Incurvaria stangei Rebel, 1903 revised combination, placed without justification in Prodoxidae, is redescribed and transferred back to the Incurvariidae. Male and female genitalia of this south-eastern alpine endemic species are illustrated for the first time. DNA barcode sequences as well as morphological traits support the revised family assignment.

Incurvariidae and Prodoxidae are two out of formerly five (van Nieukerken et al. 2011), now six (Regier et al. 2015) known families of the superfamily Adeloidea Bruand, 1850. The type-species for Incurvariidae is Incurvaria masculella (Denis & Schiffermüller, 1775) and for Prodoxidae Prodoxus decipiens Riley, 1880 (Nye and Fletcher 1991). The monophyly of both families is well supported by a morphologically based phylogenetic analysis (Nielsen and Davis 1985) and by recently published molecular studies (Mutanen et al. 2010; Regier et al. 2015).

Incurvaria stangei was described by Rebel (1903) from a single female collected in the Italian Dolomites (Südtirol, Tschierspitze, 31.vii.1902) by G. Stange. Shortly after this discovery a single male was found in the area of Triglav mountain range, currently Slovenia (Rebel 1907). Despite its extraordinary and unmistakable external appearance, this remarkable alpine species of primitive moth remained neglected for more than eight decades. Only in 1988 P. Huemer discovered a specimen and published a re-description, although without examination of genitalic structures (Huemer 1991). Nevertheless, he still treated the species in the family Incurvariidae Spuler, 1898. In contrast, Wojtusiak (1996) in the European Lepidoptera catalogue listed the species under Lampronia Stephens, 1829 in the family Prodoxidae Riley, 1881 for the first time, without giving arguments for the new combination. Subsequent publications followed this new taxonomic concept (e.g. Deutsch 2012; Huemer 2013; Karsholt and Nieukerken 2004–2011). However, Kurz (2002–2012), after dissection and careful examination of a female, reconsidered the taxonomy and informally recombined the species in Incurvaria Haworth, 1828 (Incurvariidae). This work is now supported after an extensive analysis of characters of the male and female genitalia and cytochrome c oxidase subunit I (COI-5P) DNA barcodes and we therefore now formally present this revised combination.

Our study is based on examination of ten specimens of I. stangei which are all deposited in the collections of the Tiroler Landesmuseum Ferdinandeum, Natural History Collections, Hall, Austria. Genitalia preparations followed standard techniques (Robinson 1976), with some modifications, especially the implementation of the so-called unrolling technique in male genitalia (Pitkin 1986). Although genitalia of both sexes are strongly sclerotized, male preparations were slightly stained with Mercurochrome and females with Chlorazol to highlight delicate structures. For photos, genitalia of both sexes were embedded in Euparal with ventral side up, male genitalia being unrolled. Additionally, male genitalia were photographed in their natural three-dimensional shape before spreading, both from lateral and dorsal aspect. Photos of adults were taken with a Canon EOS 5D Mark III and Canon lens MP-E 65 at 2:1, using a ring flash. Genitalia photos were taken with a microscope (Wild Heerbrugg) using a 10x objective and a 2.5x ocular. All photos were edited using the software HELICON FOCUS 4.80 and ADOBE PHOTOSHOP 6.0. For sequencing a single leg was removed, placed in a 96-well lysis plate and sent for analysis to the CCDB (Canadian Centre for DNA Barcoding, University of Guelph, Canada) where DNA extraction, PCR amplification, and sequencing were performed following standard high-throughput protocols (DeWaard et al. 2008). A Neighbor-Joining (NJ) tree of 26 European species of Incurvariidae and Prodoxidae plus a species of the type genus Prodoxus Riley, 1880 was constructed using MEGA 7 (Kumar et al. 2016). This tree is based on 132 barcode sequences which are available in the public dataset “DS-LEPIINCU Lepidoptera - Incurvarioidea” in the Barcode of Life Datasystems BOLD (Ratnasingham and Hebert 2007). Node confidences were assessed using 500 bootstrap replicates.

Abbreviations:

NHMW Naturhistorisches Museum, Wien, Austria.

TLMF Tiroler Landesmuseum Ferdinandeum, Innsbruck, Austria.

The family concept of Incurvariidae and Prodoxidae and consequently the correct combination of genera and species remained obscure through most of the 20^th century. Even in some recent literature the European genera of Prodoxidae were lumped in the family Incurvariidae (Karsholt et al. 1995). However, already Nielsen (1982, 1985) convincingly separated these families based on diagnostic characters, primarily of male and female genitalia morphology. Accordingly, males of Prodoxidae can be recognized by the presence of well-defined pectinifers of the valva, whereas in the female genitalia, Prodoxidae possess a pair of stellate signa in the corpus bursae and a posteriorly well-rounded sternum VII and a triangular tergum VII. Furthermore, the larvae of Prodoxidae – as far as known – are endophagous in herbs and shrubs, whereas nearly all larvae of Incurvariidae are free living in self-constructed cases.

The male genitalia of I. stangei lack the family characteristic pectinifers typical of Prodoxidae and the more flattened, scale-shaped spines of the valva are grouped together. These characteristics support the placement of I. stangei in Incurvariidae. Similarly the female genitalia of I. stangei, which completely lack stellate signa, perfectly fit into the concept of Incurvariidae as proposed by Nielsen (1982). Furthermore, sternum and tergum VII are both of rectangular shape, characteristic for Incurvariidae. In contrast, a posteriorly well-rounded sternum VII and a triangular tergum VII is considered diagnostic for Prodoxidae (Nielsen 1982).

A Neighbor Joining tree analysis was derived from DNA barcode sequences (COI) of selected European species of Incurvariidae and Prodoxidae with I. stangei nesting in Incurvariidae. Considering a mean interspecific distance of 8.5% (min. 1.2%, max. 13.7%) in Incurvariidae, I. stangei shows a remarkable distance (12%) to its closest species, Paraclemensia acerifoliella (Fitch, 1854), from North America, while it is over 12.3% to Incurvaria praelatella (Fig. 6).

Figure 6. 

Neighbor-Joining tree based on DNA barcodes (658 bp fragment of the mt COI gene) of European Incurvariidae and Prodoxidae. The node support values in percentages are based on 500 bootstrap replicates.

We are particularly grateful to Paul Hebert and his team at the Canadian Centre for DNA Barcoding (Guelph, Canada) whose sequencing work was enabled by funding from the Government of Canada to Genome Canada through the Ontario Genomics Institute. We are also grateful to the Ontario Ministry of Research and Innovation and to NSERC for their support of the BOLD informatics platform. We are indebted to the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano - South Tyrol for helping to fund the project “Genetische Artabgrenzung ausgewählter arktoalpiner und boreomontaner Tiere Südtirols”. Marko Mutanen (Oulu, Finland) kindly granted us full access to his valuable sequences of Incurvariidae and Prodoxidae in BOLD. We are finally grateful to Petra Schattanek for the photograph of the habitat. Last but not least we owe gratitude to Mikhail Kozlov (University of Turku, Finland) and Erik van Nieukerken (Naturalis, Leiden, The Netherlands) for helpful comments.