Alpha taxonomy of the genus Kessleria Nowicki, 1864, revisited in light of DNA-barcoding (Lepidoptera, Yponomeutidae)

Abstract The taxonomy of Kessleria, a highly specialized montane genus of Yponomeutidae with larval host restriction to Saxifragaceae and Celastraceae (Saxifraga spp. – subgenus Kessleria; Saxifraga spp. and Parnassia spp. – subgenus Hofmannia), is revised based on external morphology, genitalia and DNA barcodes. An integrative taxonomic approach supports the existence of 29 species in Europe (the two known species from Asia and North America are not treated herein). A full 658 bp fragment of COI was obtained from 135 specimens representing 24 species, a further seven sequences are >560 bp. Five new species are described: Kessleria cottiensis sp. n. (Prov. Torino, Italy; Dep. Hautes Alpes, France), Kessleria dimorpha sp. n. (Dep. Alpes-de-Haute-Provence, France), Kessleria alpmaritimae sp. n. (Dep. Alpes-Maritimes, France), Kessleria apenninica sp. n. (Prov. Rieti, Prov. L´Aquila, Italy), and Kessleria orobiae sp. n. (Prov. Bergamo, Italy).


Introduction
The genus Kessleria Nowicki, 1864 is one of the striking examples of long underestimated or neglected diversity in the generally well known fauna of European Lepidoptera. This deficiency of knowledge is reflected in the fact that only 9 out of the 29 European species were described before 1960, and 18 species, or two-thirds of the fauna, after 1990 (Friese 1960, Huemer and Tarmann 1992, 1993. The genus is exceptional in Lepidoptera due to its highly specialized host-plant relationship with the Saxifragaceae, a habit shared e.g. by a section of the Curculionidae genus Dichotrachelus (Merigalli et al. 2013). Whereby the large majority of species is restricted to Saxifraga, two species feed on the herbaceous Celastraceae genus Parnassia, long considered as Saxifragaceae (The Angiosperm Phylogeny Group 2009). Through these host-plant relationships, Kessleria is a genus characteristic of mountain regions in the northern hemisphere, reaching high altitudes of about 3000 m in the European Alps and only exceptionally occurring below 600 m. In such alpine environments adult morphology may be adapted to rough climatic conditions with female brachyptery observed in several families of Lepidoptera (Sattler 1991), including minimum five species of Kessleria. Two major revisions (Friese 1960, Huemer andTarmann 1992) are primarily based on external and internal morphology of adults, supplemented by ecological data. These authors established a stable and undisputed alpha taxonomy of Kessleria, which has been in use for the last two decades. Recently discovered suspected morphospecies led to the implementation of molecular methods as an additional tool in species delimitation. Sequences of the COI barcode region (Hebert et al. 2003(Hebert et al. , 2009) confirmed the species status of the vast majority of previously described taxa, and helped in resolving suspected cryptic species-complexes and in delimiting five species new to science.

Material and methods
Extensive descriptions and diagnoses of previously described European species of Kessleria including keys to males and females, colour figures of adults, black-and-white figures of male and female genitalia, last abdominal segments, illustrations of wing venation and figures of larval habits and habitats have been published by Huemer andTarmann (1992, 1993) and are not repeated here.
Our study was initially based on morphology of the extensive material published in detail by Huemer and Tarmann (1992), and about 100 additional specimens, with DNA barcode sequences as an additional tool for delimitation of cryptic species. Most of the material was set and dried according to standard practice, some were spread, and a few only pinned. Genitalia preparations followed standard techniques for microlepidoptera (Robinson 1976), adapted for Kessleria e.g. by the manual eversion of cornuti (Huemer and Tarmann 1992). Wing venation was not considered for new species de-scriptions as it proved irrelevant for alpha taxonomy in the genus in the earlier revision by Huemer and Tarmann (1992).
We tried to obtain DNA barcode sequences, a 658 base-pair long segment of the 5' terminus of the mitochondrial COI gene (cytochrome c oxidase 1), from 150 specimens, three from LMK and ZMUO respectively, and 144 from TLMF. DNA samples (from a single dried leg) were prepared according to the accepted standards. Legs from 150 specimens of Kessleria were processed at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) using their standard high-throughput protocol described in deWaard et al. (2008). Successfully sequenced voucher specimens are listed in Suppl. material 1 together with species names, sample-IDs, process-IDs, BINs, COI-5P sequence length, and trace counts. Sequences were submitted to GenBank during printing stage; GenBank accession numbers further details including complete voucher data and images can be accessed in the public dataset "Lepidoptera of Europe -Kessleria" http://dx.doi. org/10.5883/DS-LEAKE in the Barcode of Life Data Systems (BOLD; Ratnasingham and Hebert 2007). Degrees of intra-and interspecific variation in the DNA barcode fragment were calculated under the Kimura 2-parameter (K2P) model of nucleotide substitution using analytical tools in BOLD systems v3.0. (http://www. boldsystems.org). A neighbor-joining tree of DNA barcode data of European taxa was constructed using Mega 5 (Tamura et al. 2011) under the K2P model for nucleotide substitutions. In taxonomic delimitation, we applied principles of integrative taxonomy (Padial et al. 2010) and considered a barcode divergence of roughly 2% supported by at least one morphological character indicating species distinctiveness. We acknowledge that any threshold value of genetic distinctiveness is artificial and should not alone be used as indicating species status (cf. Collins and Cruickshank 2013), for which reason we considered 2% genetic difference associated with at least one morphological character indicating species integrity in the sense of e.g. General Lineage Species Concept (deQueiroz 1998) and Phylogenetic (diagnostic) Species Concept (Cracraft 1989), which both are applicable in delimiting also allopatric populations.
Photographs of the adults were taken with an Olympus SZX 10 binocular microscope and an Olympus E 3 digital camera, and processed using the software Helicon Focus 4.3 and Adobe Photoshop CS4 and Lightroom 2.3. Genitalia photographs were taken with an Olympus E1 Digital Camera from Olympus BH2 microscope.
Measurements were taken with a micrometer eyepiece.

Abbreviations of institutional collections BMNH
Natural History Museum (British Museum, Natural History) London, United Kingdom LMK Landesmuseum Kärnten, Klagenfurt, Austria Sequencing resulted in a full barcode fragment of 658 bp for 135 specimens, covering 24 species. A further seven sequences that were longer than 560 bp were included in the analysis. A single short sequence of 307 bp was not considered, and sequencing failed for seven voucher specimens. Mean intraspecific divergence is 0.61%. It ranges from 0-4.27%, exceeding 2% only in three species, which, however, may include further cryptic diversity (e.g. K. alpicella, K. albanica and K. inexpectata) and should be tested accordingly with more material (Table 1, Fig. 1). On the contrary, interspecific divergence in the genus is much higher with a mean divergence of 10.38% and maximum of 16.22%. Interspecific divergence to the nearest neighbour ranges from 1.86-9.29%, with the only exception being K. inexpectata and K. helvetica, which overlap in DNA barcode (Table 1, Fig. 1). From sequence analysis of 20 Kessleria species based on at least three sequences, 17 species are delimited by a minimum of one to a maximum of 10 diagnostic characters whereas K. inexpectata, K. cottiensis and K. alpmaritimae have no diagnostic character (Table 1).

Kessleria alternans-group
The K. alternans-group is characterized by strong sexual dichroism and to a lesser extent dimorphism, with females being smaller and lighter, but not strongly brachypterous (Figs 2-11). The genitalia are characterized by the strong reticulate sculpture of the apical part of the phallus (Figs 12-21) and the ductus bursae, which is extended into the corpus bursae . Larval host-plants, as far as known, belong to the small-leaved Saxifraga spp., particularly the S. oppositifolia-complex, and to broadleaved congeners such as S. paniculata. Five species belong to this group: K. alternans, K. wehrlii and the new taxa K. cottiensis, K. dimorpha and K. alpmaritimae. Compared to K. wehrlii and K. alpmaritimae, the whitish suffusion of the forewing is largely reduced. The ochre-brown markings, which are well present in K. alternans, are almost completely absent. The female of K. cottiensis is also distinctly smaller than K. alternans, with a forewing length of 6.13 mm (n=8) on average vs. 7.58 mm (n=11), whereas females of K. cottiensis and K. alpmaritimae are indistinguishable. K. wehrlii is insufficiently known from only a single worn female specimen. Compared to the genetically nearest neighbour K. dimorpha, which is similar in forewing length (6.0 mm, n=4), the hindwings are slightly less reduced and the ground colour of the forewing is much lighter. Diagnostic characters in genitalia are generally weak. The male genitalia differ from K. alpmaritimae by the medially strongly widened valva, from K. alternans by the more slender cornuti, from K. wehrlii by three instead of four cornuti, and from the nearest neighbour K. dimorpha by the distinctly longer phallus (1.65-1.70 mm vs. 1.32-1.36 mm) (Figs 12-21). The female genitalia show no diagnostic characters to related species of the K. alternans-group (Figs 22-31).
Description. Male (Fig. 4). Head covered with erected whitish hair-like scales; antennae dark grey-brown, indistinctly lighter ringed; thorax and tegulae dark greybrown. Forewing length 7.0-8.8 mm (Ø 7.59 mm; n=14); ground colour dark greybrown, mottled with whitish scales, particularly in distal half, forming indistinct patches in fold and on costa at about 4/5; irregular black dots on veins and few brown scales in medial part of wing; oblique blackish fascia at about 1/3 to 1/2 indistinct; termen mixed whitish-grey, dark grey-brown in apical part; fringes light grey, darker in apical area. Hindwing dark grey, fringes with dark grey base, distal part light grey. Female (Fig. 5). Head covered with erected whitish hair-like scales; antennae greybrown, distinctly ringed whitish; thorax and tegulae whitish. Forewing length 5.8-6.3 mm (Ø 6.13 mm; n=8); ground colour whitish, mottled with black scales, particularly along veins and in tornal part, patches of brown scales in medial part of wing from base to end of cell; oblique blackish fascia at about 1/3 to 1/2 indistinct, separated into larger dash-like patch and reduced dot; termen whitish with some dark grey-brown mottling in apical part; fringes greyish-white, darker in apical area.
Female genitalia (Figs 23,28). Genitalia ca. 4.9 mm in length; papilla analis large, densely covered with long setae; apophysis posterior rod like, ca. 0.70 mm, about length of apophysis anterior; apophysis anterior rod like; posterior part bifurcated with straight dorsal and inwardly curved ventral branch; lamella postvaginalis with large sclerotized mediolateral patches, covered with microtrichia, medial area less sclerotized, posteriolateral part with hump, covered with some long setae; ostium bursae membranous; antrum weakly sclerotized, funnel-shaped; ductus bursae very long, about 2.8 mm, from entrance of ductus seminalis to transition into corpus bursae covered with finely granulous sculpture, particularly in posterior and anterior part, ductus bursae extended into posterior part of corpus bursae, entrance to corpus bursae weakly widened; corpus bursae well delimited, about 1.2 mm in length, ovoid, with small plate-like signum.
Molecular data. The average intraspecific divergence of the barcode region is 0.0% (n=5). The minimum distance to the nearest neighbour K. dimorpha is 1.86%, whereas the minimum divergence to K. alternans, K. alpmaritimae and K. wehrlii ranges from 2.65% and 2.98% to 3.63%, respectively.
Etymology. The species name refers to the type locality in the Cottian Alps (Alpi Cozie, Alpes cottiennes).
Distribution (Fig. 32). Only known from a small area in the southwestern Alps (Cottian Alps) of Italy and France. An alleged K. alternans from the Graian Alps (Huemer and Tarmann 1992) likely refers to K. cottiensis, but the specimen in question could not be re-examined.
Ecology. Host-plant and early stages unknown. The adults were collected in late July. The flight period can most likely be further prolonged, depending on snow coverage and elevation. A specimen collected earlier during the summer, on June 9 th , by Jäckh in Valle delle Finestre (Huemer and Tarmann 1992) probably belongs to K. cottiensis. The adults were collected during the day, flying freely in the morning hours and flushed out from their resting places with a bee-smoker. The species occurs in alpine grassland interspersed with calcareous rocks. Vertical distribution: from about 1700 m to 2150 m.

Kessleria dimorpha
Diagnosis. K. dimorpha resembles other taxa of the K. alternans-group in wing markings and colour (Figs 2-11), but the male differs from the genetically nearest neighbour K. cottiensis by the on average distinctly larger forewing length of 8.25 mm (n=6) vs. 7.59 mm (n=14). Larger species are K. wehrlii with forewing length 8.75 mm (n=13) and K. alternans with 8.55 mm (n=26), whereas K. alpmaritimae with 8.05 mm (n=6) is of similar size. Furthermore, K. wehrlii and K. alpmaritimae have a much more prominent whitish suffusion on the forewing, whereas the ochre-brown markings of K. dimorpha rather resemble K. alternans. The female of K. dimorpha reflects a tendency to reinforced brachyptery and is distinctly smaller than K. alternans with a forewing length of only 6.0 mm (n=4) on average vs. 7.58 mm (n=11), whereas females of K. cottiensis and K. alpmaritimae are strongly suffused with whitish scales. K. wehrlii is insufficiently known from only a single worn female specimen. The male genitalia differ from all other taxa of the K. alternans-group by the distinctly shorter phallus with <1.40 mm vs. a minimum of 1.50 mm in other species . The female genitalia show no diagnostic characters to related species of the K. alternans-group .
Description. Male (Fig. 6). Head covered with ochre-brown hair-like scales; antennae almost unicolorous dark grey-brown; thorax and tegulae mixed dark greybrown and ochre-brown. Forewing length 8.0-8.4 mm (Ø 8.25 mm; n=6); ground colour dark grey, intensively mottled with light grey, ochre-brown and whitish scales, white medial patch in fold; black dots particularly on costal and subcostal veins; black patch near base and at end of cell, oblique blackish fascia at about 1/3 to 1/2 reduced to large patch in fold; termen mixed dark and light grey; fringes basally dark grey, distal part whitish-grey, darker in apical area. Hindwing dark grey, fringes with dark grey base, distal part whitish-grey.
Female (Fig. 7). Head covered with erected whitish hair-like scales; antennae greybrown, indistinctly lighter ringed; thorax and tegulae whitish. Forewing length 6.0 mm (Ø 6.0 mm; n=4); ground colour whitish, mottled with dark grey and black, particularly along fold and in tornal part, few black dots along costal and subcostal vein, small patches of ochre-brown scales in medial part of wing particularly in fold and at end of cell; oblique blackish fascia at about 1/3 to 1/2 indistinct, separated into larger dash-like patch and reduced dot; termen mixed whitish and dark grey; fringes whitish-grey, with dark grey basal part near apex. Hindwing grey, fringes whitish-grey with darker basal part.
Female genitalia (Figs 24,29). Genitalia ca. 4.9 mm in length; papilla analis large, densely covered with long setae; apophysis posterior rod like, ca. 0.72 mm, about length of apophysis anterior; apophysis anterior rod like; posterior part bifurcated with straight dorsal and inwardly curved ventral branch; lamella postvaginalis with large sclerotized mediolateral patches, covered with microtrichia, medial area less sclerotize, posterolateral part with hump, covered with some long setae; ostium bursae membranous; antrum weakly sclerotized, funnel-shaped; ductus bursae long, ca. 2.3 mm, posterior part from entrance of ductus seminalis anterior and anterior part covered with finely granulous sculpture, medial part with weak and hardly discernible sculpture, ductus bursae extended into posterior part of corpus bursae, entrance to corpus bursae weakly widened; corpus bursae well delimited, about 1.4 mm in length, ovoid, with small plate-like signum.
Molecular data. The average intraspecific divergence of the barcode region is low with 0.08%, ranging from a minimum of 0% to a maximum of 0.15% (n=4). The minimum distance to the nearest neighbour K. cottiensis is 1.86%, whereas the minimum divergence to K. alternans, K. alpmaritimae and K. wehrlii ranges from 3.15% and 3.64% to 4.3%, respectively.
Etymology. The species name refers to the remarkable sexual dimorphism. Distribution (Fig. 32). Only known from the type locality, the French side of Col Agnel (Cottian Alps), close to the Italian border.
Bionomics. Host-plant and early stages unknown. Based on the type locality, the host-plant is most likely Saxifraga cf. oppositifolia. The adults have been collected in early August during the early morning hours from about 7-10a.m. at low temperatures between 2-5 °C. Males were flying actively during this period in search for females. Both sexes were later found in copula, often sitting on cushions of their suspected hostplant. A single female was found at light, attracted from its nearby habitat and crawling upwards to the light tower, but unable to fly actively. From personal observations of PH, it is likely that the slightly reinforced brachyptery of K. dimorpha is combined with flightlessness. The species occurs in rocky habitat on siliceous soil. Vertical distribution: about 2800 m.
Remark. Fringes of the examined females seem partially lost and thus may lead to a biased impression of the extent of wing reduction.
Diagnosis. K. alpmaritimae resembles other taxa of K. alternans-group in wing markings and colour (Figs 2-11), but the male with average forewing length of only 8.05 mm (n=6) is distinctly smaller than K. wehrlii with 8.75 mm (n=13) and K. alternans with 8.55 mm (n=26) and larger than K. cottiensis with only 7.59 mm (n=14). K. dimorpha with an average forewing length of 8.25 mm (n=6) is similar in size, but clearly differs by the largely reduced whitish suffusion of the forewing, a character stage which also applies to K. cottiensis and K. alternans, whereas K. wehrlii is intensely mottled whitish. The female with forewing length of only 6.06 mm (n=3) is distinctly smaller than that of K. alternans with 7.58 mm (n=11), but hardly separable from other species in size. Compared to the genetically nearest neighbour K. dimorpha, the hindwings are less reduced and the ground colour of the forewing is much lighter. The female of K. wehrlii is insufficiently described due to limited material, and the females of K. cottiensis and K. alpmaritimae are indistinguishable. The male genitalia differ from K. cottiensis by the medially weakly widened valva, from K. alternans by the more slender cornuti, from the nearest neighbour K. wehrlii by three instead of four cornuti, and from K. dimorpha by the distinctly longer phallus (1.52-1.58 mm vs. 1.32-1.36 mm) (Figs 12-21).  The female genitalia show no diagnostic characters to related species of the K. alternans-group (Figs 22-31).
Female genitalia (Figs 26, 31). Genitalia ca. 4.9 mm in length; papilla analis large, densely covered with long setae; apophysis posterior rod like, ca. 0.76 mm, about length of apophysis anterior; apophysis anterior rod like; posterior part bifurcated with straight dorsal and inwardly curved ventral branch; lamella postvaginalis with large sclerotized mediolateral patches, covered with microtrichia, medial area less sclerotized, posterolateral part with hump, covered with some long setae; ostium bursae membranous; antrum weakly sclerotized, funnel-shaped; ductus bursae very long, ca. 2.4 mm, from entrance of ductus seminalis to transition into corpus bursae covered with finely granulous sculpture, particularly in posterior and anterior part, ductus bursae extended into posterior part of corpus bursae, entrance to corpus bursae weakly widened; corpus bursae well delimited, about 1.2 mm in length, ovoid, with largely reduced plate-like signum.
Molecular data. The average intraspecific divergence of the barcode region is 0.0% (n=6). The minimum distance to the nearest neighbour K. wehrlii is 1.87%,  whereas the minimum divergence to K. cottiensis, K. dimorpha and K. alternans ranges from 2.98% and 3.64% to 3.75%, respectively.
Etymology. The species name is a made-up word which refers to the area of the type locality, the Alpes Maritimes. Distribution (Fig. 32). Only known from the type locality, the Marguareis Massif, in the French Alpes Maritimes.
Ecology. Host-plant and early stages unknown. The adults have been collected in the last third of July during the day, flying freely in the morning hours and flushed out from their resting places with a bee-smoker. The species occurs in rocky habitat on calcareous soil. Vertical distribution: from about 2100 m to 2650 m. Remarks. K. alpmaritimae described here was already suspected to be distinctive from K. alternans by Huemer and Tarmann (1992), who illustrated adults (Figs 8-9).

Kessleria apenninica-group
The K. apenninica-group s.str. only includes the new species K. apenninica which is characterized e.g. by slender forewings. From characters of the male genitalia, such as the short cornuti, closer relatives are likely to be the Iberian K. diabolica, K. brevicornuta, K. brachypterella and K. pyrenaea, which all differ in adult morphology (see Figs 33-38, and Huemer and Tarmann 1992). These species together with K. apenninica may form a larger species-group, but at present material is scarce and supporting molecular data are lacking. The nearest neighbour of the new species with a full DNA barcode is tentatively attached to K. pyrenaea and considered for the differential diagnosis.  Diagnosis. K. apenninica is characterized by unusually slender forewings and a pure white colour with black pattern. Species from the K. apenninica-group are externally unmistakably distinguishable from one another both by wing pattern and colour (Figs 33-34, and Huemer and Tarmann 1992), but the genitalia of males are similar (Figs 35-38). However, in K. apenninica the saccus is distinctly shorter than in all other species with 0.23 vs. minimum 0.29 mm.
Description. Male (Fig. 33). Head covered with whitish hair-like scales; antennae almost unicolorous dark grey with light grey apex; thorax and tegulae mixed dark grey and whitish. Forewing length 5.8-6.9 mm (Ø 6.4 mm; n=4); forewing slender; ground colour white, mottled with black; black dots on veins and in terminal area; black patch near base and oblique blackish fascia at about 1/3 to 1/2; fringes white with indistinct dark grey fringe line. Hindwing dark grey, fringes with dark grey base, distal part white.
Female genitalia. Unknown. Molecular data. K. apenninica splits into two geographically separated haplogroups, which in our examination -based on limited material -did not reveal any morphological differences. The average intraspecific divergence of the barcode region is considerable with 1.05%, ranging from a minimum of 0% to a maximum of 1.69% (n=4). The minimum distance to the nearest neighbour K. pyrenaea is 5.47%.
Etymology. The species name refers to the Apennines where all type specimens have been collected.
Distribution. Only known from the Apennines in Central Italy. Ecology. Host-plant and early stages unknown, but the species probably feeds on an unidentified broad-leaved Saxifraga species growing on steep rocks. The adults have been collected in the last third of July from light. The species occurs in rocky habitat on calcareous soil. Vertical distribution: from about 2100 m to 2200 m.

Kessleria albescens-group
The K. albescens-group is characterized by small and predominantly whitish-coloured species without obvious sexual dichroism or dimorphism (Figs 39-48). The male genitalia are recognizable by the strongly spinous sacculus, the long and stout saccus, and particularly the phallus with two long cornuti with bases of similar length (Figs 49-58). The female genitalia are characterized by the curved entrance of the ductus bursae and the finely granulated sculpture of the entire ductus bursae (Figs 59-66). Larval host-plants, as far as is known, belong to broad-leaved Saxifraga spp., e.g. S. paniculata and S. incrustata. Five species are known: K. albescens, K. inexpectata, K. helvetica, K. klimeschi and the new species K. orobiae.
Diagnosis. K. orobiae largely resembles other taxa of the K. albescens-group in wing markings and colour, and cannot be unambiguously separated (Figs 39-48). Similarly, female genitalia exhibit no significant diagnostic characters for discrimination at species level (Figs 59-66, and Huemer and Tarmann 1992). The most reliable diagnostic characters in the species-group are found in the male genitalia (Figs 49-58). K. orobiae differs from K. klimeschi, K. albescens and K. helvetica by a much shorter saccus (0.32 mm vs. minimum 0.38-0.58 mm) which is only about half the length of the valva compared to the minimum 0.75 times the length of the valva in the other species. In K. inexpectata the saccus is slightly longer and furthermore without the apical widening of K. orobiae, K. albescens and K. helvetica. The two needle-shaped cornuti are similar in all species, with the exception of K. klimeschi with only one needle-shaped and one sub-ovate cornutus, and K. helvetica with cornuti of about 0.50 mm in length.
The female genitalia largely resemble other species of the K. albescens-group with only quantitative differences, such as a longer ductus bursae than in K. albescens (1.6 mm vs. 1.3-1.4 mm), and the overall length of genitalia which exceeds K. klimeschi (4.2 mm vs. 3.5 mm).
Description. Male (Fig. 45). Head covered with white hair-like scales; antennae ringed dark grey and whitish; thorax and tegulae mixed dark grey and white, distally predominantly white, particularly tegulae. Forewing length 6.7-7.3 mm (Ø 7.03 mm; n=6); ground colour whitish-grey, intensively mottled with blackish-grey spots all over wing, few ochre-brown scales in dorsal part; blackish-grey patches at base of costa and at end of cell, oblique blackish-grey fascia at about 1/3 to 1/2 narrow and indistinct, medially separated; termen whitish-grey; fringes white, basally with distinct blackishgrey cilia line, apex with small dark grey tip. Hindwing dark grey, fringes with dark grey base, distal part white.
Female (Fig. 46). As male. Head covered with white hair-like scales; antennae ringed dark grey and whitish; thorax and tegulae mixed dark grey and white, distally predominantly white, particularly tegulae. Forewing length 5.9-6.6 mm (Ø 8.18 mm; n=5); ground colour whitish-grey, intensively mottled with blackish-grey spots all over wing, few ochre-brown scales in dorsal part; blackish-grey patches at base of costa and at end of cell, oblique blackish-grey fascia at about 1/3 to 1/2 narrow and indistinct, medially separated; termen whitish-grey; fringes white, basally with distinct blackishgrey cilia line, apex with small dark grey tip. Hindwing dark grey, fringes with dark grey base, distal part white.
Male genitalia (Figs 55-56). Socii long and slender, with apical spine; anterior margin of tegumen with medial process; gnathos broadly tongue-shaped, smooth; valva slender, length 0.60 mm, maximum width 0.18 mm; densely covered with long hairs in medial part and short setae on ventromedial margin, apical part ventrally convex, costa strongly sclerotized without dentation; sacculus oval, weakly confined,    (Figs 61, 65). Genitalia ca. 4.2 mm in length; papilla analis large, densely covered with long setae; apophysis posterior rod like, ca. 0.70 mm, about length of apophysis anterior; apophysis anterior rod like; posterior part bi- furcated with straight dorsal and inwardly curved ventral branch; ventral branch descending into patch like sclerite; lamella postvaginalis with sclerotized lateral patches, covered with microtrichia, medially membranous, posterolaterally with hump, covered with some long setae; ostium bursae membranous; antrum weakly developed, ring-like; ductus bursae long, about 1.6 mm, weakly curved in posterior part, from entrance of ductus seminalis to almost transition to corpus bursae densely covered with finely granulous sculpture; sculpture in posterior part nearly bacillary, distally increasingly granulous, entrance to corpus bursae widened, without sculpture; corpus bursae about 1.7 mm in length, saccate, posterior part folded, without signum. Molecular data. The average intraspecific divergence of the barcode region is low with 0.31%, ranging from a minimum of 0.15% to a maximum of 0.46% (n=5). The minimum distance to the nearest neighbour K. albescens is 2.66%, whereas the minimum divergence to K. inexpectata, K. helvetica and K. klimeschi ranges from 3.14% and 3.46% to 9.53%, respectively.

Female genitalia
Etymology. The species name refers to the Orobian Alps (Alpi Orobie) in northern Italy, where the type locality is situated. Distribution (Fig. 67). Only known from Zambla Alta -Plassa and few nearby localities in the Orobian Alps (Prov. Bergamo, Italy).
Ecology. The larval habits are insufficiently known, but based on our observations, the larva lives in the shoots and as a leaf-miner in basal leaves of Saxifraga paniculata and Saxifraga sp. Mined leaves are partially spun together and covered with a fine silken web. The adults have been collected from the Saxifraga-cushions or nearby rock during the day. In the first few hours of the night they have been observed with a head-lamp flying actively around the larval habitat or sitting near the host-plant. The adult is on the wing from late June to mid-August, depending on altitude and snow coverage. Bred specimens date from mid to late July. K. orobiae occurs in rocky habitat both on calcareous and silicous soil. Vertical distribution: from about 1100 m to 2100 m.

Established European species of Kessleria
A brief overview of established species lists including original description, type locality, type material, references of published images of adults and images of genitalia, and hitherto unpublished molecular data. For extensive generic and species descriptions and diagnoses, see Huemer and Tarmann (1992).
Remarks. Huemer and Tarmann (1992) already recognized and described a remarkable amount of individual and geographical variation. At that time, this variation was considered as intraspecific, and the authors hesitated to describe further species.
Remarks. Huemer and Tarmann (1992) already recognized and described a considerable amount of individual and geographical variation, particularly in phenotypic appearance, but the authors hesitated to describe further species due to the lack of diagnostic genitalia characters. Molecular data suggest possible cryptic diversity, but further investigations are required.  fig. 229 (female genitalia). Molecular data. The average intraspecific divergence of the barcode region is low with 0.04%, ranging from a minimum of 0% to a maximum of 0.15% (n=8). The minimum distance to the nearest neighbour K. alpmaritimae is 6.39%.

Kessleria klimeschi Huemer & Tarmann, 1992
Kessleria klimeschi Huemer and Tarmann 1992: 47 fig. 232 (female genitalia). Molecular data. The average intraspecific divergence of the barcode region is unknown (n=1). K. helvetica overlaps in the barcode with a haplogroup of topotypical K. inexpectata, but diagnostic morphological characters indicate species status. The minimum distance to a further haplogroup of K. inexpectata is 1.77%.

Kessleria inexpectata Huemer & Tarmann, 1992
Kessleria inexpectata Huemer and Tarmann 1992: 45 fig. 231 (female genitalia). Molecular data. K. inexpectata splits into two major haplogroups. The average intraspecific divergence of the barcode region within the haplogroup of topotypical specimens is low with 0.16%, ranging from a minimum of 0% to a maximum of 0.32% (n=4) whereas the average intraspecific variation within the second haplogroup is considerable with 0.84% (maximum 1.68%). The mean intraspecific divergence of the entire sample is 1.42% (maximum 2.18%). The haplogroup of the topotypical population overlaps with K. helvetica. The minimum distance to K. orobiae is 3.14%.  fig. 236 (female genitalia). Molecular data. The average intraspecific divergence of the barcode region is low with 0.43%, ranging from a minimum of 0% to a considerable maximum of 1.28% (n=20). The minimum distance to the nearest neighbour Zelleria celastrusella Kearfott, 1903, from North America is 6.22%, and the minimum distance to the congeneric K. fasciapennella is 7.21%.  fig. 237 (female genitalia). Molecular data. The average intraspecific divergence of the barcode region is low with 0.04%, ranging from a minimum of 0% to a maximum of 0.15% (n=8). The minimum distance to the nearest neighbour Zelleria celastrusella Kearfott, 1903, from North America is 6.58%, and the minimum distance to the congeneric K. saxifragae is 7.21%.

Discussion
Our study proves the advantage of an integrative taxonomic approach, initially based on morphology, with molecular data supplemented as an additional tool for delimitation of cryptic species. Even within genera of European Lepidoptera which had been considered as well explored, cryptic diversity seems much more widespread than hitherto estimated. Recent molecular studies have proven the existence of a remarkable amount of cryptic species in several genera or species-groups, e.g. Callisto (Kirichenko et al. 2015), Stigmella (Nieukerken et al. 2012), Olethreutes (Segerer et al. 2010), Elachista (Mutanen et al. 2013), Eulamprotes (Huemer et al. 2013), Sattleria (Huemer andHebert 2011, Huemer andTimossi 2014), Caryocolum  and Coleophora (Baldizzone et al. 2014, Tabell and. Similarly, the proportion of unnamed species in Kessleria is high, adding about 20% to the hitherto described species diversity, not including several further yet unresolved possible candidates of cryptic diversity. We expect to find such additional overlooked taxa in e.g. K. alpicella and K. albanica, but additional material is needed to resolve this. Outside Europe, the species diversity of Kessleria cannot even be estimated at the present time, with the Chinese K. nivosa (Meyrick, 1938) as the only known congeneric species from Asia, and K. parnassiae (Braun, 1940), a close relative of K. fasciapennella, from North America. Particularly the Asian fauna of Kessleria may prove diverse, e.g. indicative of which is an extraordinary diversity of potential Kessleria host-plants in China with 216 out of about 450 worldwide known species of Saxifraga (139 endemic) and 63 out of 70 Parnassia spp. (49 endemic) (Jintang et al. 2001).
Most of the newly described species belong to complexes of closely related species with strictly allopatric distribution patterns. K. cottiensis, K. dimorpha and K. alpmaritimae are morphologically and genetically most similar to K. alternans and K. wehrlii, forming a separate species group in Kessleria (Fig. 32). Similarly, K. orobiae along with K. albescens, K. klimeschi, K. helvetica and K. inexpectata belong to a species-group of cryptic allopatric taxa (Fig. 67). All these taxa are extremely similar in external and internal morphology. This is a phenomenon well known from the related genus Yponomeuta, namely the Y. cagnagellus species-complex which includes morphologically virtually indistinguishable species (Bakker et al. 2008), which furthermore often share DNA barcodes. Evolution and reproductive isolation in this genus was likely driven by specific host-plant associations and sex pheromones (Menken 1981, Menken et al. 1992, Menken 1996, Löfstedt 1991, Turner et al. 2010. Unlike Yponomeuta, barcode sharing seems to be a rare exception in Kessleria, only observed in K. helvetica and K. inexpectata so far, and indicating possible introgression or recent speciation. DNA barcode divergence to the nearest neighbour is considerable in Kessleria with roughly 2-3% distance in sister species, rising to about 6-9% between morphologically well separated taxa (Table  1, Fig. 1). If reflected by at least one supplementing morphological character stage we consider barcode divergence of roughly 2% as taxonomically relevant. These taxa are described as cryptic species and not subspecies, although such decisions are prone to subjectivity Mayr in press, Mutanen et al. 2012).
A similar extent of interspecific divergence in allopatric sister species is also known from other Lepidoptera with geographically restricted alpine distribution patterns, e.g. Sattleria (Huemer and Hebert 2011) and Sciadia (Huemer and Hausmann 2009). The timing of radiation of these and Kessleria is unknown, but estimations of substitution rates of COI indicate that divergences of 1.0-2.5% correspond to divergence times of roughly one million years (Kandul et al. 2004. Even though such estimations should be considered with caution, they indicate that several well separated species of Kessleria may have diverged already in the lower Pliocene (5.3 mya-1.8 mya) while others, such as four out of five newly described species, are possibly of younger origin. Speciation in these taxa was likely reinforced by climatic oscillations in the late Pliocene and during the Pleistocene, with unglaciated but highly isolated refugial areas, particularly in the southern Alps, an area well documented as a hotspot for endemic Lepidoptera (Huemer 1998). The widespread female flightlessness may have been crucial for reducing dispersal and interrupting gene flow, particularly in maternally inherited mitochondrial DNA, thereby expediting the speciation processes. Host-plant relationship itself seems of limited importance for speciation processes in Kessleria as host specificity is moderately pronounced and host-plants are regularly much more widespread than their consumers. E.g. Saxifraga paniculata as one of the major host-plants of species of the K. albescens-group is widely distributed in the Alps and other European mountain systems, but the Kessleria spp. are allopatrically distributed in a small section of the southern Alps. Summing up, further in-depth phylogenetic studies will be necessary to finally identify drivers of speciation in Kessleria.