P. barrassoi was described in 2006 from a single male collected at 2,530 metres in the Maiella Mountains of Abruzzo, Italy. That specimen became the holotype. A female originally associated with the species was subsequently found to be a misidentified and unusually small specimen of Colostygia aqueata. A male collected on Monte Terminillo in 2004, about 100 kilometres from the type locality, was barcoded in work reported in 2019 and showed 8.9% sequence divergence from P. incultaria. That isolated genetic result supported recognition of a separate Central Italian taxon but did not establish its wider distribution. The later study brought the type anatomy, mitochondrial barcodes, nuclear genomic data, adult morphology and ecological records together to reassess populations assigned to the P. incultaria–P. barrassoi complex.

Researchers combined external characters with dissections of male and female genitalia, observations of phenology and vertical distribution, and DNA evidence. They measured anatomy with a calibrated microscope eyepiece and examined prepared genitalia and abdomens. For barcoding, they extracted DNA from one or two legs of dried specimens and analyzed a 658-base-pair region of the mitochondrial cytochrome c oxidase I gene, or COI. Sequence divergences were calculated with the Kimura two-parameter model and analyzed using a neighbor-joining algorithm. High-throughput sequencing was also used for older museum material. In parallel, ddRAD sequencing generated genome-wide single-nucleotide polymorphisms, or SNPs, for maximum-likelihood phylogenetic analysis and population-clustering tests. The authors used these independent evidence streams together because the two moths have no constant and reliable external wing feature that permits separation in the field.

The mitochondrial results divided the tested specimens into two clearly separated groups. The Monte Terminillo specimen produced a 658-base-pair sequence assigned to BOLD bin ACJ5976. Six specimens from Austria, southwestern Bavaria and northern Italy shared that bin and were only 1.1% distant from the Central Italian sequence. Their nearest species was P. incultaria, represented in the comparison by 19 specimens from Germany, Austria and northern Italy in bin AAF5044, with a reported barcode distance of 8.5%. Two specimens attributed to P. incultaria from the vicinity of Gastein in Salzburg were also successfully sequenced using high-throughput methods and fell unambiguously into the P. incultaria bin. Thus, the barcode evidence separated the taxa while also connecting Alpine populations with the Central Italian reference material.

Male genital anatomy strongly supported the DNA assignments. The newly attributed Alpine and Pyrenean populations closely matched the Italian holotype of P. barrassoi. In this species, the valvae are comparatively narrow, with their dorsal and ventral margins approximately parallel, and the costa narrows toward a finely tapered apex. Its aedeagus bears five to nine small cornuti measuring approximately 0.1 to 0.25 millimetres, none expanded at the base. P. incultaria has a much broader valval costa that dilates toward its apex and terminates in a small, beak-shaped sclerotized process bearing a conspicuous tuft of setae. Its aedeagus has five to seven stouter cornuti, with the distalmost generally the largest, curved and dilated at the base. The diagnostic contrasts were assessed in 7 male P. barrassoi and 23 male P. incultaria dissections.

Female genitalia supplied additional but subtler distinctions. In the six P. barrassoi females examined, the apophyses are fine and tapered, while the elongated colliculum is almost parallel-sided for most of its length. Its reported length was 0.37–0.5 millimetres in most specimens, with one measuring 0.6 millimetres. The corpus bursae is small and often globular, and the scobinate signum is small and round, with weak lateral spinules measuring 0.10–0.17 millimetres in diameter. Female P. incultaria has a generally longer colliculum of 0.6–0.7 millimetres and a larger, more elongate and often triangular signum of 0.13–0.3 millimetres, accompanied by stout lateral spinules. The sexes therefore differed in several reproductive structures, although the strongest visible separation was in the male genitalia.

Genome-wide data independently recovered the same two lineages. The ddRAD workflow generated an average of 1.54 million reads per individual, of which 84.8% remained after stringent quality filtering. A de novo assembly recovered 1,042 putative orthologues shared by more than three samples, covering 193,994 base pairs in total. This dataset contained 1,678 SNPs, including 200 parsimony-informative sites. A reference assembly mapped against the genome of the distantly related moth Operophtera brumata yielded a smaller final dataset of 50 retained loci across more than three individuals. Maximum-likelihood trees constructed independently from the de novo and reference datasets produced congruent arrangements, with the P. incultaria and P. barrassoi lineages receiving 100% bootstrap support. Population-clustering analysis likewise identified two discrete genetic groups corresponding to the two species. Only one of the eight individuals tested carried the bacterial parasite Wolbachia.

The revision also addressed the uncertain name-bearing material of P. incultaria. Its original description was based on two males and one female from the Alps. Two collectors were mentioned, with Gastein in Salzburg associated with the material, but the type specimens were presumed destroyed and could not be traced in museums in London or Berlin. Because P. incultaria cannot be separated externally from P. barrassoi and both may occur around Gastein, the authors designated a neotype to preserve nomenclatural stability. The chosen male was collected at Bad Hofgastein, Schlossalm, at 2,000 metres on 6 August 1975. High-throughput sequencing recovered a 658-base-pair fragment that clustered unambiguously with P. incultaria, fixing the name to a documented, genetically identified individual.

Ecological and seasonal records suggest reasons why P. barrassoi may have escaped attention. It is a montane, univoltine species recorded from approximately 700 metres in Bavarian and Austrian valleys to 2,500 metres in the Maiella Mountains. Other sites span about 1,000–1,300 metres in the Bavarian and Austrian Alps, 1,500–2,100 metres in the French Alps and Pyrenees, 1,800 metres on Monte Terminillo and 2,450 metres on the Adamello. Its central Italian specimens were recorded in mid- and late July, and its Pyrenean and French Alpine flight period extends from early July to early August. Alpine records, however, range from mid-April to late May, with one eastern Tyrolean specimen from mid-June and northern Italian specimens from late June. Where the two species coexist, P. barrassoi generally flies earlier than P. incultaria, whose Alpine populations usually fly from early June to early August. The larval stages and host plants of P. barrassoi remain unknown, although its sister species feeds on Primulaceae in early larval stages and on Saxifragaceae later, and representatives of both plant families occur at P. barrassoi collection sites.

On the present evidence, P. barrassoi is confirmed in the central Apennines, the Pyrenees, the French Alps, the Adamello area of northern Italy, eastern and northern Tyrol in Austria, and southernmost Bavaria in Germany. These confirmed records consist of distinct subareas, and the authors considered incomplete recording a more likely explanation than a genuinely fragmented distribution. They predicted that additional populations could remain in the northern Apennines and the western Alps, including Switzerland, Piedmont and Valle d’Aosta, and noted that the species’ early flight period and close resemblance to P. incultaria could make it easy to overlook. That wider range remains a prediction rather than a documented finding. Within the studied material, however, the combined barcode, nuclear-genomic, anatomical and ecological evidence establishes P. barrassoi as a distinct species occurring alongside P. incultaria across previously unrecognized parts of Central and Western Europe.

Subject of Research: Taxonomic identification and European distribution of the geometrid moth Perizoma barrassoi

Article Title: DNA barcoding and genomics reveal Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006 as new for the fauna of Central Europe ( Lepidoptera , Geometridae , Larentiinae )

Article References: Hausmann, A., Huemer, P., Lee, K. M., & Mutanen, M. (2021). DNA barcoding and genomics reveal Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006 as new for the fauna of Central Europe (Lepidoptera, Geometridae, Larentiinae). Nota Lepidopterologica, 44, 17-28. https://doi.org/10.3897/nl.44.58871

Image Credits: AI Generated

DOI: 10.3897/nl.44.58871

Keywords: Perizoma barrassoi, DNA barcoding, ddRAD sequencing, genome-wide SNPs, cryptic species, Geometridae, Lepidoptera, moth taxonomy, genital morphology, European fauna, barcoding, genomics