Research Article |
Corresponding author: Peter Huemer ( p.huemer@tiroler-landesmuseen.at ) Academic editor: Mark Metz
© 2020 Peter Huemer, Ole Karsholt, Leif Aarvik, Kai Berggren, Oleksiy Bidzilya, Jari Junnilainen, Jean-François Landry, Marko Mutanen, Kari Nupponen, Andreas Segerer, Jan Šumpich, Christian Wieser, Benjamin Wiesmair, Paul D. N. Hebert.
This is an open access article distributed under the terms of the CC0 Public Domain Dedication.
Citation:
Huemer P, Karsholt O, Aarvik L, Berggren K, Bidzilya O, Junnilainen J, Landry J-F, Mutanen M, Nupponen K, Segerer A, Šumpich J, Wieser C, Wiesmair B, Hebert PDN (2020) DNA barcode library for European Gelechiidae (Lepidoptera) suggests greatly underestimated species diversity. ZooKeys 921: 141-157. https://doi.org/10.3897/zookeys.921.49199
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For the first time, a nearly complete barcode library for European Gelechiidae is provided. DNA barcode sequences (COI gene – cytochrome c oxidase 1) from 751 out of 865 nominal species, belonging to 105 genera, were successfully recovered. A total of 741 species represented by specimens with sequences ≥ 500bp and an additional ten species represented by specimens with shorter sequences were used to produce 53 NJ trees. Intraspecific barcode divergence averaged only 0.54% whereas distance to the Nearest-Neighbour species averaged 5.58%. Of these, 710 species possessed unique DNA barcodes, but 31 species could not be reliably discriminated because of barcode sharing or partial barcode overlap. Species discrimination based on the Barcode Index System (BIN) was successful for 668 out of 723 species which clustered from minimum one to maximum 22 unique BINs. Fifty-five species shared a BIN with up to four species and identification from DNA barcode data is uncertain. Finally, 65 clusters with a unique BIN remained unidentified to species level. These putative taxa, as well as 114 nominal species with more than one BIN, suggest the presence of considerable cryptic diversity, cases which should be examined in future revisionary studies.
Europe, cryptic diversity, DNA barcoding, revision, species delimitation
The megadiverse family, Gelechiidae, includes approximately 4,700 known species and perhaps a similar number of undescribed taxa (
Over the last two decades, the Gelechiidae have received increasing attention as a result of two monographs that treated approximately half the known European species (
The lack of an updated checklist for European Gelechiidae (see
One major challenge was the difficulty in accessing specimens suitable for molecular analysis, reflecting the rarity of many species. In addition, DNA quality of the specimens was another very important limitation as sequence recovery from older specimens of rare taxa was either partial or failed completely even with protocols that employed high-throughput sequencers to analyze short amplicons. In some cases, efforts were made to recollect taxa that lacked a sequence record.
Voucher material was obtained from Europe (Fig.
Distribution map of examined material of Gelechiidae (extra-European material partially mapped). SimpleMappr (http://www.simplemappr.net).
Many institutions and private collectors contributed to the dataset (see below), supplemented by DNA barcodes from earlier studies.
INDO Inatura, Dornbirn, Austria
LMK Landesmuseum Kärnten, Klagenfurt, Austria
NMS Naturmuseum Südtirol, Bozen, Italy
RCAH Research Collection Alfred Haslberger, Teisendorf, Germany
RCER Research Collection Emily Requena Miret, Gurb, Spain
RCGB Research Collection Giorgio Baldizzone, Asti, Italy
RCGT Research Collection Giovanni Timossi, Oderzo, Italy
RCHW Research Collection Hartmut Wegner, Adendorf, Germany
RCIB Research Collection Ian Barton, Cambs, United Kingdom
RCIR Research Collection Ignác Richter, Malá Čausa, Slovakia
RCJD Research Collection Jordi Dantart, Barcelona, Spain
RCJJ Research Collection Jari Junnilainen, Vantaa, Finland
RCJK Research Collection Jari-Pekka Kaitila, Vantaa, Finland
RCJL Research Collection Gérard Labonne, Montpellier, France
RCJN Research Collection Jacques Nel, La Ciotat, France
RCJS Research Collection Jan Skyva, Prague, Czech Republic
RCJSC Research Collection Jürg Schmid, Illanz, Switzerland
RCKB Research Collection Kai Berggren, Kristiansand, Norway
RCKN Research Collection Kari and Timo Nupponen, Espoo, Finland
RCMC Research Collection Martin Corley, Faringdon, U.K.
RCOB Research Collection Oleksiy Bidzilya, Kiev, Ukraine
RCOR Research Collection Oliver Rist, Vienna, Austria
RCPB Research Collection Peter Buchner, Schwarzau am Steinfeld, Austria
RCPL Research Collection Peter Lichtmannecker, Adlkofen, Germany
RCRH Research Collection Robert Heckford, Plympton, Plymouth, U.K.
RCRHE Research Collection Richard Heindel, Günzburg, Germany
RCSP Research Collection Serge Peslier, Perpignan, France
RCTG Research Collection Thomas Guggemoos, Ohlstadt, Germany
RCTM Research Collection Toni Mayr, Feldkirch, Austria
RCTV Research Collection Thierry Varenne, Nice, France
RCWS Research Collection Wolfgang Stark, Trübensee, Austria
RCZT Research Collection Zdenko Tokár, Šal’a, Slovakia
A single leg was removed from each specimen and placed in a 96-well lysis plate that was submitted for analysis at the CCDB (Canadian Center for DNA Barcoding, University of Guelph, Canada) where DNA extraction, PCR amplification, and sequencing were performed following standard high-throughput protocols (
Levels of intra- and interspecific variation in the DNA barcode fragment were calculated under the Kimura 2-parameter (K2P) model of nucleotide substitution using analytical tools in BOLD systems v4.0 (http://www.boldsystems.org). Fifty-three Neighbor-Joining trees (Maximum Composite Likelihood method, default settings), most including representatives of a single genus, were constructed using MEGA X (
Identification success was assessed by the Barcode Index Number (BIN) system as implemented on BOLD (
DNA barcode sequences were recovered from 5986 specimens representing 751 of the 865 species of Gelechiidae described from Europe (Suppl. material
Species | Mean intra-spec. | Max intra-spec. | Nearest species | Dist. NN |
Bryotropha affinis | 0.17 | 0.77 | Bryotropha umbrosella | 0 |
Bryotropha umbrosella | 1.76 | 3.63 | Bryotropha affinis | 0 |
Iwaruna biguttella | 0.78 | 2.02 | Iwaruna klimeschi | 0 |
Iwaruna klimeschi | 0 | 0 | Iwaruna biguttella | 0 |
Teleiodes brevivalva | 0.46 | 0.46 | Teleiodes vulgella | 0 |
Teleiodes italica | 0.32 | 0.62 | Teleiodes vulgella | 0 |
Teleiodes vulgella | 0.17 | 0.5 | Teleiodes italica | 0 |
Xenolechia aethiops | 0.08 | 0.16 | Xenolechia lindae | 0 |
Xenolechia lindae | 0 | 0 | Xenolechia aethiops | 0 |
Xenolechia pseudovulgella | N/A | 0 | Xenolechia aethiops | 0 |
Scrobipalpa alterna | N/A | 0 | Scrobipalpa lutea | 0.35 |
Scrobipalpa lutea | N/A | 0 | Scrobipalpa alterna | 0.35 |
Acompsia antirrhinella | 1.39 | 1.39 | Acompsia tripunctella | 0.46 |
Acompsia tripunctella | 2.59 | 6.4 | Acompsia antirrhinella | 0.46 |
Dirhinosia cervinella | 0.14 | 0.32 | Dirhinosia interposita | 0.46 |
Dirhinosia interposita | 0 | 0 | Dirhinosia cervinella | 0.46 |
Monochroa arundinetella | 0.05 | 0.15 | Monochroa suffusella | 0.47 |
Monochroa suffusella | 0.52 | 1.07 | Monochroa arundinetella | 0.47 |
Scrobipalpula psilella | 0.21 | 0.64 | Scrobipalpula seniorum | 0.53 |
Scrobipalpula seniorum | N/A | 0 | Scrobipalpula psilella | 0.53 |
Anacampsis blattariella | 0.48 | 2.99 | Anacampsis populella | 0.56 |
Anacampsis populella | 0.22 | 1.41 | Anacampsis blattariella | 0.56 |
Teleiopsis albifemorella | 0.62 | 1.42 | Teleiopsis rosalbella | 0.61 |
Teleiopsis bagriotella | 0.91 | 2.66 | Teleiopsis diffinis | 0.61 |
Teleiopsis diffinis | 1.43 | 3.26 | Teleiopsis bagriotella | 0.61 |
Teleiopsis rosalbella | 0.22 | 0.46 | Teleiopsis albifemorella | 0.61 |
Thiotricha coleella | N/A | 0 | Thiotricha subocellea | 0.67 |
Thiotricha subocellea | 0.74 | 1.4 | Thiotricha coleella | 0.67 |
Stomopteryx lineolella | N/A | 0 | Stomopteryx nugatricella | 0.77 |
Stomopteryx nugatricella | 0 | 0 | Stomopteryx lineolella | 0.77 |
Scrobipalpula diffluella | 0.54 | 1.2 | Scrobipalpula tussilaginis | 0.8 |
Scrobipalpula tussilaginis | 0.17 | 0.46 | Scrobipalpula diffluella | 0.8 |
Scrobipalpa arenbergeri | 0.49 | 0.77 | Scrobipalpa mercantourica | 0.92 |
Scrobipalpa artemisiella | 0.6 | 2.5 | Scrobipalpa stangei | 0.92 |
Scrobipalpa mercantourica | N/A | 0 | Scrobipalpa arenbergeri | 0.92 |
Scrobipalpa salicorniae | 0.16 | 0.46 | Scrobipalpa salinella | 0.92 |
Scrobipalpa salinella | 0.28 | 0.92 | Scrobipalpa salicorniae | 0.92 |
Scrobipalpa stangei | 0.15 | 0.31 | Scrobipalpa artemisiella | 0.92 |
Sattleria melaleucella | 1.11 | 1.87 | Sattleria pyrenaica | 0.93 |
Sattleria pyrenaica | 2.6 | 3.65 | Sattleria melaleucella | 0.93 |
Scrobipalpa halymella | N/A | 0 | Scrobipalpa stabilis | 0.93 |
Scrobipalpa stabilis | N/A | 0 | Scrobipalpa halymella | 0.93 |
Intraspecific DNA barcode variation in the 741 named species with sequences ≥500 bp averaged 0.54%, but this may be an underestimate as sample sizes for 224 taxa were low and only represented by singletons. In respect to the distribution of mean intraspecific DNA barcode variation: 73.1% of sequenced species had variation ranging from 0–1%, 15.8% between 1–2%, 6.3% between 2–3%, and 4.8% > 3%.
Contrastingly, barcode gap analysis resulted in mean distances of 5.58% (maximum 12.75%) to the Nearest Neighbor (NN) with only 5.68% of all species showing a NN distance of 0–1% (Table
Species | no. of BINs |
---|---|
Aproaerema anthyllidella | 3 |
Aproaerema karvoneni | 3 |
Aroga velocella | 3 |
Brachmia dimidiella | 3 |
Bryotropha desertella | 3 |
Bryotropha umbrosella | 3 |
Caryocolum alsinella | 3 |
Caryocolum marmorea | 3 |
Caryocolum tischeriella | 3 |
Chionodes fumatella | 3 |
Chionodes viduella | 3 |
Hypatima rhomboidella | 3 |
Isophrictis meridionella | 3 |
Megacraspedus binotella | 3 |
Metzneria aprilella | 3 |
Metzneria artificella | 3 |
Neofaculta ericetella | 3 |
Oxypteryx baldizzonei | 3 |
Parachronistis albiceps | 3 |
Ptocheuusa paupella | 3 |
Stomopteryx flavipalpella | 3 |
Teleiodes flavimaculella | 3 |
Teleiodes luculella | 3 |
Teleiopsis paulheberti | 3 |
Aroga flavicomella | 4 |
Caryocolum amaurella | 4 |
Caryocolum fibigerium | 4 |
Caryocolum peregrinella | 4 |
Caryocolum vicinella | 4 |
Ephysteris promptella | 4 |
Gelechia sabinella | 4 |
Isophrictis anthemidella | 4 |
Megacraspedus imparellus | 4 |
Metzneria metzneriella | 4 |
Mirificarma cytisella | 4 |
Athrips amoenella | 5 |
Isophrictis kefersteiniellus | 5 |
Megacraspedus brachypteris | 5 |
Monochroa nomadella | 5 |
Sattleria pyrenaica | 5 |
Acompsia tripunctella | 6 |
Caryocolum schleichi | 6 |
Oxypteryx libertinella | 7 |
Stomopteryx remissella | 8 |
Megacraspedus lanceolellus | 20 |
Megacraspedus dolosellus | 22 |
In total, 5877 sequences were assigned to a BIN. These records were assigned to 992 BINs that belong to 788 putative taxa (Suppl. material
Altogether 668 (92.4%) of 723 named species have one or more unique BINs, while 55 species (7.6%) share a BIN with up to four species (Table
Species | BIN |
Acompsia antirrhinella / A. pyrenaella / A. tripunctella | BOLD:AAJ5937 |
Anacampsis blattariella / A. populella | BOLD:AAD3256 |
Aproaerema albipalpella / A. cincticulella | BOLD:ACB8811 |
Aristotelia brizella / A. confusella | BOLD:AAJ1682 |
Athrips pruinosella / A. spiraeae | BOLD:AAD2577 |
Caryocolum arenbergeri / C. blandulella | BOLD:AAV7765 |
Dirhinosia cervinella / D. interposita | BOLD:ACB0757 |
Iwaruna biguttella / I. klimeschi / I. robineaui | BOLD:AAU3602 |
Metzneria fulva / M. torosulella | BOLD:ADM4637 |
Monochroa arundinetella / M. suffusella | BOLD:AAF9390 |
Monochroa palustrellus / M. saltenella | BOLD:AAF2711 |
Sattleria melaleucella / S. pyrenaica | BOLD:AAC5037 |
Scrobipalpa alterna / S. lutea | BOLD:ADR5476 |
Scrobipalpa amseli / S. hyssopi | BOLD:ADL8424 |
Scrobipalpa artemisiella / S. stangei | BOLD:AAE9838 |
Scrobipalpa halymella / S. stabilis | BOLD:AAV9005 |
Scrobipalpa salicorniae / S. salinella | BOLD:AAF1193 |
Scrobipalpula diffluella / S. psilella / S. ramosella / S. seniorum / S. tussilaginis |
BOLD:AAF1106 |
Stomopteryx lineolella / S. mongolica / S. nugatricella | BOLD:ACB3380 |
Teleiodes brevivalva / T. italica / T. vulgella | BOLD:AAE9855 |
Teleiopsis albifemorella / T. rosalbella | BOLD:AAB6930 |
Teleiopsis bagriotella / T. diffinis / T. paulheberti | BOLD:ACE4927 |
Teleiopsis bagriotella / T. diffinis | BOLD:ACE6105 |
Xenolechia aethiops / X. lindae / X. pseudovulgella | BOLD:AAE1445 |
High levels of ‘intraspecific’ barcode variation often reflect overlooked species, but there is no fixed level of divergence that indicates species status. Furthermore, deep barcode splits can also arise as a result of the inadvertent recovery of pseudogenes, as a consequence of hybridisation, or Wolbachia infection (
33 species of European Gelechiidae with a maximum intraspecific barcode divergence > 5%.
Species | Mean intra-spec. | Max intra-spec. |
---|---|---|
Megacraspedus dolosellus | 7.49 | 13.76 |
Megacraspedus lanceolellus | 7.37 | 12.51 |
Monochroa sepicolella | 5.15 | 9.78 |
Megacraspedus brachypteris | 4.36 | 7.82 |
Stomopteryx remissella | 2.69 | 7.47 |
Ephysteris diminutella | 3.87 | 7.15 |
Sophronia sicariellus | 1.34 | 7.06 |
Caryocolum cauligenella | 1.86 | 7.00 |
Acompsia pyrenaella | 3.58 | 6.92 |
Caryocolum saginella | 2.17 | 6.86 |
Dichomeris rasilella | 3.31 | 6.67 |
Monochroa nomadella | 3.72 | 6.58 |
Caryocolum schleichi | 3.93 | 6.47 |
Acompsia tripunctella | 2.59 | 6.40 |
Megacraspedus teriolensis | 3.07 | 6.38 |
Caryocolum fibigerium | 3.41 | 6.31 |
Chionodes fumatella | 2.6 | 6.30 |
Oxypteryx baldizzonei | 3.9 | 6.29 |
Oxypteryx wilkella | 1.5 | 6.29 |
Dichomeris juniperella | 2.82 | 6.24 |
Parapodia sinaica | 2.97 | 5.95 |
Megacraspedus balneariellus | 3.97 | 5.95 |
Mirificarma burdonella | 5.9 | 5.9 |
Caryocolum peregrinella | 3.56 | 5.71 |
Caryocolum alsinella | 2.11 | 5.60 |
Oxypteryx libertinella | 2.65 | 5.48 |
Aproaerema suecicella | 2.43 | 5.44 |
Megacraspedus imparellus | 4.05 | 5.43 |
Isophrictis anthemidella | 2.92 | 5.3 |
Catatinagma trivittellum | 5.24 | 5.24 |
Pexicopia malvella | 1.1 | 5.23 |
Acompsia maculosella | 2.16 | 5.19 |
Ephysteris promptella | 3.31 | 5.12 |
In some recently revised taxa with high, geographically structured intraspecific barcode divergence such as Megacraspedus (
A further group of unrevised species in our dataset includes 65 unidentified DNA barcode clusters which were assigned to separate BINs (Table
Taxon | BIN |
---|---|
Anarsia | BOLD:ADE9567 |
Anarsia | BOLD:ADE9710 |
Apatetris | BOLD:AAV7596 |
Apatetris | BOLD:ABA4360 |
Aproaerema | BOLD:AAT9258 |
Aproaerema | BOLD:ACF7323 |
Aproaerema | BOLD:ADG7311 |
Aproaerema | BOLD:ADL8444 |
Aproaerema | BOLD:ADL9068 |
Aproaerema | BOLD:ADL9069 |
Aristotelia | BOLD:AAU2122 |
Aristotelia | BOLD:AAV7599 |
Aristotelia | BOLD:ABV2430 |
Aristotelia | BOLD:ACC2990 |
Aristotelia | BOLD:ACK0360 |
Aristotelia | BOLD:ADC8189 |
Aristotelia | BOLD:ADK9648 |
Aristotelia | BOLD:ADL8520 |
Aristotelia | BOLD:ADL8769 |
Aristotelia | BOLD:ADL9120 |
Aristotelia | BOLD:ADM4599 |
Aristotelia | BOLD:ADY0927 |
Brachmia | BOLD:ADM5065 |
Caulastrocecis | BOLD:ADM1812 |
Caulastrocecis | BOLD:ADR7056 |
Chrysoesthia | BOLD:ADM8914 |
Chrysoesthia | BOLD:ADN7772 |
Dichomeris | BOLD:ADI2574 |
Epidola | BOLD:ADF2272 |
Gelechia | BOLD:ADF0061 |
Gelechiidae | BOLD:ADO2643 |
Isophrictis | BOLD:ADF3165 |
Isophrictis | BOLD:ADI3246 |
Ivanauskiella | BOLD:ACB0708 |
Megacraspedus | BOLD:ACZ8654 |
Megacraspedus | BOLD:ADY4582 |
Mesophleps | BOLD:AAU3614 |
Mesophleps | BOLD:ADM4492 |
Metzneria | BOLD:ABW1820 |
Metzneria | BOLD:ACB3385 |
Metzneria | BOLD:ADM8252 |
Monochroa | BOLD:ACF6594 |
Monochroa | BOLD:ACS5726 |
Monochroa | BOLD:ACW2532 |
Monochroa | BOLD:ADL7906 |
Monochroa | BOLD:ADL9322 |
Monochroa | BOLD:ADR3927 |
Neofriseria | BOLD:ADR5460 |
Ochrodia | BOLD:ACE0260 |
Oxypteryx | BOLD:ACR9491 |
Oxypteryx | BOLD:ACS7858 |
Oxypteryx | BOLD:ACS7859 |
Psamathocrita | BOLD:ADF0071 |
Psamathocrita | BOLD:ADL7901 |
Ptocheuusa | BOLD:AAV7056 |
Scrobipalpa | BOLD:AAV4547 |
Scrobipalpa | BOLD:ACT3383 |
Scrobipalpa | BOLD:ACT4605 |
Scrobipalpa | BOLD:ADF0070 |
Scrobipalpa | BOLD:ADG5400 |
Scrobipalpa | BOLD:ADL6932 |
Scrobipalpa | BOLD:ADL7117 |
Sophronia | BOLD:ADF5021 |
Stomopteryx | BOLD:ADM5270 |
Telphusa | BOLD:ADM5148 |
During the past decade, several national DNA barcoding campaigns have led to the development of an increasingly well-parameterised DNA barcode library for European Lepidoptera. However, these projects have mainly focused on the fauna of central and northern Europe. As a consequence, genetic coverage for species in the Mediterranean region remains patchy. Reflecting this fact, continent-wide analysis has only considered a few groups so far, such as Nepticulidae (van Nieukerken pers. comm.), Gracillariidae (Lopez-Vaamonde pers. comm.), Elachistinae (
The current DNA barcode library makes it clear that the Gelechiidae is a particularly good example of the serious gaps in the knowledge of European biodiversity. Nearly a quarter of current fauna has been described since 1990 (Fig.
In the present study, DNA sequences revealed a high level of possible cryptic diversity in European Gelechiidae, despite extensive revisionary work over the last decades (see e.g.,
The intraspecific DNA barcode variation is reflected in some taxa as allopatric divergence, but in other cases, it reflects sympatric deep splits. However, few of these species have received detailed taxonomic assessment such as the recent comprehensive study on Megacraspedus (
By providing coverage for 751 species of European Gelechiidae, the current DNA barcode library represents the largest release in terms of species diversity for any family of Lepidoptera on this continent. The results reveal unexpected genetic diversity in many taxa as well as numerous unidentified taxa. This indicates that the alpha-taxonomy of this family, still requires serious attention despite one-quarter of the known species described after 1990. The current results indicate that the Gelechiidae remain one of the most taxonomically challenging families of Lepidoptera in the World as complete coverage of even European fauna will require extensive effort. However, the DNA barcode library generated in this study will allow these revisionary studies to target groups that are particularly problematic, accelerating the documentation of the fauna.
We are very grateful to the entire team at the Canadian Centre for DNA Barcoding (Guelph, Canada), whose sequencing work was enabled by funding to PDNH from Genome Canada through Ontario Genomics. We also thank the Ontario Ministry of Research and Innovation and NSERC for their support of the BOLD informatics platform. PH is particularly indebted to the Promotion of Educational Policies, University and Research Department of the Autonomous Province of Bolzano - South Tyrol for helping to fund 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)”. The work of JS was supported by the Ministry of Culture of the Czech Republic (DKRVO 2019-2023/5.I.a, National Museum, 00023272). MM thanks Finnish Academy, Finnish Cultural foundation and Kone foundation for financial support to the Finnish Barcode of Life (FinBOL) project.
We gratefully acknowledge the support with material donated to our institutional collections by several colleagues, which was of crucial value for our analysis and to several colleagues mentioned in the list of private and institutional collections) for supporting our DNA barcoding approach.
Philipp Kirschner (Innsbruck, Austria) most kindly helped with the construction of Fig.
This study was only possible due to contributions from many colleagues. As such it provides an impetus for closer co-operation among the community of taxonomists working on Gelechiidae and similarly ‘difficult’ groups of other micro-moths.
Barcoded species
Data type: species data
NJ trees 1–53
Data type: species data
Specimen data to NJ trees 1–53
Data type: specimen data