Research Article |
Corresponding author: Peter Huemer ( p.huemer@tiroler-landesmuseen.at ) Academic editor: Mark Metz
© 2022 Kai Berggren, Leif Aarvik, 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:
Berggren K, Aarvik L, Huemer P, Lee KM, Mutanen M (2022) Integrative taxonomy reveals overlooked cryptic diversity in the conifer feeding Batrachedra pinicolella (Zeller, 1839) (Lepidoptera, Batrachedridae). ZooKeys 1085: 165-182. https://doi.org/10.3897/zookeys.1085.76853
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During efforts to generate DNA barcodes for all European Lepidoptera, Batrachedra pinicolella (Zeller, 1839) was found to comprise two genetically distinct clusters. Morphological investigation and results from two nuclear markers and ddRAD sequencing furthermore support the existence of two distinct taxa which we treat as two separate species, B. pinicolella and B. confusella sp. nov. A lectotype for B. pinicolella is designated. Available data indicate that the biology of both species also differs, with Picea abies (L.) Karsten as a proved host-plant for B. pinicolella and Pinus sylvestris L. for B. confusella sp. nov. Both species are mainly distributed on the European continent with B. pinicolella occurring in boreal parts of North and Central Europe and introduced to Canada, reflecting a boreo-montane distribution pattern. Batrachedra confusella sp. nov. is more widely distributed in temperate Northern and Central Europe.
Boreo-montane, ddRAD sequencing, DNA barcoding, Europe, Gelechioidea, new species, nuclear genes, Pinaceae
During the last two decades, aided by DNA barcoding, several cryptic species have been discovered among European Lepidoptera (
DNA barcodes refer to a 658 base-pair long fragment of the mitochondrial cytochrome c oxidase subunit 1 (CO1). Legs from 49 specimens of the involved species pair were prepared according to the prescribed standards and successfully processed at the Canadian Centre for DNA Barcoding (CCDB, Biodiversity Institute of Ontario, University of Guelph) to obtain DNA barcodes and using the standard high-throughput protocol described in
We attempted to obtain data of six nuclear genes, wingless, CAD (carbamoyl-phosphate synthetase), EF-1a (elongation factor 1 alpha), MDH (malate dehydrogenase), RpS5 (ribosomal protein S5), and IDH (isocitrate dehydrogenase), for specimens representing the two clusters of B. pinicolella. Five of these specimens represented BIN AAF0077 and three BIN AAF0078. Nuclear sequences were obtained for both clusters only for two of these genes, EF-1a and MDH. A few sequences of CAD and wingless were also recovered, but representing of only one of the BINs, and were therefore not considered in this study. We also studied the presence of Wolbachia bacteria in all eight specimens using ftsZ ja wsp markers (
We used DNA aliquots that were extracted at CCDB for ddRAD-seq library preparation. The quantity of DNA extracts was checked using PicoGreen kit (Molecular Probes). The ddRAD library was implemented following protocols in
To infer maximum likelihood (ML) tree, we used IQ-TREE (
Dissections of genitalia followed
KBE Collection of Kai Berggren, Kristiansand, Norway
Holotype
1♂ Ø, Moss: Rygge, Sildebauen, 59.3268°N; 10.7101°E; 9.vii.1980; L. Aarvik leg.;
Norway 1♂ AAY, Arendal: Havsøy; 10.vii.2015; K. Berggren leg.; BOLD sample ID:
Sweden 1♂ Öland, Borgholm: Byrums Sandvik; 23.vii.1985; K. Berggren leg.; KBE prep. 13527; KBE. 1♂ Öland, Borgholm: Byrums Sandvik; 23.vii.1985; K. Berggren leg.; KBE prep. 6142; KBE.
Denmark 1♂ EJ, Anholt; 1.viii.1975; E. S. Nielsen leg., Lundquist prep. 2050;
Austria 1♀, Oberösterreich, NP Kalkalpen, Spering-Lackerbodenstraße, ca 700 m; 16.vii.2004; J. Wimmer leg; genitalia in glycerine;
Germany 1♀, Bavaria, Inning/A, 550 m, mid vii.1970 Zürnbauer F. leg.; genitalia in glycerin PH;
Italy 1♂ Südtirol, Oberrasen, Biotop Rasner Möser S, 1100 m, 5.vii.2015, P. Huemer leg.; DNA Barcode ID
Armenia 1♂, Tavush Province, Diljan, 1395 m; 13.vii.2011; O. Karsholt leg.; BOLD sample ID: ZMUC00029754;
Batrachedra confusella sp. nov. (Fig.
Sequences of B. pinicolella and B. confusella sp. nov. form two well-defined clusters which received separate Barcode Index Numbers (BIN-codes): AAF0078 and AAF0077 respectively. The DNA barcode region of B. confusella sp. nov. shows 0.95% intraspecific divergence and a minimum divergence of 6.82% to its closest relative B. pinicolella (Fig.
We generated a genomic dataset from nine individuals of B. pinicolella using ddRAD sequencing. We obtained 1.24 million reads per individual on average after quality filtering steps. After clustering 88% sequence similarity, we recovered 19,292 clusters per sample were retained with an average of 82.26 per sample for cluster depth (Table
Species | SampleID | Reads passed filter | Clusters at 88% | Coverage | Retained loci | Consensus loci |
---|---|---|---|---|---|---|
B. confusella | MM22066 | 1520904 | 29400 | 40.06 | 11608 | 4488 |
B. confusella | MM22068 | 1624893 | 31776 | 45.71 | 12030 | 4346 |
B. confusella | MM23464 | 2976135 | 62919 | 43.18 | 19568 | 4557 |
B. confusella |
|
1353160 | 16809 | 60.85 | 5292 | 2563 |
B. confusella |
|
624001 | 11396 | 47.99 | 3177 | 917 |
B. pinicolella | MM06705 | 808698 | 4251 | 163.44 | 572 | 149 |
B. pinicolella | MM21052 | 971904 | 6440 | 131.25 | 1305 | 200 |
B. pinicolella | MM21054 | 931540 | 7060 | 118.22 | 1344 | 370 |
B. pinicolella |
|
370227 | 3579 | 89.61 | 611 | 155 |
Total | 1242385 | 19292 | 82.26 | 6167 | 1972 |
Maximum-likelihood tree inferred from the ddRAD SNP data. 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 red clusters referring to Batrachedra confusella, the blue clusters to B. pinicolella. Each bar represents one individual and colours represent the proportion of the individuals that belong to each of the genetic cluster.
Male (Fig.
Female. Externally similar to male.
Male genitalia (Figs
Female genitalia (Fig.
The species’ name, confusella, indicates the confusion with its sister species, B. pinicolella.
Due to the confusion of the two species, the biology is insufficiently known. Batrachedra confusella sp. nov. is the well-known species affecting Pinus, and it has been found in several localities of pure pine forests. However, a female beaten from an artificial afforestation of Larix at a lowland locality in Switzerland without Pinus in the nearby surroundings (Bryner in litt.) also belongs to B. confusella sp. nov., indicating that Larix may be an additional host-plant. Batrachedra pinicolella, in contrast, seems to be restricted to forests of Norway spruce (Picea abies), which is considered to be the host-plant. This hypothesis is proved by a dissected Finnish female specimen bred from Picea.
Batrachedra pinicolella.
Finland 1♂ PPe, Oulu; 7.vii.2011; M. Mutanen leg.; L. Aarvik prep. 2015.024; BOLD sample ID: MM21054;
Norway 1♀ AAI, Bygland: Heddevika; 29.vii.2008; K. Berggren leg.; BOLD sample ID:
Switzerland 1♀, BE, La Neuveville, Ligeresse, 810 m, 4.vii.2002, Larix decidua; leg. R. Bryner; coll. R. Bryner.
Austria 1♂ Vorarlberg, Gaschurn-Partenen, Schuttfluren Lifinar, 1150 m, 31.vii.2018; P. Huemer leg.; BOLD sample ID:
Germany 1♀ Baden-Württemberg, Schwarzwald, Buchenberg, 21.vii.1954; H.G. Amsel leg; genitalia in gylcerine;
Italy 1♂ Südtirol, Oberrasen, Biotop Rasner Möser S, 1100 m, 5.vii.2015, P. Huemer leg.; DNA Barcode ID
The species pair B. confusella sp. nov. and B. pinicolella is widely distributed in Europe but seems to be absent from the Mediterranean (http://www.faunaeur.org; accessed on 25.iv.2021). However, as former records have been summarized among the latter taxon, a detailed study of distribution of both species is required for future studies. From our sequenced and/or genitalized material, B. confusella sp. nov. together with its major hostplant, Pinus sylvestris, is distributed in the temperate zones between the Alps in the South and Fennoscandia in the north. Batrachedra pinicolella in concordance with its major host-plant, Picea abies, shows a boreo-montane distribution pattern with isolated records from the Alps and mountainous areas of Central Europe as well as northern and north-western Europe. A barcoded specimen of B. confusella sp. nov. from Armenia (coll.
Because of their external similarity, the two species have until present been confused. This is in spite of the fact that they have different host plants. Specimens collected in localities with Pinus (and no Picea present) belong to B. confusella sp. n., i.e. from Byrum Sandvik, Öland in Sweden, where only Pinus is present and from Belteviga and Bråvann, Kristiansand, Norway. In Finland and Austria, B. pinicolella has been collected in places where only Picea grows but rarely both species have been found in syntopy. Further breeding experiments are needed to confirm additional hostplants, e.g., in the genera Abies and Larix. The issue dealt with in the present paper is not unique. DNA barcoding has revealed that there still exist numerous taxonomic problems in the European lepidopteran fauna; see for instance
Paul D.N. Hebert and the entire team at the Canadian Centre for DNA Barcoding (Guelph, Canada) are gratefully acknowledged for continuous support with sequencing work. The study was partially funded by the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano - South Tyrol with grants 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)”. DNA barcoding in Finland was supported by the Kone Foundation, Finnish Cultural Foundation, and the Academy of Finland through their grants to the Finnish Barcode of Life (FinBOL) and Finnish Biodiversity Information Facility (FinBIF). We cordially thank David Lees (