Research Article
Research Article
The Grasshopper Paradigm in damselflies: evidence for phalanx-like postglacial recolonization of Europe from a Balkan refugium in Platycnemis pennipes Pallas (Odonata: Zygoptera: Platycnemidae)
expand article infoThomas J. Simonsen§, Marie Djernæs§, Ole Fogh Nielsen§, Kent Olsen§
‡ Aarhus University, Aarhus, Denmark
§ Natural History Museum Aarhus, Aarhus, Denmark
Open Access


We explore haplotype diversity, phylogeography and phylogenetic relationships of the damselfly Platycnemis pennipes in Europe based on 618 bp DNA from the mitochondrial gene COI. A haplotype network analysis shows that the species is divided into two haplotype groups. One is restricted to the Italian Peninsula, while the other is found from the Black Sea region across eastern and central Europe to Scandinavia, England, and southwestern France. This pattern is recovered in a Bayesian phylogenetic analysis. Genetic distance (K2P) between the two groups is approximately 1.5%, while within-group variation is an order of magnitude lower. An analysis of the molecular variance (AMOVA) shows that variation between the two groups account for more than 96% of the total variation within the dataset, adding to the evidence that they have been isolated for a considerable amount of time. The pattern we find is similar to the so-called Grasshopper Paradigm in European phylogeography, where a species has recolonized Europe after the last glaciation from a glacial refugium in the southeast, while other refugial populations in the Iberian and Italian peninsulas have remained isolated to this day. In P. pennipes there is only an isolated refugial population in Italy as the species does not have current populations in the Iberian Peninsula. By comparing the genetic distance between the two groups to a previously published divergence time analysis of European Odonata we estimate that they have likely been isolated since the onset of the Saale Glaciation ca 400 ky ago.

Key words

Phylogeography, Glacial refugia, expansion, Europe, Zygoptera

1. Introduction

The past decades have seen a number of studies on molecular diversity, systematics and phylogeography of European Odonata. Weekers et al. (2001) used the highly variable nuclear ribosomal ITS region to explore the phylogeny and diversity of European Calopteryx species. They found an overall east-west division at the species level, but also a clear separation between C. splendens splendens (Harris, 1780) and the Italian subspecies C. splendens caprai Conci, 1956. Sadeghi et al. (2010) in an AFLP based study of C. splendens found a complex pattern that only partially corresponds to the current subspecific taxonomy. They found indication of a post-glacial recolonization of southern Europe from the southeast. Guan et al. (2013) studied the phylogeny of the genus Pyrrhosoma Charpentier, 1840 (red damselflies) based on the ITS region and the mitochondrial gene COI with special focus on the status and position of the endangered P. elisabethae Schmidt 1948. They found that while P. elisabethae was indeed monophyletic it grouped within the widespread species P. nymphula (Sulzer, 1776) and—surprisingly—closely related to P. nymphula specimens from Morocco, clearly indicating that more studies on this species complex are needed. Hinojosa et al. (2017) found a distinct pattern—albeit with low variation—in the ITS region and COI of the skimmer Sympetrum vulgatum (Linnaeus, 1758) with the three Western Palearctic subspecies S. vulgatum vulgatum (most of Europe), S. vulgatum ibericum Ocharan, 1985 (Iberian Peninsula), and S. vulgatum decoloratum Selys, 1884 (Anatolia) appearing separated and distinct. In that study, they suggested that the three subspecies represented three different glacial refugia in the Balkans, the Iberian Peninsula, and Anatolia, respectively. Schneider et al. (2015) and Simonsen et al. (2020) studied the Aeshna cyanea (Müller, 1794) and A. vercanica Schneider et al., 2015 species complex. Their results indicate that A. cyanea colonized Western and Central Europe from the Iberian Peninsula, which had previously been colonized from the Maghreb region in Northern Africa. In contrast to the studies above, which provide detailed information on the diversity and distributional patterns of Western Palearctic Odonata, other studies have found little or no variation within the Western Palearctic region. Bernard et al. (2011) found evidence for a rapid expansion from East Asia through western Siberia into West Palearctic in Nehalennia speciosa Charpentier, 1840 with little variation across its range. Kohli et al. (2018; 2021) found very little variation and no phylogeographic pattern in the circumpolar Somatochlora sahlbergi Trybom, 1889 across its range. While Kohli et al. (2021) generally found clear distinction between Nearctic and Palearctic populations in Aeshna juncea (Linneaus, 1758), Aeshna subarctica Walker, 1908, Libellula quadrimaculata Linneaus, 1758, and Sympetrum danae (Sulzer, 1776), as well as some internal Nearctic population structure and occasional distinction between Japan and the remaining Palearctic, they found little variation and no internal structure in the Western Palearctic for either four species.

The molecular diversity and phylogeography of Odonata in the Western Palearctic thus appear highly variable with the potential to illuminate a number of phylogeographic patterns. European phylogeography is currently dominated by four paradigms associated with expansion from the three peninsular refugia, on the Iberian, Italian and Balkan peninsulas (see Hewitt 1999; 2004; Schmitt 2007 for review and details): the Hedgehog Paradigm with postglacial expansion from the Iberian, Italian and Balkan peninsulas; the Bear Paradigm with postglacial expansion from the Iberian Peninsula and an eastern refugium (not necessarily the Balkan Peninsula); the Butterfly Paradigm with postglacial expansion from the Italian and Balkan peninsulas; and the Grasshopper Paradigm with postglacial expansion from the Balkan Peninsula. Especially the Iberian and Italian peninsulas may show historic connections to the North African Maghreb regions (see Husemann et al. 2014; Simonsen et al. 2020 and references therein). In addition to the four paradigms and North African refugia, two other patterns appear to be important in European phylogeography (e.g., Schmitt 2007). First, there is a growing body of evidence for refugia in Europe north of the Pyrenees, the Alps, and the Balkans (e.g., Ursenbacher et al. 2006; Schmitt 2007; Schmitt et al. 2007; Gratton et al. 2008; Varga & Schmitt 2008; Hammouti et al. 2009; Simonsen & Huemer 2014). Second, it has been hypothesized that a considerable number of European species originate in the East Palearctic (de Lattin 1967). Even though the latter has been challenged (e.g., Schmitt 2007), some of the studies listed above (Bernard et al. 2011; Kohli et al. 2018; 2021) indicate an East Palearctic origin of some West Palearctic odonate species.

Here we present a phylogeographic study of the damselfly Platycnemis pennipes (Pallas, 1771) in Europe based on the mitochondrial gene COI. P. pennipes is a widespread species in continental Europe (Fig. 1) except for the Iberian Peninsula, and central and northern Scandinavia and Finland. It is also missing from Ireland, northern England and Scotland, and most Mediterranean islands. Outside Europe it occurs in Anatolia, the Caucasus, and extends eastwards into western Siberia and northern Kazakhstan as far east as the Yenisei River (Boudot et al. 2015). Generally, the taxonomy of P. pennipes is considered uncomplicated, but a separate subspecies, P. penniepes nitidula (Brullé, 1832), occurs in the coastal regions of Montenegro, Albania and Greece where it may hybridize with the nominal subspecies (Boudot et al. 2015). Throughout its range P. pennipes is found associated with a variety of slow flowing and standing waters with surface and edge vegetation (Nielsen 1998; Askew 2004; Boudot et al. 2015). At least in Denmark it is considered a species with low mobility.

Figure 1. 

Approximate distribution of Platycnemis pennipes (inserted photo) in Western Palearctic (based on Boudot et al. 2015), and approximate sample sites for specimens included in this study. Small circles indicate single specimens, while large circles indicate multiple specimens. For details see Table 1.

While there are problems associated with using COI as a single genetic marker for diversity and phylogeography studies (e.g. Kondandaramaiah at al. 2013; Brunet et al. 2017; Roe et al. 2017), several of the studies mentioned above demonstrate that it can be used (albeit with some caution) in Odonata. Utilising COI also allow us to combine new data with the extensive, and rapidly growing, DNA barcode data that are already available online. Recently, two major studies of European Odonata (Galimberti et al. 2020; Geiger et al. 2021) have made a considerable number of European odonatan COI sequences available. By combining these and other publicly available sequences with several new sequences we are able to analyse an extensive geographical dataset of a European damselfly to address key question about European phylogeography.

2. Material and Methods

2.1. Material

We sampled 43 specimens of Platycnemis pennipes from Europe and one additional specimen from Caucasus (Georgia). The new samples were augmented with 17 samples from Genbank or Barcode of Life (BOLD). Nine samples were from Galimberti et al. (2020), six were from Geiger et al. (2021), one was from the Darwin Tree of Life project (, and one was unpublished but available in BOLD’s public database. All specimen data are provided in Table 1. All specimens sequenced for this study are deposited at the Natural History Museum Aarhus. Approximate sample localities are shown in the distribution map in Fig. 1. The sequence from the Darwin Tree of Life project is part of a full reference genome. The full mitochondrial genome for this specimen was downloaded from Genbank and the relevant part of COI was identified in Mega 11 (Tamura et al. 2021) and saved as a fasta file.

Table 1.

Platycnemis pennipes specimens used in this study with sampling localities, voucher designations, Genbank and BOLD accession numbers, and voucher deposits for all samples when available, and references for sequences not generated in this study, localities for such samples are from BOLD, Genbank or the Welcome Sanger Institute Darwin Tree of Life Projects webpage. The asterisks (*) indicated that the COI sequence used here was extracted from a full mitochondrial genome available at Genbank. WSI: Welcome Sanger Institute; NMW: Naturhistorisches Museum Wien; UL: University of Lodz; UMB: University of Milano Bicocca.

Country Region Voucher Haplotype Source Genbank BOLD Deposit
Denmark South Jutland ENT-DNA-22 B2 New MN913173 DANOD015-22 NHMA
Denmark South Jutland ENT-DNA-23 B2 New MN913174 DANOD016-22 NHMA
Denmark South Jutland ENT-DNA-24 B1 New MN913179 DANOD017-22 NHMA
Denmark South Jutland ENT-DNA-25 B1 New MN913180 DANOD018-22 NHMA
Denmark South Jutland ENT-DNA-26 B1 New MN913181 DANOD019-22 NHMA
Denmark South Zealand ENT-DNA-27 B2 New MN913175 DANOD020-22 NHMA
Denmark South Zealand ENT-DNA-28 B2 New MN913176 DANOD021-22 NHMA
Denmark South Zealand ENT-DNA-29 B2 New MN913170 DANOD022-22 NHMA
Denmark East Jutland ENT-DNA-30 B1 New MN913182 DANOD023-22 NHMA
Denmark East Jutland ENT-DNA-31 B2 New MN913177 DANOD024-22 NHMA
Denmark East Jutland ENT-DNA-169 B2 New MN913171 DANOD153-22 NHMA
Denmark East Jutland ENT-DNA-170 B2 New MN913172 DANOD154-22 NHMA
Denmark East Jutland ENT-DNA-258 B1 New MN913183 DANOD231-22 NHMA
Denmark South Zealand ENT-DNA-259 B1 New MN913184 DANOD232-22 NHMA
Denmark South Zealand ENT-DNA-260 B2 New MN913178 DANOD233-22 NHMA
Sweden Södermanland ENT-DNA-762 B1 New MN913185 DANOD640-22 NHRM
Sweden Södermanland ENT-DNA-763 B1 New MN913186 DANOD641-22 NHRM
Sweden Skåne ENT-DNA-764 B1 New MN913187 DANOD642-22 NHMA
Sweden Skåne ENT-DNA-765 B1 New MN913188 DANOD643-22 NHMA
Germany Slesvig-Holstein ENT-DNA-912 B1 New MN913189 DANOD776-22 NHMA
Germany Slesvig-Holstein ENT-DNA-913 B1 New MN913190 DANOD777-22 NHMA
Germany Slesvig-Holstein ENT-DNA-914 B1 New MN913191 DANOD778-22 NHMA
Germany Slesvig-Holstein ENT-DNA-915 B1 New MN913192 DANOD779-22 NHMA
Germany Slesvig-Holstein ENT-DNA-916 B1 New MN913193 DANOD780-22 NHMA
Georgia Batumi ENT-DNA-917 B4 New MN913210 DANOD781-22 NHMA
Romania Voila ENT-DNA-918 B1 New MN913194 DANOD782-22 NHMA
Romania Voila ENT-DNA-919 B3 New MN913207 DANOD783-22 NHMA
Romania Voila ENT-DNA-920 B3 New MN913208 DANOD784-22 NHMA
Romania Voila ENT-DNA-921 B3 New MN913209 DANOD785-22 NHMA
Romania Voila ENT-DNA-922 B6 New MN913206 DANOD786-22 NHMA
France Sorgues ENT-DNA-923 B1 New MN913195 DANOD787-22 NHMA
France Sorgues ENT-DNA-924 B1 New MN913196 DANOD788-22 NHMA
France Sorgues ENT-DNA-925 B1 New MN913197 DANOD789-22 NHMA
France Sorgues ENT-DNA-926 B1 New MN913198 DANOD790-22 NHMA
France Sorgues ENT-DNA-927 B1 New MN913199 DANOD791-22 NHMA
Bulgaria Varna ENT-DNA-1011 B7 New MN913211 DANOD818-22 NHMA
Bulgaria Varna ENT-DNA-1013 B1 New MN913200 DANOD819-22 NHMA
Bulgaria Varna ENT-DNA-1014 B1 New MN913201 DANOD820-22 NHMA
Bulgaria Varna ENT-DNA-1015 B1 New MN913202 DANOD821-22 NHMA
Romania Vadu ENT-DNA-1179 B1 New MN983216 DANOD936-22 NHMA
Romania Vadu ENT-DNA-1180 B1 New MN983217 DANOD937-22 NHMA
Germany Baden-Württemberg ENT-DNA-1222 B1 New MN913203 DANOD949-22 NHMA
Germany Baden-Württemberg ENT-DNA-1223 B1 New MN913204 DANOD950-22 NHMA
Germany Baden-Württemberg ENT-DNA-1224 B1 New MN913205 DANOD951-22 NHMA
England Kent SAMEA9065986 B3 WSTLP OW121859* WSI
Austria Vienna Odo0016 B5 BOLD AODON016-20 NMW
Germany North Rhine-Westphalia ZFMK-TIS-2010623 B1 Geiger et al. (2021) MW490449 GODO023-18 ZFAK
Germany North Rhine-Westphalia ZFMK-TIS-2010635 B1 Geiger et al. (2021) MW490180 GODO027-18 ZFAK
Germany North Rhine-Westphalia ZFMK-TIS-2010638 B1 Geiger et al. (2021) MW490514 GODO030-18 ZFAK
Netherlands Drentsche Aa RMNH.INS.228274 B1 Geiger et al. (2021) KF369498 ODOPH289-13 RMNH
Poland Wadolek Lake ODOPL_149 B1 Geiger et al. (2021) MW490351 PLSW015-20 UL
Poland Wadolek Lake ODOPL_184 B1 Geiger et al. (2021) MW490494 PLSW050-20 UL
Italy Taranto MIB:ZPL:08479 A4 Galimberti et al. (2020) MT298597 ZPLOD679-20 UMB
Italy Trento MIB:ZPL:08480 A1 Galimberti et al. (2020) MW377872 ZPLOD680-20 UMB
Italy Torino MIB:ZPL:08481 A1 Galimberti et al. (2020) MT298595 ZPLOD681-20 UMB
Italy Vercelli MIB:ZPL:08483 A1 Galimberti et al. (2020) MT298594 ZPLOD683-20 UMB
Italy Lecco MIB:ZPL:08485 A2 Galimberti et al. (2020) MT298600 ZPLOD685-20 UMB
Italy Grosseto MIB:ZPL:08487 A1 Galimberti et al. (2020) MT298599 ZPLOD687-20 UMB
Italy Campobasso MIB:ZPL:08490 A3 Galimberti et al. (2020) MT298602 ZPLOD690-20 UMB
Italy Lazio MIB:ZPL:08491 A1 Galimberti et al. (2020) MT298601 ZPLOD691-20 UMB
Montenegro MIB:ZPL:08634 B8 Galimberti et al. (2020) MT298598 ZPLOD834-20 UMB

2.2. Methods

2.2.1. Laboratory procedures

DNA was extracted at Department of Biology, Aarhus University (AU), Denmark using either E.Z.N.A. Tissue DNA Kit (Omega BIO-TEK) or DNeasy Blood & Tissue Kit (Qiagen). The E.Z.N.A. Tissue DNA Kit protocol was followed with some modifications: samples were incubated at 42°C for 18–23 hours during lysis, steps 5 and 6 in the protocol were skipped, and samples were incubated with Elution Buffer for 5–10 min at 70°C and eluted once in 200 μl. The DNeasy Blood & Tissue Kit protocol was applied with modification following Krosch & Cranston (2012) and using a lower lysis temperature combined with a longer lysis time: samples were incubated at 42°C for 20 hours during lysis, elution buffer AE was heated to 60°C prior to elution, samples were incubated with buffer AE for 10 min at 60°C and eluted once in 100 μl.

We used the following PCR protocol for COI: 95° C, 2 min; then 35–45 cycles of 95°C, 30 s; 45°C, 30 s; 72°C, 1 min and a final extension of 72°C for 5 min using the primers from Simonsen et al. (2020) OdoF2 (with universal tail, M13-FP): TGTAAAACGACGGCCAGTTTTCT­A­C­AAAYCAYAARGATATTGG (tail in boldface italics); and OdoR3 (with universal tail, M13R-pUC): CAGGAAACAGCTATGACTAAACYTCTGGRTG­RCCAAARAATCA (tail in boldface italics). All samples were sequenced at Macrogen Europe using the Sanger Method. Contigs and consensus sequences were obtained using DNA Baser Sequence Assembler v5.8.0 (Heracle Biosoft, Romania). We checked the identity of all sequences using BLAST on GenBank and/or BOLD (Barcode of Life Data base) Identification System. GenBank and BOLD accession numbers are listed in Table 1.

2.2.2. Haplotype network and phylogenetic analyses

All obtained COI sequences were aligned in Mega 11 (Tamura et al. 2021) using the built in Muscle algorithm. The resulting alignment was checked in Mesquite v. 3.03 (Maddison & Maddison 2015). As haplotype network analyses are highly susceptible to missing data, we trimmed the dataset so that all sequences were the same length. We constructed a minimum spanning haplotype network (Blandelt et al. 1999) in PopART (Leight & Bryant 2015) following Kohli et al. (2021) and Simonsen et al. (2021).

We analyzed phylogenetic patterns in MrBayes 3.2 (Ronquist et al. 2012) using Platycnemis acutipennis Selys, 1841 (Genbank accession# GU644640) as outgroup. Based on the model finding function in Mega 11 (Tamura et al. 2021), we set the model for molecular evolution to HKY. The analysis was run for 50 million generations with sampling every 10,000 generations. After evaluation, the log files in Tracer v1.7.2 (part of the BEAST package: Bouckaert et al., 2019) the first 25% were discarded as burnin. The resultant tree was examined and visualised in FigTree 1.4.4 (Rambaut 2018).

2.2.3. Assessment of genetic diversity

Based on the results from the haplotype network and phylogenetic analyses, we divided the dataset into two haplotype groups as indicated in Fig. 2 and calculated the genetic distance within and between the two groups based on the Kimura-2 parameter (K2P) (Kimura 1980) in Mega 11 (Tamura et al. 2021). The distance values are provided in Table 2. To assess genetic divergence between and within these groups and sample sites (countries or regions as indicated in Table 1), we carried out a nested AMOVA test (Excoffier et al. 1992) as implemented in PopART. The results are provided in Table 3.

Figure 2. 

Minimum spanning haplotype network of Platycnemis pennipes COI sequences. The number of mutations between groups are indicated by bars. Groups and haplotypes are indicated as described in the text and Table 1.

Table 2.

Estimates of evolutionary divergence over sequence pairs within and between groups as indicated in the text based on the Kimura-2 Parameter.

Europe Italy
Europe 0.0015
Italy 0.0150 0.0021
Table 3.

Summary of molecular variance analysis (AMOVA). The percentage of molecular variance (%variation) is provided, together with appropriate ɸ-statistics. The statistical significance of each value is based on 1000 permutation.

Variation df Sigma² % variation ɸ-statistics p
Among groups 1 40.400 96.5 0.985 < 0.001
Among sample localities 13 0.836 2.0 0.566 = 0.012
Within sample localities 46 0.642 1.5 0.965 < 0.001

3. Results

3.1. Phylogenetic and haplotype network analyses

The 61-specimen dataset of P. pennipes was trimmed to 618 bp COI to avoid missing or ambiguous data. The minimum spanning network analysis in PopART (Fig. 2) revealed a striking pattern with two well-separated and distinct groups. One group (haplotype group A, n = 8, four haplotypes, numbered A1–4) comprises all specimens from Italy, while the other group (haplotype group B, n = 53, eight haplotypes, numbered B1–8) (hereafter called the European group) comprises all other specimens from Georgia in the east, to southern France and the UK in the west, and to southern Scandinavia in the north. The Italian group is overall homogenous with only a few base pair differences between some of the specimens. The European group is somewhat more heterogenous, although the heterogeneity is skewed: most specimens form a homo­genous group with little internal variation, one Bulgarian specimen (ENT-DNA-1011, haplotype B7) and a single specimen from Montenegro (ZPLOD834-20, haplotype B8) display considerable variation, both compared to each other and to the majority of the specimens in the group. The alignment is provided as File S1.

For the phylogenetic analysis in MrBayes, we added a COI barcode 648 pb sequence of P. acutipennis as outgroup to the original 658 bp P. pennipes alignment. The phylogenetic tree (Fig. 3) displays a pattern very similar to the haplotype network. The Italian and European groups are both monophyletic although only the former receive strong support. The two outlaying specimens in the European group (ENT-DNA-1011 and ZPLOD834-20) are deeply subordinate within that group but placed as sister taxa on a reasonably long branch. The alignment is provided as File S2.

Figure 3. 

Tree from the 50 million generation analysis of Platycnemis pennipes COI sequences in MrBayes with the outgroup removed. Groups are indicated as discussed in the text. Relevant posterior probability values are shown below a branch.

3.2. Genetic diversity

The average Kimura-2 Parameter distance within each haplotype group (Table 2) is very low and ranges from 0.0015 in the European group to 0.0021 in the Italian group. The distance between the two groups is 0.015 and as such approximately an order of magnitude higher than within groups. The AMOVA-test (Table 3) shows that variation among the two major groups explains almost all the genetic variance (96.5%).

4. Discussion

Our results reveal a highly distinctive pattern with two clearly separated haplotype groups of P. pennipes in Europe—one restricted to Italy, and one found in the rest of the species’ distribution area in Europe from Georgia to the UK and from southern France to Scandinavia. This clearly indicates that P. pennipes remained in at least two independent glacial refugia in Europe during the Weichsel Glaciation (Gibbard & Cohen 2008) (Fig. 4), and perhaps even before that. The genetic variation within and between the two haplotype groups in P. pennipes (Table 2) are much higher than in S. vulgatum (Hinojosa et al. 2017) or N. speciosa (Bernard et al. 2011), but similar to A. cyanea (Schneider et al. 2015; Simonsen et al. 2020), and Orthetrum cancellatum (Linnaeus, 1758) and O. coerulescens (Fabricius, 1798) (Simonsen et al. 2021). If the rate of evolution in P. pennipes is comparable to that found in A. cyanea by Simonsen et al. (2020), the difference between the two haplotype groups in P. pennipes (0.015) corresponds roughly to the difference between North African and European A. cyanea (0.013—Simonsen et al. 2020, table 2). The split between the two latter groups was estimated to have occurred ca. 400 ky ago (Simonsen et al. 2020, fig. 3), meaning that the split between the two haplotype groups in P. pennipes could date back to the onset of the Saale Glaciation, rather than the onset of the Weichel Glaciation (Gibbard & Cohen 2008).

Figure 4. 

Potential glacial refugia and post-glacial dispersal route as discussed in the text. Blue indicates an Italian refugium and dispersal routes. Red indicates a southeastern refugium and dispersal routes. Question marks indicate uncertain dispersal routes as discussed in the text. The pink area indicates a potentially larger southeastern refugium that encircles the Black Sea.

One of these refugia was obviously in Italy as haplotype group A is restricted to that area. As the greatest variation in haplotype group B is found in the east and southeast, we infer that the second refugium was in the Balkan-Black Sea region of Europe. This pattern is directly comparable to the so-called Grasshopper Paradigm, so named because the pattern is found in the grasshopper Pseudochorthippus parallelus (Zetterstedt, 1821) (Hewitt 1999; 2004; Schmitt 2007). According to these authors P. parallelus recolonized Europe north of the Alps and the Pyrenees from a refugium in the northern Balkans with isolated populations still existing in the southern Balkans/Anatolia as well as in the Iberian Peninsula and the Italian Peninsula. P. pennipes does not occur in the Iberian Peninsula (Boudot et al. 2015), but the Italian population is isolated from the rest of Europe, and it does not appear to have crossed the Alps, possibly because it rarely occurs more than 1000 m above sea level (Boudot et al. 2015). Our regional sampling from the southern Balkans and Anatolia is too limited to allow us to infer the extent of the Balkan refugium for P. pennipes with certainty, but the presence of a specimen with a haplotype very similar to Romanian specimens in Georgia (ENT-DNA-917) could indicate that it extended to the eastern shores of the Black Sea (Fig. 4). While we cannot exclude that a single, large Balkan refugium existed during the Weichsel Glaciation, the presence of two genetically highly distinct specimens from Bulgaria (ENT-DNA-1011, haplotype A7) and Montenegro (ZPLOD834-20, haplotype A8) could indicate that several subrefugia existed in the region, possibly in the south as in P. parallelus. As mentioned earlier, the distinct subspecies P. pennipes niti­dula occurs in costal Montenegro as well as Albania and Greece. It is therefore also possible that ZPLOD834-20 represents this subspecies. However, neither the original publication (Galimberti et al. 2020) nor the record on BOLD mention subspecific status for the specimen. The low genetic variation in the European group—with the exception of the two Balkan specimens mentioned above—indicate a rapid, phalanx-like post-glacial expansion (Fig. 4) as seen in several common butterfly species (see Schmitt 2007 for a review). The low genetic variation in the European group does not allow us to assess how P. pennipes colonized southern Scandinavia or the British Isles (Fig. 4). To reach southern Scandinavia it may have used the area that is today Denmark as steppingstones, or it may have crossed the Baltic Sea directly, either by long-distance dispersal or at a time when the sea levels were lower. Similarly, it may have reached the British Isles via the ancient landmass Doggerland (Lambeck 1995) as hypothesized for the ghost moth Hepialus humuli (Linnaeus, 1758) by Simonsen & Huemer (2014), or it may have crossed the English Channel either by long-distance dispersal or reached what is today southern England before the English Channel formed 10,000-8000 yrs ago (Lambeck 1995).

In addition to P. paralellus, several European species show a similar post-glacial dispersal pattern, and Hewitt (2004) stated that the Balkans comprise the main refugium for many European species. The two tree species Fagus sylvatica Linnaeus, 1753 and Alnus glutinosa (Linnaeus, 1790), and the water newt Triturus cristatus (Laurenti, 1768) all show a pattern with expansion across Europe from the Balkans, with populations remaining isolated in the Iberian Peninsula and the Italian Peninsula (Demesure et al. 1996; King & Ferris 1998; Wallis & Arntzen 1989; Hewitt 1999; 2004; Schmitt 2007). Ursenbacher et al. (2006) found that in the viper Vipera berus (Linnaeus, 1758) there is a distinct Italian refugium clearly separated from a Balkan/Northern European group, while Sztencal-Jablonka et al. (2015) found potential support for a southeastern refugium in the smooth snake Coronella austriaca Laurenti, 1768. In Lepidoptera, Louy et al. (2013) found a clear split between Italian and Balkan populations in the butterfly Coenonympha rhodopensis Elwes, 1900, while Simonsen & Huemer (2014) found deep division between Italian (and southern Austrian) and northern (including northern Austrian) populations in H. humuli. Simonsen & Huemer (2014) concluded that H. humuli likely survived the Weichsel Glaciation in peripheral alpine refugia to the south, northwest, north, and northeast of the Alps. They did, however, mention the possibility that the northeastern refugium could have extended further to the southeast.

Within Odonata several different patterns have been elucidated in the Western Palearctic region. Weekers et al. (2001) found in a study of the highly variable nuclear ITS in C. splendens that there was a clear gap between Italy and the rest of the Western Palearctic, indicating an isolated Italian refugium. In an AFLP study of the same species, Sadeghi et al. (2010) found indication of a southeastern refugium. The two studies thus point to a pattern similar to what we find here. Hinojosa et al. (2017) similarly found evidence for an isolated Iberian refugium, an Anatolian refugium, and a Balkan refugium in S. vulgatum with post-glacial expansion to the rest of Europe from the latter. In contrast to these studies, Guan et al. (2013) found in a phylogenetic study of the genus Pyrrhosoma with focus on the relationship of the widespread P. nymphula and the endangered south Balkan endemic P. elisabethae that P. elisabethae is closer related to P. nymphula from Morocco than either are to P. nymphula for the Balkans or western Europe. Schneider et al. (2015) and Simonsen et al. (2020) found a complex pattern in A. cyanea with sequential glacial refugia in the Caucasus, the Maghreb region in Northern Africa, and the Iberian Peninsula, all related to different glacial events in the Quaternary. Simonsen et al. (2021) found complex and admixed patterns in O. cancellatum and O. coerulescens in Europe. However, they did find evidence for post-glacial expansion from an Italian refugium in both species, as well as evidence for post-glacial expansion from a Balkan refugium in O. cancellatum. In O. coerulescens they found some evidence of contact between the Italian refugium and the Balkan region, as well as evidence for east-west Mediterranean contact. Simonsen et al. (2023) found evidence in COI for the current division of the small emerald spreadwing Lestes virens (Charpentier, 1825) into the subspecies L. virens virens and L. virens vestalis Rambur, 1842, probably originating from a southwestern and a combined Italian/Balkan refugium respectively. However, they also found evidence for a separate Sicilian/Mediterranean refugium. Finally, Bernard et al. (2011), and Kohli et al. (2018; 2021) found no internal pattern in N. speciosa, and A. juncea, A. subarctica, S. sahlbergi, L. quadrimaculata and S. danae, respectively, but rather evidence for colonization of the Western Palearctic region from the east.

5. Authors’ contributions

T.J.S. and K.O. designed the study, secured funding and collected material. M.D. carried out laboratory work, data mining and the initial analyses, and drafted parts of the text. O.F.N. provided information on biology and natural history, and collected material. T.J.S. carried out the bulk of the analyses and drafted the text. All authors contributed to the Discussion and the final version of the paper.

6. Funding

Funding was provided by The Research Fund under the Danish Ministry for Culture (Grant: FORM.2015-0023), the 15 June Foundation (Grant: 2015-A-89), Schjøtz-Christensen’s Memorial Fund (Denmark), and the EU Synthesys Program (SE-TAF-5543).

7. Competing interests

The authors have declared that no competing interests exists.

8. Acknowledgements

We are grateful to the following people for support: Arne Drews (Nature Conservation Schleswig-Holstein, Germany), Rasmus Hovmöller (Swedish Museum of Natural History, Sweden), Michel Papazian (France), Boyan Zlatkov (Bulgarian Academy of Science), and Mogens Holmen† (Denmark) for providing specimens for DNA extractions; Guillaume Poirier (French Ministry for Environment) for help with registering specimens with the French Nagoya office. Annie Brandstrup (Aarhus University) assisted MD in the DNA lab. We thank an anonymous reviewer and the editor who made a number of suggestions for improvements on an earlier version of the text, which greatly improved the final version of the manuscript.

9. References

  • Bernard R, Heiser M, Hochkirch , Schmitt T (2011) Genetic homogeneity of the Sedgling Nehalennia speciosa (Odonata: Coenagrionidae) indicates a single Würm glacial refugium and trans-Palaearctic postglacial expansion. Journal of Zoological Systematics and Evolutionary Research 49: 292–297.
  • Bouckaert R, Vaughan TG, Barido-Sottani J, Duchêne S, Fourment M, Gavryushkina A et al. (2019) BEAST 2.5: An advanced software platform for Bayesian evolutionary analysis. PLoS computational biology 15: e1006650.
  • Boudot J-P, Rimšaité J, Bernard R (2015) Platycnemis pennipes. In: Boudot J-P, Kalkman VJ (Eds) Atlas of the European Dragonflies and Damselflies. KNNV Publishing, The Netherlands. 85–86.
  • Brunet BMT, Blackburn GS, Muirhead K, Lumley LM, Boyle B, Lévesque RC, Cusson M, Sperling FAH (2017) Two’s company, three’s a crowd: new insights on spruce budworm species boundaries using genotyping-by-sequencing in an integrative species assessment (Lepidoptera: Tortricidae). Systematic Entomology 42: 317–328.
  • de Lattin G (1967) Grundriss der Zoogeographie. Verlag Gustav Fischer, Stuttgart, Germany, 602 pp.
  • Demesure B, Comps B, Petit RJ (1996) Chloroplast DNA phylogeography of the common beech (Fagus sylvatica L.) in Europe. Evolution 50: 2515–1520.
  • Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479–491.
  • Galimberti A, Assandri G, Maggioni D, Ramazzotti F, Baroni D, Bazzi G, Casiraghi M (2021) Italian odonates in the Pandora’s box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale. Molecular Ecology Resources 21: 183–200.
  • Geiger MF, Kolbmüller S, Assandri G, Chovanec A, Ekrem T, Fischer I, Moriniere J (2021) Coverage and quality of DNA barcode references for Central and Northern Europe Odonata. PeerJ 9: e11192.
  • Gratton P, Konopiński MK, Sbordoni V (2008) Pleistocene evolutionary history of the Clouded Apollo (Parnassius mnemosyne): genetic signatures of climate cycles and a ‘time-dependent’ mitochondrial substitution rate. Molecular Ecology 17: 4248–4262.
  • Guan Z, Dumont HJ, Yu X, Han B-P, Vierstraete A (2013) Pyrrhosoma and its relatives: a phylogenetic study (Odonata: ­Zygoptera). International Journal of Odonatology 16: 247–257.
  • Hammouti N, Schmitt T, Seitz A, Kousch J, Veith M (2010) Combining mitochondrial and nuclear evidences: a refined evolutionary history of Erebia medusa (Lepidoptera: Nymphalidae: Satyrinae) in Central Europe based on the COI gene. Journal of Zoological Systematics and Evolutionary Research 48: 115–125.
  • Hewitt GM (2004) Genetic consequences of climatic oscillations in the Quaternary. Philosophical Transactions of the Royal Society of London Series B: Biological Sciences 359: 183–195.
  • Hinojosa JC, Martin R, Maynou X, Vila R (2017) Molecular taxonomy of the Sympetrum vulgatum (Odonata: Libellulidae) complex in the West Palaearctic. European Journal of Entomology 114: 373–378.
  • Husemann M, Schmitt T, Zachos FE, Uirich W, Habel JC (2014) Palaearctic biogeography revisited: evidence for the existence of a North African refugium for Western Palaearcticbiota. Journal of Biogeography 41: 81–94.
  • King RA, Ferris C (1998) Chloroplast DNA phylogeography of Alnus glutinosa (L.) Gaertn. Molecular Ecology 7: 1157–1161.
  • Kimura M (1980) A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111–120.
  • Kodandaramaiah U, Simonsen TJ, Bromilow S, Wahlberg N, Sperling FAH (2013) Deceptive single-locus taxonomy and phylogeography: Wolbachia mediated discordance between morphology, mitochondria and nuclear markers in a butterfly species. Ecology and Evolution 3: 5167–5176.
  • Kohli M, Sahlén G, Kuhn WR, Ware JL (2018) Extremely low genetic diversity in a circumpolar dragonfly species, Somatochlora sahlbergi (Insecta: Odonata: Anisoptera). Scientific Reports 8: 15114.
  • Kohli M, Djernæs M, Sanchez Herrera M, Sahlen G, Pilgrim E, Simonsen TJ, Olsen K, Ware J (2021) Comparative phylogeography uncovers evolutionary past of Holarctic dragonflies. PeerJ 9: e11338.
  • Krosch MN, Cranston PS (2012) Non-destructive DNA extraction from Chironomidae, including of fragile pupal exuviae, extends analysable collections and enhances vouchering. CHIRONOMUS Newsletter on Chironomidae Research 25: 22–27.
  • Lambeck K (1995) Late Devensian and Holocene shorelines of the British Isles and North-Sea from models of glacio-hydro-isostatic rebound. Journal of the Geological Society 152: 437–448.
  • Louy D, Habel JC, Abadjiev S, Schmitt T (2013) Genetic legacy from past panmixia: high genetic variability and low differentiationin disjunct populations of the Eastern Large Heath butterfly. Biological Journal of the Linnean Society 110: 281–290.
  • Nielsen OF (1998) De Danske Guldsmede. Danmarks Dyreliv 8. Steenstrup, Denmark, 280 pp.
  • Roe A, Dupuis J, Sperling FAH (2017) Molecular dimensions of insect taxonomy in the genomics era. Pp. 547–573 in: Insect Biodiversity: Science and Society Vol. 1, 2nd ed (eds R. G. Foottit, P. H. Adler). John Wiley & Sons, Ltd, Chichester, UK.
  • Ronquist F, Huelsenbeck J, Teslenko M (2012) Draft MrBayes version 3.2 Manual: Tutorials and Model Summaries. Online publication available from the authors.
  • Sadeghi S, Kyndt T, Dumont HJ (2010) Genetic diversity, population structure and taxonomy of Calopteryx splendens (Odonata: Calopterygidae). European Journal of Entomology 107: 137–146.
  • Schneider T, Schneider E, Schneider J, Vierstraete A, Dumont HJ (2015) Aeshna vercanica sp. nov. from Iran with a new insight into the Aeshna cyanea group (Odonata: Aeshnidae). Odonatologica 44: 81–106.
  • Simonsen TJ, Huemer P (2014) Phylogeography of Hepialus humuli (L.) (Lepidoptera: Hepialidae) in Europe: short distance vs. large scale postglacial expansions from multiple Alpine refugia and taxonomic implications. Insect Systematics and Evolution 45: 209–249.
  • Simonsen TJ, Olsen K, Djernæs M (2020) The African-Iberian connection in Odonata: mtDNA and ncDNA based phylogeography of Aeshna cyanea (Müller, 1764) (Odonata: Aeshnidae) in Western Palaearctic. Arthropod Systematics and Phylogeny 78: 309–320.
  • Simonsen TJ, Djernæs M, Nielsen OF, Olsen K (2021) A tale of two Skimmers: complex relationships between DNA barcoding, distributions and taxonomy in European Orthetrum cancellatum and O. coerulescens. International Journal of Odonatology 24: 316–331.
  • Simonsen TJ, Djernæs M, Nielsen OF, Olsen K (2023) COI diversity supports subspecific diversion in Western European Lestes virens (Zygoptera: Lestidae), but hints at further Mediterranean complexity. International Journal of Odonatology 26: 18–26.
  • Sztencel-Jabłonka A, Mazgajski TD, Bury S, Najbar B, Rybacki M, Bogdanowicz W, Mazgajska J (2015) Phylogeography of the smooth snake Coronella austriaca (Serpentes: Colubridae): evidence for a reduced gene pool and a genetic discontinuity in Central Europe. Biological Journal of the Linnean Society 115: 195–210.
  • Ursenbacher S, Carlsson M, Helfer V, Tegelstrom H, Fumagalli L (2006) Phylogeography and Pleistocene refugia of the adder (Vipera berus) as inferred from mitochondrial DNA sequence data. Molecular Ecology 15: 3425–3439.
  • Wallis GP, Arntzen JW (1989) Mitochondrial-DNA variation in the crested newt superspecies: limited cytoplasmic gene flow among species. Evolution 43: 88–104.
  • Weekers PHH, de Jonckheere J, Dumont H (2001) Phylogenetic relationships inferred from ribosomal ITS sequences and biogeographical patterns in representatives of the genus Calopteryx (Insecta: Odonata) of the West Mediterranean and adjacent West European zone. Molecular Phylogenetics and Evolution 20: 89–99.

Supplementary materials

Supplementary material 1 

File S1

Simonsen TJ, Djernæs M, Nielsen OF, Olsen K (2023)

Data type: .nex

Explanation note: DNA alignment as a NEXUS file used for the PopART analyses.

This dataset is made available under the Open Database License (­licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (41.03 kb)
Supplementary material 2 

File S2

Simonsen TJ, Djernæs M, Nielsen OF, Olsen K (2023)

Data type: .nex

Explanation note: DNA alignment as a NEXUS file used for the MrBayes analysis.

This dataset is made available under the Open Database License (­licenses/odbl/1.0). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (41.67 kb)
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