Vol. 23(1): 153-162 Wrocław, 30 IV 2012

Molecular data on the systematic position of (: )

Józef Razowski1 & Sebastian Tarcz2 1Department of Invertebrate Zoology, Institute of Systematics and Evolution of , Polish Academy of Sciences, Kraków 31-016, Sławkowska 17, Poland. 2Department of Experimental Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków 31-016, Sławkowska 17, Poland. Corresponding autor: e-mail:[email protected]

Abstract. Due to the uncertain relationships between Bactrini and , we propose a preliminary molecular approach as a tool for resolving this taxonomic problem. There are no phylogenetic studies on the Bactrini or Endotheniini based on DNA variation of the mitochondrial cox1 gene. The present paper is the first comparative molecular study of representatives of the mentioned tribes. Based on molecular data, the synonymy of Endotheniini (genus ) with Bactrini (genus ) is confirmed.Bactra and Endothenia are compared with five genera of . Altogether, 16 of are examined. The paper constitutes a part of the project concerning a broad comparison of the system of Tortricidae based on morphological and molecular characteristics.

Key words: entomology, molecular biology, Lepidoptera, Tortricidae.

Introduction

A taxonomic system based on morphology of Tortricidae has been improving in the last 150 years but, like most other systems of groups, is far from definitive. Recent studies relate mainly to the systematic problems within the specific genera, such as species relationships. Nevertheless, the classification of many species to the various tribes and even genera is doubtful. A preliminary study based on comparative analysis of the cox1 gene fragment in the tribe (Razowski et al. 2010) showed an opportunity to improve the system within the tribes and genera. Thus, there is a possibility of revising the system of relationships of species and genera. 154 Józef Razowski, Sebastian Tarcz

The tribe Bactrini was described by Falkovitsh (1962) and includes the cosmopolitan genus Bactra Stephens, 1829. It was separated from Olethreutinae as a group parallel to Olethreutini on the basis of absence of a scent organ in the male. Falkovitsh considered the lack of male scent organs in Bactra as a primary distinguishing character. According to Diakonoff (1973), Bactrae (= Bactrini) consists of 6 genera. Bactra was usually subdivided into 7 subgenera, recently synonimized by Horak (2006). Endotheniini was erected by Diakonoff (1973) for Endothenia as Endotheniae, a subtribe of Olethreutini. Endotheniae included Endothenia and Molybdocrates, a new genus from the Moluccan Islands. Other genera, viz., Alloendothenia Oku, 1963 and Neothenia Diakonoff, 1973 proved synonymous. Horak & Brown (1991) tentatively included Endothenia in Gatesclarkeanini implicating that Endotheniini is a of the latter. Kuznetzov & Stekolnikov (1984), however, treated Endotheniini and Gatescklarkeanini as sister groups. Dang (1990) revised the status of Bactrini and placed Endothenia in this tribe based on four synapomorphies shared between Bactra and Endothenia. These included the following genital characters: the presence of a spiny terminal part of the strongly curved uncus with a dorso-basal concavity; the short, ventrally directed tegumen; the peculiar position of valva with reduced procesus basalis; and the reduction of setae of the distal part of the tarsus. Horak (2006) put forward more evidence for the mo- nophyly of Bactrini by adding the single, similar signum, the boring mode of feeding of the larvae, and the character of pheromones. Horak & Brown (1991) found that the pheromone (Z10-14 carbon acetate) of one species of Bactra is also an attractant for Endothenia. The supposition of Falkovitsh (1962) that the lack of scent organs in Bactra is a primary (diagnostic) character cannot be accepted, as in the closely related genus Endothenia a distinct pedal scent organ occurs. Bactrini was given a rather generalized position with several plesiomorphic characters. Horak (2006) discussed some of the characters and mentioned their distribution within the . They are either widely distributed in Olethreutinae or are not plesiomorphies (e.g. the feeding mode of the larvae). The tribal status of Bactrini was accepted by subsequent authors (e.g. Kuznetsov & Stekolnikov 1977) and only Diakonoff (1973) regarded it as a subtribe of Olethreutini. Horak (2006) based on several possible synapomorphies and the presence of the tibial scent organ of Endothenia and the Neopotamiae suggested that Bactrini “could well be subordinate within the Olethreutini, close to Neopotamia and Gatesclarkeana-groups.” Despite her preliminary cladistic analysis, she refrained to propose a new status for the group until a molecular analysis was conducted. Razowski (2003) treated Bactrini and Olethreutini as separate tribes and Endotheniina as a subtribe of the latter. There are a few studies on the molecular diversity of Tortricidae. Most evaluate the population structure of economically important species. The earliest analyses were based on allozyme comparisons. The degree of genetic differentiation of the larch budmoth diniana was studied using 24 allozyme loci (Emelianov et al. 1995). Restric- tion fragment analysis was applied to evaluate the genetic structure of viridana (Schroeder & Degen 2008) and economically important Tortricidae from South Africa Molecular data on the systematic position of Bactrini 155

(Timm et al. 2010). However, analysis of intra-specific relationships among Torticidae taxa are mostly based on comparisons of mitochondrial genome fragments, especially the cytochrome oxidase gene (cox1). The phylogenetic relationships of Argyrotaenia francisca were determined by analysis of a mitochondrial DNA fragment containing the genes cox1 and cox2 (2300 bp) (Landry et al. 1999). A similar analysis using the cox1 segment was carried out by comparing the sequences obtained from closely related species of the genus Archips (Kruse & Sperling 2001, 2002). A 940 bp cox1 fragment was ap- plied to discriminate between two types of orana (different food plants) in the Adoxophyes species complex (Lee et al. 2005). The mitochondrial cox1 gene was proposed as a universal DNA barcode (Hebert et al. 2003a, 2003b) and used for the first time in butterfly species identification in the Astraptes fulgerator species complex (Hebert et al. 2004). Application of the DNA barcode fragment confirmed polyphagy in mermerodes, because identical haplotypes of cox1 were obtained from multiple host plant families (Hulcr et al. 2007). Barcoding with part of the cox1 gene (460bp) successfully distinguished between two genera of New Zealand leafroller but in some cases within-species polymorphism was greater than between-species divergence (Langhoff et al. 2009). Because of the uncertain relationships between Bactrini and Endotheniini, we pro- posed a preliminary molecular approach as a tool for resolving this taxonomic problem. The present paper is the first comparative molecular study of representatives of the studied tribes.

Material and methods

The material was collected chiefly in southern Poland and is preserved in the Insti- tute of Systematics and Evolution of Animals, PAS, Cracow. Unfortunately, we could not obtain material from the Neopotomia and Gatesclarkeana groups, hence Bactra and Endothenia are compared only with five genera of Olethreutini. A representative of the tribe Tortricini, , was used as the outgroup. Our molecular study is based on 17 species belonging to eight genera listed in Table 1. We selected five genera of Olethretini ( , , , , Sricoris) and compared them with Bactra and Endothenia. The type species of the four studied genera and additionally at least two congeners were included with the exceptions of Olethreutes and Argyroploce.

Molecular methods DNA was usually extracted from two hind legs of dry specimens because we could not completely destroy the museum material using other parts of the bodies (e.g. the entire tagmata). On the other hand the collection of new, rare species was possible. The material was less than ten years old. Specimens older than ten years usually gave insufficient results. The best results were obtained from specimens preserved for one to three years. Genomic DNA was isolated without protocol modification using the NucleoSpin Tissue Kit (Macherey-Nagel, Germany). To elute purified DNA we applied 100μl of 156 Józef Razowski, Sebastian Tarcz

Elution Buffer (EB) onto the silica membrane. To amplify a fragment of the mitochon- drial cox1 gene (650bp) the following primer pair designed for Lepidoptera was used: LEP-F1, 5’-ATTCAACCAATCATAAAGATAT-3’; and LEP-R1, 5’-TAAACTTCTG- GATGTCCAAAAA-3’. They are universal primers used for species identification in DNA barcoding (HEBERT et al. 2004). PCR amplification of both markers was carried out in a final volume of 40 μl containing: 4μl of DNA, 1.5 U Taq-Polymerase (EurX, Poland), 0.6μl 20mM of each primer, 10x PCR buffer, 0.8μl of 10mM dNTPs in a Mastercycler ep (Eppendorf, Germany). The amplification protocol was the same as in Hebert et al. (2004). To check amplification, 10μl of each PCR product was electro- phoresed in 1% agarose gels for 45 min at 85V with a DNA molecular weight marker (Mass Ruler Low Range DNA Ladder, Fermentas, Lithuania). For purification of PCR reactions we used NucleoSpin Extract II (Macherey-Nagel, Germany). In some of the

Table 1. Species of Tortricidae used in present studies. Tortrix viridana was used as an outgroup.

DNA Lp. Tribe Genus Species Origin cox1 acc Voucher 1. TORT121 Bactrini Małoszyn, Poland JF730056

2. TORT123 Bactrini Bactra lacteana Kosyń, Poland JF730057

3. MM06531 Bactrini unknown GU828745 Puszcza 4. TORT108 Bactrini Niepołomicka, JF730058 Poland Kraków-Tyniec, 5. TORT101 Bactrini JF730059 Poland 6. TORT131 Olethreutini Hajnówka, Poland JF730060

7. TORT132 Olethreutini Kojszówka, Poland JF730061 Brzoskwinia, 8. TORT127 Olethreutini JF730062 Poland 9. TORT070 Olethreutini Lucynów, Poland JF730063 Brzoskwinia, 10. TORT103 Olethreutini JF730064 Poland Kraków-Nowa 11. TORT109 Olethreutini JF730065 Huta, Poland Brzoskwinia, 12. TORT104 Olethreutini JF730066 Poland Puszcza 13. TORT102 Olethreutini Olethreutes subtilana Niepołomicka, JF730067 Poland Brzoskwinia, 14. TORT105 Olethreutini lacunana JF730068 Poland Brzoskwinia, 15. TORT106 Olethreutini Syricoris rivulana JF730069 Poland Kraków-Nowa 16. TORT112 Olethreutini Syricoris siderana JF730070 Huta, Poland 17. Ac13 Tortricini Tortrix viridana Kraków, Poland GU989457 Molecular data on the systematic position of Bactrini 157

PCR reactions apart from main band additional sub-bands were obtained. In these cases 30μl of each PCR product was separated on a 1.8 % agarose gel (100V/60min). Then, the band representing the examined fragment was cut out and purified. Cycle sequencing was done in both directions with application of the BigDye Terminator v3.1 chemistry (Applied Biosystems, USA). Primers LEP-F1 and LEP-R1 were used for sequencing. Each sequencing reaction was carried out in a final volume of 10μl containing: 3μl of template, 1μl of BigDye (1/4 of standard reaction), 1μl of sequencing buffer, 1μl of 5mM primer. Sequencing products were precipitated using Ex Terminator (A&A Biotechnology, Poland) and separated on an ABI PRISM 377 DNA Sequencer (Applied Biosystems, USA). Sequences are available in the GenBank database (for accession numbers see Table 1).

Data analysis Sequences were examined by eye using Chromas Lite (Technelysium, Australia) to evaluate and correct chromatograms. Alignment and consensus of the studied sequences were performed using Clustal W (Thompson et al. 1994) in the BioEdit program (Hall 1999) and checked manually. All obtained sequences were unambiguous and were used for analysis. Phylograms were constructed for the studied fragments in MEGA version 5.0 (Tamura et al. 2007), using the Neighbor-Joining method (NJ) (Saitou & Nei 1987). The NJ analysis was performed using p-distance, Kimura 2-parameter and Maximum Composite Likelihood correction model (Kimura 1980) by bootstrapping with 1000 replicates (Felsenstein 1985). The haplotype diversity, nucleotide diversity and analysis of variable nucleotide positions were calculated in DnaSP v. 5.10.01 (Lib- rado & Rozas 2009). The nucleotide frequencies were computed with MEGA version 5.0 (Tamura et al. 2004, 2007).

Results and Discussion

A total of 17 sequences of the gene encoding cytochrome oxidase subunit I (606 bp) from species of Bactrini and Olethreutini were used in this study. Of these, 15 were newly obtained, while the remaining sequences were taken from GenBank - for Bactra lancealana - and from a previous study - for Tortrix viridana – (Razowski et al. 2010). The interspecific haplotype diversity value wasH d=1. Nucleotide diversity amoun- ted to π=0.0928. The nucleotide frequencies were A=0.313, T=0.369, C=0.164 G=0.154 and revealed a high proportion of A-T pairs which corresponds with typical charac- teristics of mitochondrial DNA. Mean divergence over all studied Tortricidae sequence pairs was p=0.093/SE=0.007 (p-distance/standard error). Mean divergence over all studied Bactrini sequence pairs was p=0.083/SE=0.008 and Olethreutini (p=0.080/SE=0.006). More detailed information about divergences between particular species are presented in Table 2. We found 175 variable positions across all studied species in the cox1 fragment, 121 of which were parsimony informative (68 with two variants, 46 - three variants, 158 Józef Razowski, Sebastian Tarcz 17. 0.017 0.017 0.015 0.016 0.017 0.017 0.017 0.016 0.017 0.016 0.018 0.016 0.016 0.014 0.015 0.014 16. 0.011 0.011 0.011 0.013 0.012 0.012 0.014 0.014 0.013 0.013 0.012 0.013 0.015 0.012 0.010 0.107 15. 0.011 0.011 0.116 0.012 0.012 0.010 0.013 0.015 0.014 0.014 0.012 0.012 0.013 0.014 0.012 0.066 14. 0.011 0.012 0.013 0.013 0.015 0.015 0.015 0.012 0.013 0.013 0.014 0.013 0.012 0.066 0.070 0.107 13. 0.013 0.013 0.013 0.014 0.016 0.015 0.013 0.014 0.015 0.014 0.015 0.015 0.084 0.077 0.084 0.129 species. Analyses were conducted using the

12. 0.117 0.110 0.015 0.014 0.014 0.016 0.016 0.015 0.015 0.014 0.014 0.015 0.013 0.100 0.109 0.128 11. 0.014 0.014 0.013 0.015 0.015 0.014 0.015 0.014 0.015 0.014 0.086 0.105 0.103 0.095 0.088 0.137 10. 0.114 0.117 0.014 0.013 0.013 0.014 0.016 0.015 0.015 0.012 0.012 0.108 0.098 0.088 0.074 0.138 9. 0.116 0.014 0.014 0.013 0.015 0.015 0.016 0.014 0.010 0.084 0.120 0.109 0.088 0.084 0.085 0.142 mtDNA fragment mtDNA (lower-left) are shown. Standard error estimate(s) are cox1 8. 0.014 0.013 0.012 0.014 0.016 0.015 0.015 0.062 0.085 0.099 0.107 0.090 0.075 0.070 0.070 0.123 7. 0.111 0.111 0.110 0.116 0.015 0.014 0.014 0.014 0.010 0.009 0.108 0.106 0.106 0.101 0.088 0.137 6. 0.111 0.111 0.118 0.112 0.015 0.015 0.014 0.015 0.008 0.041 0.125 0.125 0.106 0.109 0.096 0.133 5. 0.115 0.113 0.116 0.119 0.116 0.119 0.016 0.015 0.015 0.015 0.043 0.061 0.131 0.126 0.099 0.141 4. 0.114 0.114 0.118 0.110 0.014 0.013 0.012 0.102 0.104 0.127 0.139 0.097 0.100 0.095 0.099 0.127 3. 0.113 0.110 0.008 0.007 0.078 0.103 0.103 0.081 0.090 0.090 0.103 0.086 0.075 0.063 0.074 0.120 2. 0.119 0.113 0.114 0.006 0.034 0.093 0.107 0.100 0.102 0.108 0.107 0.094 0.092 0.091 0.089 0.141 1. 0.111 0.119 0.113 0.118 0.022 0.032 0.101 0.125 0.102 0.098 0.106 0.098 0.089 0.087 0.089 0.147 Celypha rufana Tortrix viridana Tortrix Bactra lacteana Celypha striana Bactra furfurana Syricoris rivulana Apotomis inudana Syricoris siderana Bactra lancealana Celypha cespitana Apotomis sauciana Syricoris lacunnana Olethreutes arcuellus Olethreutes Argyroploce arbutella Argyroploce Apotomis sororculana Endothenia ericentana Endothenia marginana shown above the diagonal. All results are based on the pairwise analysis of 17 sequences of studied Tortricidae p-distance in MEGA5. 1. 2. 3. 4. 5. 6. 7. 8. 9. 11. 10. 12. 13. 14. 15. 16. 17. Table 2. Table The number of base substitutions per site from analysis between sequences Molecular data on the systematic position of Bactrini 159

6 - four variants). Among species (N=5) belonging to Bactrini there were 99 variable positions (34 parsimony informative); Olethreutini (N=11) differed in 140 (73 parsimony informative). A total of 17 haplotypes were found among the studied species. The proposed phylogenetic reconstruction produced two main groups on the phylogenetic tree (Fig.1). One group contains genera of Olethreutini (Apotomis, Ar- gyroploce, Celypha, Olethreutes, Syricoris) and the other group - genera of Bactrini (Bactria, Endothenia). The phylogenetic tree revealed that Bactra and Endothenia are closely related and form a monophyletic clade with two groupings - Bactra (bootstrap support 100 p-distance; 100 K2P, 100 MCL) and Endothenia (bootstrap support 88 p-distance; 85 K2P, 91 MCL). The two groupings represent the tribe Bactrini which has priority over Neopotamiae and Gatescklarkeanini. The latter could be eventually incorporated within Bactrini on the basis of further molecular studies (if the supposition by Horak & Brown (1991) based on morphology is confirmed or treated separately).Bactra and Endothenia represent, on the basis of the present study, sister genera. Almost all species of Bactra show great external and genital similarity throughout their entire area of distribution and in many cases are difficult to identify. Their biology and host plant preferences also contribute to this state of affairs. The arrangement of the species obtained in this study essentially fits that proposed by Razowski (2003) based chiefly on external anatomy. The species of Endothenia are morphologically and biologically more variable than representatives of Bactra. The examined European species are very closely related to one another and the proposed arrangement agrees with that of Razowski (2003). It is in congruence with our results in which mean genetic distance between Endothenia species is greater (p=0.081) than across Bactra (p=0.046). Based on the examination of 16 species and five genera we suggest that Bactrini and Olethreutini represent two distinct but not subordinate groupings (bootstrap support 45

1. Phylogenetic tree constructed for 16 species of Bactrini and Olethreutini (Tortrix viridana, Tortricini as an outgroup). The evolutionary history was inferred using the neighbor joining method. The bootstrap con- sensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The evolutionary distances were computed using the p-distance method and are in the units of the number of base differences per site. The analysis involved 17 nucleotide sequences. There were a total of 606 positions in the final dataset. 160 Józef Razowski, Sebastian Tarcz p-distance; 48 K2P, 45 MCL). Olethreutini are more specialized than Bactrini. A more complete study on the Olethreutini genera may confirm this supposition. The studied genera should, however, constitute a sufficient sample to characterize the tribe of Olethreutini. In the system based on morphology there are many problems in distinguishing the genera and often most species are grouped under one name, Olethreutes. In our approach, Olethreutes, Argyroploce, and Apotomis represent a clade (bootstrap support 26 p-distance; 27 K2P, 24 MLC) sister to the clade of Celypha + Syricoris. The obtained arrangement of genera is congruent with that of Razowski (2003). In the present analyses a stronger bootstrap support is revealed for species across particular genera than between genera. A similar situation was observed across European Tortricini (Razowski et al. 2010). This could be caused by the outstanding variability of the amplifiedcox1 fragment which confirms its utility as a barcode marker. An excep- tion occurs in the case of Syricoris siderana which is in one clade with Celypha. Low support (12 p-distance; 11 K2P, 12MLC) for the Celypha + Syricoris clade confirms that these two genera are close to one another. Hd=1 confirms the great variability of thecox1 fragment and its utility for barcoding. Investigations like the one presented above, are just the beginning to accumulate and there are no comprehensive studies, at least in this (economically important) fa- mily of Lepidoptera. Applying of DNA fragments give a possibility of revision and confirmation of the taxon placements across Tortricidae.

Conclusions

Endotheniini and Bactrini are synonymous, confirming the diagnosis by Dang (1990). Bactra is more generalized than Endothenia and Bactrini are more primitive than Olethreutini. Our proposed systematic arrangement of the examined genera is as follows:

Tribe Bactrini Genus Bactra Genus Endothenia Tribe Olethreutini Genus Apotomis Genus Argyroploce Genus Celypha Genus Olethreutes Genus Syricoris

Acknowledgements The authors are grateful to Mr Witold Zajda, Department of Invertebrate Zoology, Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków for providing and preparing material for DNA isolation. Molecular data on the systematic position of Bactrini 161

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