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 (: )

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- (one species examined) and (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 , 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, 1775 Poland KF493842 myrtillana Italy, 4. TLMFLep02051 Enarmoniini Ancylis TREITSCH,1830 SouthTyrol,Etschtal JF859643 uncella 5. TORT081 Enarmoniini Ancylis DENIS &SCHFFERMÜLLER, 1775 Poland KF493843 unculana 6. TORT082 Enarmoniini Ancylis HAVORTH,1811 Poland KF493844 unguicella Italy, 7. TLMFLep02053 Enarmoniini Ancylis LINNAEUS,1758 SouthTyrol,Etschtal JF859645 8. DA04 Enarmoniini HÜBNER,1825 SCOPPOLI,1763 Poland KF493845 Blastesthia mughiana 9. TORT115 Eucosmini OBRASCOV,1960 ZETTERSCHEDT,1868 Poland KF493846 turionella 10. TORT071 Eucosmini Blastesthia LINNAEUS,1758 Poland KF493847 Epinotia granitana Italy, 11. TLMFLep02358 Eucosmini HÜBNER,1825 HERRICH-SCHÄFFER,1851 SouthTyrol,Etschtal JF859912 tetraquetrana 12. TORT079 Eucosmini Epinotia HAVORTH,1811 Poland KF493848 Cydia nigricana 13. TORT098 Grapholitini HÜBNER,1825 FABRICIUS,1794 Poland KF493849 INNAEUS Canada, 14. 04HBL006698 Grapholitini Cydia pomonella L ,1758 Ontario,Puslinch GU093187 strobilella Italy, 15. TLMFLep02100 Grapholitini Cydia LINNAEUS,1758 SouthTyrol,Etschtal JF859687 Grapholita caecana 16. TORT092 Grapholitini TREITSCH,1829 SCHLÄGER,1848 Poland KF493850 molesta Canada, 17. Jflandry1667 Grapholitini Grapholita BUSCK,1916 Ontario,Vineland GU096466 ALSINGHAM asphodelana Canada, 18. 10-JDWBC-2183 Grapholitini W , EARFOTT HM863928 1879 K ,1907 BritishColumbia Pammene albuginana Italy, 19. TLMFLep02164 Grapholitini HÜBNER,1825 GUENÉE,1845 SouthTyrol,Etschtal JF859748 argyrana Italy, 20. TLMFLep02374 Grapholitini Pammene HÜBNER,1796-99 SouthTyrol,Etschtal JF859925 fasciana Italy, 21. TLMFLep02160 Grapholitini Pammene LINNAEUS,1761 SouthTyrol,Etschtal JF859744 Apotomis inundana 22. TORT131 Olethreutini HÜBNER,1825 DENIS &SCHFFERMÜLLER, 1775 Poland,Hajnówka JF730060 sauciana 23. TORT132 Olethreutini Apotomis FRÖLICH,1828 Poland,Kojszówka JF730061 sororculana 24. TORT127 Olethreutini Apotomis ZETTERSCHEDT,1839 Poland,Brzoskwina JF730062 Celypha cespitana 25. TORT103 Olethreutini HÜBNER,1825 HÜBNER,1814-17 Poland,Brzoskwina JF730064 rufana Poland, 26. TORT109 Olethreutini Celypha SCOPPOLI, 1763 Kraków-NowaHuta JF730065 striana 27. TORT104 Olethreutini Celypha DENIS &SCHFFERMÜLLER, 1775 Poland,Brzoskwina JF730066 Olethreutes subtiliana 28. TORT102 Olethreutini HÜBNER,1822 FALKOVITSH,1958 Poland JF730067 Pseudatteria ALSINGHAM heliocausta 29. TORT039 Polyorthini W , OGNIN Ecuador JX144973 1913 D ,1912 30. TORT040 Polyorthini Pseudatteria chrysanthema MEYRICK,1912 Ecuador JX144974 94 J.RAZOWSKI,S.TARCZ haplotype diversity, nucleotide diversity and vari- decided to present only one tree reconstructed by able nucleotide positions was done with DnaSP Bayesian Inference (Fig. 1). The Enarmoniini clus- v5.10.01 (LIBRADO &ROZAS 2009). Analysis of ter is well separated and is composed of a mono- nucleotide frequencies, p-distance estimation and phyletic group containing seven studied Ancylis identification of substitution model (GTR+G+I for species and a distinct branch represented by Enar- COI mtDNA fragments) for ML analysis were monia formosana. On the COI tree, the Enarmoni- donewithMegav5.2(TAMURA et al.2004,2011). ini cluster appears between a group composed by two tribes: Eucosmini and Grapholitini and the more generalized Olethreutini. Although all tribes Results and Discussion are well defined on the tree, it is difficult to hy- pothesize about their reciprocal relationships be- Besides a fragment of the COI gene, several nu- cause there was low or even no bootstrap support clear markers (ITS2 rDNA, EF1a, wingless and for basal nodes of the studied trees (only posterior CAD genes) were tested on the presently studied probabilities are acceptable – see Figure 1). This museum material. However, we failed to obtain could be connected with the COI gene which is good quality sequences or even amplicons of these a fast evolving genome fragment, however in the genome fragments, probably because of the poor qual- present analysis we were not able to obtain DNA ity of DNA isolated from dry legs of specimens. sequence for other, more slowly evolving molecu- lar markers. Therefore our current analysis, based on the COI fragment only, shows the possibility of applying TheEnarmoniiniDIAKONOFF,1953(synonyms: this part of the genome as a molecular marker not Ancylisidii PIERCE &METCALFE, 1922, Anchy- onlyforTortricidaespeciesbutalsoforinitialtribe loperidae STAINTON, 1859) was given a variable delineation. In some cases we obtained unresolved position within the Olethreutinae and their mono- phylogenies. This may be connected with a fast phyly was also controversial. The most recent and rate of evolution of the studied COI fragment and important interpretations of their systematics and also may be caused by the close relationship and phylogeny are as follows. lack of monophyly of some of the genera (for ex- KUZNETZOV and STEKOLNIKOV (1973) based on ample within Cydia and Grapholita). A total of the musculature of the male genitalia placed Enar- 30 sequences of the gene encoding cytochrome moniini (under the name Ancylidini) in the super- oxidase subunit I (606 bp) from species of Enar- tribe Eucosmidii as a sister group of Eucosmini + moniini, Eucosmini, Grapholitini, Olethreutini Laspeyresiini (= Grapholitini); similarly, RAZOWSKI and Polyorthini (outgroup) were used in this study (1976a) divided Eucosmini into Enarmoniina and (Table 1). Eucosmina + Grapholitina; KUZNETZOV and STE- KOLNIKOV (1977) divided Eucosmidii into Enar- The interspecific haplotype diversity value was moniii and Grapholitini and the latter into Hd=1, indicating substantial variability of the stud- Grapholitae and Dichroramphae; the same authors ied DNA fragment. Nucleotide diversity amounted B (KUZNETZOV &STEKOLNIKOV 1984) used only to =0.11106. The nucleotide frequencies were a different nomenclature of Eucosmidii in an analogi- A=0.308, T=0.374, C=0.163 G=0.155 and re- cal phylogenetic tree: Enarmoniini, Eucosmini, vealed a high proportion of A-T pairs which corre- Laspeyresiina and Lipoptychina; and SAFONKIN sponds to typical characteristics of (2007) in his phylogeny based on pheromones di- mitochondrial DNA. Mean divergence over all vided Olethreutinae into Gatesclarkeanidii con- studied Tortricidae (N=30) sequence pairs was sisting of Gatesclarkeanini and Endotheniini and p=0.111/SE=0.008 (p-distance/standard error). Olethreutidii in which he placed three tribes, viz., The mean divergence over all studied Enarmoniini Microcorsini, Bactrini and Olethreutini. The sister (N=8) sequence pairs was p=0.097/SE=0.008; Eu- group of Olethreutidii is Grapholitidii composed cosmini (N=4) (p=0.073/SE=0.008); Grapholitini with Enarmoniini, Eucosmni and Grapholitini (N=9) (p=0.096/SE=0.007) and Olethreutini (N=7) tribes. In all the mentioned publications (except (p=0.092/SE=0.007). In the analyzed dataset we for RAZOWSKI 1976a and SAFONKIN (2007) Enar- found211variablepositionsacrossallstudiedspe- moniini are included in Eucosmini or Grapholitini cies in the COI fragment, 180 of which were parsi- and essentially followed the earlier proposals by monyinformative(98withtwovariants,61–three HEINRICH (1923) and HORAK and BROWN (1991). variants, 21 – four variants). A total of 30 haplo- In the most recent molecular approach (REGIER et al. types were found among the studied species. 2012) Enarmonnini was represented by a single All constructed trees (NJ, MP, ML, BI) showed genus Ancylis, which formed a clade separated the existence of four well separated clusters which both from Eucosmini and Grapholitini but was represent particular studied tribes (Enarmoniini, closer to them than to Olethreutini + Endotheniini Eucosmini, Grapholitini and Olethreutini), so we + Bactrini. These authors suggested that their data SystematicPositionofEnarmoniini 95

“do strongly support removal of Enarmoniini from Enarmonia, which is the type genus of the tribe, Eucosmini”. seems to be regarded as a more generalized genus At present the systematics of the Enarmoniini is than Ancylis. The phylogenetic relationships of its not yet resolved. The only recent approach con- seven species, based on sequences of the COI cerning the Australian fauna was revealed by mtDNA fragment, differ from that based on mor- HORAK (2006) because in RAZOWSKI (1976b) the phology, chiefly genital characters. In conclusion, diagnosesofthegeneraarenotsupportedbyaphy- future projects using comparative analysis of logenetic discussion. In this paper two Palaearctic DNA fragments should be verified by an analysis genera with their type-species were studied (only ofseveralgenitalcharactersaswellasaphylogeny one in REGIER et al. 2012). One representative of based on pheromones.

Fig. 1. Phylogenetic tree constructed for 8 species of Enarmoniini, 4 species of Eucosmini, 9 species of Grapholitini and 7 speciesofOlethreutini(twospeciesofPolyorthiniwereusedasanoutgroup). The tree was constructed on the basis of a comparison of sequences from mitochondrial COI fragment using the Bayesian inference method. Bootstrap values for neighbor joining, maximum parsimony analysis, maximum likelihood, and posterior probabilities for Bayesian inference are presented. Bootstrap values smaller than 50% (posterior probabilities <0.50) are not shown. Dashes represent no bootstrap or posterior value at a given node. All positions containing gaps and missing data were eliminated. Phylogenetic analyses were conducted using MEGA 5.2 (NJ/MP/ML) and MrBayes 3.1.2 (BI). The analysis involved30nucleotidesequences.Therewereatotalof606positionsinthefinaldataset. 96 J.RAZOWSKI,S.TARCZ

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