Ent. Tidskr. 132 (2011) DNA barcoding in species complexes of Stigmella DNA barcoding resolves species complexes in Stigmella salicis and S. aurella species groups and shows additional cryptic speciation in S. salicis (Lepidoptera: Nepticulidae) ERIK J. VAN NIEUKERKEN, MARKO MUTANEN & CAMIEL DOORENWEERD Nieukerken, E.J. van, Mutanen, M. & Doorenweerd, C.: DNA barcoding resolves species complexes in Stigmella salicis and S. aurella species groups and shows additional cryptic speciation in S. salicis (Lepidoptera: Nepticulidae). [DNA analyser visar artuppdeln- ingen inom Stigmella salicis- och S. aurella grupperna och att det finns ytterligare kryptiska arter inom S. salicis (Lepidoptera: Nepticulidae).] – Entomologisk Tidskrift 132 (4): 235-255. Uppsala, Sweden 2012. ISSN 0013-886x. We sequenced the mitochondrial barcoding marker COI and nuclear marker EF1-alpha for most Nordic and other European species of the Stigmella salicis and S. aurella species groups. In the S. salicis group both markers confirm the synonymy ofS. lappovimella with S. zelleriella. Specimens previously identified as Stigmella salicis and S. vimineticola are shown to form a complex of several cryptic species for which the taxonomy needs to be worked out. The species previously recorded as S. vimineticola from Norway represents probably an unnamed species. In the S. aurella group, the oligophagous Rosaceae feed- ers S. aurella and S. poterii are confirmed to be each a single oligophagous species. The synonymy between Stigmella ulmariae from Filipendula ulmaria and S. filipendulae from Filipendula vulgaris is corroborated. E.J. van Nieukerken & C. Doorenweerd, NCB Naturalis, PO Box 9517, NL-2300 RA Leiden, Netherlands, [email protected] M. Mutanen, Zoological Museum, Department of Biology, PO Box 3000, FI-90014 Univer- sity of Oulu, Finland, [email protected] The Lepidoptera fauna of northern Europe, in- 1974, the latest posthumously published: Svens- cluding Sweden, is amongst the best studied in son 2011). Furthermore he was interested in un- the world and with a very long history of study ravelling difficult species groups, resulting in a as well thanks particularly to Linnaeus, but also number of species descriptions (e.g. Svensson other early naturalists. Whereas in Linnaeus’ 1966, 1976), and had the strong opinion that a time the smallest moths were still mostly un- taxonomist should be careful when considering known, we now have an unprecedented know- lumping different forms into one species: in this ledge of their taxonomy, biology and distribution way one might lose important ecological infor- as, for instance, shown in the first two volumes mation. He called himself a splitter at the species dealing with Microlepidoptera in the prestigious level, in contrast to a conservative attitude to- series Nationalnyckeln (Bengtsson et al. 2008, wards higher classification (a lumper of genera) Bengtsson & Johansson 2011). Ingvar Svens- (Svensson 1992). son was a leading person in the study of Swedish His interest in species complexes and host- smaller moths, with his constant stream of annual plant races in leafminers often lead to heated de- updates of the checklist (starting with Svensson bates, e.g. during the biannual conferences of the 235 Erik J. van Nieukerken, Marko Mutanen & Camiel Doorenweerd Ent. Tidskr. 132 (2011) Societas Europaea Lepidopterologica, almost all cies complex, as suggested earlier by finding of which he visited between 1978 and 2009. In different forms in Norway and Sweden (Aarvik this paper we will consider some species com- et al. 2001, Aarvik et al. 2003, Bengtsson et al. plexes that had our joint interest and for which 2008). The extent of this complex, however, is Ingvar also brought material together for mol- remarkable. ecular research. After it appeared to be impos- DNA barcoding has shown in the last seven sible to get allozyme data from his material, years to be a useful addition for understanding it luckily could still be used for DNA analysis species complexes and cryptic species in Lepi- many years later. doptera (e. g. Hebert et al. 2004, Decaens & The genus Stigmella Schrank, 1802 is the Rougerie 2008, Hausmann et al. 2009, Huemer largest genus in the Nepticulidae, comprising & Hebert 2011, Ivanova et al. 2009, Segerer et to date 391 named species worldwide (Diškus al. 2010, Vaglia et al. 2008, Wilson et al. 2010). & Puplesis 2003, van Nieukerken 2010b), of We have started building a DNA barcode data- which 107 are known from Europe (van Nieu- base for Nepticulidae (van Nieukerken 2007, kerken 2011). The genus is rather homogeneous 2010a), and in Ectoedemia Busck, 1907 its in morphological characters, including the geni- usefulness is shown for European species (van talia, making it difficult to separate it into clear- Nieukerken et al. 2012), at the same time dem- cut subgenera. Instead, informal species groups onstrating that in one species complex, the E. are widely recognised and used in European lit- rubivora complex, only the nuclear gene EF1- erature (Emmet 1976, Johansson 1971, Johans- alpha was able to separate the species and not son & Nielsen 1990, van Nieukerken 1986), and the common barcode marker COI. Here we many of these probably represent monophyletic combine two datasets of Stigmella, one cover- entities, although a full phylogenetic study of ing a large part of the European fauna and also the genus is not yet available. Two of these spe- including sequences of EF1-alpha, and another cies groups are the subject of this paper. basically covering the Finnish fauna. We have The Stigmella salicis and aurella groups con- chosen to include EF1-alpha sequences to have tain several species or species complexes that an independent marker from a different genome have been the subject of many debates. Whereas (nuclear versus mitochondrial) in order to have a Emmet (1976) still recognised all host races better supported pattern and to rule out artefacts of Stigmella aurella (Fabricius, 1775) and of and anomalies of one marker (van Nieukerken S. poterii (Stainton, 1857) as different species, et al. 2012). later it became clear by conventional methods This study should not be seen as a taxonomic that these species had a broader host range than revision of these groups since not enough mate- previously thought (Johansson & Nielsen 1990, rial is yet available to study morphological char- Klimesch 1981). Here we corroborate these acters properly. It merely serves to indicate the findings by DNA barcodes and confirm a further complex nature of the taxonomy of these groups synonymy in the group. The Stigmella salicis and to encourage collecting and studying espe- group is a more complex one, with some species cially Salix feeding Stigmella. Only with a good complexes feeding on the same range of Salix representation of adult material from throughout species and some on a different range. One of Europe and from many hosts, paired with DNA these was of particular interest to Ingvar Svens- sequences, such a revision is possible. son: the pair S. zelleriella (Snellen, 1875) and S. lappovimella (Svensson, 1976). In this case, the Material and methods DNA barcode confirms what has been thought Material. Material used is very diverse, many by different authors (Johansson & Nielsen 1990, sequences were derived from larvae that were van Nieukerken 1983), that this particular pair sampled from their leafmines, and for which the is just one variable species. Ingvar knew about mines usually remain as voucher. Larvae were these results and had accepted them. Interest- stored in ethanol 96-100%, usually in a minus ingly, another species that he did not study so 80 freezer, but those collected by Ingvar Svens- much in detail, S. salicis, appears to form a spe- son had been several years in a minus 20 freezer 236 Ent. Tidskr. 132 (2011) DNA barcoding in species complexes of Stigmella in Lund University before they were sent to in volumes of 25µl, containing 0.4µM of each us (still in their mines) in ethanol 70%. These primer, 50µM dNTP, 1x Qiagen PCR buffer, mines were dried after taking out the larvae. 1.25 units of Qiagen Taq polymerase and 1µL Adults used for sequencing were either reared DNA template. All samples were sequenced in or collected as adult. Two methods of extrac- both directions on an aBi 3730 XL by Macro- tion were used: in Leiden usually the DNA was gen europe. SeQuencher 4.2 software was used extracted non-destructively from the abdomen to align the forward and reverse sequences, to when preparing genitalia slides (Knölke et al. manually check for ambiguities in the chro- 2005), but for the Finnish material usually one matograms and to export contigs. The sequences hindleg was used. of both markers contain no gaps or stop codons. The labels of each specimen in the figured The sequences, primer details as well as all trees are composed of the voucher registry chromatograms are posted on the BOLD web- number, (where RMNH.INS. is the registry for site. The EF1-α primers have been optimized for insects in the collection of the NCB Naturalis Nepticulidae (van Nieukerken et al. 2012) and (Leiden) and MM stands for Marko Mutanen can amplify a section of 482 bp. collection), the (initial) species name, the ISO Tree building. Neighbor-joining trees were code for the country and the hostplant name for created in paup* 4.0b10 (Swofford 2003) using larvae and reared adults. In total we sequenced uncorrected P distance rather than the frequently COI from 145 specimens (102 in the salicis used K2P distance (Srivathsan & Meier 2011). group and 43 in the aurella group) and EF1-al- Different trees were created for the two groups pha from 63 specimens (50 in the salicis group and two markers, using two representatives from and 13 only in the aurella group). No type mate- the other group as outgroup. Neighbor-joining rial was included in the sequenced material. trees serve to display the sequences by similar- Detailed data of the material and sequences ity, where the scale bar at the bottom can be used (including GenBank accession numbers) are to measure the difference between sequences.