Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006

Hausmann, Axel, Huemer, Peter, Lee, Kyung Min & Mutanen, Marko, 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, pp. 17-28 : 17

Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006

Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006: 95:31, figs 1a, b, 2a, b (Locus typicus: Italy, Abruzzo: Maiella Mts., Valle Cannella, Manzini-Hütte). Holotype male (coll. Zahm/ ZSM, examined and dissected, DNA barcoding failed). Associated female paratype a misidentification of Colostygia aqueata ( Hübner).

Material examined.

Italy: Holotype male, central Italy, Abruzzo, Maiella, Valle Cannella, Manzini-Hütte, 2530 m, 23.vii.1988, leg. N. Zahm, coll. ZSM (gen.prp. ZSM G 20836; DNA barcode sample ID BC ZSM Lep 54528, 77874 (failures); 1 female, central Italy, Lazio, Rieti, Mt. Terminillo, 1800 m, 18.vii.2004, DNA barcode sample ID BC ZSM Lep 82927, gen.prp. ZSM G 15840; 2 males, Trentino, Adamello, Rifugio Mandrone, 2450 m, 26.vi.2014, leg. T. Mayr, DNA barcode sample ID TLMF Lep 25428 (RCTM); France: 1 male, France, Hautes Alpes, Pelvoux, [2000 m], 1.-10.viii.1972, leg. L. Hinterholzer, gen.prp. ZSM G 21237 ( ZSM); 1 male, France, Alpes Maritimes, Col des Champs, [2100 m], 5.vii.1931, [leg. C. Herbulot], gen.prp. ZSM G 21238 ( ZSM); 1 female, France, Alpes Maritimes, Cayolle, 2100 m, 31.vii.1975, leg. Lukasch, gen.prp. ZSM G 21259 ( ZSM); 1 female, France, Hautes Alpes, la Moutieres, 2100 m, 3. viii.1975, leg. Lukasch ( ZSM); 1 female, France, Valloire, 1500 m, 31. vii.1954, leg. G. Bernardi ( ZSM); 1 male, France, Pyrenees orientales, Porté, 1650-2100 m, 25.VI.-21. vii.1948, leg. H. de Toulgoet, gen.prp. ZSM G 14302 ( ZSM); 1 female, France, Pyrenees orientales, Col de Puymorens, 1900 m, 10.vii.1975, leg. G. Behounek, gen.prp. ZSM G 21255 ( ZSM); Germany: 1 female, Germany, Bavaria, Allgäu, Oberstdorf, Oytal, Oybach E Oytalhaus, 1036 m, 01.vi.2014, leg. D. Doczkal, DNA barcode sample ID BC ZSM Lep 86512, gen.prp. ZSM G 21249 (malaise trapping, ZSM); 2 males, Germany, Bavaria, Eschenlohe, [700 m], 12.vi.1977, leg. L. Hinterholzer, gen.prp. 21229 ( ZSM); 3 females, Germany, Bavaria, Bergen, Weißachental, 700 m, 24.v.1980, leg. A. Beyerl ( ZSM); Austria: 16 males and females, Austria, North Tyrol, Riedenberg / Landl, 800-900 m, 16.v-19.vi.1974-1979 leg. Scheuringer, Wolfsberger, gen.prp. ZSM G 21228, 21248 ( ZSM); 1 male, Austria, northern Tyrol, Ötztaler Alps, Feichten, 1300 m, late May 1986, leg. E. Scheuringer, gen.prp. ZSM G 21233 ( ZSM); 1 male, id., late May 1992 ( ZSM); 1 female, id., early June 1983, gen.prp. ZSM G 21257 ( ZSM); 1 female, id., leg. [coll.] Wolfsberger ( ZSM); 2 males, Austria, North Tyrol, unterhalb Farst / Umhausen N, 1100 m, 10.iv.2017, leg. P. Huemer, DNA barcode sample ID TLMF Lep 22852, 22853 ( TLMF); 1 male, id., 28.v.2013, DNA barcode sample ID TLMF Lep 12589; 1 male, Austria, East Tyrol, Matreier Tauerntal, Aussergschloess Umgebung, 1700 m, 14.vi.2013, leg. P. Huemer, DNA barcode sample ID TLMF Lep 13024 ( TLMF).

External characters and abdomen

(Fig. 1 ). See description in Müller et al. (2019). The newly attributed Alpine populations with wingspan of 17-21 mm in males, 19-22 mm in females. Ground colour whitish pale grey, silky shining. Wing pattern varying in a similar way as in the sister species P. incultaria , no constant and reliable differential feature in wing pattern and coloration was found that would allow discrimination between the two sibling species. Palpi short, length 0.5-0.7 mm, hardly exceeding frons. Male antennae ciliate-setose, flagellum dorsally chequered grey and white.

Male genitalia (n = 7; Figs 3-5 ). The genitalia of the newly attributed Alpine populations are well matched to those of the holotype, i.e. showing comparatively narrow valvae, ventral and dorsal margins approximately parallel, costa of valva narrow from base to apex, at apex narrowly tapering (cf. Müller et al. 2019). Anal tube strongly developed, spinulose at tip. Head of (fused) labides comparatively large. Aedeagus with five to nine small cornuti of 0.1 mm (basalmost) to 0.25 mm (distalmost), cornuti not dilated at base. Length of aedeagus 0.9-1.1 mm.

Female genitalia (n = 6; Figs 9-11 ). The genitalia of the newly attributed Alpine populations are well matched to those of the central Italian female as figured in Müller et al. (2019). Apophyses fine, tapered at apices. Colliculum (in earlier publications referred to as 'ductus bursae’ but homology with colliculum postulated in Mironov 2003) elongate, length 0.37-0.5 mm, in one specimen 0.6 mm), parallel-sided almost over the whole length. Corpus bursae small, often globular. Scobinate signum small, round, lateral spinules weak, diameter 0.10-0.17 mm.

Differential diagnosis.

In external appearance (wing shape, wing coloration, pattern), the newly attributed populations of P. barrassoi from the Alps and the Pyrenees do not exhibit significant and constant differences from the sympatric P. incultaria . The latter differs in male genitalia (n = 23; Figs 6-8 ) with much broader costa of valva which is dilated towards apex and ending in a small, sclerotized, beak-shaped process and bearing a conspicuous setal tuft. Aedeagus with stout cornuti (5-7), the distalmost being the largest, usually curved and dilated at base. Female genitalia of P. incultaria (n = 10; Figs 12-14 ) are similar to those of P. barrassoi , but in the former the colliculum is longer (0.6-0.7 mm), signum larger, more elongate, often triangular, length 0.13-0.3 mm, lateral spinules stout.

Distribution.

Recorded in central Italy (locus typicus: Maiella mountains; Mt. Terminillo), Pyrenees, French Alps, and in a section of the central part of the Alps with records, so far, restricted to eastern and northern Tyrol (Austria) and to southernmost Bavaria (Germany), as a sympatric, sibling species of P. incultaria (see examined material and genetic data). Furthermore an isolated occurrence was detected in northern Italy (Adamello).

Phenology.

Univoltine, the two central Italian records are from mid-July and late July, in the Pyrenees and French Alps the species flies from early July to early August. The remaining records from the Alps, however, refer to much earlier dates, from mid-April to late May, the single specimen from eastern Tyrol in mid-June and specimens from northern Italy in late June. Therefore, when occurring sympatrically, P. barrassoi flies earlier than P. incultaria whose Alpine populations are usually on the wing from early June to early August.

Biology.

The larval stages are unknown. Larvae of the sister species P. incultaria feed on Primulaceae (first stages), later instars also on Saxifragaceae. Representatives of both plant families are abundant at the collecting sites of P. barrassoi .

Habitat.

Montane. Collected on karstic slopes at 2500 m in the Maiella and 1800 m on Mt. Terminillo. In French Alps and Pyrenees from 1500 m up to 2100 m and in northern Italy (Adamello) occurring at 2450 m. In the Bavarian and Austrian Alps collected from much lower elevations in valleys from 1000 m up to 1300 m (Bavaria; northern Tyrol) (n = 29) with only one specimen from eastern Tyrol recorded at 1700 m, while the vertical distribution of sympatric P. incultaria ranges from 1700 m up to 3200 m, with very few exceptions.

Genetic data

(DNA barcodes and nuclear genomic data). The specimen from Mt. Terminillo was DNA barcoded with an HTS approach, resulting in a 658 bp sequence including an 89 bp n-gap. BIN: BOLD:ACJ5976 (n = 1 from central Italy). Six specimens from Austria, south-western Bavaria and northern Italy are BIN-sharing, at a distance of only 1.1% from central Italian P. barrassoi . The genetic divergence from P. incultaria is strongly correlated with the differential features in genitalia and bionomic traits (see above). Nearest species: P. incultaria (8.5%; n = 19 from Germany, Austria and northern Italy; BIN BOLD:AAF5044). The distinctness of Alpine P. barrassoi and P. incultaria at species level was confirmed by ddRAD-sequencing involving nuclear loci ( Mutanen et al. 2017). Two specimens of P. incultaria from the type locality near Gastein (Austria, Salzburg) could be sequenced, using the HTS approach for old museum material and revealed to belong to BIN BOLD:AAF5044, confirming the suggestion of species identity as P. incultaria as inferred from the comparatively high elevation (1800-2000 m).

Data exploration and phylogenetic analyses based on SNP data.

We generated a genome-wide set of genetic clusters from 8 individuals of Perizoma incultaria using ddRAD sequencing, and used this data set to perform phylogenetic analyses. We obtained 1.54 million reads per individual on average, of which 84.8% were retained after stringent quality filtering steps. After filtering and clustering at 95% sequence similarity using ' de novo ' assembly method, we recovered 1,042 putative orthologues shared across more than three samples, for a total length of 193,994 base pairs (Fig. 21A ). These data include 1,678 SNPs, of which 200 are parsimony informative. For the ‘reference’ assembly, an average of 1,298 reads per sample was mapped to the Operophtera brumata genome, while an average of 107,289 reads per sample remained unmapped. Because the reference species is only distantly related to Perizoma , the sample 'BC ZSM Lep 84546' was dropped out in the reference assembly due to the lack of mapped reads to the reference genome. After filtering, 222 clusters per sample were obtained, with 45.13 per sample for cluster depth on average. The final dataset from the reference assembly consisted of 50 recovered loci across more than three individuals (Fig. 21B ). Phylogenetic analysis using the concatenated ddRAD dataset produced robust support for the relationship between the individuals (Fig. 21 ). The ML tree based on the reference assembly produced a remarkably congruent tree with the tree of de novo assembly. In both ML trees, the two revealed lineages correspond to cluster A ( P. incultaria , marked with green) and cluster B ( P. barrassoi , marked with orange) that was supported by 100% bootstrap support values. STRUCTURE also identified two discrete clusters (Fig. 21 ). Only one of the eight examined samples (' TLMF Lep 00264') was Wolbachia infected.

Figures

Figure 21. Maximum likelihood trees inferred from the ddRAD data matrices based on (a.) de novo assembly and (b.) reference assembly method against Operophtera brumata genome (GCA _ 001266575). Bootstrap support values are indicated above the branches and only the values> 50 % are shown. The barplot shows the assignments of individuals into two genetic clusters, the green clusters referring to Perizoma incultaria, the orange clusters to P. barrassoi. Each bar represents one individual and colors represent the proportion of the individuals that belong to each of the genetic cluster. Samples infected with Wolbachia are marked with an asterisk.

Figures 1, 2. Adults (females). 1. Perizoma barrassoi (Austria, Riedenberg); 2. Perizoma incultaria (Germany, Hirschberg). Scale bars: 1 cm.

Figures 3 - 8. Male genitalia. 3 - 5. Perizoma barrassoi (3. Austria, Riedenberg; 4. Germany: Eschenlohe; 5. France, Pyrenees, Porte); 6 - 8. Perizoma incultaria (6. Austria, Brandenberg; 7. Italy, Friuli, Altiplano Montasio; 8. Italy, southern Tyrol, Sulden). Scale bar: 1 mm.

Figures 9 - 14. Female genitalia. 9 - 11. Perizoma barrassoi (9. Austria, Feichten; 10. French Alps: Cayolle; 11. France, Pyrenees, Col de Puymorens); 12 - 14. Perizoma incultaria (12. Germany, Hochfelln; 13. Germany, Koblat; 14. Italy, Mt. Grappa). Scale bar: 1 mm.