J Mol Evol (2004) 58:280–290 DOI: 10.1007/s00239-003-2550-2 The Utility of the Neglected Mitochondrial Control Region for Evolutionary Studies in Lepidoptera (Insecta) Marta Vila,1,2 Mats Bjo¨ rklund1 1 Department of Animal Ecology, Evolutionary Biology Centre, Uppsala University, Norbyva¨ gen 18 D, SE-752 36, Uppsala, Sweden 2 IUX-Edificio de Servicios Centrais de Investigacio´ n, University of A Corun˜ a, Campus de Elvin˜ a, E-15071, A Corun˜ a, Galicia, Spain Received: 24 February 2003 / Accepted: 15 September 2003 Abstract. The insect mitochondrial control region are straightforward over one part of the CR. The (=AT-rich-region) is a rarely used genetic marker in combination CR+COI appears to be a very prom- phylogeographic studies and population genetic sur- ising phylogenetic tool to resolve fast-evolving veys. Reasons for this are that the high AT content species-level phylogenies. and the presence of tandem repeats and indels pose technical and analytical problems. We provide a new Key words: mtDNA control region — Cyto- pair of primers and the first taxonomically wide-scale chrome oxidase I — Insecta — Lepidoptera — description of control region (CR) structure in an Erebia — Indels — Structure — Phylogeography insect order after sequencing it in 31 lepidopteran — Phylogeny species. We assessed levels of variation occurring in the CR and cytochrome oxidase I (COI) by se- quencing and comparison. Intrapopulation analyses Introduction in five species of butterflies showed that CR was more variable than COI. Interpopulation variation from During the last decade there has been a remarkable three populations of Erebia triaria and E. palarica increase in population genetic and phylogeographic was slightly lower in the CR than COI with regard to studies using DNA sequence data. This has been single-nucleotide polymorphisms, but the results were greatly aided by the availability of effective markers. concordant between both markers and highly con- One popular marker is the mitochondrial control gruent with regard to population differentiation. region (CR), which has been used in many animal Using 15 species of Satyrinae we found that the CR taxa (Merila¨ et al. 1997; Donaldson and Wilson 1999; has the same, or stronger, phylogenetic resolution as Rankin-Baransky et al. 2001; Larizza et al. 2002). COI. Our results indicate that the CR can be of im- In Insecta, however, the CR remains a largely portance in addition to COI in population genetic unused and little-known genetic marker in most of studies. Alignments for the whole CR are direct and the more than 30 extant orders (Roehrdanz and unambiguous at the intraspecific level. Indels show Degrugillier 1998; Caterino et al. 2000; Lessinger and phylogenetic signal, but make this marker more Azeredo-Espin 2000; Schultheis et al. 2002a; complex to use than COI for higher phylogenetic Mardulyn et al. 2003). In Lepidoptera, the informa- analyses. Nevertheless, alignments at the generic level tion on the CR is almost null; it has been described for seven species of Lycaenids by Taylor et al. (1993) and sequenced for two moths (McKechnie et al. Correspondence to: Mats Bjo¨ rklund; email: mats.bjorklund@ 1993a,b). Thus, CR data from only nine species of ebc.uu.se Lepidoptera appear in sequence databases, although 281 the whole mitochondrial genomes are given for of the genus Erebia, one Maniola and four species of Bombyx mori (Lee et al. 2000) and B. mandarina Coenonympha, and compare the topologies obtained (Yukuhiro et al. 2001). by using both CR and COI sequences to evaluate The usefulness of the CR, also known as the AT- their relative power in resolving species and genus rich region, for species-level phylogenies and popu- level phylogenies. Inter-family level analyses are be- lation-level studies in Insecta has been questioned or yond the scope of this paper due to the problems in remains controversial (Taylor et al. 1993; Zhang and weighting large indels, needed for phylogenetic Hewitt 1997; Caterino et al. 2000). The remarkable analyses, at such taxonomic level. lack of CR sequences in Insecta may be due to (i) difficulties in amplification and/or sequencing, (ii) the Materials and Methods assumption that CR might not evolve faster than other regions of the mtDNA in insects (Zhang and Specimens Hewitt 1997), and (iii) putative strong selective con- straints related to its extreme richness in AT and to Individuals from Erebia triaria (n = 60) and E. palarica (n = 60) the presence of highly conserved structural elements were hand-netted in Galicia (NW of Spain) between 1998 and 2000 (Zhang and Hewitt 1997). Drosophila is the subject of and preserved in 95% ethanol until analysis. The average distance more studies about the insect CR (Clary and between the three locations was 96 km for E. triaria and 46 km for Wolstenholme 1987; Monnerot et al. 1990; Monforte E. palarica. Both species occur in spatial sympatry in Courel and et al. 1993; Inohira et al. 1997). The objective of these Queixa, although their flight periods do not coincide. For conser- vation purposes and to avoid the risk of amplifying sperm DNA has mainly been to survey its structure and evolution, from the female spermatophores, only males were caught. Other not variation at the population level or higher, with species were either collected by the authors themselves or provided the exception of Brehm et al. (2001). There are only six by colleagues (see Appendix I for collectors and locations). studies using the CR as a genetic marker at the pop- ulation level in Insecta: McKechnie et al. (1993a, b), DNA Extraction, Amplification, and Sequencing Zhang and Hewitt (1996), Atkinson and Adams (1997), Mardulyn (2001), and Schultheis et al. Most genomic DNA was extracted from adult thorax tissue using (2002b), besides Vandewoestijne and Nice et al. (un- the DNAeasy Tissue Kit (Qiagen) or a Chelex protocol. Legs published). (Parnassius apollo) or larvae (Prodoxus decipiens and P. quinque- The aim of this paper is to evaluate the population punctellus) were used when thorax was not available. Amplifications were performed in 30-ll volumes containing 1· genetic and phylogenetic information contained by PCR Buffer II (Applied Biosystems), 2–2.5 mM MgCl2, 1.5 U the lepidopteran CR. First, we describe the structure AmpliTaq polymerase (Applied Biosystems), a 1 mM concentration of CR at the generic and family levels in Lepidoptera. of GeneAmp dNTP (Perkin Elmer), a 0.7 lM concentration of each Thus, we have sequenced 29 species ranging across primer J6 (Zhang et al. 1995) + Lep12S (Taylor et al. 1993) or five families of butterflies and two Prodoxidae moths. SeqLepMet + LepAT2B (this paper) and 50–70 ng of DNA. Primer SeqLepMet (50 TGA GGT ATG ARC CCA AAA GC 30) lies in the Second, we survey the usefulness of the CR as a ge- 50 end of the tRNA-methionine gene, whereas primer LepAT2B netic marker at the intraspecific level. We focus on (50 ATT AAA TTT TTG TAT AAC CGC AAC 30), antisense to the amount of variability found within and among SeqLepMet, is located toward the 50 extreme of the 12S rRNA gene. three populations of Erebia triaria and E. palarica Primers SeqLepMet and LepAT2B correspond in their 30 ends to (Nymphalidae: Satyrinae). We compare CR se- positions 10,361 and 9667, respectively, in the Bombyx mori mtDNA sequence (Lee et al. 2000). CR PCR started with 95°C for 2 quences with those from cytochrome oxidase I (COI) min, followed by 35 cycles at 94°C for 60 s, 51°C for 90 s, and 65°C since the latter is the main marker used at the intra- for 60 s, and a final step at 65°C for 7min. DNA extractions from 5- specific level of analysis in Insecta (Caterino et al. year-old dried Erebia triaria were not suitable for PCR amplifica- 2000; e.g., Juan et al. 1998; Bogdanowicz et al. 2000; tion of the CR and hardly yielded any COI products. Shufran et al. 2000; Althoff et al. 2001; Williams COI length is 1.5 kilobases (kb) but we focused on its second half following the variability survey presented by Lunt et al. (1996) 2002). In addition, we have sequenced both markers in other Insecta. The fragment used for intraspecific surveys in in a single population from five more widespread Erebia triaria and Erebia palarica (786-bp length) started at posi- species. Overall, the mean sequence diversity of these tion 717 of COI. For species-level phylogenies, a fragment of about seven species sheds some light on the degree of vari- 1250 bp (starting at position 313) was used. COI (PCR product, 2 ability expected from both markers at the intrapop- kb) was amplified in 30-ll reaction volumes containing 1·PCR, 2 mM MgCl2, 1 U Taq, 1 mM dNTP, a 1 lM concentration of each ulation and intraspecific level. primer C1-J-1751 (Simon et al. 1994) and C2-N-3662 (Crozier and Third, we compare the genetic divergence of the Crozier 1993), and 50–70 ng of DNA. The PCR cycle differed from CR among 15 Satyrinae species with that obtained that of the CR only in annealing at 53°C and extension for 90 s. from COI since the latter is also frequently used for PCR products were electrophoresed on 2% agarose gels and phylogenetic analyses in Lepidoptera (e.g., Brower visualized under UV light after ethidium bromide staining. Prod- ucts were purified with Microcon PCR Centrifugal Filter Devices 1994; Zimmermann et al. 2000; Rubinoff and Sper- (Amicon–Millipore) and used as template for direct sequencing on ling 2002). We perform phylogenetic analyses using an Applied Biosystems ABI-310 DNA sequencer. All PCR prod- both parsimony and distance methods on ten species ucts were sequenced in both directions.