A new pygmy leafmining moth , Stigmella tatrica sp . n . , from the alpine zone of the Tatra Mountains ( Lepidoptera , Nepticulidae )

Stigmella tatrica sp. n. is described from moths taken in the alpine zone of the Tatra Mountains in Slovakia. The new species is similar to several other montane species of Stigmella Schrank, 1802 in the S. aurella (Fabricius, 1775) group in external characters and male genitalia; its closest relative is S. dryadella (O. Hofmann, 1868). It is indistinguishable from S. tormentillella (Herrich-Schäffer, 1860) by the colour and pattern elements of the forewing; however, it differs in the male genitalia in the shape and number of cornuti, in the female genitalia by long apophyses with protruding ovipositor, and by COI barcodes. Immature stages are unknown, but Dryas octopetala L. (Rosaceae) is the possible hostplant. The long ovipositor suggests an unusual, possibly hidden place for oviposition. The male and female adults and genitalia of both sexes are figured and photographs of the habitat are provided.


Introduction
The pygmy leafmining moths or Nepticulidae have around 300 species in Europe, the great majority of which are leafminers, feeding on woody plants and they are abundant in forested areas (van Nieukerken et al. 2016).Overall they do not form an important aspect of the diversity of high mountain faunas (but see Stonis et al. (2016) for a group of Stigmella Schrank, 1802 species occurring in the high Andes).In Europe few Nepticulidae are adapted to high altitudes, and apart from some common species with large altitudinal ranges, only a few species of Stigmella are alpine specialists, particularly a group of species feeding on herbaceous Rosaceae (Klimesch 1981;Johansson and Nielsen 1990;Laštůvka and Laštůvka 1997).
The high altitude Stigmella species of the Tatra Mountains, especially above the tree line, have been poorly studied and all records reside in a handful of publications.In the Polish part of the alpine zone Stigmella species were first observed by Toll (1948) and Borkowski (1970).Buszko et al. (2000) mentioned three Stigmella species from the alpine grasslands: S. pretiosa (Heinemann, 1862), S. aeneofasciella (Herrich-Schäffer, 1855), andS. dryadella (O. Hofmann, 1868).From the Slovak part of the Tatras, only one faunal contribution is known: Gregor (1986) found mines of S. dryadella in mid-October in the Belianske Tatra Mountains, Kvetnica and Bujačí Vrch Mountain at altitudes between 1500 and 1900 m (the number of mines and hostplant were not given).Most European alpine Nepticulidae belong to the Stigmella aurella (Fabricius, 1775) group, a Holarctic group of 18 named species (12 in Europe), all but one of which feed on Rosaceae, particularly herbs and shrubs such as Rubus.
In late July 2005, while inventorying the Lepidoptera fauna of the Western Tatra Mountains, the first author with his friend found four males of an unknown Stigmella species in alpine grasslands at altitudes between 1800 and 1900 m.Detailed study of two specimens by the second author was inconclusive, because of the relatively high similarities of their external appearance and male genitalia to closely related Stigmella species in the S. aurella group.
Later on, additional males were found in the eastern part of the Tatras, Belianske Tatra Mts at altitudes of ca.2000 m.These specimens were sent to the third author for DNA analysis.In 2016 the first author's last visit to the locality brought success in the form of a large series of specimens, including two females.Individuals of the new species were observed flying around the low cover of high-altitude plant communities with Dryas octopetala L.An examination of the female genitalia of this tiny moth, as well as the analysis of the DNA barcodes, showed that it represents a hitherto undescribed Stigmella species.

Material and methods
All specimens were taken as adults during daylight, either using a net or caught directly from the leaves of Dryas octopetala into small glass vials.The genitalia were dissected in the usual way for small Lepidoptera, the preparations being stored in glycerol in small plastic vials or embedded in Euparal on glass.The drawings of the male and female were made by the second author using water colours.Drawings of the genitalia were made by the first author using Indian ink on transparent sheets.Photographs of the type locality were taken using digital cameras Canon PowerShot G11 and Nikon Coolpix P600.
For methodology of DNA barcodes we refer to papers of the third author and his colleagues (van Nieukerken et al. 2012a;Doorenweerd et al. 2015).The specimen data for barcoded specimens are given in the public BOLD dataset DS-STIGTATR.Many of these barcodes were earlier published by van Nieukerken et al. (2012b).For the alpine species Stigmella stelviana (Weber, 1938) and S. geimontani (Klimesch, 1940) we were only able to obtain short barcodes of 146 base pairs.Tree-building was performed with PAUP and Phylip plugins in Geneious R8.1.8,as outgroup we used a specimen of Stigmella tityrella (Stainton, 1854).
The nomenclature of species follows the "Revised classification and catalogue of global Nepticulidae and Opostegidae" (van Nieukerken et al. 2016).Description.Adult, male (Fig. 1).Wingspan 4.0-5.0mm.Head: frontal tuft black, collar with black lamellar scales.Antenna black, with 32-35 segments.Scape white.Thorax and tegula glossy black.Ground colour of forewing brown with dark golden sheen; dark violet metallic reflection around postmedial fascia and towards the forewing apex.Fascia shining silver, wider at costal margin.Fringe grey.Hindwing grey, no androconial scales.Abdomen brown to black with whitish scales on margins of segment and whitish anal tufts.

Abbreviations
Female (Fig. 2).Wingspan 4.0-4.5 mm.Antenna shorter, with 26 segments.Forewing markings as male.Abdomen with remarkable protruding ovipositor, no anal tufts.5).Vinculum moderately long, anteriorly slightly bilobed.Uncus very broad, with posterior processes well separated, each bilobed.Gnathos with long and separated posterior processes.Valva rectangular with very short and blunt distal process.Transtilla broad with relatively short sublateral processes.Phallus cylindrical, slightly longer than genital capsule, vesica with long series of pointed cornuti of different sizes and three separate thorn-shaped cornuti near phallotrema.
Diagnosis.Stigmella tatrica is somewhat similar to several other montane species in the Stigmella aurella group in forewing colour and pattern and in male genitalia.Externally it is indistinguishable from S. tormentillella (Herrich-Schäffer, 1860).Carpathian S. dryadella differs from the new species by more glossy forewings, a wider violet area before fascia and paler head and collar.In the male genitalia the new species is most similar to S. geimontani, S. stelviana and S. aeneofasciella, but it differs significantly from these externally.Unlike S. tatrica, S. tormentillella and S. dryadella have only one large cornutus in their vesica.In the female genitalia S. tatrica differs considerably from other species in the group by the long and strong apophyses, forming a protruding ovipositor.
Biology.The early stages of the new species are unknown.The adults were observed flying over low cover of high-altitude plant communities with the presence of Dryas octopetala and resting or quickly moving on leaves of that plant.Adults were collected during daylight hours between 29 th July and 4 th August (light collecting was not attempted).This likely represents a single annual generation.
Distribution and habitat .Stigmella tatrica is so far known only from the alpine zone of the Eastern and Western Tatra Mountains.In the Eastern Tatra it was found in the Belianske Tatra Mountains (Belianske Tatry), Zadné Jatky Mountain and Bujačí Vrch Mountain at an altitude of 1800-2010 m.In the Western Tatra it was taken in the Red Mountains (Červené Vrchy), Stoly Rocks at Temniak Mountain, near the Slovak-Polish border at an altitude of about 1800-1900 m.   (Šibík et al. 2004;Kliment et al. 2010).
Etymology.The specific name tatrica, an adjective, is derived from the Tatra Mts, where the new species was discovered.Molecular data (Fig. 11).We obtained COI barcodes from legs of four specimens, two of which were partial barcodes.The Barcode Index Number is BOLD:ACU7181.The maximum K2P intraspecific distance for a full barcode is 1.5%.The nearest neighbour is Stigmella tormentillella at a distance between 6.2 and 7.1%.Both in Neighbour-Joining and Maximum Likelihood analyses S. tatrica groups consistently with S. dryadella, but bootstrap support is lacking.Borkowski (1970) described Stigmella geimontani tatrensis from the Polish Tatra Mts, reared from Geum montanum L. (Rosaceae).Later he synonymised it with S. pretiosa (see Borkowski 1975).

Remarks
In the paper from 1970 he recorded also S. stelviana (as ssp.crantziella) and S. dryadella from the Tatras.He found mines of S. stelviana on Potentilla crantzii Crantz & Fritsch (Rosaceae) from the end of August to mid of September at altitudes 1200-1800 m but no adult was reared from these.The mines could therefore also have been made by larvae of S. tormentillella that are feeding on various Potentilla spp.He also mentioned mines of S. dryadella on Dryas octopetala.They were observed in September and in May at an altitude of about 1800 m.The aforementioned record by Gregor (1986) of S. dryadella mines was also not supported by reared adults.

Discussion
The discovery of Stigmella tatrica shows that the montane fauna of Central European Nepticulidae is still insufficiently studied and can provide surprises.We expect that S. tatrica can also be found in the Polish part of the Tatra and other parts of the Carpathians, e.g. in Romania; currently it seems to be an endemic species for the Carpathians.Although the partial mitochondrial gene COI as used for DNA barcodes is usually not sufficient for a robust phylogenetic analysis, both analytical methods invariably group S. tatrica with S. dryadella, and this together with the fact that S. tatrica has usually been collected on or near Dryas octopetala, suggest that the two species are sister taxa whose ancestor shifted to Dryas.The protruding ovipositor suggests that S. tatrica has an unusual, possibly hidden oviposition site, and potentially rather different leafmines compared to S. dryadella, or even a different feeding mode.

Figure 11 .
Figure 11.Maximum likelihood tree of European species of the Stigmella aurella group, showing the position of Stigmella tatrica in red.Small figures represent bootstrap values, after 100 replicates.Bootstrap values below 50 are not given.A specimen of Stigmella tityrella is used as outgroup.Nomenclature follows van Nieukerken et al. (2016).