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DNA Barcodes for the Northern European Tachinid Flies (Diptera: Tachinidae)

DNA Barcodes for the Northern European Tachinid Flies (Diptera: Tachinidae)

  • Jaakko L. O. Pohjoismäki, 
  • Jere Kahanpää, 
  • Marko Mutanen
PLOS
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Abstract

This data release provides COI barcodes for 366 species of parasitic flies (Diptera: Tachinidae), enabling the DNA based identification of the majority of northern European species and a large proportion of Palearctic genera, regardless of the developmental stage. The data will provide a tool for taxonomists and ecologists studying this ecologically important but challenging parasitoid family. A comparison of minimum distances between the nearest neighbors revealed the mean divergence of 5.52% that is approximately the same as observed earlier with comparable sampling in Lepidoptera, but clearly less than in Coleoptera. Full barcode-sharing was observed between 13 species pairs or triplets, equaling to 7.36% of all species. Delimitation based on Barcode Index Number (BIN) system was compared with traditional classification of species and interesting cases of possible species oversplits and cryptic diversity are discussed. Overall, DNA barcodes are effective in separating tachinid species and provide novel insight into the taxonomy of several genera.

Introduction

The Tachinidae are one of the most species rich families of Diptera, with almost 10,000 described species worldwide [1]. Of some 880 species reported from Europe, 328 have been recorded from Finland [2]. The latter number includes the following recent additions to the Finnish fauna: Parasetigena silvestris (Robineau-Desvoidy), Admontia maculisquama (Zetterstedt), Lecanipa bicincta (Meigen), Trigonospila ludio (Zetterstedt), Winthemia speciosa (Egger), Carcelia puberula Mesnil, Lydella thompsoni Herting, Peribaea setinervis (Thomson), Synactia parvula (Rondani) and Billaea fortis (Rondani). Siphona variata Andersen has proved to be a misidentification and the species has been removed from the Finnish checklist.

Where known, all tachinids are obligate parasitoids of other arthropods and as such have great ecological importance [3]. As tachinid community size and structure are influenced by a number of biological variables, their species diversity offers a useful proxy to assess habitat intactness and quality [46]. Moreover, tachinids are important natural enemies of many ecologically important pest species, such as nun moths, Lymantria spp. (Lepidoptera: Lymantriidae) [7,8], the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Pyralidae) [9,10] and earwigs, Forficula sp. (Dermaptera) [11].

Because of their species richness, morphological diversity and varying characters, many tachinid species are challenging to determine even for experts. Whereas the European fauna is rather well known, difficulties in classification and the poor quality of the early taxonomic work makes especially the determination of the tropical tachinid species impassable without the study of the type specimens [12]. While excellent resources into the identification of the European species and Palearctic genera are existing [13,14], DNA barcodes based on the 658 bp mitochondrial cytochrome oxidase I gene (COI) sequence [15] could prove to be valuable in helping non-specialists in species identification as well as enabling the identification of early developmental stages. The latter is especially interesting, as it permits the assessment of parasitoid diversity and the study of local food webs by sampling hosts [16,17]. Besides using COI barcodes to uncover cryptic host differentiation in tachinids [18], a comprehensive COI barcode library can provide rough identification of taxa even if the actual species identity remains unsolved. Because of their multiple utilities and ease of use, DNA barcodes have become an integral part of modern ecology [19].

The presented dataset provides reference barcodes for 366 mainly north European tachinids. The barcode library has been collected as a part of the Finnish Barcode of Life (FinBOL, www.finbol.org) initiative and represents projects opening data release as well as the first comprehensive collection of DNA barcodes for European tachinids. We explore the performance of DNA barcodes and Barcode Index Numbers (BINs) in discriminating species and discuss several species groups showing barcode-sharing or extraordinary intraspecific variation.

Materials and Methods

A total of 1,136 specimens belonging to 397 species of Tachinidae were included in the analysis. The majority of the samples were from the personal collection of JLOP, supplemented by specimens donated or loaned by other researchers and institutions (Table 1 and S1 Table). J.P. identified the majority of the specimens, using the available literature [13,14,2022] and, in doubtful cases, with the help of specialists mentioned in the acknowledgments. 814 specimens have been collected from Finland, 163 from Germany, 57 from France, 22 from Greece and 16 from Italy and lower numbers from several other countries. Majority of the species occur in the northern Europe, with the exception of some rare Palearctic species, which were included in the study as being possible the only opportunity to DNA barcode these species. Notably, many tachinid species have a wide distribution range, enabling us to directly compare for example Mediterranean populations with the Finnish. Full specimen details, storing institutions and GenBank accession numbers are provided in the S1 Table. Taxonomic and collection information as well as voucher photographs are also available through individual specimen pages within the public dataset DS-TACFI (dx.doi.org/10.5883/DS-TACFI) in the BOLD (Barcode of Life Data Systems, www.boldsystems.org) barcode data repository [dataset numbers are to be released upon the acceptance of the manuscript]. Larger sets of specimens were photographed in the University of Oulu and a single leg was removed and sent in a 96-well plate for DNA extraction and sequencing to the Canadian Center for DNA Barcoding (CCDB).

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Table 1. List of species and the number of specimens from which at least a partial COI sequence was recovered.

Abbreviations for the countries of origin: B–Belgium, CH–Switzerland, DE–Germany, E–Spain, F–France, FIN–Finland, GB–Great Britain, GR–Greece, I–Italy, NL–The Netherlands, RUS–Russia, SE–Sweden. Singleton countries are written in full. Length of the longest COI sequence together with the number of ambiguous bases is indicated. The list follows the taxonomic order of Herting & Dely-Draskovitz [29].

https://doi.org/10.1371/journal.pone.0164933.t001

The CCDB’s sequencing protocol is described in detail in deWaard et al. [23]. The primer pair LepF1 and LepF2 is primarily used to amplify the barcode region in Tachinid flies, but, in cases of failure, other primer sets were also attempted. Full primer details, laboratory reports, trace files, sequences and GenBank accession numbers can be retrieved from the sequence page of each record in BOLD. Before the sequences were uploaded to BOLD, several validation steps were conducted in CCDB to detect possible cases of contaminations, pseudogenes (NUMTs) and chimeric sequences. Sanger sequencing trace electropherograms were reviewed for quality, excising sequences associated with a mean trace quality “phred” score below 30 and where more than 10% of the bases showed a quality score below 20 after trimming of the primer sequences. Sequences that met these quality criteria were reviewed to excise those that are likely pseudogenes (NUMTs) or chimeric in origin. Pseudogenes were detected by comparing each sequence to a Hidden Markov Model [24] of the COI protein [25]. Some rare specimens were individually processed and sequenced following standard protocols in the Department of Environmental and Biological Sciences, University of Eastern Finland. Records were placed in the TACFI project that was administrated via the Barcode of Life Database (BOLD) www-interface using the available on-line tools.

Sequences were aligned using the standard BOLD nucleotide sequence alignment tool, and for full NJ analyses subsequently slightly edited using Mega 6 [26]. Since there is no length variation in the barcode fragment among the analyzed Tachinidae, alignment was straightforward. We used Neighbor Joining (NJ) method to examine and visualize genetic patterns revealed by the data. A tree constructed with BOLD under Kimura-2-parameter (K2P) substitution model and BOLD alignment was built primarily to show BIN assignments for the specimens and species. To test the effects of substitution model for the full data, the NJ analyses were conducted separately under the K2P and uncorrected p-distance models using Mega 6. Bootstrap node support values were calculated based on 500 replicates. Four specimens with less than 300 bp fragment were not included in Mega analyses because of lack of overlapping data with other specimens. Additionally, NJ trees for some specific species groups showing interesting patterns in barcodes were built in BOLD under K2P model and BOLD alignment. Distance statistics, including mean and maximum intraspecific divergence, distance to the nearest neighbor were retrieved using the Barcode gap analysis tool available in BOLD. Barcode Index Number (BIN) operational taxonomic units are automatically created in BOLD for sequences that fulfill minimum requirements. In short, sequences are initially clustered by employing a fixed 2.2% threshold of uncorrected p-distance, and refined into the final BINs by Markov clustering. Further details for BINs are provided in Ratnasingham and Hebert, 2013 [27].

Results and Discussion

Over 200 bp DNA barcode was successfully obtained from 925 specimens representing of 366 species of Tachinids. Over 400 bp and over 600 bp sequence was recovered for 923 and 879 specimens, respectively. Sequences of sufficient length and quality for BIN assignment (usually >500 bp sequences of high quality) were assigned to 329 operational taxonomic units (OTUs) as based on Barcode Index Numbers (BINs) (Fig 1 and S1 Fig, Table 1 and S1 Table). Substitution model had some effects on the overall tree topology, especially orderings of higher clades, but little effect at the lower levels, i.e. sister-group relationships between and within the species (S2 and S3 Figs). 82% success rate for the specimens and 93% for the species can be regarded extraordinary, considering that >99% of the material were pinned dry specimens. The oldest pinned specimen from which full barcode sequence was retrieved, was collected in 1980. On one hand, we were unable to obtain PCR products from some species, such as Rondania fasciata (Macquart), despite numerous attempts with freshly collected material. We expect poor primer binding likely been involved in such cases. The data covers 85% of the Finnish fauna and all genera except Policheta, Ligeria, Ligeriella and Peteina. Additionally, we were able to cover several rare European genera, such as Alsomyia, Trichactia, Pandelleia, Chaetovoria and Strongygaster. The sequences for Linnaemya, Lydina, Lypha, Peleteria, Nowickia and Tachina have been released as a part of a previous study [28], but are listed also here for the completeness.

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Fig 1. Overview of the tachinids sequenced in the FinBoL project.

(A) Accumulation curve for the 366 species, corresponding 329 BINs. (B) The FinBoL project produced DNA barcodes for 280 Finnish tachinid species (85% of the species recorded in Finland) and for 86 non-Finnish species, which most are present in the adjacent countries. No samples or successful barcodes were obtained for 48 species on the Finnish checklist. Example species: Female Carcelia bombylans Robineau-Desvoidy, Espoo, Finland.

https://doi.org/10.1371/journal.pone.0164933.g001

Identification performance of the DNA barcodes

The included 366 species of Tachinidae show a mean minimum K2P divergence of 5.51% to the nearest neighbor (range 0.00–14.35%, SD = 0.62, SE = 0.03) (S2 Table). This is on average 22.7 times the mean of maximum intraspecific variation (0.24%, range 0.00–7.58, SD = 3.51, SE = 0.18), demonstrating the general presence of a barcoding gap between species. This estimate of barcoding gap is however highly exaggerated by the presence of many singletons in our data. With singletons excluded, the mean barcoding gap drops to 13.8 times the mean of maximum intraspecific variation. This value is still an overestimate since the sampling was inadequate for providing a reliable estimate of total extent of intraspecific variation. Evidently, the true intraspecific variation is on average much less than the mean distance to the nearest neighbor. Moreover, the identification performance is negatively affected by several operational factors likely involved in our data as well [30]. For example, cases where a taxonomically accepted species actually consists of cryptic species highly exaggerates the estimate of intraspecific variation. Similarly, it is possible that our data include unjustified species, misidentifications, small sequencing and alignment errors, i.e. various operational factors, which all diminish the estimation of identification success of DNA barcodes.

The observed divergence of 5.51% between the species is slightly less than what has previously been reported in Lepidoptera (mean divergence among 2,577 species 5.73% [31]), and much less than in Coleoptera (mean divergence among 1,872 species 11.99% [32]) with similar sampling effort and geographic coverage. Comparisons of mean intraspecific divergences are slightly biased by different sampling, but differences between the insect groups remain evident, and are reflected to the protein level as well [31]. It has been suggested that COI evolution rate is generally correlated with the metabolic rate, with groups having high metabolic rate (such as Lepidoptera) tending to show slow evolution rate in COI compared to those having low metabolic rate (such as Coleoptera) [33]. This is in good accordance with our results, since many dipterans, including Tachinids, are strong fliers and are likely characterized with very high metabolic rate. Additionally, when compared to the ancient beetle families [34], Tachinidae are evolutionarily young, having underwent rapid diversification no earlier than the Oligocene [35] and therefore also explaining the large difference between the species divergence in the two groups.

Full barcode sharing (K2P distance to nearest neighbor = 0) was observed in 13 species pairs or triplets and between 28 species (7.36%) (S2 Table). In 53 species (14.4%), the divergence to the nearest neighbor was less than 1% and in 79 species (21.5%) less than 2%. These values are clearly higher than what was observed in beetles in the same region [32] since among 1,872 species of beetles only 1.6% showed full barcode sharing and 4.9% less than 2% divergence to the nearest neighbor. This difference is likely true and linked to the generally much larger interspecific distances in beetles than in tachinid flies.

Taxa sharing BINs

Considering their recent evolutionary origin [35], BIN OTUs performed generally well for separating the studied taxa. However, some morphologically clearly separable species in Exorista, Nilea, Eumea (all Exoristinae), Peleteria, Nowickia, Macquartia (all Tachininae), Gymnosoma and Leucostoma (Phasiinae) share BIN as they had identical or nearly identical COI sequence (Fig 2, S2 Table). Interestingly, at least the members of Exorista sg. Adenia seem to be also poorly separable by nuclear genes [36]. Surprisingly, the species in morphologically difficult genus Siphona, had distinct BINs with the exception of S. maculata Staeger and S. collini Mesnil (Fig 3). BIN sharing occur in many taxa and may even be common in some groups [32,37], although the underlying mechanisms vary [38,39]. Whereas the Exorista species are undoubtedly evolutionary young, it is interesting that the Greenlandic Peleteria aenea Staeger and P. rubescens Robineau-Desvoidy from Mediterranean France are also almost inseparable (S1S3 Figs, discussed previously in [28]). Siphona collini and S. maculata can be distinguished by a number of external characters, but for example their genital features are less descript and variable [40]. As they have a similar distribution with differing flight times, it is not impossible that the two could represent spring and high summer forms of the same species. It is clear that issues such as this can be only resolved by genome-wide analysis such as RAD sequencing [41] or meticulous study of the species’ biology.

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Fig 2. Examples of species or species complexes with poor BIN separation.

(A) Exorista mimula (Meigen) COI sequence is embedded within the E. rustica (Fallén) sequences in the NJ trees. Same applies to E. fasciata (Fallén) and E. larvarum (L.), whereas similarly closely related E. grandis (Zetterstedt) and E. sorbillans (Wiedemann) are distinctly different. Notice also the differentiation between the Finnish and the Mediterranean specimens of E. deligata Pandelle. (B) Whereas E. mimula and E. rustica can be reliably determined only using male genital characters, E. fasciata (upper) and E. larvarum (lower) are clearly separable by various morphological characters and habitat preference. Other morphologically distinct species sharing BINs are (C) Eumea linearicornis (Zetterstedt) and E. mitis (Meigen), Nilea hortulana (Meigen) and N. innoxia Robineau-Desvoidy, (D) Gymnosoma spp. (E) Macquartia dispar (Fallén) and M. viridiana Robineau-Desvoidy as well as (F) Leucostoma spp. Scale bar: 2% sequence difference.

https://doi.org/10.1371/journal.pone.0164933.g002

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Fig 3. NJ tree of Siphona COI sequences.

Contrary to the expectations, BINs have a good resolution in this morphologically variable genus, whose members are notoriously challenging to determine, only expectation is the S. collini Mesnil–S. maculata Staeger pair. Some duplicates omitted from the NJ tree for clarity, see S2 and S3 Figs for the full data. Example species: Male Siphona setosa Mesnil, Jämijärvi, Finland.

https://doi.org/10.1371/journal.pone.0164933.g003

BIN variation: Geography and putative cryptic species

While the aforementioned species exhibited barcode sharing, some species showed significant sequence divergence within or between different geographical regions. The most extreme example is Microsoma exiguum (Meigen) for which three distinct haplotypes with maximum divergence of 2.99% were detected; Mediterranean, Central- and Northern European (Fig 4). M. exiguum is the only known member of its genus in the Palearctic region. The flies are small (<3.0 mm) and live as parasitoids of adult weevils (Coleoptera: Curculionidae). Their small size alone could have implications on their dispersal ability, resulting in geographical differentiation. However, as the Central European haplotype (locality 5) was present also in southern Finland (localities 3 and 4), which is much more distant from Germany than the Mediterranean France, it is likely that the differentiation is explained by other biological factors, such as host specialization [42]. Exorista deligata Pandellé represents a similar case, although the difference and/or the sample size is not big enough to separate BINs (maximum intraspecific divergence 1.24%, n = 3) and that only two different haplotypes were found (Fig 2). E. deligata has an interesting discontinuous range, being present in the Mediterranean countries and Scandinavia, but absent from the Central Europe [43]. As far as known, they are specialized parasitoids of bagworm moths (Lepidoptera: Psychidae) and it is unlikely that the Finnish and Mediterranean subpopulations share any common host species [14]. As a comparison, Peleteria rubescens (Robineau-Desvoidy), Tachina fera (L.), Thriathria setipennis (Fallén) and Cylindromyia brassicaria (Fabricius) collected from the same locations exhibited less or no variation in their COI sequences (Fig 4). All these species are rather common across Europe and are likely to be generalists in their host use.

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Fig 4. Geographic variation in tachinid COI sequences.

(A) The northern Finnish (map locations 1–2) Microsoma exiguum (Meigen) belong to a different BIN cluster than the specimens from southern Finland, Central Europe (map locations 3–6) and Mediterranean France (map location 7). Note that the specimens from Provence represent a different haplogroup than the Central European ones, although the difference is not enough to split the BIN. Similar differentiation was not observed for (B) Tachina fera (L.), (C) Mintho rufiventris (Fallén), Thriarthria setipennis (Fallén) and (D) Cylindromyia brassicaria (Fabricius) collected from the same locations. Example species: Male Microsoma exiguum, Friedberg, Germany. Scale bar: 1% sequence difference.

https://doi.org/10.1371/journal.pone.0164933.g004

COI barcodes also revealed deep intraspecific splits in the Finnish populations of Gymnocheta viridis (Fallén) and Medina collaris (Fallén), which seem to be also associated with habitat preference and morphology (JP, personal observations) (Fig 5). The identity of the different forms is currently under investigation. It should be noted that similar examples exist to lesser extent in some other genera, where variation within the species is not quite enough to differentiate BINs (S1 Fig).

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Fig 5. Intraspecific BIN splits in Finnish Tachinidae.

(A) Finnish Gymnocheta viridis (Fallén) are split into two separate BIN clusters with a minimum divergence of 1.41% between them and a maximum divergence of 0.62% within the clusters. The G. sp. nr. viridis is also ecologically separable from the true G. viridis with all records being solely from the northern and eastern Finnish bog habitats, whereas the latter is almost purely a meadow species. (B) The Finnish Medina collaris (Fallén) specimens are similarly falling into two separate BINs. Coincidentally to G. viridis example also M. sp. nr. collaris are confined to bog habitats. Both Medina species have one rear bristle on their forelegs, a character state not present in other European representative of the genera. Example species: Male Gymnosoma sp. nr. viridis, Lieksa, Finland. Scale bar: 1% sequence difference.

https://doi.org/10.1371/journal.pone.0164933.g005

Possible taxonomic implications

Although COI sequences are normally highly similar among the species within a genus, there are some occasions where the species from one genus are embedded among the species of another. As NJ can cluster unrelated sequences accidently, it is meaningful to compare only closely related taxa. Tachinids are notoriously rich in genera and can be that some of these sequence associations reflect unjustified splitting of genera by taxonomists. This is likely to be the case with Wagneria-Kirbya and Billaea-Dinera as well as Phorocera and Parasetigena, latter which originally belong to Phorocera (Fig 6). As of note, Dinera sp. nr. fuscata is a widespread species in Central Europe, which has been previously confused with Dinera carinifrons (Fallén). The European specimens differ from the Oriental D. fuscata Zhang & Shima and their taxonomic status needs to be solved [44]. The true D. carinifrons has apparently declined drastically and is probably extinct in Finland [2].

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Fig 6. Possible taxonomic conflicts within tachinid genera.

(A) Kirbya moerens (Meigen) is embedded within the COI sequences of the closely related Wagneria, whereas the other Voriini genera form their own clusters. Voria is thought only to be represented by V. ruralis (Fallén) in the Palearctic. However, the COI of a specimen from S-Agean Greece differs significantly (by 4.91%) from the northern European examples and could represent a species of its own. Similar to Kirbya–Wagneria case, also (B) Billaea–Dinera and (C) Phorocera–Parasetigena have mixed COI clusters. Example species: Male Billaea kolomyetzi, Ruokolahti, Finland. Scale bar: 2% sequence difference.

https://doi.org/10.1371/journal.pone.0164933.g006

Whereas the previous examples might indicate unjustified splitting of genera there are also opposite examples. For instance, Oswaldia reducta does not share much similarity with the other three species of Oswaldia, but is in all comparisons closer to other Blondelini, such as Belida (Fig 7 and S1S3 Figs). Similarly, Phebellia seems to be split into two distinct lineages. While COI can be useful in identifying possible taxonomical conflicts, the proper revision of the genera needs be based on additional genetic markers and morphological characters.

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Fig 7. Deep divergence within tachinid genera.

(A) COI from Oswaldia reducta (Villeneuve) has more sequence similarity with genera other than Oswaldia. (B) Species of Phebellia fall into two distantly related clusters, which also do not represent the proposed split of the genus into Phebellia s. str. and Prooppia [45].

https://doi.org/10.1371/journal.pone.0164933.g007

Concluding Remarks

We provide here the first comprehensive collection of DNA barcodes for the European Tachinidae. Simultaneously the collection represents the first ever data release of Diptera from the FinBoL initiative. The DNA barcodes provided here permit the identification of the majority of the Finnish fauna and are likely to suffice for all of the common European species. Acknowledging the taxonomic difficulties, a great deal of care has been taken to confirm the species determinations and the data should provide a good reference for taxonomical and ecological studies using tachinids in the future. Importantly for tachinids, which are often unidentifiable as wet samples, pinned specimens proved to be perfectly adequate as source material for DNA barcoding.

Supporting Information

S1 Fig. BOLD taxon ID Tree, constructed with neighbor-joining method and under K2P evolutionary model, of all samples as taken from BOLD.

BIN clusters given in different colours.

https://doi.org/10.1371/journal.pone.0164933.s001

(PDF)

S2 Fig. A neighbor-joining tree of near-full data constructed with Mega 6 under K2P model of nucleotide substitution.

Node bootstrap support values based on 500 replicates are shown.

https://doi.org/10.1371/journal.pone.0164933.s002

(EMF)

S3 Fig. A neighbor-joining tree of near-full data constructed with Mega 6 under P-distance model of nucleotide substitution.

Node bootstrap support values based on 500 replicates are shown.

https://doi.org/10.1371/journal.pone.0164933.s003

(EMF)

S1 Table. List of specimens in alphabetical order with sample location, BOLD process ID, BOLD sample ID and GenBank access number.

The list includes also the failed specimens. Note that the taxonomy in BOLD follows O’Hara and Wood [45], treating Ramonda as the synonyme of Periscepsia.

https://doi.org/10.1371/journal.pone.0164933.s004

(XLSX)

S2 Table. Barcode cap analysis of tachinid species included in the study.

Mean and maximum intraspecies variation, distance to the nearest neighbor (NN) and the nearest species are given.

https://doi.org/10.1371/journal.pone.0164933.s005

(XLSX)

Acknowledgments

Dr. Hans-Peter Tschorsning, Stuttgart (DE), Dr. Joachim Ziegler, Berlin (DE), Dr. Theo Zeegers, Soest (NL) and Mr. Christer Bergström, Uppsala (SE) are thanked for their valuable help with identifications as well as donating rare species for the project. Dr. Matti Koivikko, Tampere (FIN), kindly helped in curating and sampling specimens for sequencing. We would also like to express our gratitude to all those people who contributed in donating samples for the project: Mr. Antti Haarto (FIN), Mr. Kaj Winqvist (FIN), Mr. Kari Varpenius (FIN), Mr. Jari Flinck (FIN), Mr. Antonio Rodriquez (FIN/E), Mr. Chris Raper (GB), Mr. Steve Downes (GB), Mr. Henrik Gyurkovics (HU) and Mr. Leif Karlsson (SE). We are very grateful to staff at the Biodiversity Institute of Ontario for their continuous help in generating sequences, entering data into BOLD and aiding the curation of this information. We thank Dr. Valerie Levesque-Beaudin and Ms. Megan A. Milton for the help with GenBank submissions and BOLD dataset.

Author Contributions

  1. Conceptualization: JP MM.
  2. Data curation: JP MM.
  3. Formal analysis: JP MM.
  4. Funding acquisition: MM.
  5. Investigation: JP JK MM.
  6. Methodology: JP MM.
  7. Project administration: MM.
  8. Resources: JP JK MM.
  9. Supervision: JP MM.
  10. Validation: JP JK MM.
  11. Visualization: JP.
  12. Writing – original draft: JP JK MM.
  13. Writing – review & editing: JP JK MM.

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