Research Article |
Corresponding author: Axel Hausmann ( hausmann.a@snsb.de ) Academic editor: Sven Erlacher
© 2021 Axel Hausmann, Peter Huemer, Kyung Min Lee, Marko Mutanen.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Hausmann A, Huemer P, Lee KM, 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
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Perizoma barrassoi Zahm, Cieslak & Hausmann, 2006, previously considered a central Italian endemic, shows a much wider distribution with additional records from the Alps and the Pyrenees, in partial sympatry with its sister species, Perizoma incultaria (Herrich-Schäffer, 1848) which is widespread in the Alps, Pyrenees, Carpathians and Dinaric mountains. The disruptive genetic patterns of both species involve the COI barcode gene as well as nuclear genomic data and are confirmed by correlated differential features in male and female genitalia. To fix nomenclatural stability a neotype is designated for P. incultaria.
In his outstanding revision of European Perizomini and Eupitheciini,
Three years after the publication of Mironov’s monograph,
The study is based on more than 250 specimens of Perizoma incultaria and P. barrassoi, including 46 dissections, 29 DNA barcodes and ddRAD-data for eight specimens. The material is housed at the following collections:
RCTM Research Collection Toni Mayr, Feldkirch, Austria;
The taxa were delimited on the basis of combining data from various sources: morphology, bionomy (mainly phenology, partly vertical distribution) and DNA barcodes. The genitalia and the abdomens were prepared following methods described by
For the DNA barcoding analyses, one or two legs were removed from each dried specimen and transferred to lysis plates. DNA extraction, amplification, and sequencing of the barcode region of the mitochondrial cytochrome c oxidase I (COI) gene (658 base pairs) were carried out in the Canadian Centre for DNA Barcoding, Guelph, Ontario, Canada (CCDB), using standard high throughput protocols (
We used DNA aliquots that were extracted at the Canadian Centre for DNA Barcoding (CCDB) following laboratory protocols used routinely in CCDB as explained in
To infer maximum likelihood (ML) trees, we used RAxML v.8.2.0 (
To check for the presence of bacterial parasite Wolbachia, we sequenced two markers, FstZ and Wsp, using primers and laboratory procedures of
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/
Italy: Holotype male, central Italy, Abruzzo, Maiella, Valle Cannella, Manzini-Hütte, 2530 m, 23.vii.1988, leg. N. Zahm, coll.
(Fig.
Male genitalia
(n = 7; Figs
Female genitalia
(n = 6; Figs
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
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).
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.
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.
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.
(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 (
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.
The shape and pattern of the wings of the barcoded, Central Italian female collected at Monte Terminillo (just 100 km from the type locality of P. barrassoi in the central Apennines) clearly showed it to belong to the Perizoma incultaria/barrassoi species complex although the large geographical distance of 500 km from the nearest Alpine populations of P. incultaria and the scarcity of high alpine habitats in the northern Apennines supported the initial hypothesis of conspecificity of that Central Italian specimen with P. barrassoi. This hypothesis was corroborated by the fact that the male genitalia of the holotype from the Maiella mountains agree perfectly with the male genitalia of one of the Alpine DNA barcode clusters, the latter corresponding to the barcoded central Italian female.
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.
Larentia incultaria was described from two males and a single female collected in the Alps (
The discovery of a cryptic species of Geometridae in the Alps and Pyrenees comes as a surprise, although, recently, a similar case has been discovered in the genus Perizoma when P. juracolaria (Wehrli, 1919) was recognized as a cryptic species differing from its sister species, P. obsoletata (Herrich-Schäffer, 1838) in morphology and DNA barcodes (
Perizoma barrassoi shows, according to current data, an unusual distribution pattern which is presumed to be due to a patchy recording. The distinctly isolated sub-areas in parts of the Alps and in the Pyrenees as well as in the central Apennines seem implausible when compared with the zoogeographical patterns of other geometrids inhabiting alpine and sub-alpine habitats, e.g. the above mentioned P. juracolaria. There is a large probability that P. barrassoi has still unrecorded relic populations in the northern Apennines and it will show a wider distribution in the western Alps including Switzerland, Piedmont and Valle d’Aosta. In particular, the early flight time, which starts shortly after the snowmelt, as well as the morphological similarity with P. incultaria are obvious reasons for the possibility that the species has simply been overlooked in many places.
We are very grateful to the staff at the Canadian Centre for DNA Barcoding for sequence analysis. Paul D.N. Hebert, Evgeny Zakharov and many other colleagues of the Barcode of Life project (Centre for Biodiversity Genomics, Guelph, Canada) contributed to the success of this study. The data management & analysis system BOLD was provided by Sujeevan Ratnasingham (CBG, Guelph). PH is indebted to the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano – South Tyrol for funds to the projects “Genetische Artabgrenzung ausgewählter arktoalpiner und boreomontaner Tiere Südtirols” and “Erstellung einer DNA-Barcode-Bibliothek der Schmetterlinge des zentralen Alpenraumes (Süd-, Nord- und Osttirol)”. We also thank Lauri Kaila, Jörg Gelbrecht and Hossein Rajaei for giving valuable comments to the manuscript. Gernot Embacher and Patrick Gros (Haus der Natur Salzburg, Austria) kindly helped with material of P. incultaria from the type locality, Toni Mayr (Feldkirch, Austria) with Italian samples of P. barrassoi. We thank Laura Törmälä for the invaluable help in the molecular laboratory and CSC – IT Centre for Science, Finland for computational resources. This work was supported by the Academy of Finland (grant number 277984) to MM and the Kvantum Institute to KML.