ESM Annex 3 Species pairs with difficult morphology-based identification that can be reliably separated through DNA barcoding

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1 ESM Annex 3 Species pairs with difficult morphology-based identification that can be reliably separated through DNA barcoding Leptidea sinapis (Linnaeus, 1758) Leptidea reali Reissinger, 1989 Fig. S1. Representative wing vouchers of barcoded Leptidea sinapis and L. reali specimens. a-b. L. sinapis (males) of the spring brood. c. L. sinapis (male) of the summer brood. d-e. L. reali (males) of the spring brood. f. L. reali (male) of the summer brood. Scale bar is 10 mm. Fig. S2. Lateral view of representative male genitalia of barcoded Leptidea sinapis (a) and L. reali (b). Scale bar is 0.5 mm. Wing morphology: As numerous studies have already shown (e.g. Lorković 1993; Mazel 2000, 2001; Vila et al. 2003), reliable identifications are not possible based on this character alone due to high intraspecific variability that broadly overlaps between the two taxa (fig. S1a-f). Genitalia: Previous studies involving morphometry of the genitalia showed that the length of the saccus and phallus are the best characters that allow for the separation of the males, and the length of the ductus bursae for females (e.g. Lorković 1993; Kristal & Nässig 1996; Mazel & Leestmans 1996, 1999; Martin et al. 2003; Vila et al. 2003; Fumi 2008). Therefore, for male specimens, short phallus and saccus indicate that the specimen is L. sinapis (fig. S2a), and long phallus and saccus indicate that it is L. reali (fig. S2b). We measured these elements and normalized them by vinculum width, to minimize the effect of the general size of the specimen. Bivariate scatter plot (fig. 2a of the main text), showed the presence of two clearly separated morphotypes attributable to L. sinapis and L. reali. For female specimens, short ductus bursae corresponds to L. sinapis, while a long one

2 corresponds to L. reali. We analyzed this character for the two barcoded female specimens and found that they both were L. sinapis. DNA barcoding: Two clusters displaying a minimum between-cluster distance of 2.8% were obtained (fig. 2b of the main text). The results obtained through genitalia examination perfectly matched the distribution of the specimens in the two DNA barcode clusters showing that DNA barcoding can reliably identify the two species. Comments: Some studies applying morphometry of the male genitalia for the species pair L. sinapis L. reali found slight overlaps between the lengths of the two characters (e.g. Martin et al. 2003; Verovnik & Glogovčan 2007; Fumi 2008) and the existence of natural hybrids has been proposed (Verovnik & Glogovčan 2007). However, Fumi (2008) showed that, by employing additional genitalic characters (especially the vinculum width) and performing multivariate statistics, 100% correct classification is achieved even in datasets involving intermediates based only on phallus and saccus length. While specimens with intermediate morphology might exist, normalizing the variables by vinculum width should improve the separation of the two species by eliminating the effect of size. Leptidea sinapis is common and widespread in Romania. L. reali however, based on published data (Rákosy 1996) and our observations, seems to be much more localized and usually associated to humid and relatively cold areas. In the sampled localities, we always found L. reali in sympatry with L. sinapis.

3 Colias alfacariensis Ribbe, 1905 Colias hyale (Linnaeus, 1758) Fig. S3. Representative wing vouchers of barcoded Colias alfacariensis and C. hyale. a-c. males of Colias alfacariensis; d. female of C. alfacariensis. e-g. males of C. hyale. h. female of C. hyale. Scale bar is 10 mm. Fig. S4. Antero-posterior view of representative male genitalia of Colias alfacariensis (a-b) and C. hyale (cd). Phallus is shown below in lateral view. Scale bar is 1 mm. Wing morphology: Below we present the diagnostic characters often invoked in literature (e.g. Higgins et al. 1991; Lafranchis 2004; Slamka 2004; Tolman & Lewington 2008) and analyze them based on our barcoded material. The extension of the basal shading of the forewings upperside (less marked in C. alfacariensis, where it continues to some extent along the inner margin) found not to be constant enough (e.g. compare figs S3b,c with fig. S3e, where this character would rather assign fig. S3e to C. alfacariensis and figs S3b,c to C. hyale).

4 The colour of the discoidal orange spot on the hindwings upperside (brighter orange in C. alfacariensis) it is a subjective character as colour is variable according to habitat type and locality, as well as by the condition of the specimen (a slightly worn out adult may display a paler orange spot). The outer margin of the forewing (slightly rounded in C. alfacariensis, straight in C. hyale) not constant enough (e.g. compare figs S3b,c with figs S3e,f where the specimens of C. alfacariensis have a straighter outer margin). The overall colour of the upperside of the wings (bright lemon yellow in C. alfacariensis and pale lemon yellow in C. hyale) - it is a very subjective character as colour is variable according to habitat type and locality, as well as by the condition of the specimen (a slightly worn out adult may look paler yellow). Therefore, the reliable separation between C. alfacariensis and C. hyale based on wing morphology is hazardous, excepting the occasions in which several of the characters mentioned above can be found in a certain specimen. Such cases however appear not to be very frequent. Moreover, females (figs S3d,h) are even more similar and no reliable diagnostic character has been observed by us. Male genitalia: No constant diagnostic characters have been found based on our dataset (fig. S4a-d). We agree to the opinion expressed by Higgins (1975) who mentioned that the characters recorded by Niculescu (1963) may not be well marked in all specimens and therefore do not allow for the reliable separation of the two taxa. Fig. S5. Neighbour-joining tree of COI barcodes of Romanian Colias alfacariensis and C. hyale with bootstrap support values >50% indicated.

5 DNA barcoding: The resulting tree (fig. S5) shows two well-diverged clades that can be attributed to C. hyale and C. alfacariensis, based on the few morphologically typical specimens. The six specimens in the C. hyale clade have identical barcodes and their minimum interspecific distance with respect to the sister C. alfacariensis clade is 2.65%. Because morphology-based identification is difficult in this group, we compared our sequences with those present in GenBank (results not shown) and found that the general tree topology is maintained, confirming our interpretation. With these data, we conclude that DNA barcoding is able to discriminate between the two species. Comments: The two species were reported to differ in larval morphology so that, apart from DNA barcoding, they may be safely identified during larval stage (Slamka 2004; Tolman & Lewington 2008). According to our data, in Romania C. hyale prefers more humid and colder habitats compared to C. alfacariensis, although the separation in habitat niches is not at all absolute.

6 Melitaea athalia (Rottemburg, 1775) Melitaea aurelia Nickerl, 1850 Melitaea britomartis Assmann, 1847 Fig. S6. Wing vouchers of representative barcoded specimens of Melitaea athalia (a-e), M. aurelia (f-j) and M. britomartis (k, l). Scale bar is 10 mm. Wing morphology: It is useful only to a limited extent for discriminating between these three species. One of the most useful features is the thin marginal band on the hindwings underside which is usually of the same colour or slightly darker than the adjacent lunules in M. athalia (fig. S6a-e), while in M. aurelia (fig. S6f-j) and M. britomartis (fig. S6k,l) it is darker (more orange) (Higgins et al. 1991; Lafranchis 2004; Slamka 2004; Tolman & Lewington 2008). However, the three species display pronounced variability overlapping at interspecific level so that wing-based identification is often difficult, especially between M. aurelia and M. britomartis. Male genitalia: It allows for reliable separation between the three taxa. One of the most useful features is related to the sub-unci of the tegumen: well developed (long) in M. athalia (fig. S7a,d), short and thick in M. britomartis (fig. S7b,e) and very small or absent in M. aurelia (fig. S7c,f). Moreover, the ostium-keel of the phallus is especially distinctive in M. aurelia, where it has an upturned apex and a number of cornuti on each side (fig. S7c,f) (Issekutz & Kovács 1954; Higgins 1975). DNA barcoding: The three taxa appear as sister species and form distinct clusters (fig. S8). The minimum interspecific distances are as follows: M. athalia M. aurelia, 4.3%; M. athalia M. britomartis, 2.65%; M. aurelia M. britomartis, 4.9%. The sample composition of the clusters is in perfect accordance to the identifications obtained based on genitalic examination and we therefore conclude that DNA barcoding can reliably differentiate between these three species. Comments: in Romania, depending on locality, often two or even all three species can be sympatric, increasing identification difficulties in the field based on wing morphology.

7 Fig. S7. Detail of representative male genitalia (tegumen with sub-unci and terminal part of the phallus in lateral view) of M. athalia (a, d), M. britomartis (b, e) and M. aurelia (c, f). Scale bar is 0.5 mm. Fig. S8. Neighbour-joining tree of COI barcodes of Romanian Melitaea athalia, M. britomartis and M. aurelia with bootstrap support values >50% indicated.

8 Plebejus argyrognomon (Bergsträsser, 1779) Plebejus idas (Linnaeus, 1761) Fig. S9. Wing vouchers of representative barcoded Plebejus argyrognomon and P. idas. a, b. males of P. argyrognomon. c, d. males of P. idas. Scale bar is 10 mm. Fig. S10. Detail of representative male genitalia (labides and falces) of Plebejus argyrognomon and P. idas. a. P. argyrognomon. b. P. idas. Scale bar is 0.5 mm. Fig. S11. Labides and falces of male genitalia of P. argyrognomon with measured variables used in morphometrical analyses indicated. PpF proximal part of falces; DpF distal part of falces. Scale bar is 0.5 mm. Wing morphology: For the Romanian specimens, we found that wing morphology is insufficient to allow for a safe separation between these two species. Characters invoked in literature such as the ground colour of the upperside and underside of the wings and the shape of the black lunules bordering the orange submarginal spots (said to be usually sharper in P. idas), do not seem to be constant enough to allow for reliable identifications (fig. S9a-d). Difficulties in separating the two species based on wing morphology have been acknowledged in literature (e.g. Higgins et al. 1991). Male genitalia: It allows for species level identification. The proximal and distal parts of the falces are more or less equal in P. idas, while in P. argyrognomon the distal part of the falces is considerably longer (Higgins 1975) (fig. S10a,b). We measured these characters for the barcoded specimens (fig. S11) and found that this feature points out two groups attributable to P. argyrognomon and P. idas (fig. S12). DNA barcoding: These species formed two clusters whose sample composition corresponded to identifications based on male genitalia (16 P. argyrognomon and two P. idas) (fig. S13). Maximum intraspecific variability was very low in both species (0.15% in P. argyrognomon and 0% in P. idas). The minimum interspecific distance was also low (0.46%) but, given the very low levels of intraspecific variability (especially for P. argyrognomon where the sample size is rather large), it seems that, for Romanian specimens, DNA barcoding can reliably separate the two species. Comments: In Romania, P. argyrognomon and P. idas can be sometimes found in sympatry.

9 Fig. S12. Bivariate scatter plot using the distal part of falces (DpF) and the proximal part of falces (PpF) as discriminative characters. Circles Plebejus argyrognomon; triangles P. idas. Fig. S13. Neighbour-joining tree of COI barcodes of Romanian Plebejus argyrognomon and P. idas with bootstrap support values >50% indicated.

10 Aricia agestis ([Denis & Schiffermüller], 1775) Aricia artaxerxes (Fabricius, 1793) Fig. S14. Wing vouchers of representative barcoded Aricia agestis (a-d) and A. artaxerxes (e, f). Scale bar is 10 mm Fig. S15. Detail of representative male genitalia (labides and falces) of Aricia agestis (a, c) and A. artaxerxes (b, d). Scale bar is 0.5 mm. Wing morphology: Reliable identification is often not possible based on wing morphology. Although A. agestis usually has more developed submarginal orange spots (fig. S14a,b) compared to A. artaxerxes (fig. S14e, f), there are cases in which A. agestis has these spots little developed (fig S14c, d), especially in specimens from colder areas or higher elevations. Male genitalia: No characters of the male genitalia allow for a reliable separation between the two species. The ratio between the labides and the falces (fig S15a-d) seems to be not useful in the case of A. agestis and A. artaxerxes (Higgins 1975). DNA barcoding: Two clusters (fig. S16) correspond to the two species, based on specimens collected in typical areas. The minimum interspecific distance is 2%. With the exception of one sample (collected at ca. 800 m), all A. artaxerxes specimens were collected between m in habitats that are typical for this species. The specimens of A. agestis were collected between m. The current data suggest that DNA barcoding is able to reliably separate these two species. Comments: Apart from molecular techniques, A. agestis and A. artaxerxes can be sometimes identified by taking into account wing morphology combined with habitat information. In Romania A. agestis flies from near sea level to probably about 1400 m (published data on the upper altitudinal limit are to be regarded with reserves due to possible confusions with A. artaxerxes). A. artaxerxes typically flies above 1300 m, although exceptions have been found (e.g. sample EZRMN was collected at ca. 800 m). Therefore, although identification up to species level is possible for A. agestis specimens flying at low elevations, the situation is different for mountainous areas where there is overlap in altitudinal ranges (and often in wing morphology). In such cases, molecular markers such as COI represent the most reliable identification method.

11 Fig. S16. Neighbour-joining tree of COI barcodes of Romanian A. agestis and A. artaxerxes with bootstrap support values >50% indicated.

12 References Fumi, M Distinguishing between Leptidea sinapis and L. reali (Lepidoptera: Pieridae) using a morphometric approach: impact of measurement error on the discriminative characters. Zootaxa 1819, Higgins, L. G The Classification of European butterflies. London: Collins. Higgins, L., Hargreaves, B. & Lhonoré, J Guide complet des papillons d Europe et d Afrique du nord. Paris: Delachaux et Niestlé. Issekutz, L. & Kovács, L Melitaea britomartis Assmann, with Special Regard to its Occurrence in Hungary. Annales Historico-Naturales Musei Nationalis Hungarici 5, Kristal, P. M. & Nässig, W. A Leptidea reali Reissinger, 1989 auch in Deutschland und einigen anderen europäischen Ländern (Lepidoptera: Pieridae). Nachrichten des entomologishen Vereins Apollo N. F. 16, Lafranchis, T Butterflies of Europe. Paris: Diatheo. Lorković, Z Leptidea reali Reissinger, 1989 (= lorkovicii Real, 1988), a new European species (Lepid., Pieridae). Natura Croatica 2, Martin, J., Gilles, A. & Descimon, H Species concepts and sibling species: the case of Leptidea sinapis and Leptidea reali. In Butterflies: ecology and evolution taking flight (eds. Boggs, C. L., Watt, W. B. & Ehrlich, P. R.), pp Chicago University Press. Mazel, R Le polymorphisme de deux espèces-jumelles Leptidea sinapis L. et L. reali Reissinger en France (Lepidoptera: Pieridae). Première partie. Linneana Belgica 17, Mazel, R Le polymorphisme de deux espèces-jumelles Leptidea sinapis L. et L. reali Reissinger en France (Lepidoptera: Pieridae). Deuxième partie. Linneana Belgica, 18, Mazel, R. & Leestmans, R Relations biogéographiques, écologiques et taxinomiques entre Leptidea sinapis Linné et L. reali Reissinger en France, Belgique et régions limitrophes (Lepidoptera: Pieridae). Linneana Belgica 15, Mazel, R. & Leestmans, R Séparation biométrique des Leptidea sinapis L., L. morsei Fenton et reali Reissinger. Linneana Belgica 17, Niculescu, E. V Fam. Pieridae (Lepidoptera). Fauna R.P.R. 11. Bucureşti: Ed. Academiei R.P.R. Rákosy, L Leptidea reali Reissinger, 1989 (Lepidoptera: Pieridae) specie nouă pentru fauna României. Bul. inf. Soc. lepid. rom. 7, Verovnik, R. & Glogovčan, P Morphological and molecular evidence of a possible hybrid zone of Leptidea sinapis and L. reali (Lepidoptera: Pieridae). European Journal of Entomology 104, Vila, R., Viader, S. & Jubany, J Leptidea sinapis (Linnaeus, 1758) i L. reali (Reissinger, 1989): dues espècies «bessones» a Catalunya i Andorra (Lepidoptera: Pieridae). Butll. Soc. Cat. Lep. 90, Slamka, F Die Tagfalter Mitteleuropas - östlicher Teil. Published by the author. Tolman, T. & Lewington, R Collins Butterfly Guide. London: Collins.