Monopis jussii, a new species (Lepidoptera, Tineidae) inhabiting nests of the Boreal owl (Aegolius funereus)

Abstract Monopis jussii Kaila, Mutanen, Huemer, Karsholt & Autto, sp. nov. (Lepidoptera, Tineidae) is described as a new species. It is closely related to the widespread and common M. laevigella ([Denis & Schiffermüller], 1775), but differs in its distinct COI DNA barcode sequences, four examined nuclear loci as well as details in forewing coloration and pattern. Most reared specimens of M. jussii have emerged from the nest remnants of the Boreal owl (Aegolius funereus (Linnaeus, 1758)), but also nests of the Ural owl (Strix uralensis Pallas, 1771) and the Great tit (Parus major Linnaeus, 1758) have been observed as suitable habitats. Based on the present knowledge, the new species has a boreo-montane distribution as it is recorded only from northern Europe and the Alps. Several extensive rearing experiments from Strix spp. nest remnants from southern Finland did not produce any M. jussii, but thousands of M. laevigella, suggesting that the species is lacking in the area or, more unlikely, that the nest of these owl species do not serve as good habitat for the new species. This unexpected species discovery highlights, once again, the usefulness of DNA barcoding in revealing the cryptic layers of biodiversity. To serve stability we select a neotype for Tinea laevigella [Denis & Schiffermüller], 1775, and discuss the complicated synonymy and nomenclature of this species.


Introduction
The lepidopteran fauna of Central and North Europe has been investigated for a longer time and more intensively than that of any other region in the world. Consequently, discoveries of species new to the region are nowadays uncommon and usually involve expansive or invasive species. Large-scale efforts to build taxonomically comprehensive regional DNA barcode reference libraries have, however, resulted in a boost in discoveries of overlooked species during the last 15 years, as demonstrated by the increase of new species descriptions e.g. in the family Gelechiidae by . Characteristic to the new discoveries is that they often concern unexpected cases of cryptic diversity among well-known and often widespread species. Examples of such recent findings, originally detected as deep intraspecific splits in DNA barcode sequences, include Leptidea reali Reissinger, 1990(Dinca et al. 2011, Olethreutes subtilana (Falkovitsh, 1959) (Segerer et al. 2010), Phalonidia udana (Guenée, 1845) (Mutanen et al. 2012a), Epinotia cinereana (Haworth, 1811) (Mutanen et al. 2012b), Nemophora scopolii Kozlov, Mutanen, Lee & Huemer, 2016(Kozlov et al. 2017, several Elachista spp. (Mutanen et al. 2013) and Hoplodrina alsinides (Costantini, 1922)  .
There are many more additional cases of potential cryptic diversity in European Lepidoptera, as dozens of species show high levels of genetic polymorphism in their mitochondrial DNA (Mutanen et al. 2016. While polymorphism in the mitochondrial DNA may result from multiple other phenomena, including mitochondrial introgression and retained ancestral polymorphism, many of those cases are likely to result from cryptic diversity. An intraspecific split of the mitochondrial DNA being reflected in the nuclear genome in sexually reproducing species and in sympatry would strongly suggest the presence of cryptic diversity, because, unlike mitochondrial DNA, nuclear DNA is subject to genetic recombination. From this starting point, we sequenced four nuclear markers of Monopis laevigella ( [Denis & Schiffermüller], 1775), a widespread and common species of tineid moths, showing a deep sympatric genetic split in its DNA barcode region in Europe (Gaedike 2019). Despite the limited number of analyzed specimens, the results provided unequivocal genetic support for the presence of two biologically distinct species. Subsequent morphological examination revealed consistent differences in the adult wing patterns, providing additional support for the overlooked cryptic diversity. Additionally, based on the presently available data, the two species show overlapping, but different ranges and based on the present knowledge, also a different ecology. Based on these grounds, we here describe one of the taxa as new to science.

Material and methods
The material examined was acquired from the following collections:

ITJ
Research Terminology of genitalia follows Robinson and Nielsen (1993) and Gaedike (2019). Preparation of genitalia generally follows the method outlined by Robinson (1976). Male genitalia were mounted in dorso-ventral position as it was considered to best show shapes of diagnostic structures, even if the shape of the gnathos is not optimally expressed. Male genitalia were stained using Eosin, female genitalia as well as abdominal pelts of both sexes using Chlorazol black. Structures were embedded in Euparal. Images were edited using Corel PHOTO-PAINT (2019).
Species of Tineidae have been systematically sequenced for the standard barcode region of the mitochondrial COI (cytochrome c oxidase subunit 1) in the connection of ongoing regional or national DNA barcoding projects in the Alps (Lepidoptera of the Alps campaign) and Finland (FinBOL). DNA barcode sequencing was conducted at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) using standard Sanger protocols as explained in deWaard et al. (2008). We successfully sequenced 87 specimens of Monopis representing twelve species, the newly described species included. Five European species of Monopis (M. luteocostalis Gaedike, 2006, M. henderickxi Gaedike & Karsholt, 2001, M. christophi Petersen, 1957, M. pallidella Zagulajev, 1955and M. barbarosi (Koçak, 1981) were not included in this sampling. Each of them is morphologically clearly distinct from M. jussii sp. nov. (Gaedike 2019). Full collection and taxonomic data as well as voucher photographs, DNA sequences and GenBank accession numbers of all these specimens are available in the Barcode of Life Data Systems (BOLD; Ratnasingham and Hebert 2007) in the public dataset DS-MONOJUS at https://dx.doi.org/10.5883/DS-MONOJUS. Collection data of the specimens are also given in Table 1. Some of the COI sequences used in this study were previously published in Mutanen et al. (2016), the others are novel.
Four nuclear genes, carbamoylphosphate synthase domain protein (CAD), elongation factor 1 alpha (EF-1a), cytosolic malate dehydrogenase (MDH) and wingless, were sequenced at the University of Oulu, Finland. These genes were chosen primarily based on the high amplification success rate in other Tineidae, but also based on our previous experience on their general good functionality to provide useful taxonomic information between closely related species. In these analyses, three specimens of M. laevigella and two specimens of M. jussii, all collected from Finland, were included. Legs of dry and pinned adult specimens were used for extraction of genomic DNA with DNeasy Blood & Tissue Kit (Qiagen). We largely followed the sequencing protocol by Wahlberg and Wheat (2008), but PCR clean-up was carried out with ExoSAP-IT (Affymetrix, Santa Clara, CA, USA) and Sephadex columns (Sigma-Aldrich, St. Louis, MO, USA). Additionally, sequencing was performed using an ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). Sequences were checked and edited using BioEdit software (Hall 1999). The sequences were uploaded to a VoSeq database (Peña and Malm 2012). The same dataset was used to generate fasta files for Neighbor-Joining analyses. Minimum genetic p-distance barcode divergence between M. laevigella and M. jussii was calculated using analytical tools in BOLD Systems v. 4.0 (http://www.boldsystems.org). Neighbor-joining trees for the barcode region for all included Monopis species and specimens as well as four nuclear genes for five analyzed specimens of M. laevigella and M. jussii were constructed under p-distance model using Mega 7.0 (Kumar et al. 2016). The trees were stylized using CorelDraw v. 20.0.0.633.

Results
DNA sequencing resulted in a barcode of 552 bp or longer for 81 specimens. All except seven specimens yielded a full-length (654 bp) barcode. BOLD's barcode gap analysis showed that all included species have highly species-specific DNA barcodes with the mean of minimum divergences (p-distance model) to the nearest species being 10.01% (range 4.43-17.58%) (Figure 1). The minimum divergence between M. laevigella and M. jussii is 4.43%.
For each nuclear gene, data of only a single specimen of two analyzed M. jussii specimens were retrieved. Informative (i.e. data from both species available) sequence lengths by genes were as follows: CAD: 336 bp, EF-1a: 410 bp, MDH: 334 bp, wingless: 307 bp. Genetic p-distances between the two species were: CAD: 2.1%, EF-1a: 2.2%, MDH: 1.5%, and wingless: 4.1%. As a rule, the specimen of M. jussii formed a sister to the two or three specimens of M. laevigella (Figure 2). Diagnosis. Monopis jussii sp. nov. is externally close to M. laevigella, but the forewing appears darker, as it is less mottled with pale scales, especially along the margins (Figures 4, 5). Fringes are yellow and with a clear fringe line in M. laevigella but grey and without the fringe line in M. jussii. Besides the genetic markers, the forewing col- our is indeed the best clue to separate these species. There is nevertheless some variation, especially in M. laevigella. Both male and female genitalia vary considerably, as do those of M. laevigella. The variation in all characters of genitalia overlaps between these species, and, apparently, they cannot be identified by genital characters. For variation of M. laevigella see also Gaedike (2019). Moreover, M. weaverella (Scott, 1858)  Description. Forewing length 5.8-8.5 mm (n = 8 ♂ and 8 ♀) (note that the specimens are reared which may have affected their size). Maxillary palpus, labial palpus and head ochreous yellow; outer side of labial palpus with dark grey scales, second segment distally bristled. Scape of antenna ochre with pecten formed of bristle-shaped scales, pedicel and flagellum dark brown. Thorax dark grey, dorsomedially variably intermixed or entirely with pale ochre scales; tegula dark grey, apically often paler grey or ochre. Fore and mid leg inwardly ochre, outwardly leaden grey, apex of tibia and tarsal segments ochre. Hind leg inwardly pale, outwardly ochre, intermixed with grey scales; spurs and apex of tibia and tarsal articles ochre. Forewing dark grey, variably mottled with pale grey scales; costa narrowly and variably sometimes ochre; basal scales of termen with alternating pale ochre and grey scales, distal scales of termen unicolorous grey, contrast between distally paler basal scales and darker distal scales giving an impression of faint fringe line; silvery grey spot somewhat basal of middle of wing length at fold. Hind wing bluish grey with somewhat darker grey veins; fringe basally narrowly ochre, otherwise grey. Underside of wings grey with ochre margin; underside of hindwing dark grey along costal margin. Abdomen leaden grey, basal segments ventrally more or less ochre.

Monopis jussii
Male genitalia (Figure 6). Uncus elongate, triangular, laterally with long, hairlike scales, distally pointed, bifid. Gnathos arms angled in the middle, tapered toward hook-shaped apex. Basal and distal margins of tegumen reinforced, U-shaped, anter- The arrows indicate differences in fringe colour (yellow/grey), fringe line (present/absent; chequered/nonchequered) and forewing costa (many white scales between the costa and the dorsal spot/few white scales between the costa and the dorsal spot).
ior margin more deeply. Shape of valva highly variable, gradually varying from ovoid and basally broadest to somewhat elongate and medially widest; distally round. Every aspect of saccus variable; straight or somewhat undulate, apically little or very much widened; length also very variable. Phallus straight and nearly parallel-sided, slightly widened at basal 1/3; length compared to that of saccus impossible to establish due to variation in length of saccus. Phallus distally inserted in cylindrical, internally spinose anellus. Vesica distally densely spinose, devoid of cornuti.  (Figures 7-9). Papilla analis membranous, elongate, distally round, with a few setae. Apophysis posterioris as long as segments 7+8, posteriorly starting as continuation of papilla analis, slender, anteriorly slightly widened, apex cut. Apophysis anterioris 1/3 length of and slightly stouter than apophysis posterioris, twice as long as 8 th segment, distally not widened. Ovipositor telescopic, with two retractile nodes; with a few stout setae. Ventral pseudapodemes (sensu Davis and Robinson 1999) not decipherable. Tergum 8 posteriorly somewhat sclerotized. Ostium a widely U-shaped opening, laterally bordered as posteriorly curved rim, laterad shallowly emarginated in posterior direction, emargination with a few long setae; devoid of microtrichia but minutely granulose. Length of antrum variable, narrowed toward colliculum; colliculum tubular, length variable, 2-4 times as long as wide, usually narrowed in the middle. Ductus bursae between colliculum and corpus bursae membranous, as long as apophysis anterioris. Corpus bursae oval, 3 times as long as wide; in approximately the middle to posterior 1/3 ca. 12 elongate, sharply spicular or dentate signa forming transverse band.

Female genitalia
Genetic characterisation. Clearly distinguishable by its DNA barcode from all other species of Monopis barcoded globally so far (Figure 1). Genetically the closest species with a minimum divergence of 4.43% is M. laevigella. Intraspecific divergence among four barcoded specimens from Finland and Italy is 0.15%. Additionally, the species show 1.5-4.1% interspecific divergence in the nuclear genes of CAD, EF-1a, MDH and wingless (Figure 2).
Etymology. The species is dedicated to Dr Juhani (Jussi) Itämies, a Finnish expert of Lepidoptera who, as far as we know, is the first to have reared this species. He has Distribution. From our available observations M. jussii seems to have a boreomontane distribution pattern. It is widely distributed in Finland and also recorded from Norway (Finnmark) and Sweden (Härjedalen). Records from the Alps seem rare with a proved, barcode-based locality in the Italian Dolomites and two further unpublished records (ZSM, A. Segerer) in the Bavarian Alps.
Biology. So far reared on five different occasions from the nest bottoms of the Boreal owl (Aegolius funereus). Two specimens in the collection of ZMUO have been reared from the nest of the Ural owl (Strix uralensis) and one specimen from the nest  of the Great tit (Parus major). Additionally, three reared specimens of two different rearing events do not state anything about the origin. One specimen has been found in a vacated house. Thirteen specimens in coll. ZMUO and a specimen from the Italian Alps in coll. TLMF have been collected in the wild between 17 June to 21 July, which matches well with the flight time of other Monopis species of these regions.

Taxonomic remarks on Monopis laevigella
Monopis jussii sp. nov. is most closely related to M. laevigella and can easily be confused with that species (see above). We therefore re-evaluate available names in the M. laevigella species group.

Neotype selection
Tinea laevigella was described from an unspecified number of specimens collected in the area of Vienna, Austria ( [Denis & Schiffermüller], 1775). The collection was later deposited in the "Hof-Naturalien-Kabinett" and destroyed by fire during the Vienna Rebellion on 31 st of October 1848 (Speta 2003). Since this species can be confused with M. jussii sp. nov. and several other congeneric taxa we designate as neotype a male specimen from Austria to preserve stability ( Figure 10). It is labelled "AUSTRIA occ. Nordtirol / Brandenberg / Tiefenbachklamm / 11°51'52"E, 47°29'4"N / 645 m, 16.6.2013 / leg. Huemer" "DNA Barcode / TLMF Lep 10354" (TLMF). Tinea rusticella was figured twice by Hübner in the eighth volume of his Sammlung europäischer Schmetterlinge, first it was validly described on page 61, pl. 3, fig. 17 (1796) and later a different species was figured on pl. 49, fig. 339 (1813). Hübner (1825) considered them conspecific, and he referred to both figures when he erected the monotypic genus Monopis. Zeller (1852) fig. 17 as Recurvaria rustica, which is an unjustified emendation and thus an objective synonym of Tinea rusticella (Hübner, 1796) fig. 17, proposed the name Tinea saturella in synonymy with Tinea rusticella. Because Tinea saturella was described in synonymy with Tinea rusticella it was always considered a synonym of that species (viz. Monopis rusticella), but because Haworth referred only to Hübner's fig. 17 (and not to fig. 339) it is an objective junior synonym of Tinea rusticella Hübner, 1796, and thereby a subjective junior synonym of Haplotinea insectella (Fabricius). However, as the name Tinea saturella has never been made available under the provision of Art. 11.6. of the Code (ICZN 1999) and adopted as the name of a taxon before 1961, we consider it as unavailable.
Although Monopis Hübner 1825 was described as a monotypic genus, it is based on a partly misidentified species. We consider Zeller (1852) as First Reviser of Tinea rusticella Hübner, restricting the name to the species now (and also by Zeller 1852) known as Monopis laevigella ( [Denis & Schiffermüller], 1775).

Discussion
Compared with many other groups of Lepidoptera, the species diversity of Tineidae is generally poorly investigated. Hundreds of species deposited in museum collections remain undescribed (Robinson 2009). It is likely that many more species remain entirely undiscovered globally. The European fauna is comparatively well understood, and the fauna of the entire continent has recently been taxonomically reviewed in two monographs (Gaedike 2015, 2019). New species discoveries are uncommon, particularly for central and northern parts of Europe. An example of a recent species discovery is that of Monopis neglecta Šumpich & Liška, 2011, a species that morphologically is nearly indistinguishable from M. weaverella (Scott, 1858) (see Gaedike 2019). While no genetic data were provided for M. neglecta in the original description, the DNA barcode sequences provided in the present study confirm its status as a separate species from M. weaverella. It is encouraging that although the species of Tineidae are often difficult to tell apart from each other morphologically, no cases of barcode sharing in the European fauna are known. Evidently, therefore, DNA barcoding provides an efficient way to investigate their diversity in less thoroughly explored areas as well.
Based on the available distributional data, Monopis jussii has a much more limited range than M. laevigella. It is possible, if not likely, that it is a member of boreo-montane faunal elements, being distributed in the boreal region on the one hand and in the Alps below the timberline on the other hand. It is likely absent from the lowlands of Central Europe. It would not be surprising if the species turns out to be present in other European mountain systems and the eastern Palearctic. Based on the large number of examined museum specimens from the ZMUO and MZH collections, the species is widely present in northern Finland south to ca. 64° N but becomes much scarcer towards the more southern localities. The southernmost verified records from Finland are from the province of Tavastia australis (ca. 61° N).
Based on our own and other experiences (Robinson 2009, Gaedike 2019), Monopis laevigella is not strict regarding the source of its food, but it seems to prefer cavitybreeding birds, possibly because their nests are usually dry. Several extensive rearing experiments of nest bottoms of various birds, mostly the Tawny Owl (Strix aluco Linnaeus, 1758) and the Ural Owl (S. uralensis Pallas, 1771), from southern Finland have yielded large numbers of M. laevigella, which is usually present in every nest in large numbers. In an experiment by MM in 2017 with 13 nest bottoms of Strix spp., probably thousands of M. laevigella emerged. Among several dozen pinned specimens sampled from each nest, none represents M. jussii. Other species that are regularly or often present in the nests of Strix spp. in Finland are Niditinea striolella (Matsumura, 1931) (usually emerges in great numbers too), Tinea svenssoni Opheim, 1965 (present in almost all nests), Tinea steueri Petersen, 1966 (not present in every nest) and Monopis fenestratella (Heyden, 1863) (present in most nests but is cryptic in behaviour). While it is possible that M. jussii has stricter habitat requirements and that it has a strong preference for the Boreal Owl, we find this possibility unlikely. The Boreal owl, the Ural owl, as well as the Great tit are all cavity breeders, rendering the nest conditions between these species very similar. In rearing conditions, tineids are not selective for the origin of food and readily feed on mammal hairs too. It is more likely that Monopis jussii has been reared mostly from the nests of the Boreal owl just because it is a more common owl species within the moth's main distribution in Finland than either of the two Strix species present in Finland. Further rearing experiments, optimally systematically from different species of birds, would bring additional valuable information on the habitat requirements of M. jussii and several other species of Tineidae.
Monopis laevigella has a Holarctic distribution Pohl 2018, Gaedike 2019). Many specimens of this species have been barcoded from the Nearctic region, both from Canada and the U.S.A. They fall in two clusters, both of which are highly distinct from the clade consisting of M. jussii and the Palearctic M. laevigella (data only partially public in BOLD). In the Neighbor-Joining trees neither of these clusters is placed as sister to the Palearctic M. laevigella + M. jussii clade, suggesting that they represent distinct taxa and even that their closest relative is not M. laevigella. However, due to the limited phylogenetic information content of the DNA barcode region, verification of both scenarios requires more rigorous and thorough taxonomic and phylogenetic scrutiny.