﻿Thiotrichasumpichi sp. nov. – a new species of Thiotrichinae (Lepidoptera, Gelechiidae) from south-eastern Europe

﻿Abstract Thiotrichasumpichisp. nov. is described from Greece and Croatia. The systematic position of the new species within Thiotricha is discussed based on external and genitalia characters and from DNA barcodes of the mitochondrial COI gene (cytochrome c oxidase 1). Adults, details of external morphology, and male and female genitalia of the new species are illustrated.


Introduction
Despite continuing efforts over many years, the taxonomy of European Gelechiidae is still incompletely covered and several genera and cryptic species groups await revision, probably with dozens of undescribed species . One of the unresolved problems relates to an externally rather characteristic, small grey species with black markings from Greece and Croatia which has been known to the authors for many years. However, the generic and even tribal/subfamily assignment has remained uncertain. This taxon was first published as a genus and species close to Caulastrocecis Chrétien, 1931 by Huemer and Karsholt (2017), however, without detailed analysis of morphology and genetics. Extensive DNA barcoding of this species indicated a systematic placement near Thiotricha Meyrick, 1886. As a result of our detailed morphological study of this undescribed species we came to the conclusion that despite rather unusual admixture of external and genitalia characters, it shares several synapomorphies with the genus Thiotricha. In this contribution we therefore establish a new species of Thiotricha to accommodate this, locally rather abundant, species distributed in both the mainland and islands of Greece, and in Croatia.
Male and female genitalia were dissected and prepared using standard methods (Huemer and Karsholt 2010). Male genitalia were spread using the unrolling technique as described by Pitkin (1986) and Huemer (1988). Pinned specimens and details of external morphology were photographed with a Canon EOS 5DS R DSLR camera (O. Bidzilya). Slide-mounted genitalia were photographed with a Canon EOS Rebel T5 DSLR camera mounted on an Olympus U-CTR30-2 trinocular head combined with a Carl Zeiss compound microscope (O. Bidzilya). For each photographed specimen, sets of 5-10 images were taken at different focal planes and focus-stacked using Helicon Focus 6 with the final image edited further in Adobe Photoshop CS5. Photographs of adults were taken with a Zeiss Stemi 508 KMAT stereo microscope, genitalia photographs with a Zeiss Axiolab 5 microscope, both adapted to an Olympus OM-D Mark III camera. Stacked photos were edited using Helicon Focus 4.8 and Adobe Photoshop 6.0 (P. Huemer).
The terminology of genitalia follows Lee et al. (2021) and Ponomarenko (2005). Tissue samples (a single hind leg) of 26 specimens of Thiotricha were prepared for DNA barcoding (deWaard et al. 2008), a method which usually allows accurate species identification from a 658 base-pair long segment of the mitochondrial COI gene (cytochrome c oxidase 1), and successfully processed at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph). In addition, a single private sequence > 600 bp was made available to us in the Barcode of Life Data Systems (BOLD) (Ratnasingham and Hebert 2007;Ratnasingham 2018). Details including complete voucher data and images of our specimens can be accessed in the public dataset "New species of Thiotrichinae" (dx.doi.org/10.5883/DS-THIOSUMP) in BOLD. Sequences from the dataset were submitted to GenBank. All sequences were assigned to the Barcode Index Numbers (BIN), algorithm-based operational taxonomic units that provide an accurate proxy for the true species. BINs were automatically calculated for records in BOLD that comply with the DNA barcode standard (Ratnasingham and Hebert 2013). Follow-up species identification strictly followed available reference sequences in BOLD. Degrees of intra-and interspecific variation of DNA barcode fragments were calculated under the Kimura 2 parameter model of nucleotide substitution using analytical tools of BOLD systems v. 4.0. (http://www.boldsystems.org). Calculation of intraspecific distance was furthermore normalized with BOLD calculation tools to reduce bias in sampling at the species level. A Neighbour-Joining tree was constructed using the Kimura two-parameter model in MEGA7 (Kumar et al. 2016).

Molecular analysis
DNA barcoding resulted in a BIN concordant barcode fragment of >600 bp for 25 specimens and a single sequence of 413 bp for four European species of Thiotricha. Sequences revealed moderately low intraspecific, but significantly higher interspecific genetic distances (Table 1, Fig. 1). Whereas intraspecific divergence is unknown in T. wollastoni (Walsingham, 1884), mean divergence ranges from 0.52 to 0.8% in the other species. All species are attached to a unique BIN with the exception of T. subocellea (Stephens, 1834) where barcodes grouped in two BINs (Ratnasingham and Hebert 2013). In contrast, minimum interspecific divergence is 3.62% in T. subocellea and T. majorella (Rebel, 1910) whereas the distance of T. wollastoni and T. sumpichi sp. nov. to the nearest neighbour in Thiotricha is c. 10-11%. No DNA barcode is available for T. coleella since the only published sequence was based on a misidentified specimen ).  ence of black spots rather than streaks. The male genitalia are characterized by having the valva distinctly broadened in the basal half, strongly edged posteromedial emargination of the vinculum and a short broadly rounded saccus. Other species of Thiotricha have unmodified or weakly broadened basally valva, gently edged posterior margin of the vinculum and short, usually subtriangular saccus. Segment VIII in the male is distinct by having a very short band-shaped tergum and large subtriangular sternum with broadened basal lobes separated by broadly rounded medial emargination, and narrow posterior portion with short apical incision. In T. subocellea segment VIII is similar in general, but the sternum is deeper bifurcated. Comparatively short (shorter than segment VIII) apophyses anteriores, antrum with narrow band of small triangular sclerites and long, slender longitudinal ridge-shaped signum are characteristic for the female genitalia of the new species. Thiotricha subocellea has somewhat similar female genitalia, but apophyses anteriores are longer that segment VIII, the antrum lacks sclerites and the signum is crescent-shaped.
Etymology. The new species is dedicated to Jan Šumpich (NMPC) in recognition of his contribution to the study of Palaearctic Lepidoptera.
Biology. Host plant and immature stages unknown. Adults have been observed flying from late August to early November and were collected in large number at light (P. Huemer). The habitat is insufficiently known, but the species has been observed in a wide spectrum of different vegetation, mainly in open oak and maple forests intermixed with Mediterranean macchia (Fig. 21).

Discussion
The species dealt with above has a restricted distribution in south-eastern Europe where it is locally common. The new species is small and grey, but the shape of the black markings on the forewings is distinctive. It has been known to lepidopteran specialists for several decades, but it could not be described until now because of uncertainty of its placement in the system of Gelechiidae. Instead, it was known by specialists as "Caulastrocecis sp." (e.g., Huemer and Karsholt 2017), mainly based on external characters. During a recent revision of that genus (now Ptycerata Ely, 1910) (Bidzilya and Karsholt 2021) it became clear that the present species is not even distantly related.
We have been able to show that the new species fits into the subfamily Thiotrichinae. This is supported both by morphology and DNA barcode analyses, which, despite considerable divergences, group the new species nearest to European species of Thiotricha (Huemer 1993) (Fig. 1). Thiotrichinae are characterized by morphological characters such as a well-developed pterostigma in the forewing, a large sternum VIII and a reduced tergum VIII, valvella with a pair of digitate processes with one or a few apical setae, signum a plate (e.g., sickle-shaped, jar-shaped or occasionally missing), and the male antennae with rather long cilia (shorter in Polyhymno Chambers, 1874) (Karsholt et al. 2013;Gregersen and Karsholt 2022). The present species shares most of these characters.
Thiotrichinae is a moderately small subfamily of Gelechiidae, with about 180 described species in seven genera (Lee et al. 2021) and is most diverse in Asia. Since the description of the subfamily in 2013, they have been the subject of several studies (e.g., Lee et al. 2018Lee et al. , 2021Kyaw et al. 2019;Lee and Hayden 2019). Lee et al. (2021) presented a phylogeny of the subfamily with a revision of the generic classification based on molecular and morphological analyses. Based on that we are able to suggest a placement for the new species in Thiotricha.
Thiotrichinae currently comprises seven genera: Thiotricha, Polyhymno Chambers, 1874, Macrenches Meyrick, 1904, Calliprora Meyrick, 1914, Pulchrala Lee & Li, 2021, Tenupalpa Lee & Li, 2021and Palumbina Rondani, 1876(Lee et al. 2021. Thiotricha sumpichi sp. nov. differs from these genera by the wide basal lobe of the valva in the male genitalia and by the lack of pecten on the antennal scape which is characteristic for other Thiotrichinae genera. Despite these autapomorphies, the new species shares with Thiotricha the following characters: (1) antennal ciliation in the male; (2) absence of Rs4 vein in the forewing; (3) male sternum VIII broadened at the base with a tendency to be bifurcate apically; (4) vestigial coremata; (5) elongate slender signum; and (6) modified base of the valva. We therefore tentatively include the new species in Thiotricha. However, Thiotricha is rather diverse morphologically and in need of revision. Several DNA barcode lineages require additional molecular data and extensive analysis of morphology (Fig. 1). In a phylogeny based on both morphology and molecular data of several taxa presented by Lee et al. (2021) Thiotricha clusters into two main groups. One includes the type species of the genus T. thorybodes Meyrick, 1886 from New Zealand. In the other group we find the European T. subocellea (Stephens, 1834) which is type species of the genus Reuttia Hofmann, 1898 and all other continental European congeners (Huemer 1993). It is beyond the scope of the present study to evaluate if Thiotricha should be split into several genera. nating specimens to ZMUC. Collecting permits were supplied by the Greek Ministry for the Environment, Energy and Climate Change and P. Huemer particularly thanks K. Agapitou and D. Dason. Furthermore, we are grateful to reviewers and subject editor for critical remarks on an earlier version. Martin Corley (U.K.) kindly commented on and improved the English language of the manuscript.

Conflict of interest
The authors have declared that no competing interests exist.

Ethical statement
No ethical statement was reported.

Funding
The work of O. Bidzilya was supported by the Ukrainian State Budget Program "Support for the Development of Priority Areas of Scientific Research" (Code: 6541230).

Author contributions
Contributions to this research are as follows: P. Huemer: molecular research analysis, discussion on the results of the molecular research, preparation of Table 1