentrated salt solution (SEUTIN et al. 1991) or 95% ethanol in the field. DNA was extracted from a tiny portion of each wing membrane sample using either Chelex (WALSH et al. 1991), a modified salting out procedure (MILLER et al. 1988) or a Qlagen DNA extraction kit following the manufacturer’s protocol. Control region mtDNA was amplified using two 22-bp primers, P and F (WILKINSON and CHAPMAN 1991). The P primer begins at position 15975 in the human proline tRNA gene (ANDERSON et al. 1981), while the F primer ends at position Repeat Array Evolution in Bat mtDNA 16425 in a conserved sequence region found in the middle of the control region (Figure 1, (SOUTHERN et al. 1988). Doublestranded amplifications using PCR were performed as described in WILKINSON and CHAPMAN (1991) using AmpliTaq (Perkin Elmer) and 40 cycles of 95” for 1 min, 55” for 1.5 min, and 72” for 2 min in a Peltier thermal cycler. Amplification products were purified and concentrated using either ethanol precipitation or a silica gel-based method (Geneclean kit, QIAEX or Qlagen PCR-prep kit) following the manufacturers’ instructions. Double-stranded PCR products were sequenced by the dideoxy chain termination method using either y””SATP and Sequenase 2.0 (Amersham) or by cycle sequencing with Thermosequenase (Amersham) using flourescent labeled primers and automated sequencers (LI-COR automated sequencer in Erlangen or an AB1 automated sequencer at the Molecular Genetics Instrumentation Facility at the University of Georgia). Cycle sequencing was performed according to the manufacturer’s protocol. A nested primer (P* 5’-CCCCACCATCAACACCCAAAGCTGA-3’) was used to sequence PCR products generated with primers C and F (WILKINSON and CHAPMAN 1991) in a single direction for S. bilineata, H. diad m , R affinis, R sedulus, R fmmequinum, T. teniotis, M. suilla, K. papillosa, M. schreibersi, E. nilssoni, E. serotinus, M. myotis, M. bechsteini, P. pipistrellus, P. kuhli, P. nathwii, V. murinus, N. leisleri, N. lasiopterus, and N. gouldii. The three megadermatid sequences were provided by J. WORTHINGTON-WILMER. Control region sequence was obtained in both directions using both the P and F primers to initiate the sequencing reaction for the remaining 18 species. R1 repeat estimation and comparison: The number of R1 repeats in the arrays of homoplasmic and heteroplasmic individuals was inferred by comparing PCR product sizes to a 100-bp ladder after agarose gel electrophoresis and ethidium bromide staining under UV. Expected repeat length was estimated from sequence information for each species. To test for differences in the frequencies of heteroplasmic and h o m e plasmic genotypes between species, we used contingency chisquare tests. We also used analysis of variance (ANOVA) to determine if the number of R1 repeats differed between heteroplasmic and homoplasmic individuals among the eight vespertilionid species for which the DNA of eight or more individuals was amplified. When determining repeat number we assumed that heteroplasmic individuals contained equal amounts of each repeat array detected on the gel. Sequence comparison and analysis: All sequences were aligned with the help of the Higgins algorithm using the program MACDNASIS and were improved by subsequent manual alignment. When more than one individual of a species was sequenced, a consensus sequence was generated and then used for among species comparisons. In species having multiple R1 repeats, the flanking single copy region, as well as the first and last repeats (Figure l ) , were aligned in a similar way. Throughout this paper we refer to the repeat nearest the central portion of the control region as the first repeat because it undergoes replication first. The last repeat refers to the repeat nearest the tRNA-Pro gene. Because prior studies of bats (WILKINSON 1992; WILKINSON and CHAPMAN 1991) with R1 tandem repeats have demonstrated that some process, such as competitive strand displacement (BUROKER et al. 1990), homogenizes repeat sequences in the middle of the array, we compared sequences between species with variable numbers of repeats by aligning all repeats between the first and last repeat to generate a single middle repeat consensus sequence. This method resulted in a consensus sequence for each of eight species of vespertilionine bats with repeats consisting of a first, middle and last repeat and flanking single copy sequences on each side of the repeats (Figure 2). 1037