PL-ISSN0015-5497(print),ISSN1734-9168(online) FoliaBiologica(Kraków),vol.62(2014),No2 Ó InstituteofSystematicsandEvolutionofAnimals,PAS,Kraków, 2014 doi:10.3409/fb62_2.91 Molecular Variability of the COI fragment Supports the Systematic Position of Enarmoniini within the Subfamily Olethreutinae (Lepidoptera: Tortricidae) JózefRAZOWSKI andSebastianTARCZ Accepted February 19, 2014 RAZOWSKI J., TARCZ S. 2014. Molecular variability of the COI fragment supports the systematic position of Enarmoniini within the subfamily Olethreutinae (Lepidoptera: Tortricidae). Folia Biologica (Kraków) 62: 91-96. The Tortricidae, a globally distributed family of Lepidoptera, consists of approximately 10000 described species, of which a large number do not have clearly defined taxonomic positions. In the present paper the systematics of Enarmoniini based on molecular data is compared to systematics based on morphology. Two genera of Enarmoniini were used for analysis: the type-genus Enarmonia (one species examined) and Ancylis (7 species examined). A comparison of a 606 bp homologous fragment of the COI mitochondrial gene revealed that Enarmoniini form a cluster distinct from Olethreutini (3 genera and 7 species examined), Eucosmini (2 genera, 4 species) and Grapholitini (4 genera, 9 species). In our opinion the molecular studies combined with previously obtained morphological data should facilitate a more natural classification system of this relatively poorly explored family of Microlepidoptera. Altogether, 30 species of Tortricidae were examined. Key words: Enarmoniini, Olethreutinae, Tortricidae, Lepidoptera, molecular variability, mitochondrial COI. Józef RAZOWSKI, Department of Invertebrate Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, 31-016 Kraków, S³awkowska 17, Poland. E-mail: [email protected] Sebastian TARCZ, Department of Experimental Zoology, Institute of Systematics and Evolu- tion of Animals, Polish Academy of Sciences, 31-016 Kraków, S³awkowska 17, Poland. E-mail: [email protected] The classification of species using genomic in- 2002; LANDRY et al.1999)orthepopulationstruc- formation is particularly significant in the case of ture of Tortricid pests (SCHROEDER &DEGEN 2008; described taxa in which morphological character- TIMM et al. 2010). A preliminary study based on com- istics do not provide an unambiguous answer. parative analysis of the COI gene fragment resolved An example of this is the family Tortricidae, Lepi- uncertain relationships between the tribes Bactrini doptera (BROWN 2005), a group consisting of and Endotheniini (RAZOWSKI &TARCZ 2012) as about 10000 described species. A taxonomic sys- well as reassessed the systematic position of the tem based on morphological characters of mem- Neotropical genus Orthocomotis (RAZOWSKI et al. bers of this family has been improved for more 2013). than 150 years, but the inclusion of many genera to tribes is doubtful. Therefore, a comparative analy- Enarmoniini is a rather small tribe of Tortricidae sis of genome fragments provides an opportunity in the subfamily Olethreutinae. There was no con- for taxonomic progress and determination of the sensus as to its systematic and phylogenetic posi- relationships among various taxa. tion. The present molecular data confirm the recent suppositions by HORAK (2006) and RAZOWSKI Currently, the most popular molecular marker is (1976a, 2003) that Enarmoniini are more closely a part of the mitochondrial COI gene which was related to Olethreutini than to Eucosmini or other proposed as a universal DNA barcode (HEBERT et al. tribes of Olethreutinae. 2003) and used successfully in butterfly species identification in the Astraptes fulgerator species Two genera were examined: Enarmonia consists complex (HEBERT et al. 2004). Recent studies based of four Palaearctic species whilst Ancylis has onacomparisonofthe COI fragment relate mainly a worldwide distribution with approximately 130 to systematics within the genera of the Tortricidae, representatives. Moreover 20 other genera and such as species relationships (KRUSE &SPERLING about 60 species are known from Australia 92 J.RAZOWSKI,S.TARCZ (HORAK 2006). Until now, there was no conclu- plification protocol was the same as in (HEBERT et al. sive evidence based on morphological characteris- 2004). tics that allowed classifying Enarmoniini to any of In order to assess the quality of the amplification, these tribes, or to demonstrate that it is a separate PCR products were electrophoresed in a 1% aga- tribe. Although a multilocus attempt at Tortricidae rose gel for 45 min at 85 V with a DNA molecular classification has been made (REGIER et al. 2012), weightmarker(MassRulerLowRangeDNALad- only one species of the tribe Enarmoniini was ana- der, Thermo Scientific, USA). NucleoSpin Gel lyzed. Furthermore, DNA sequence variability and PCR Clean-up (Macherey-Nagel, Germany) within the studied tribe has not been characterized. was used for purifying PCR products. In some The present survey provides molecular evidence PCR products, additional sub-bands were ob- of the separate position of Enarmoniini within the tained apart from the main band. In these cases, 30 subfamily Olethreutinae as well as a preliminary Fl of each PCR product was separated on 1.8% analysis of intra-tribe relationships. agarose gel (100 V/60 min) with a DNA molecular weightmarker(MassRulerLowRangeDNALad- der, Live Technologies, USA). Then the band rep- Material and Methods resenting the examined fragment was cut out and purified. Cycle sequencing was done in both directions Material with the application of BigDye Terminator v3.1 chemistry (Life Technologics, USA). Primers The specimens were preserved dry. Due to prob- LEP-F1 and LEP-R1 were used for sequencing. lems associated with obtaining good quality DNA Each sequencing reaction was carried out in a final suitable for molecular analysis, COI sequences of volume of 10 Fl containing: 3 Fl of template, 1 Fl other species were taken from GenBank. Two rep- of BigDye Master Mix (1/4 of standard reaction), resentatives of the tribe Polyorthini were used as 1 Fl of sequencing buffer, 1 Fl of 5 mM primer. Se- the outgroup. A list of examined taxa arranged al- quencing products were precipitated using Ex phabetically is presented in Table 1. No permits were required for collection of these specimens, Terminator (A&A Biotechnology, Poland) and and no endangered species were used. separated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequences are avail- able in the GenBank database (for accession num- Molecular methods bersseeTable1).Asapositivecontrolof PCRand sequencing, a successfully amplified and se- DNA was extracted from two hind legs of dry quenced COI fragment from Olethreutes subtili- specimens because we could not completely de- ana (Table 1) from previous analyses was used. stroy the museum material using other parts of the bodies (e.g. the entire tagmata). The examined specimens were not older than 10 years and were Data analysis first identified by comparison of the genitalia. Specimens older than ten years usually gave insuf- Sequences were examined using Chromas Lite ficient results. The best results were obtained from (Technelysium, Australia) to evaluate and correct 1-3 year old individuals. chromatograms. The alignment of the studied se- quences was performed using ClustalW (THOMPSON Genomic DNA was isolated without protocol et al. 1994) within the BioEdit software (HALL 1999). modification using the NucleoSpin Tissue Kit Phylograms were constructed for the studied frag- (Macherey-Nagel, Germany). To elute purified DNA mentswithMegav5.2(TAMURA et al.2011)using we applied 100 Fl of elution buffer onto the silica neighbor-joining – NJ, (SAITOU &NEI 1987), maxi- membrane. To amplify a fragment of the mito- mumparsimony – MP, (NEI &KUMAR 2000), and chondrial COI gene (650bp) the following primer maximum likelihood – ML, (FELSENSTEIN 1981). pair designed for Lepidoptera was used: LEP-F1, NJ analysis was performed using the Kimura 2- 5’-ATTCAACCAATCATAAAGATAT-3’; and parameter correction model (KIMURA 1980) by LEP-R1, 5’-TAAACTTCTGGATGTCCAAAAA-3’. bootstrapping with 1000 replicates (FELSENSTEIN These are universal primers used for species iden- 1985). MP analysis was evaluated with the min- tification in DNA barcoding (HEBERT et al. 2004). mini heuristic parameter (at level 2) and bootstrap- PCR amplification for all analyzed DNA frag- ping with 1000 replicates. Bayesian inference (BI) ments was carried out in a final volume of 40 Fl was performed with MrBayes 3.1.2 (RONQUIST & containing 30 ng of DNA, 1.5 U Taq-Polymerase HUELSENBECK 2003); the analysis was run for (EURx, Poland), 0.8 Fl of 20 FM of each primer, 5,000,000 generations and trees were sampled 10 × PCR buffer, and 0.8 Fl of 10 mM dNTPs in every 100 generations. All trees were constructed a Mastercycler ep (Eppendorf, Germany). The am- with TreeView 1.6.6 (PAGE 1996). Analysis of SystematicPositionofEnarmoniini 93 Table 1 Speciesof Tortricidaeusedinthepresentstudy.Twospecies of tribe Polyorthini were used asanoutgroup.Forbetterorientation,specimensforwhich COI sequences were obtained in the present study are highlighted in gray No.DNAVoucher Tribe Genus Species Origin COI acc Ancylis apicella Italy, 1. TLMFLep02146 Enarmoniini HÜBNER,1825 DENIS &SCHFFERMÜLLER, 1775 SouthTyrol,Etschtal JF859730 laetana Italy, 2. TLMFLep02145 Enarmoniini Ancylis FABRICIUS,1775 SouthTyrol,Etschtal JF859729 mitterbacheriana 3. TORT083 Enarmoniini Ancylis DENIS &SCHFFERMÜLLER,