We investigated the distribution and species circumscription of Nychiodes obscuraria Villers (1789) and Nychiodes ragusaria Millière (1884). The morphology of male and female genitalia of Calabrian populations (southern Italy) revealed them to belong to N. ragusaria while specimens from Basilicata, Molise and the rest of Italy turned out to be N. obscuraria. Although the DNA barcodes showed a divergence of 1.55% between typical N. obscuraria and N. ragusaria, 9 out of 28 Calabrian specimens of N. ragusaria shared the N. obscuraria haplotype, and sometimes both haplotypes were found at the same site. Genetic introgression appears as the most probable scenario to explain the observed pattern. A correct identification for the investigated species pair should be based on morphology instead of DNA barcode analysis because of the observed mitonuclear discordance at mountain sites. Further studies are needed to clarify the occurrence of this discordance at high elevations only.

The genus Nychiodes comprises 27 species (Rajaei et al. 2022), currently assigned to the Boarmiini, a tribe of geometrid moths including more than 5,000 species (Müller et al. 2019; Murillo-Ramos et al. 2019). The genus is distributed in the Palaearctic region from Western Europe and North Africa to Iran, Afghanistan, and Pakistan. Nychiodes species are large, robust moths, forming a diverse group based on external morphology, without well-defined differential characters (Lederer 1853; Müller et al. 2019). Species are large-sized, with females being slightly larger than males. Wings are light brown to beige, dark grey, or with shades of brown, wing tip is slightly darker. Nychiodes species are mainly characterized by prominent ante- and postmedial lines on the forewing (Müller et al. 2019). Discal spots are usually present on all wings, of medium size. Antennae are dorsally covered with a mixture of yellowish and brown scales, bipectinate in both sexes, branches long in males, and short in females. The proboscis is absent. Despite the conspicuousness of this genus, its morphological variability makes it difficult to identify and classify species. Therefore, the study of genitalia structures is essential for a clear diagnosis of the species. Based on the morphology of the genitalia, the genus Nychiodes has been divided into three distinct species-groups: obscuraria species-group, waltheri species-group, and amygdalaria species-group (Wehrli 1929a, b; Müller et al. 2019). However, this grouping should not be considered as a phylogenetic concept until confirmed by a thorough molecular phylogenetic study. The obscuraria species-group can be diagnosed on the basis of the following characteristics according to Müller et al. (2019): In male genitalia, the costa of the valva is sclerotized to the subapical part, humped medially; the apex of the upper ampulla is covered with setae; the lower ampulla is not setose, pointed; the process of the sacculus is pointed; the tip of the aedeagus bears an acute triangular extension. In the female genitalia, the ductus bursae is long and membranous (equal to or longer than corpus bursae); lamella postvaginalis is strongly extended laterally and sclerotized. The following European species are included in this group: N. obscuraria Villers (1789), N. ragusaria Millière (1884), N. andalusiaria Millière (1865), N. notarioi Hermosa (2005), N. mauretanica Wehrli (1929c), N. hispanica Wehrli (1929c). The work focuses on two species of the genus Nychiodes, N. obscuraria and N. ragusaria. In Italy, the presence of N. obscuraria is confirmed in Valle d’Aosta, Piedmont, Lombardy, Trentino, South Tyrol, Veneto, Liguria, Emilia, Romagna, Tuscany, Umbria, Marche, Abruzzo, Molise, Puglia, Basilicata (Parenzan and Porcelli 2007). In southern Italy there are individuals of different size and with more or less marked wing pattern. The species is widespread and frequent, especially in hilly and mountainous areas, flying from early June to late July (Parenzan 1994). N. ragusaria, on the other hand, was previously considered an endemism of Sicily but Parenzan (1994) reported N. ragusaria as a new for the peninsula, occurring in xerothermic sites, both coastal and mountainous and being common from late June to mid-September. N. ragusaria has been confirmed in several locations in Calabria. In Calabria we found some individuals with contrasting mitochondrial DNA (mtDNA) and morphology. When two species externally similar exhibit significant variation in colouration, mtDNA analysis is often used for correct identification. However, in this particular case, mtDNA alone was insufficient. Dissection of the genitalia revealed discrepancies between the mitochondrial and morphological characteristics of some individuals collected from certain localities.

The aim of the work is to analyze a series of samples collected in different parts of Italy, in particular: (1) to investigate the potential presence of N. obscuraria in Calabria (southern Italy), through DNA barcoding and morphology of genitalia; (2) to refine the boundaries between both species as potential hybridization zones.

One hundred and sixty-eight specimens were analyzed, used for comparison of internal characters combined with DNA data and distribution patterns. Standard protocols (e.g., Ivanova et al. 2006) were used for DNA extraction and amplification of the ‘barcode’ fragment (658 base pairs of the 5’ terminus) of mitochondrial Cytochrome C Oxidase subunit I (COI). DNA extraction, PCR and sequencing were performed at the Canadian Centre for DNA Barcoding (CCDB, Guelph). All samples used for DNA analysis are presented in Table 1 along with their label data and sample ID. All sequences and metadata are available on BOLD in the public dataset DS-NYCHSITAL. MEGA X software (Kumar et al. 2018) was used to reconstruct maximum likelihood (ML) tree (Fig. 6) (using the K2P model: Kimura 1980). Genetic p-distances were calculated using the barcode gap analysis function on BOLD. The illustration of distributional data was created using Google Earth Pro (ver. 7.3.6.9345 for Mac). Male and female genitalia were prepared according to standard procedures (e.g., Robinson 1976), embedded in Euparal and photographed with an Olympus E-M1 camera, stacking the different focus layers with Helicon Focus 6 software.

The species studied were from the following collections.

CREA-FL Collection of the Research Centre for Forestry and Wood (Rende, Italy).

SNSB-ZSM Bavarian State Collection of Zoology (Munich, Germany).

CAS Collection of Andrea Sciarretta (Molise, Italy).

The two species studied exhibit very similar external morphology. However, examination of the genitalia allows for clear discrimination between N. obscuraria and N. ragusaria. Our results reveal a case of mitonuclear discordance as the conflict between morphology and molecular data demonstrates. In nine of the 24 Calabrian samples examined of N. ragusaria, BIN sharing was found with N. obscuraria, while the morphology of the genitalia clearly indicated N. ragusaria. In addition, we observed a case of exact haplotype sharing between N. obscuraria from Basilicata (Lagonegro, Italy) and N. ragusaria from the Pollino Massif area, being around 40 km apart. Therefore, in this case, discrimination at a specific level must rely on morphology, which clearly delimits the two species. The P-distance between the two haplotype clusters of N. ragusaria from southern Italy (including N. obscuraria from Basilicata) is 1.55% in the BOLD DNA barcode gap analysis.

Haplotype sharing as observed for the pair Nychiodes obscuraria – N. ragusaria also occurs in other taxa in the Geometridae family. In the allopatric Eupithecia manniaria and E. conterminata, haplotype sharing was reported between Scandinavian and Alpine populations separated by a very large distance (Scalercio et al. 2021). In the sympatric Scopula frigidaria and S. ternata, identical haplotypes were found at a distance not lower than 580 km (Hausmann et al. 2013). Given the observed geographic pattern of the pair Nychiodes obscuraria – N. ragusaria, we can hypothesize the occurrence of genetic introgression from N. obscuraria into N. ragusaria as also observed in the species pair Perizoma hydrata – P. affinitata (Hausmann et al. 2011). The slight divergences, and - in one case - also identical haplotypes, suggest that introgression has probably taken place several times, possibly also recently. Incomplete lineage sorting is unlikely because of the geographic distribution of the haplotypes. In fact, both haplotypes were found only in a small geographic area close to the potential contact zone of both species. Interestingly, we found that the obscuraria haplotype was found only at mountain sites south of the Pollino Massif, suggesting some kind of physiological advantages for mountain populations of ragusaria. Based on mitochondrial data this cannot be studied in further detail, but physiological studies and genomics would allow assessing the altitudinal segregation of mitonuclear discordance in much higher detail.

Regarding the geographical distribution, Figs 4, 5 demonstrate that N. ragusaria is present from Sicily to the Pollino Massif, while N. obscuraria is distributed from Basilicata, Latium, and Molise to northern Italy and France. Further research and more material with additional dissections and DNA barcodes is required to investigate the exact boundaries of the distribution areas of both species, in order to verify or reject a potential sympatric presence of both species in south-western Basilicata, elucidating further the patterns of species circumscription. Additionally, experimental investigation of the potential occurrence of hybridisation is necessary to better understand the discordance between genetics and morphology, which has – so far - only been observed in the Calabrian mountains above 840 m.

Figure 4. 

Distribution map of Nychiodes ragusaria and N. obscuraria. Google Earth Pro (ver. 7.3.6.9345).

Figure 5. 

Contact area between Nychiodes ragusaria and N. obscuraria. Google Earth Pro (ver. 7.3.6.9345).

Figure 6. 

Maximum likelihood tree of 33 French and Italian specimens of the Nychiodes obscuraria species-group, with N. mauretanica (Tunisia) as outgroup. Built with Mega X, Kimura 2 parameter, 100 replicates, bootstraps indicated on nodes. Scale bar: 0.5%.

We are very grateful to Paul Hebert, Evgeny Zakharov and Sujeevan Ratnasingham (CBG, CCDB University of Guelph, Canada) for support in DNA sequencing and analysis. Andrea Sciarretta (University of Molise, Campobasso, Italy) provided some very useful data from Molise. Bernd Müller (Berlin, Germany) and Gabriele Fiumi (Nature Studies Society of Romagna, Agricultural Sciences University of Bologna) for the genetic sequences of some specimens. University “Mediterranea” of Reggio Calabria, Italy gave financial support in the framework of the PhD program of the first author. The project was partially funded under the National Recovery and Resilience Plan (NRRP), Mission 4 Component 2 Investment 1.4 - Call for tender No. 3138 of 16 December 2021, rectified by Decree n.3175 of 18 December 2021 of Italian Ministry of University and Research funded by the European Union – NextGenerationEU; Award Number: Project code CN_00000033, Concession Decree No. 1034 of 17 June 2022 adopted by the Italian Ministry of University and Research, CUP 83D21014060006, Project title “National Biodiversity Future Center - NBFC”.We gratefully acknowledge the support of the Societas Europaea Lepidopterologica for covering the costs of processing the article (APCs).