Ent. Tidskr. 138 (2017) Cryptic Leptidea species in Finland DNA barcoding reveals widespread occurrence of Leptidea juvernica (Lepidoptera: Pieridae) in southern Finland SAMULI LEHTONEN*, ILARI LEHTONEN, ANSSI TERÄS, JUHA VARRELA, PASI VIRTA & EERO J. VESTERINEN Lehtonen, S., Lehtonen, I., Teräs, A., Varrela, J., Virta, P. & Vesterinen, E.J.: DNA bar- coding reveals widespread occurrence of Leptidea juvernica (Lepidoptera: Pieridae) in southern Finland. [DNA-analys visar att ängsvitvingen Leptidea juvernica är utbredd i stora delar av Finland.] – Entomologisk Tidskrift 138(2): 151-159. Uppsala Sweden, 2017. ISSN 0013-886x. DNA barcoding was used to identify 54 specimens of butterfly genus Leptidea collected from various parts of southern Finland in 2011-2013. Results reveal the presence of both the widespread Leptidea sinapis and its cryptic congener L. juvernica from several loca- tions throughout the southern Finland. Our sampling also reveals different habitat prefer- ences between these species in Finland: specimens collected from open, disturbed habitats were mainly identified as L. juvernica, whereas specimens from forest habitats were all found to represent L. sinapis. A morphometric analysis revealed that L. juvernica and L. sinapis hardly differ by their fore wing shape, although males and females seem to differ from each other. Our attempts to DNA barcode selected museum specimens failed and we were not able to verify historical presence of L. juvernica in Finland. The recently increased observations of Leptidea butterflies in large numbers in unusually open habitats across the southern Finland together with our findings suggests ongoing rapid expansion of L. juvernica in Finland. S. Lehtonen, Biodiversity Unit, University of Turku, FI-20014 Turku, Finland. E-mail: [email protected], Corresponding author I. Lehtonen, Finnish Meteorological Institute, P.O.Box 503, FI-00101 Helsinki, Finland A. Teräs, Biological Collections of Åbo Akademi University, Zoological Museum, Univer- sity of Turku, FI-20014 Turku, Finland J. Varrela, Department of Oral Development and Orthodontics, University of Turku, FI-20014 Turku, Finland P. Virta, Department of Chemistry, University of Turku, FI-20014 Turku, Finland Turku, FI-20014 Turku, Finland E.J. Vesterinen, Department of Biology, University of Turku, FI-20014 Turku, Finland; Spatial Foodweb Ecology Group, Department of Agricultural Sciences, University of Hel- sinki, FI-00014 Helsinki, Finland The emergence of molecular systematics and cies throughout Europe, until it was first splitted application of DNA barcoding is helping to re- into two morphologically cryptic species (Réal vise the taxonomy and species delimitation in 1988), and not long after realised to consist of many Lepidoptera groups (e.g. Hausman et al. three genetically distinct species (Dinca et al. 2011). One of the most remarkable examples is 2011). These three species, L. sinapis, L. reali in the genus Leptidea Billberg (Pieridae). The Reissinger, 1990 and L. juvernica (Williams, species Leptidea sinapis (Linnaeus, 1758) was 1946) can be easily differentiated using DNA considered as a widespread and common spe- sequence data, yet it is very difficult to tell them 151 Samuli Lehtonen et al. Ent. Tidskr. 138 (2017) a b c d e f Figure 1. Typical habitats of the two cryptic Leptidea species in Finland: – a) open landfill area in Helsinki Vuosaari where Leptidea juvernica occured in 2012-2013, – b) thinned spruce-dominated moist forest with abundant herbs on the forest floor in Pargas, Lemlaxön. Leptidea sinapis was abundant on this site in 2013. Butterflies – c)L. juvernica male, – d) L. juvernica female, – e) L. sinapis male and – f) L. sinapis female, all DNA identified first generation individuals. Typiska habitat för de två kryptiska vitvingearterna i Finland: – a) öppen ruderatmark i Helsingfors, Nordsjö där ängsvitvingen Leptidea juvernica fanns 2012-2013, – b) gallrad örtrik granskog, Pargas, Lemlaxön där skogsvitvingen Leptidea sinapis var vanlig 2013. Fjärilarna: – c) L. juvernica hane, – d) L. juvernica hona, – e) L. sinapis hane och – f) L. sinapis hona, alla DNA identifierade exemplar från årets första generation. apart morphologically (Dinca et al. 2011, Mazel is L. juvernica, although its range is difficult to 2012). The two widely distributed and largely document because of the morphological simi- sympatric species, L. sinapis and L. juvernica larity with the very common and widespread (Fig. 1 c-f) seem to have constant differences L. sinapis. An opposite trend in the ranges of in the genitalia (e.g. Sachanowicz 2013), and it these two species has been revealed in Poland, has been suggested that the species can be sepa- where L. sinapis has declined and L. juvernica rated by the coloration of the dorsal apical spot expanded during the 20th century (Sachanowicz (Mazel 2012), or slightly different coloration of et al. 2011). In Poland, L. sinapis favour wood- wing undersides and more attenuate fore wing lands and L. juvernica open meadows, and the apex in L. juvernica (Ivonin et al. 2009). In latter species may therefore be better adapted contrast, Solovyev et al. (2015) concluded that to human altered habitats (Sachanowicz et al. neither external nor genital morphology could 2011). In the face of habitat degradation and cli- provide a reliable identification. mate change it is therefore crucial to better un- At the same time with a taxonomical overhaul derstand the distribution and population trends the distributions of many lepidopteran species of these two species across their geographical are in flux, apparently due to climate warming range (Beneš et al. 2003). In Finland, the occur- (Hill et al. 2002). In Finland, this has resulted rence of L. juvernica was first verified by geni- in new species records and northward migration tal identification from Åland Islands in the year of the lepidopteran fauna (Pöyry et al. 2009). 2000 (Suomen Perhostutkijain Seura ry 2015). One potential such climate driven expansion The first Finnish DNA barcoded specimens ofL. 152 Ent. Tidskr. 138 (2017) Cryptic Leptidea species in Finland juvernica were reported in 2013 from Lappeen- from SW Finland (AAT-2015-004 Ab: Paimio ranta, eastern Finland (Saarinen et al. 2013). 1932; AAT-2015-001 & AAT-2015-005 Ab: Since then, increasing numbers of Leptidea but- Kakskerta 1973; AAT-2015-003 Ab: Kaksker- terflies have been observed from open habitats, ta 1976; AAT-2015-006 Ab: Kakskerta 1982; previously unoccupied by Leptidea, throughout AAT-2015-002 Ab: Turku 1992). One leg was southern Finland (Saarinen 2017). In Sweden, detached from each of these specimens and used L. sinapis is a habitat generalist occurring both for DNA extraction, but we failed to produce in forests and open meadows, in contrast to L. any sequence data from them. juvernica, which is a specialist of open habitats (Friberg et al. 2008a,b). It therefore appears likely that the ongoing expansion of Leptidea in Field sampling open habitats across the southern Finland rep- During 2011-2013 we repeatedly observed resents invasion by L. juvernica. However, due Leptidea specimens in open habitats generally to the cryptic morphology the actual range and avoided by L. sinapis in Finland. These were population trends of these two species have re- suspected to belong to L. juvernica instead, and mained unverified in Finland. we opportunistically sampled specimens from Here, we have DNA barcoded 54 Leptidea these open habitats as well as from more for- specimens in order to estimate the range of L. ested sites using a butterfly sweep net. The sam- juvernica in southern Finland. The specimens pling localities are described below. were collected from several locations from Åland Islands through Finnish southern coast Finland: Al Eckerö, Torp. In total 14 first generation to East Finland during 2011-2013. It is possible specimens were collected as L. juvernica from a that L. juvernica has been part of Finnish fauna west-posing slope having a gradient of humid to already for a long time, or that the species have dry meadow-like vegetation in 2012. DNA bar- L. had short-living populations during favourable coding revealed that one of these was actually sinapis (see below). Coll. Virta. years. We therefore investigated historical col- Finland: Ab Pargas, Lemlaxön. Most specimens ob- lections in order to test the hypothesis that L. served in this location were found flying at forest juvernica is a new member of Finnish fauna. edges and were suspected to belong to L. sinapis. Furthermore, we ran a morphometric analysis to For five sampled specimen this identification was test numerical support for the perceived differ- confirmed by DNA barcoding. However, in May ence in the fore wing shape. Our results revealed 2013 one individual was collected from an un- widespread occurence of L. juvernica through- plowed harvested field, flying close to the forest out this region and suggests that the two species edge. This specimen was assumed and later con- cannot be reliably identified by their fore wing firmed to represent L. juvernica. All these speci- shape. mens represent the first generation. Coll. Karhu & coll. Lehtonen. Finland: Ab Salo, Tupuri. A single second genera- Material and methods tion specimen of L. juvernica was collected from a humid grass-dominated meadow surrounded by Sampling of natural history collections forests. Coll. Ruokonen. The butterfly collections located at the Zoologi- Finland: N Helsinki, Vuosaari. About 20 first genera- cal Museum of the University of Turku (also tion individuals of L. juvernica were observed in holding collections of Åbo Akademi) were vi- May and early June 2012 in the neighbourhood sually inspected by AT considering characteris- of a landfill hill on open habitats, both on dry and tics that have been suggested useful in telling L. moist meadow, grassland and wasteland areas. juvernica apart from L. sinapis, specifically the In 2013, four specimens were observed in May. Coll. Lehtonen. shape and darkness of the dorsal apical spot and Finland: N Sipoo. A single first generation specimen the fore wing shape (Ivonin et al. 2009, Mazel of L.