Two Italian Moth Species Blur the Line Between DNA and Anatomy

In the mountains of Calabria, two species of geometrid moths are quietly dismantling one of modern taxonomy’s most trusted shortcuts. A new study published in Nota Lepidopterologica has investigated the distribution and species boundaries of Nychiodes obscuraria and Nychiodes ragusaria, two large, robust moths of the family Geometridae that have long puzzled entomologists working in southern Italy. The findings reveal a striking case of mitonuclear discordance, a phenomenon in which an animal’s mitochondrial DNA tells a different evolutionary story than its anatomy, and they carry a practical warning for anyone who believes a DNA barcode alone can settle a species identification.

The genus Nychiodes comprises 27 species assigned to the tribe Boarmiini, a group of geometrid moths that includes more than 5,000 species and stretches across the Palaearctic region from Western Europe and North Africa to Iran, Afghanistan and Pakistan. These are conspicuous insects, with wingspans reaching roughly 48 millimetres, light brown to dark grey wings, prominent ante- and postmedial lines on the forewing and bipectinate antennae in both sexes. Yet despite their size, the species are notoriously difficult to tell apart by external appearance alone, because their morphological variability blurs the very characters that should diagnose them. For that reason, researchers have long relied on the fine architecture of the genitalia, which differs consistently between species, to draw firm taxonomic lines.

The two species at the centre of the new work occupy neighbouring territories in Italy. Nychiodes obscuraria, first described from southern France in 1789, is widespread and frequent across the peninsula, confirmed from Valle d’Aosta and Piedmont in the north down through Tuscany, Umbria, Marche, Abruzzo, Molise, Puglia and Basilicata, flying mainly from early June to late July in hilly and mountainous areas. Nychiodes ragusaria, described in 1884, was long considered an endemic of Sicily, until the entomologist Parenzan reported it as new for the mainland in 1994, occurring in xerothermic sites both coastal and mountainous and flying from late June to mid-September. The species has since been confirmed at several locations in Calabria, the mountainous toe of the Italian boot, where it approaches the range of its congener.

Because the two species are externally so similar, and because individuals vary considerably in size and wing pattern, researchers routinely turn to mitochondrial DNA analysis when colouration fails. The standard tool is DNA barcoding, which reads a 658 base pair fragment of the mitochondrial Cytochrome C Oxidase subunit I gene, the COI barcode that underpins the reference library hosted on the Barcode of Life Data System. In the new study, the team analysed 168 specimens, extracting and sequencing DNA at the Canadian Centre for DNA Barcoding in Guelph, reconstructing maximum likelihood trees with MEGA X software using the Kimura 2 parameter model, and calculating genetic p-distances with the barcode gap analysis function on BOLD. In parallel, male and female genitalia were prepared according to standard procedures, embedded in Euparal and photographed with focus stacking to reveal their diagnostic structures.

The morphological results were unambiguous. Examination of the genitalia allowed clear discrimination between the two species. In the male genitalia of N. ragusaria, the valva is extended with a triangular dorsal part that fuses apically with a digitiform extension covered in fine bristles, a character that separates it from all other European members of the obscuraria species group, while the upper ampulla is clubbed and apically spined and the aedeagus bears a needle-like cornutus slightly shorter than itself. In N. obscuraria, the medial line of the forewing is absent, an important diagnostic character against other European congeners, and the male genitalia show a broadly expanded valva with a strongly reduced sacculus process and an aedeagus larger than in any other species of the group. The female genitalia differ as well, with the lamella postvaginalis apically divided and bilobed in N. ragusaria and less sclerotized in the middle in N. obscuraria. Applying these characters to the Calabrian populations, the researchers found that every specimen belonged to N. ragusaria, while moths from Basilicata, Molise and the rest of Italy were N. obscuraria.

The DNA data, however, refused to cooperate. The barcode gap analysis showed a divergence of 1.55 percent between the typical haplotype clusters of the two species, a distance consistent with their status as separate species. Yet of the 28 Calabrian specimens of N. ragusaria barcoded, nine shared the BIN and in one case the exact haplotype of N. obscuraria from the rest of Italy and France, even though their genitalia clearly indicated N. ragusaria. Two barcode index numbers occur in Calabria, BOLD: AAE5581, shared with typical Sicilian populations of N. ragusaria, and BOLD: ACE5020, shared with N. obscuraria. The discordance went further still: one specimen of N. obscuraria from the Lagonegro area of southernmost Basilicata carried an identical haplotype to N. ragusaria from the Pollino Massif, sites roughly 40 kilometres apart. Sometimes both haplotypes were found at the same site.

The most probable explanation for this pattern, the authors argue, is genetic introgression, the movement of genes from one species into another through hybridization and repeated backcrossing. Similar haplotype sharing has been documented elsewhere in the Geometridae. In the allopatric species pair Eupithecia manniaria and E. conterminata, haplotype sharing was reported between Scandinavian and Alpine populations separated by a very large distance, while in the sympatric Scopula frigidaria and S. ternata, identical haplotypes were found at sites no closer than 580 kilometres. The slight divergences, and in one case identical haplotypes, observed in the Nychiodes pair suggest that introgression has probably taken place several times, possibly also recently. Incomplete lineage sorting, the alternative scenario in which ancestral polymorphism persists in newly separated species, is considered unlikely because both haplotypes were found only in a small geographic area close to the potential contact zone of the two species, a distribution inconsistent with widespread ancestral variation.

Perhaps the most intriguing observation is altitudinal. The obscuraria haplotype in Calabrian N. ragusaria was found only at mountain sites south of the Pollino Massif, above roughly 840 metres, and the discordance has so far been observed exclusively in the Calabrian mountains. This pattern hints at some kind of physiological advantage for mountain populations carrying the obscuraria mitochondrion, though mitochondrial data alone cannot test that hypothesis. The authors call for physiological studies and genomic analyses to assess the altitudinal segregation of the mitonuclear discordance in far greater detail, and for experimental investigation of potential hybridization to better understand the conflict between genetics and morphology.

The distributional picture that emerges from the study maps the two species from Sicily to the Alps. Nychiodes ragusaria is present from Sicily north to the Pollino Massif, while N. obscuraria is distributed from Basilicata, Latium and Molise northward through the rest of Italy and into France. Whether the two species occur sympatrically in south-western Basilicata remains an open question that will require more material, additional dissections and further DNA barcodes to verify or reject. Until then, the exact boundaries of their ranges, and the precise nature of their contact zone, remain to be elucidated.

The practical message of the study is as clear as it is counterintuitive for a genomic era: for this species pair, correct identification must be based on morphology rather than DNA barcode analysis, because of the observed mitonuclear discordance at mountain sites. The finding is a vivid reminder that DNA barcodes, powerful as they are, read only a single maternally inherited locus, while species are shaped by their whole genomes and their whole bodies. In the sunlit mountains of Calabria, where two moth species meet and mingle their genes, the anatomist’s scalpel still outperforms the sequencer, and the study of genitalia structures remains essential for a clear diagnosis of the species. Further studies are needed to clarify why this discordance appears to occur only at high elevations, a question that turns a routine taxonomic revision into an open invitation to explore how species boundaries are drawn, and redrawn, by evolution itself.

Subject of Research: Taxonomy and distribution of two closely related Nychiodes moth species in southern Italy

Article Title: Distribution and taxonomy of two closely related Nychiodes species in southern Italy ( Lepidoptera , Geometridae , Ennominae )

Article References: Latella, I., Scalercio, S., & Hausmann, A. (2024). Distribution and taxonomy of two closely related Nychiodes species in southern Italy (Lepidoptera, Geometridae, Ennominae). Nota Lepidopterologica, 47, 93-104. https://doi.org/10.3897/nl.47.118416

Image Credits: AI Generated

DOI: 10.3897/nl.47.118416

Keywords: Distribution, taxonomy, closely, related, Nychiodes, species, southern, Italy, Lepidoptera, Geometridae, Ennominae, scientific research