Taxonomic Revision of Eastern Part of Western Palaearctic Cordulegaster Using Molecular Phylogeny and Morphology, with the Description of Two New Species (Odonata: Anisoptera: Cordulegastridae)

: Taxonomy of the genus Cordulegaster Leach in Brewster, 1815 in the Eastern part of the Western Palaearctic is poorly resolved. A two-step approach was applied: sequences of mitochondrial and nuclear DNA fragments were used to sort specimens; poorly known or new taxa with their phenotypic variation were described. The existence of two traditional groups ( boltonii - and bidentata group) was conﬁrmed. Cordulegaster coronata Morton, 1916, however, belongs to a different group. Molecular-analysis supported three known and one new species ( C. heros Theischinger, 1979, C. picta Selys, 1854, C. vanbrinkae Lohmann, 1993, and C. kalkmani sp. nov.) in the boltonii- group. In the bidentata -group, all specimens from West-Turkey belonged to C. insignis Schneider, 1845, all specimens further east to a complex of four closely related species, which we name charpentieri complex ( C. amasina Morton, 1916, stat. rev., C. mzymtae Bartenev, 1929 C. charpentieri (Kolenati, 1846), stat. rev. and C. cilicia sp. nov.). The following taxa: C. insignis nobilis Morton, 1916, syn. nov., C. nachitschevanica Skvortsov and Snegovaya, 2015, syn. nov. C. plagionyx Skvortsov and Snegovaya, 2015, syn. nov. and the Caucasian subspecies C. insignis lagodechica Bartenev, 1930, syn. nov., were synonymized with C. charpentieri . Finally, we provide a key for all Western Palaearctic Cordulegaster . used for description and in the key. ia—inferior appendage, sa—superior appendage, mvt—medioventral tooth, lbt— op—occiput, opt—occipital atr—anal triangle, abdominal 10.


General Remarks on the Genus Cordulegaster
Cordulegastridae is a family, containing three genera: Cordulegaster Leach in Brewester, 1815, Anotogaster Selys, 1854 and Neallogaster Cowley, 1934. Cordulegastridae is a rather small family with 51 species [1]. They are a morphologically conserved family of robust, blackand-yellow dragonflies. The genus Cordulegaster occurs in North Africa, Europe, and South-West Asia as far east as the Himalayan region of India and Nepal with species reported also from China and Vietnam [2,3]. In addition, there is a group of Cordulegaster species in North America, with one species extending to Central America [4].

General Methods
Our molecular analysis included Genbank sequences of New World and Asian species ( Figure S2). Already published sequences of Western Palaearctic Cordulegaster are included [13,35,43]. From Central Turkey and east of this region, no sequences of Cordulegaster from the Western Palaearctic have been published so far. Thus, only the sequences published with this manuscript are available. Andy Vierstrate (A.V.) did the molecular phylogenetic work. He received only legs, with exact geographic data, however, without knowing the assumed taxa. Table 1. Cordulegaster specimens investigated are listed here with group name, species names, notes, geo-coordinates, collectors, reference, and gene bank numbers. For further details see Figure S1.

General Methods
Our molecular analysis included Genbank sequences of New World and Asian species ( Figure S2). Already published sequences of Western Palaearctic Cordulegaster are included [13,35,43]. From Central Turkey and east of this region, no sequences of Cordulegaster from the Western Palaearctic have been published so far. Thus, only the sequences published with this manuscript are available. Andy Vierstrate (A.V.) did the At that point of the study, taxonomy of this region was no more than a guess. For example, all members of the bidentata-group of the region are currently treated under C. insignis or a subspecies of this species [11]. Furthermore, some authors treated C. charpentieri or C. coronata as separate species without specifying scientific reasons [44][45][46][47].
We used at first a molecular genetic approach analysing the COI and ITS genes for constructing bootstrap maximum likelihood trees, Bayesian inference trees and haplotype networks. In addition, evolutionary distance between a pair of sequences was measured by the p-distance and the Kimura 2-P distance (K2-P). The latter methods can be translated into taxonomic ranks. In zoology, about 3% difference is considered as a good limit for species difference [48]. For odonata, a difference between 2 to 3% are discussed for species discrimination and may be lower in closely related species [49,50].
Possible hybrids were defined when specimens with their maternally inherited COI corresponding to a different species than their ITS.

Molecular Genetic Analysis
We analyzed the cytochrome c oxidase subunit I (COI) gene from the mDNA and the ITS region (ITS1, 5.8S and ITS2) from the rDNA. For details of the origin of specimens, see maps Figures 5 and 6, Figure S1 and Table 1. For the COI gene, the sequences are 568 bp long, for the ITS region the sequences are between 713 and 815 bp long, depending on the species. We used Aeshna umbrosa Walker, 1908 and Anotogaster chaoi Zhou, 1998 as outgroups.

DNA Extraction, PCR Methods, and Sequencing
Per specimen, a 1.0 mm section of a leg was transferred to a tube with 20 µL 0.05 N NaOH and 2 µL 5% Tween 20. This was heated for 15 min at 95 • C and cooled on ice. 100 µL sterile water was added to the tube and mixed. 1 to 5 µL of this solution was used in a PCR reaction. We amplified and sequenced a fragment of mDNA (the barcoding segment of the cytochrome c oxidase subunit I (COI) gene) and the entire ITS region separating the SSU and LSU region of the 18S nuclear rDNA operon comprising the ITS 1 intergenic ITS, the conserved 5.8S gene and the ITS 2 intergenic ITS, using the PCR on a 2720 Thermal Cycler of Applied Biosystems, Waltham, Massachusetts, USA. Primers used for PCR were CO1490F (5 -GGT CAA ATC ATA AAG ATA TTG G-3 ) and CO2198R (5 -TAA ACT TCA GGG TGA CCA AAA AAT CA-3 ) for the COI fragment [51]. Cycle conditions were 95 • C for 3 min followed by 45 cycles of 95 • C for 30 s, 48 • C for 30 s and 72 • C for 1 min. Primers used for amplifying the rDNA fragment are Vrain2F (5 -CTT TGT ACA CAC CGC CCG TCG CT-3 ) and 28R1 (5 -TGA TAT GCT TAA NTT CAG CGG GT-3 ) primers. Cycle conditions were 95 • C for 3 min followed by 45 cycles of 95 • C for 30 s, 54 • C for 30 s and 72 • C for 1 min. PCR products were sequenced on an ABI 3130XL automatic sequencer from Applied Biosystems with the BigDye 3.1 kit according to manufacturer instructions.

MAFFT (Multiple Alignment with Fast Fourier Transform)
Multiple sequence alignments were made with the online version of MAFFT [52].

jModelTest
The model of DNA evolution that best fit the data was determined with JMODELTEST version 2.1.10 [53]. Based on the Bayesian information criteria (BIC), the best model was chosen for Bayesian inference in MRBAYES 3.2.7a [54] and maximum likelihood analysis in PAUP 4.0a168 [55].

Tree Construction with MrBayes and PAUP
The model parameters from JMODELTEST were used in MRBAYES (nst = 6 and rates = gamma for COI; nst = 1 and rates = equal for the ITS region). The settings were: 10 million generations, a sample frequency of 1000 and a burnin value of 5000 trees. PAUP 4.0a168 was used for constructing the bootstrap maximum likelihood trees. The model parameters from JMODELTEST were used as model in PAUP (TPM2uf + G base = (0.3247 0.1447 0.1192) nst = 6 rmat = (1.8954 21.0629 1.8954 1.0000 21.0629) rates = gamma shape = 0.1580 ncat = 4 pinvar = 0 for COI, HKY + G base = (0.2011 0.2807 0.3290) nst = 2 tratio = 0.9345 rates = gamma shape = 0.3020 ncat = 4 pinvar = 0 for the ITS region). 100 bootstrap replicates (starting with a Neighbor Joining tree) were performed with a branch swapping limit set to 100,000 or 3 h (whichever comes first) per bootstrap replicate. For creating trees, Cordulegaster DNA-sequences from the West Palaearctic, from America and from E-Asia were used. Genbank was searched for credible sequences. Available sequences for COI and the ITS region approximately the same length as our own were added to the alignment. Their accession numbers are listed next to the names in the trees.

StarBeast
In addition, StarBeast, a multi-individual multi-locus species tree estimation program, using Bayesian coalescent analysis, as implemented in the BEAST package was applied for both genes [56,57]. Xml input files were created in BEAUTI v2.6.3 [57], using the HKY + Γ + I model for both markers. The following settings were used for all analysis: base frequencies 'empirical' clock model 'Strickt clock Clock.rate = 1'; tree prior 'default values (Yule Model)'; The analyses were run in BEAST v.2.6.3 [56,57]. Analyses were run for 50 million generations, sampling every 5000th generation. Tracer v. 1.7.1 [56] was used for examining the effective sample size (ESS) for parameters and determining the burn-in. Trees and posterior probabilities were summarized using TreeAnnotator v. 2.6.3 [56] and showed on the Maximum clade credibility tree, with a Posterior probability limit = 0.5 and Burnin percentage = 0. The trees were drawn in FigTree v.1.4.4 [58].

BPP
As a third program, we used BPP v. 4.3.8 for coalescent species delimitation [59]. For COI, the input species tree contained 21 species and 151 sequences. For the ITS, the input species tree contained 12 species and 53 sequences. For the combined COI and ITS, the input species tree contained 12 species and 47 sequences.

Timetree
MEGA X was used to do the Timetree analysis using the RelTime method to estimate divergence times by distinct molecular dating [60]. The estimated log likelihood value is −4139.29. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+G, parameter = 0.1688)). The analysis involved 151 nucleotide sequences. Codon positions included were 1st + 2nd + 3rd + Noncoding. There were a total of 568 positions in the final dataset.

Haplotype Analysis
Haplotype networks were built in POPART with the TCS network interference method from the COI alignment [61]. A haplotype network is the evolutionary sum of mutations that defines the current haplotypes through the DNA lineages that connect the current DNA molecules to the common ancestral DNA molecule.

Evolutionary Distance Analysis
The evolutionary distance between a pair of sequences is usually measured by the number of nucleotide or amino acid substitutions between them: p-distance. This distance is merely the proportion (P) of nucleotide sites at which the two sequences compared are different. This is obtained by dividing the number of nucleotide differences (nd) by the total number of nucleotides compared (n). This is one of the more simple methods to evaluate differences and to calculate relationships between taxa. Another established technique for evaluating DNA sequences is the Kimura 2-parameter (K2-P) index, which is conveniently expressed in percent differences between sequences. The pand K2-P distance matrix between groups was constructed in MEGA X [60]. Transitions and transversions were included, uniform rates among sites and gaps were pairwise deleted.

Morphological Analysis and Systematics
As the molecular work revealed novel taxonomic information, we had to re-describe some poorly defined and described taxa: C. amasina (Turkey), C. mzymtae (Georgia), C. charpentieri (East Anatolia, North-West Iran, Azerbaijan) with its variations in its large geographical range and select a neotype for C. charpentieri as the neotype described by Waterston belongs to C. picta and should be exchanged by a new neotype [24,62]. Furthermore, we describe the new taxa C. kalkmani sp. nov. (East Anatolia) and C. cilicia sp. nov. (South-East Turkey, Lebanon). Furthermore, the phenotypic variations in characters such size and colouration of the poorly known C. vanbrinkae (provinces Mazandaran and Golestan in Iran and Lenkoran in Azerbaijan) were compared with the closely related species C. picta (Bulgaria, Greece, Turkey, Georgia). The hybrid between C. vanbrinkae and C. picta from Armenia detected by our molecular analysis is phenotypically characterized, described, depicted and compared with C. picta and C. vanbrinkae. All specimens used for molecular analysis, description, measurements and verification of characters used for the key are listed in Table 1. More detailed remarks including notes on previous taxonomy, more detailed geographical data and methods on conservation, name-bearing types, neotype, re-description, paratype, DNA isolation, access numbers for gene bank and source are provided in Figure S1. The types, and the neotype are deposited in RMNH.
Specimens were measured (total length, abdomen length, forewing length, hindwing length) with a Vernier caliper in mm (error limit: 0.03 mm). The measurements of total length, abdomen length and wing length were rounded to the nearest millimetre. For differences in the measurements a D'Agostino-Pearson Test was performed to confirm normality of the data sets followed by a two-tailed unpaired t-test. Values are given as individual values with means and extremes. All drawings are artwork of Ole Müller (O.M.), made on graphic tablets (Wacom Intuos, Surface Book).
For the description and for the key we used the traditional morphological features of Cordulegaster shown in Figure 3.

Distribution Maps
The distribution maps were created as follows: Collecting localities of were first georeferenced using Google's Geocoding API (Application Interface). Subsequently, the resulting coordinates were plotted in ARCGIS using the geographic coordinate system WGS 1984. The source of the bac for reference was the U.S. National Park Service, publicly available through In Figures 21 and 22 we have drawn the biogeographic boundary known as Abbreviations: ia-inferior appendage, sa-superior appendage, mvt-medioventral tooth, lbtlaterobasal tooth, ho-hook, op-occiput, opt-occipital triangle, atr-anal triangle, S10-abdominal segment 10.

Distribution Maps
The distribution maps were created as follows: Collecting localities of the specimens were first georeferenced using Google's Geocoding API (Application Programming Interface). Subsequently, the resulting coordinates were plotted in ARCGIS version 10.2.2 using the geographic coordinate system WGS 1984. The source of the background map for reference was the U.S. National Park Service, publicly available through Esri services. In

Molecular Genetic Analysis of the COI and ITS Genes
The COI alignment contained 151 taxa and 568 characters of which 213 were distinct data patterns. The ITS alignment contained 53 taxa and 885 characters, of which 263 were distinct data patterns. The partition alignment with two markers (COI, ITS) contained 47 taxa and 568 characters for COI, 891 for the ITS of which 150 (COI) and 296 (ITS) were distinct data patterns.
All three programs used showed for both genes in the majority-rule consensus trees four distinct major clades: boltonii, bidentata, coronata and a New World clade, shown are the MrBayes trees (Figures 4 and S2). Western Palaearctic Cordulegaster species fall in traditional boltoniiand bidentata-groups. We observed no evidence for hybridization between members of the bidentataand the boltonii-group. C. coronata, until recently often treated as a subspecies of C. insignis, is grouped neither with bidentatanor with the boltonii-group. All three programs with both genes and combination of both genes revealed an extra clade, which we named the coronata-group (Figures 4, 5 and S3-S6). For better resolution, we separated the MrBayes COI-tree in two parts and added the haplotype analysis ( Figures 6 and 7). For better understanding of previously used names of the members of the genus in the region in relation to our molecular genetic results we introduced Table 2.

Cordulegaster boltonii-Group
In the boltonii-group of the Eastern region of the Western Palaearctic we recovered with molecular genetic analysis three known taxa (C. heros, C. picta, C. vanbrinkae) and one new taxon, which is described phenotypically below in the descriptive section (C. kalkmani sp. nov.). For the little known member of the boltonii-group C. vanbrinkae specimens from localities of all provinces from which this taxon has been reported so far (Armenia: Syunik; Azerbaijan: Lankaran; Iran: Mazandaran, Golestan) were included in this study (Figures 1 and S1, Table 1). In the case of specimens so far treated as C. picta, the whole large geographical range from East-Europe to Russian Black Sea region was covered by our molecular approach. Specimens from the East Anatolian provinces Van and Kars grouped separately from C. picta and C. vanbrinkae with both genes in MrBayes analysis . The additional two programs used for COI and a combination of COI and ITS, placed the two specimens of the new taxon apart from the other members of the boltonii-group ( Figures S3-S6). Looking at the trees for COI, the boltonii-group forms two bigger clades in all three programs applied: the "western" clade with C. boltonii, C. trinacriae, and C. princeps; and the "eastern" clade with C. heros, C. picta, C. vanbrinkae, and C. kalkmani The K2-P analysis of the COI gene revealed that the proposed new taxon (C. kalkmani) is genetically more distant from C. picta than C. vanbrinkae is from C. picta (Table 3). Thus, these specimens belong, neither to C. picta nor to C. vanbrinkae. The K2-P distance between C. picta and C. kalkmani is 5.88%, and between C. kalkmani and C. vanbrinkae 4.93% (Table 3). The presence of four eastern species in the boltonii-group was also supported by the haplotype analysis of the COI gene ( Figure 6). ity 2021, 13, x FOR PEER REVIEW 15 of 48

Possible Hybridisation in the Cordulegaster boltonii-Group
The two available specimens from the new taxon C. kalkmani showed no evidence for hybridization with other taxa. However, a specimen labeled under the name C. vanbrinkae from Armenia (Genbank MK779798) grouped in the COI analysis (PCR COI: 11586) with C. picta, while in the ITS analysis (PCR ITS: 11936) with C. vanbrinkae (Figures 4-6).

Cordulegaster coronata-Group
Specimens of supposed C. coronata from North-East Iran and Kyrgyzstan formed an extra clade in all analyses performed for both genes and the combination of the two genes (Figures 4, 5 and S3-S6). Thus, C. coronata does not group with members of the bidentatagroup neither in the COI nor in the ITS analysis, which is in contrast to phenotype analysis, in which C. coronata is similar to C. charpentieri (see below in the descriptive part). Diversity 2021, 13, x FOR PEER REVIEW 18 of 48 Figure 7. Tree of the COI-analysis for the C. bidentata-complex and C. coronata including haplotype-analysis (TCS-network made in POPART 1.7). Bayesian posterior probabilities are depicted at the nodes. Figure 7. Tree of the COI-analysis for the C. bidentata-complex and C. coronata including haplotype-analysis (TCS-network made in POPART 1.7). Bayesian posterior probabilities are depicted at the nodes.  Table): Actual used names and taxonomic rank as well as revised names and taxonomic rank, and the country of origin of the Cordulegaster specimens investigated.

Cordulegaster bidentata-Group
Within the bidentata-group, three major clades were recognized by the majority-rule consensus trees for both genes in all three programs and with the combination analysis of COI and ITS genes with the StarBeast and BPP program (Figures 4-6 and S3-S6), which we name here C. bidentata, helladica-insignis-complex, and charpentieri-complex. Specimens from West Turkey grouped together with specimens from Eastern Greece islands like Samos and Ikaria. They are separated in all trees for both genes in all three programs and with the combination analysis of COI and ITS genes with the StarBeast and BPP program from all specimens collected further east, which belong to the charpentieri-complex (Figures 4-6 and S3-S6). Thus, our molecular analysis revealed that C. insignis occurs only in the Western part of Turkey further east all specimens of the bidentata-group belong to the charpentieri-complex.

Cordulegaster charpentieri-Complex
The COI analysis with the two programs MrBayes and StarBeast support four closely related taxa in the charpentieri-complex (Figures 4, 5, 7 and S3). The majority-rule consensus tree for the COI created by the BPP program revealed a reduced topology with C. amasina and C. mzymtae together, as well as C. charpentieri and C. cilicia, as well as insignis and helladica, as well as C. picta and C. vanbrinkae, as well as C. princeps, C. trinacriae, and C. boltonii, all in one group, respectively ( Figure S5). This may indicate that the BPP program does not sufficiently distinguish more recently split taxa. Our timetree analysis supports a more recent separation of the taxa within the charpentieri-complex ( Figure S7). For a more detailed taxonomy in this complex we followed the COI analysis suggested by the majority-rule consensus trees created by the MrBayes and StarBeast program, because these analyses also differentiated the well-established taxa C. boltonii, C. trinacriae and C. princeps. Finally, we suggest the differentiation of the charpentieri-complex in four taxa: C. amasina stat. rev., C. mzymtae, C. cilicia sp. nov. and C. charpentieri stat. rev. The phenotypical description of the suggested new taxon and the re-description of the poorly described other three taxa in this charpentieri-complex are presented below in the descriptive part. The trees for COI by the BPP program, and by the combination of the two genes using the StarBeast program suggest C. amasina and C. mzymtae as well as C. charpentieri and C. cilicia as sister taxa, respectively (Figures S4 and S5) supplementary y = COI-ITS combined Starbeast, z = COI BPP).
The K2-P distances in the COI analysis between the four taxa suggested here as species are 2.96-4.24% (Table 4). For Western Palaearctic Anisoptera, a recent large analysis revealed a K2-P distance above 1.96% for a good threshold [43]. The existence of four species (C. amasina, C. mzymtae, C. cilicia and C. charpentieri) in the charpentieri-complex is also supported by the haplotype analysis ( Figure 7). To avoid further taxonomic trouble, we decided not to recognize or create subspecies within the charpentieri-complex.  Table 1). C. mzymtae the darkest member of the bidentata-group occurs eastwards from C. amasina reaching Georgia, the Russian Black Sea Coast and even until Karachay Cherkessia region in the North Caucasus (Russia) (Figures 2 and S1, Table 1).
An undescribed species, here named C. cilicia sp. nov., occurs in central Anatolia, and along the East Mediterranean Sea.
Surprisingly, there was evidence for a hybrid at the contact between C. insignis and C. cilicia. In the ITS analysis, C. cilicia from Turkey, Konya, Doganbey (Genbank MK779798) grouped in the ITS analysis (PCR ITS: 11622) with C. insignis, while in the COI analysis (PCR COI: 11570) it grouped with eight other C. cilicia (Figures 4, 5 and 7, Table 4).
C. charpentieri occurs in a large area including East-Central Anatolia, Georgia, Armenia, Azerbaijan and Iran, and most probably in North-East Iraq. This taxon is slightly more heterogeneous in the COI and haplotype analysis (Figures 4 and 7) compared to the other three species of the charpentieri-complex. However, C. nachitschevanica was in the COI analysis identical with Armenian C. charpentieri, and C. plagionyx was identical with North Iranian C. charpentieri (Figures 4 and 7). The K2-P distances are far below 2% and the haplotype analysis did not support species level (Figure 7). Therefore, we synonymized C. nachitschevanica syn. nov. and C. plagionyx syn. nov. with C. charpentieri. The COI and haplotype analysis of the Georgian specimens from Lagodechi (C. i. lagodechica) did not support a separate species or subspecies as suggested by Bartenev [30]. Therefore, we synonymized C. insignis lagodechica syn. nov. with C. charpentieri. The C. charpentieri specimens from South and South-East Iran (provinces Fars and Kerman) grouped extra in the COI analysis and haplotype analysis (Figures 4 and 7). However, the distance in the COI analysis between these specimens and C. charpentieri was less than 2%. To avoid further confusion in this group we refrain from describing further species or subspecies. For better visualization, the morphological variants were illustrated in the descriptive part below.

Cordulegaster boltonii-Group
Variation of Cordulegaster vanbrinkae and characterisation of a hybrid between C. picta and C. vanbrinkae Comments. In the molecular section, we detected a possible hybrid between C. vanbrinkae and C. picta. The specimen is from Armenia, geographically between known populations of C. picta and C. vanbrinkae. Therefore, we compared the morphology of this hybrid with C. picta and C. vanbrinkae.
As shown in Figure 8, by abdominal colour pattern the hybrid cannot be distinguished from C. vanbrinkae. C. vanbrinkae is usually darker than C. picta ( Figure 8A-C,F), although more yellow specimens of C. vanbrinkae can be found ( Figure 8C). The male appendages of the hybrid look intermediate, as the basal tooth of the superior appendages is a bit larger than in typical C. vanbrinkae ( Figure 8J-L   Center, Leiden, Netherlands, who collected the female paratype. Description of the male holotype. Head (Figure 9 E,F): anteclypeus black; postclypeus yellow; labrum yellow with thicker black margin and a short black line in the middle; frons yellow, in dorsal view with black bar at the rear edge; labium yellow, mandible black; occipital triangle black with two yellow dots ( Figure 9G); antenna and vertex black; eyes green (collector's observation). Thorax ( Figure 9A,E): anterior lobe with yellow frontal edge, median lobe of pronotum black, posterior lobe with yellow margin at the rear edge that narrows dorsolaterally; front of synthorax with broad yellow antehumeral stripes, narrowing towards pronotum leaving less than 30% black of the antehumeral region; small yellow dot on the mesanepisternum near the antealar ridge; mesepimeron and metepimeron with broad yellow bands; metepisternum black with yellow interrupted stripe-like marking reaching to one third of basal margin; metakatepisternum black; poststernum black; coxae black with yellow parts, trochanter, femora, tibiae and tarsi black.
Wing ( Figure 9E hyaline; Pt black; FW 3.8 × 0.5; HW 4.5 × 0.7; anal triangle with 3 cells; all veins black, except costa. Abdomen ( Figure 9A,E): color pattern as in Figure 9A; S1 black with two yellow markings in lateral view, S1 dorsally covered with yellow hairs, yellow marking (abdominal rings) on side of S1 semilunar; S2 to S8: yellow abdominal rings of S2 and S3 Thorax ( Figure 9A,E): anterior lobe with yellow frontal edge, median lobe of pronotum black, posterior lobe with yellow margin at the rear edge that narrows dorsolaterally; front of synthorax with broad yellow antehumeral stripes, narrowing towards pronotum leaving less than 30% black of the antehumeral region; small yellow dot on the mesanepisternum near the antealar ridge; mesepimeron and metepimeron with broad yellow bands; metepisternum black with yellow interrupted stripe-like marking reaching to one third of basal margin; metakatepisternum black; poststernum black; coxae black with yellow parts, trochanter, femora, tibiae and tarsi black.
Wing ( Figure 9E hyaline; Pt black; FW 3.8 × 0.5; HW 4.5 × 0.7; anal triangle with 3 cells; all veins black, except costa. Abdomen ( Figure 9A,E): color pattern as in Figure 9A; S1 black with two yellow markings in lateral view, S1 dorsally covered with yellow hairs, yellow marking (abdominal rings) on side of S1 semilunar; S2 to S8: yellow abdominal rings of S2 and S3 dorsally connected, rings of S4 to S8 interrupted by fine black line, yellow abdominal rings leaving a bigger part black but running onto underside; S9-S10 black.
Appendages ( Figure 9B-D,H,I): superior appendages in dorsal view slender and diverging, with pointed and slightly curved tips, nearly as long as S10; laterobasal tooth not visible in lateral view because it is covered by the rear edge of S10, prominent medioventral tooth, with a claw-shape tip only visible in lateral view, located at the base of the inferior near S10 (Figure 8B,D); inferior appendage in lateral view at the end with a single pointed hook-shaped tooth on each side ( Figure 9C,D). Inferior appendage broad, nearly parallelsided, the dorsally pointing lobes widen the inferior distally, hind margin not notched ( Figure 9I medioventral tooth, with a claw-shape tip only visible in lateral view, located at the base of the inferior near S10 (Figure 8B,D); inferior appendage in lateral view at the end with a single pointed hook-shaped tooth on each side ( Figure 9C,D). Inferior appendage broad, nearly parallel-sided, the dorsally pointing lobes widen the inferior distally, hind margin not notched ( Figure 9I

Differential diagnosis
The male inferior appendage of C. kalkmani distally much wider than in all other members of the genus. A single hook-shaped tooth in lateral view at the apex of the inferior appendage separates it from C. vanbrinkae and C. picta. These latter two species of the Eastern boltonii-group have two teeth in lateral view that are often covered by bristles and are therefore difficult to see. In C. heros the laterobasal and medioventral teeth on superior appendages are clearly more distal than in the other three Eastern species of the boltonii-group. Compared to C. vanbrinkae, C. kalkmani has a stout inferior appendage in lateral view. In addition, the medioventral teeth are more clearly visible, although are closer to S10 than in C. vanbrinkae. In C. picta the medioventral teeth are more distal than in C. kalkmani. The medioventral teeth are more clearly hooked in C. kalkmani than in C. picta. The position of the laterobasal teeth are comparable in C. picta and C. kalkmani. In contrast to C. vanbrinkae, C. kalkmani has a yellow frons as well as C. picta and C. heros. Females within the boltonii-group are impossible to separate without knowing their geographical origin.
Distribution East Anatolia and probably North-West Iran. Separated from C. picta through the Anatolian Diagonal, and further east by the Armenian Highland and the Caucasus Mountains. Cordulegaster kalkmani is separated from C. vanbrinkae by the Elburz Mountains, hybridization between C. picta and C. vanbrinkae occurs in South-East Armenia.

Redescription.
As the type is lost and the original description is poor, we selected a neotype for re-description; in addition we describe the variation of this species in its geographical range. Thorax ( Figure 11A,D): anterior lobe of pronotum with yellow front edge; median lobe black with yellow dorsolateral patterns, middorsal with two small yellow dots; posterior lobe yellow with narrow middorsal line ending in front of rear edge of lobus; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 20% black of antehumeral region; mesepimeron and metepimeron with broad yellow bands, metepisternum black with yellow triangle marking with base to wings and short yellow comma-shaped stripe ending near metastigma; metakatepisternum black; poststernum black; coxae black with yellow ventral parts, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 11A,D): colour pattern as shown in Figure 11A,B; S1 dorsal black, yellow marking on side of S2 to S9 with medium sized yellow markings leaving ca. 40% black, on dorsal view of S2 to S8 there are a pair of small yellow line-art markings on lateral distal part of segment not touching in the middle of segment, on S2 to S10 the black markings reaching lateral sides, yellow makings also present on S9 and S10.
Appendages (Figure 11C,F,G): superior appendages in dorsal view straight, slender, with pointed tips, medioventral teeth clearly visible and directed to each other, in lateral view laterobasal teeth are visible and are located near S10, in lateral view upper medioventral teeth located more ventral far away of crossing with tooth on the end of inferior appendage; inferior appendage is stout trapezoid, not notched on distal margin when viewed from above, in lateral view distally small teeth on end of the inferior appendage are visible, the three teeth form a crown-shaped complex in which the two outer teeth are fused, the inner tooth is isolated from them.  Figure 11B,D,E): anteclypeus black; postclypeus yellow; labrum yellow with black margin; frons yellow with faint black bar; labium yellow; occiput domed and yellow, occipital triangle domed, yellow with black margins; antenna and vertex black; eyes blue (collector's observation); postocular area yellow with black upper margin. Thorax ( Figure 11A,D): anterior lobe of pronotum with yellow front edge; median lobe black with yellow dorsolateral patterns, middorsal with two small yellow dots; posterior lobe yellow with narrow middorsal line ending in front of rear edge of lobus; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 20% black of antehumeral region; mesepimeron and metepimeron with broad yellow bands, metepisternum black with yellow triangle marking with base to wings and short yellow comma-shaped stripe ending near metastigma; metakatepisternum black; poststernum black; coxae black with yellow ventral parts, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 11A,D): colour pattern as shown in Figure 11A,B; S1 dorsal black, yellow marking on side of S2 to S9 with medium sized yellow markings leaving ca. 40% black, on dorsal view of S2 to S8 there are a pair of small yellow line-art markings on lateral distal part of segment not touching in the middle of segment, on S2 to S10 the black markings reaching lateral sides, yellow makings also present on S9 and S10.
Appendages (Figure 11C,F,G): superior appendages in dorsal view straight, slender, with pointed tips, medioventral teeth clearly visible and directed to each other, in lateral view laterobasal teeth are visible and are located near S10, in lateral view upper medioventral teeth located more ventral far away of crossing with tooth on the end of inferior appendage; inferior appendage is stout trapezoid, not notched on distal margin   Individuals from Kerman Province are darker, more similar to typical forms, but much larger and with pterostigma extremely narrow ( Figure 13). These individuals are the biggest in the bidentata-group, even bigger than C. heros, and are therefore among the largest dragonflies in the Western Palaearctic

Differential Diagnosis
The males have large abdominal markings, those on S9-10 often connect to form «7» shaped markings, apical dorsal pair of yellow spots usually present on S2-10, whereas they are less present in C. cilicia sp. nov. and absent in C. mzymtae. Individuals of C. charpentieri

Differential Diagnosis
The males have large abdominal markings, those on S9-10 often connect to form «7» shaped markings, apical dorsal pair of yellow spots usually present on S2-10, whereas they are less present in C. cilicia sp. nov. and absent in C. mzymtae. Individuals of C. charpentieri from South-East Iran are larger than all other members of the bidentata-group. The occiput and the postocular area are domed and yellow. The other species (C. amasina, C. mzymtae, C. cilicia) of the charpentieri-complex have the occiput and postocular area more flattened and less yellow.
Superior appendages in dorsal view are more straight and slender than in the other two species of the complex, inferior appendage is stout trapezoid, not rectangular as in C. cilicia (see below). Only the very big, yellow females of C. charpentieri from South-East Iran and the small and dark females of C. mzymtae can easily be recognized. Other females are nearly impossible to separate within the charpentieri-complex without knowing their geographical origin.

Distribution
Cordulegaster charpentieri has a wide distribution, from East Anatolia (Turkey), Georgia, Armenia, Azerbaijan and Iran to North-East Iraq.  Superior appendages in dorsal view are more straight and slender than in the other two species of the complex, inferior appendage is stout trapezoid, not rectangular as in C. cilicia (see below). Only the very big, yellow females of C. charpentieri from South-East Iran and the small and dark females of C. mzymtae can easily be recognized. Other females are nearly impossible to separate within the charpentieri-complex without knowing their geographical origin.
Distribution Cordulegaster charpentieri has a wide distribution, from East Anatolia (Turkey), Georgia, Armenia, Azerbaijan and Iran to North-East Iraq.
Cordulegaster Etymology. The name refers to the historical Roman province of Cilicia located on the south coast of Asia Minor. The range of this species exceeds the limits of this historical province to northeast and southeast.
Description of holotype. Head ( Figure 15D-F): anteclypeus black; postclypeus yellow; labrum yellow with thicker black margin; frons yellow with faint black seam; labium yellow; occiput less domed than in C. charpentieri, black and yellow occipital triangle domed, yellow with pale brown margins; antenna and vertex black; eyes turquoise (collector's observation); postocular area yellow with black upper margin. Etymology. The name refers to the historical Roman province of Cilicia located on the south coast of Asia Minor. The range of this species exceeds the limits of this historical province to northeast and southeast.
Description of holotype. Head ( Figure 15D-F): anteclypeus black; postclypeus yellow; labrum yellow with thicker black margin; frons yellow with faint black seam; labium yellow; occiput less domed than in C. charpentieri, black and yellow occipital triangle domed, yellow with pale brown margins; antenna and vertex black; eyes turquoise (collector's observation); postocular area yellow with black upper margin. Thorax ( Figure 15A,D): anterior lobe of the pronotum with yellow front edge; median lobe black with yellow dorsolateral wing-shaped patterns; posterior lobe yellow interrupted by middorsal black band; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 30% black of the antehumeral region; mesepimeron and metepimeron with broad yellow bands; metepisternum black with tiny yellow markings near base of wings; metakatepisternum black; poststernum black; coxae black with yellow ventral parts, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 15A,D): colour pattern as in Figure 15A,D; S1 dorsal black, yellow marking on side of S1 small, S2-9 with medium sized yellow markings leaving ca. 50% black, on dorsal view of S2-5 there are a pair of small, linear yellow spots present on the Thorax ( Figure 15A,D): anterior lobe of the pronotum with yellow front edge; median lobe black with yellow dorsolateral wing-shaped patterns; posterior lobe yellow interrupted by middorsal black band; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 30% black of the antehumeral region; mesepimeron and metepimeron with broad yellow bands; metepisternum black with tiny yellow markings near base of wings; metakatepisternum black; poststernum black; coxae black with yellow ventral parts, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 15A,D): colour pattern as in Figure 15A,D; S1 dorsal black, yellow marking on side of S1 small, S2-9 with medium sized yellow markings leaving ca. 50% black, on dorsal view of S2-5 there are a pair of small, linear yellow spots present on the dorsal distal part of abdominal segments not touching in middle of the segment, on S2-10 black markings reaching lateral sides, yellow markings present on S9-10.
Appendages (Figure 15B,C,G,H): superior appendages in dorsal view broad and stout with curved and inward pointing tips, the medioventral teeth clearly visible and directed with the tips slightly in direction of S10, in the lateral view the laterobasal teeth visible and located near S10, in lateral view the medioventral teeth located more ventral and far from crossing with the tooth on end of the inferior appendage; inferior appendage in ventral view stout, nearly rectangular, slightly notched on distal margin, in lateral view distally small teeth on end of the inferior appendage are visible ( Figure 15C), the three teeth form a crown-shaped complex in which the two outer teeth are fused, the inner tooth is isolated from them ( Figure 15B).

Differential diagnosis
The males are similar in colour pattern to other species of charpentieri-complex, but are usually more yellow than C. amasina and less yellow than C. charpentieri, pairs of abdominal spots on apical margins of S6-10 usually absent. The occiput less domed and less yellow than in C. charpentieri, postocular area is less domed and mostly darker than in C. charpentieri; but less dark than in C. amasina. The superior appendages in dorsal view curved, widened at base, in contrast to C. charpentieri, which has nearly parallel sided superior appendages. The medioventral teeth in dorsal view located more closely to S10 than in C. charpentieri. The medioventral teeth in lateral view located more ventral and further from crossing with the teeth on the end of the inferior appendage, as in C. charpentieri. In C. amasina the inferior appendage slightly wider at the base, in contrast to C. cilicia which has a nearly rectangular shape.
Female individuals are nearly impossible to separate within the charpentieri-complex not knowing the geographical origin, only the dark and small C. mzymtae, and the large and yellow forms of C. charpentieri are easily identified.
Distribution C. cilicia occurs east of Anamur along the Mediterranean coast, reaching Lebanon. It is also distributed to East-Central Turkey reaching Erzurum near Uzundere, where it meets C. charpentieri. Cordulegaster

Differential diagnosis
The males are usually smaller than in other species of the charpentieri-complex, C. amasina is only comparable in size to C. mzymtae. However, C. mzymtae is much darker than C. amasina. Pairs of abdominal spots on dorsal apical margin of S2-8 present in C. amasina are absent in C. mzymtae. The occipital triangle usually dark, not regular yellow as in other species of charpentieri-complex, in some individuals the occipital triangle of C. amasina and C. mzymtae has small yellowish markings, occiput not domed as in C. charpentieri, postocular area not domed and darker than in C. charpentieri and C. cicilia.
The tips of superior appendages of C. amasina are bent outwards (possibly an artefact of desiccation), not inwards like in C. charpentieri, the medioventral teeth smaller than in two other species of the group. The medioventral teeth in C. amasina are closer to S10 than in C. charpentieri. The females are smaller and darker than those of other species of Thorax ( Figure 17A,D): anterior lobe of pronotum with yellow front edge; median lobe black with yellow dorsolateral ellipsoid patterns, two yellow middorsal dots; posterior lobe yellow interrupted by a middorsal black band; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 20% black of antehumeral region; mesepimeron and metepimeron with broad yellow bands; metepisternum black with small yellow triangle near base of wings; metakatepisternum black; poststernum black; coxae, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 17A,D): colour pattern as shown in Figure 17A,D; S1 dorsally black, yellow marking on side of S2-9 with medium sized yellow markings leaving ca. 40% black, in dorsal view of S2-8 there are a pair of small, linear yellow spots present on the dorsal distal part of abdominal segments not touching in the middle of segment, on S2-10 black markings reaching sides, yellow markings also on S9-10.
Appendages (Figure 17B,C,G,H): superior appendages in dorsal view paddle-like, with tips pointing outwards, medioventral teeth visible and directed towards each other; in lateral view, laterobasal tooth visible, located near S10. In lateral view, medioventral tooth visible with a relatively broad base and a prominent hook-shaped curved tip; inferior appendage stout, inverse trapezoid, deeply notched on distal margin seen from above, in lateral view distally small teeth on end of the inferior appendage are visible ( Figure 17C), the four single teeth form a small wreath that is visible from latero-dorsal view ( Figure 17B), the single teeth inclined distally, in lateral view the caudal end of the inferior is slightly elongated to the rear in the ventral area ( Figure 17C).

Differential diagnosis
The males are usually smaller than in other species of the charpentieri-complex, C. amasina is only comparable in size to C. mzymtae. However, C. mzymtae is much darker than C. amasina. Pairs of abdominal spots on dorsal apical margin of S2-8 present in C. amasina are absent in C. mzymtae. The occipital triangle usually dark, not regular yellow as in other species of charpentieri-complex, in some individuals the occipital triangle of C. amasina and C. mzymtae has small yellowish markings, occiput not domed as in C. charpentieri, postocular area not domed and darker than in C. charpentieri and C. cicilia.
The tips of superior appendages of C. amasina are bent outwards (possibly an artefact of desiccation), not inwards like in C. charpentieri, the medioventral teeth smaller than in two other species of the group. The medioventral teeth in C. amasina are closer to S10 than in C. charpentieri. The females are smaller and darker than those of other species of charpentieri-complex. In contrast to the other species of the group, the small teeth at the caudal end of the inferior appendage form a regularly shaped crown consisting of four teeth. C. amasina shares this character with C. mzymtae. The size of the teeth varies among male individuals.
Distribution C. amasina occurs approximately from the province Kastamonu to the province Samsun and from the Black Sea Coast to central Turkey reaching the province Ankara ( Figure S1, Table 1).
Cordulegaster Description of the male. Head (Figures 18B,D): anteclypeus black; postclypeus yellow; labrum yellow with thicker black margin and a pigmented comma-shaped line in the middle; frons yellow with faint black seam; labium yellow; occiput yellow, not domed, occipital triangle black with yellowish hairs above; antenna and vertex black; eyes green (collector's observation); postocular area black.  Thorax ( Figure 18A,D): anterior lobe of pronotum black; median lobe black with yellow dorsolateral spindle-shaped patterns; posterior lobe black; front of synthorax with broad big yellow antehumeral stripes, narrowing moderately towards pronotum leaving less than 20% of antehumeral region black; mesepimeron and metepimeron with broad yellow bands; metepisternum black with small yellow dot near base of wings; metakatepisternum black; poststernum black; coxae, trochanter, femora, tibiae and tarsi black.
Abdomen ( Figure 18A,D): dorsal yellow spots on abdomen are reduced to small pairs of semi-lunar markings leaving over 90% black; S9 and S10 completely dark in dorsal and lateral view.
Appendages (Figure 18C,F,G): superior appendages in dorsal view paddle-like, with pointed tips, medioventral teeth visible and directed towards each other, in lateral view, laterobasal teeth visible, located near S10. In lateral view, medioventral teeth visible located at the first half of the superiores, with a relatively broad base, gradually narrowing, slightly pointed; inferior appendage stout, slightly trapezoid, notched on distal margin seen from above, in lateral view distally small teeth on end of the inferior appendage are visible, the four hook-shaped single teeth form a small comb that is visible from caudal view, the single teeth inclined slightly distally, the caudal end of the inferior appendages is almost rounded in lateral view.
Measurements ( located at the first half of the superiores, with a relatively broad base, gradually narrowing, slightly pointed; inferior appendage stout, slightly trapezoid, notched on distal margin seen from above, in lateral view distally small teeth on end of the inferior appendage are visible, the four hook-shaped single teeth form a small comb that is visible from caudal view, the single teeth inclined slightly distally, the caudal end of the inferior appendages is almost rounded in lateral view. Description of female. Head ( Figure 19A): much as in ♂.

General Discussion on the Genus Cordulegaster in the Western Palaearctic
The taxonomic debate on the genus Cordulegaster in the Western Palaearctic has ongoing for over one and half centuries [  C. mzymtae the darkest member of the bidentata-group occurs eastwards from C. amasina reaching Georgia, the Russian Black Sea Coast and even until Karachay Cherkessia Region in the North Caucasus (Russia) ( Figure S1, Table 1).

General Discussion on the Genus Cordulegaster in the Western Palaearctic
The taxonomic debate on the genus Cordulegaster in the Western Palaearctic has been ongoing for over one and half centuries [2,13,14,19,26,30,35,63,64]. For the Western part of the Western Palaearctic, a molecular approach exists [13]. However, for the more complex and unsettled Eastern part of the Western Palaearctic, no molecular studies have been conducted until now, and no published sequences are available so far. From the east of the Western Palaearctic, including Turkey, the Caucasus countries and Iran, several species and subspecies have been described ( Table 2). All these were based on external morphology and the colour patterns of imagines, especially of the male appendices. However, colour patterns and structure of the appendices may vary and hybrids may complicate phenotypic taxonomy. C. boltonii and C. trinacriae interbreed in a broad zone in Italy [35]. In a similar way, our data support hybridization in the boltonii-group between C. vanbrinkae and C. picta in Armenia and in the bidentata-group between C. insignis and C. cilicia sp. nov. Such hybridization supports the view that pre-and post-mating barriers are inefficient, and structural differences in the genital apparatus do not prevent gene flow. It explains the limitations of morphological characters for species recognition in this genus. Only part of the variation observed is taxonomically relevant and it is sometimes impossible to identify specimens to species. As an alternative, we undertook a molecular genetic approach, which reveals the genotype, not the phenotype. It helped us to clarify the status of several doubtful subspecies and species in this region. Furthermore, an inherent limitation of the morphological method is that not all hybrids are intermediate and can only be detected by molecular genetic approach.

Cordulegaster boltonii-Group
In the boltonii-group of the Eastern region of the Western Palaearctic, we recovered with molecular genetic analysis three known (C. heros, C. picta, C. vanbrinkae) and one new species (C. kalkmani). These four eastern species of the boltonii-group (C. heros, C. picta, C. kalkmani, and C vanbrinkae) seem to be geographically separated but may meet in contact zones ( Figure 21). The existence of the new taxon C. kalkmani was supported by the alignments of both genes separately or combined as well by the haplotype-analysis and the K2-P distances. C. heros is known from rather Central and Southeast Europe, reaching Ukraine in the North-East [65]. C. picta inhabits an area extending from North-Eastern Greece and Bulgaria, through West and North Turkey, including Samos and Lesbos in the Aegean, along the Black Sea coast as far as Russia, Georgia and Azerbaijan [16,66,67]. However, the presence of C. picta in Azerbaijan may be questioned, as the only voucher specimen is old and in bad condition; it is not clear whether it belongs to C. picta, to C. vanbrinkae or is a hybrid between them [16]. Cordulegaster picta from South-West Turkey and Samos are rather yellow, where those from the Black Sea region are much darker ( Figure 20). C. vanbrinkae is restricted to the South Caspian Sea region [68,69]. In East Anatolia, possibly reaching Armenia and North-West Iran, a new species, C. kalkmani was found. The record of C. picta by Rastegar in North-West Iran may belong to the latter species [70]. C. kalkmani seems to be geographically separated from C. picta and C. vanbrinkae by the Anatolian Diagonal and further east by the Armenian Highlands and the Caucasus (Figure 21). C. vanbrinkae and C. kalkmani seem to be separated by the Armenian Highland ( Figure 21). As mentioned before, we found hybridization between C. picta and C. vanbrinkae in Armenia. These hybrids may be phenotypically indistinguishable from one of their parents. This is the case of the hybrid from Armenia, which looks like C. vanbrinkae, causing specimens from this population to be initially described as C. vanbrinkae [71]. We found no evidence for hybridisation between C. picta and C. kalkmani and between C. vanbrinkae and C. kalkmani. The K2-P distances of the COI sequences revealed that C. kalkmani is more distant from C. picta (5.88%) than C. vanbrinkae from C. picta (3.95%). Correspondingly, the male appendices of C. kalkmani are more different from C. picta than those of C. vanbrinkae from C. picta.
This suggests that the latter two were separated more recently (see also timetree-analysis, Figure S7) and may be regarded as sister taxa. of C. boltonii; C. b. immaculifrons Selys, 1850; C. b. iberica Boudot & Jacquemin, 1995; C. b. algirica Morton, 1916, although statistically representative, may just be colour variations [13]. We conclude, including the data of Froufe, that the following species in the boltoniigroup live in the West Palaearctic: C. princeps, C. boltonii, C. trinacriae, C. heros, C. picta, C. kalkmani, and C. vanbrinkae. The division of the boltonii-group in a "western" group, with C. princeps, C. b. algirica (North Africa only), C. boltonii, C. trinacriae and an "eastern" group with C. heros, C. picta, C. kalkmani and C. vanbrinkae is supported by our molecular data. This view was already suggested by Verschuren based on the morphology of the larvae without knowing the latter two species at that time [12].

Cordulegaster bidentata-Group
In the bidentata-group of the Eastern region of the Western Palaearctic, two complexes were revealed by molecular analysis: the helladica-insignis-complex in South-East Europe and Western Turkey, and the charpentieri-complex in Eastern Anatolia, Levant, Caucasus countries and Iran ( Figure 22). These species are geographically separated by mountains of Anatolia and the Caucasus, although overlapping zones exist in East Anatolia ( Figure  22). As widely accepted, the nominotypic C. insignis inhabits Western Turkey and eastern Greece islands like Samos. However, the molecular analysis of all specimens collected east of the Marmara and Aegean region grouped away from C. insignis using both programs for both genes separately or combining the COI and ITS genes. All the eastern specimens of the bidentata-group were therefore summarized here in the charpentieri-complex. Hybridization may occur also between C. boltonii and C. heros as both are locally syntropic [72], and may also occur in still undocumented contact zones in Greece or Bulgaria between C. heros and C. picta, and even in Ukraine and Russia north of the Black Sea.
Thus, we highlight broad variation in colour pattern in some taxa. Male appendages, often used as diagnostic, have limits for discriminating species, and the distinction between intraspecies variation and significant differences is often hard to make. This was a source of confusion in the past. The work by Froufe showed that all European subspecies of C. boltonii; C. b. immaculifrons Selys, 1850; C. b. iberica Boudot & Jacquemin, 1995; C. b. algirica Morton, 1916, although statistically representative, may just be colour variations [13]. We conclude, including the data of Froufe, that the following species in the boltonii-group live in the West Palaearctic: C. princeps, C. boltonii, C. trinacriae, C. heros, C. picta, C. kalkmani, and C. vanbrinkae. The division of the boltonii-group in a "western" group, with C. princeps, C. b. algirica (North Africa only), C. boltonii, C. trinacriae and an "eastern" group with C. heros, C. picta, C. kalkmani and C. vanbrinkae is supported by our molecular data. This view was already suggested by Verschuren based on the morphology of the larvae without knowing the latter two species at that time [12].

Cordulegaster bidentata-Group
In the bidentata-group of the Eastern region of the Western Palaearctic, two complexes were revealed by molecular analysis: the helladica-insignis-complex in South-East Europe and Western Turkey, and the charpentieri-complex in Eastern Anatolia, Levant, Caucasus countries and Iran ( Figure 22). These species are geographically separated by mountains of Anatolia and the Caucasus, although overlapping zones exist in East Anatolia ( Figure 22). As widely accepted, the nominotypic C. insignis inhabits Western Turkey and eastern Greece islands like Samos. However, the molecular analysis of all specimens collected east of the Marmara and Aegean region grouped away from C. insignis using both programs for both genes separately or combining the COI and ITS genes. All the eastern specimens of the bidentata-group were therefore summarized here in the charpentieri-complex. The COI analysis of the charpentieri-complex with two programs is in favour of four species (C. amasina, C. mzymtae, C. cilicia, and C. charpentieri). The K2-P distance between these four taxa is 2.96-4.24% (Table 4), in agreement with a full species level [43,45,46]. The ITS analysis by the MAFT program, however, put them all together indicating a more recent diversification in this taxa complex (supported by our timetree, Figure S7). This may be due to special glacial events in Anatolia as discussed below. For several aspects, including the conservation of highly endangered and unique biotopes, we would suggest to hold up these three traditional and the new taxa.
Morton introduced C. amasina as a race, based on five males from Amasya, Turkey [26]. As our results support the occurrence of a separate species along the western part of Black Sea Coast, we adopted the existing name for this lineage. Further east, reaching Georgia and Russia, C. mzymtae occurs. This taxon was sometimes treated as a subspecies of C. insignis [10,11]. More extensive fieldwork along the Black Sea Coast may clarify the exact distribution borders between C. insignis, C. amasina, and C. mzymtae. The separation between these taxa may be due to the glacial events discussed in a recent paper on banded newts in this region [73]. The COI analysis of the charpentieri-complex with two programs is in favour of four species (C. amasina, C. mzymtae, C. cilicia, and C. charpentieri). The K2-P distance between these four taxa is 2.96-4.24% (Table 4), in agreement with a full species level [43,45,46]. The ITS analysis by the MAFT program, however, put them all together indicating a more recent diversification in this taxa complex (supported by our timetree, Figure S7). This may be due to special glacial events in Anatolia as discussed below. For several aspects, including the conservation of highly endangered and unique biotopes, we would suggest to hold up these three traditional and the new taxa.
Morton introduced C. amasina as a race, based on five males from Amasya, Turkey [26]. As our results support the occurrence of a separate species along the western part of Black Sea Coast, we adopted the existing name for this lineage. Further east, reaching Georgia and Russia, C. mzymtae occurs. This taxon was sometimes treated as a subspecies of C. insignis [10,11]. More extensive fieldwork along the Black Sea Coast may clarify the exact distribution borders between C. insignis, C. amasina, and C. mzymtae. The separation between these taxa may be due to the glacial events discussed in a recent paper on banded newts in this region [73].
We did not find any published name for another clade defined by molecular data in the charpentieri-complex, and therefore we name it C. cilicia. The name refers to the historical province of Cilicia located on the south coast of Asia Minor. However, the range of this species exceeds the limits of this historical province to northeast and southeast and it inhabits East Anatolia and extends along the East-Mediterranean Sea reaching Lebanon. C. cilicia and C. charpentieri meet in Central-East Anatolia in the province of Kars ( Figure 22). C. cilicia is well separated in the COI and haplotype analysis, and the K2-P distance to its next relative C. charpentieri is 3.22% and in agreement with species level for Anisoptera [43].
C. charpentieri is a widespread species occurring in Central-East Anatolia, Armenia, Georgia, and Iran. It is most likely also present in northern Iraq taking the description by Asahina into account [74]. C. charpentieri exhibits some variation in the colour-pattern across its range; therefore, we have depicted representative examples in this study. The size of this species is also variable, and the largest animals of this taxon exist in the South and South-Eastern Zagros Mountains. These individuals belong with C. heros and Anax immaculifrons to the largest dragonflies of the Western Palaearctic.
Anatolia emerges as a hotspot for the charpentieri-complex. Here, three refugia meet and interact: the Caucasus, Irano-Anatolian and the Mediterranean [40,41]. Two refugia, one in western and one in Eastern Anatolia, separated by the Anatolian diagonal, have been suggested, based on non-genetic data [75,76]. More have been found by genetic analysis of different animal groups [77,78]. The lake system was present in central Anatolia during the Pliocene and the inhabitability of the Central Anatolian Plateau in the glacial maxima of the Pleistocene probably broke up previously continuous faunal ranges leading to subspeciation [79]. Vicariant events related to the formation of the Anatolian diagonal and the orogenesis of the mountain chains in southern and Eastern Anatolia are what led to current distribution patterns of animals like Cordulegaster. The Anatolian diagonal was originally described for plants, but also applies to animals [41,42,77,78]. In the case of the charpentieri-complex it separates C. amasina from C. cilicia and the western Taurus, nominal C. insignis from all others. The Eastern Anatolian Mountains and the Armenian Highland separate C. charpentieri from C. amasina, and C. cilicia ( Figure 22). However, hybridization may have occurred and may still occur in postglacial contact zones ( Figure 22).
For C. magnifica, described by Bartenev based on a single female of unknown origin, we did not find any support in the investigated region [28]. The type is lost. The description fits to most females of 'mid-yellow' Cordulegaster of the bidentata-group in the East Mediterranean, the Middle East and the South Caucasus. In view of its unknown origin and as no other specimen or living population is known, it is not possible to ascribe it to any taxon and the name should be deleted.
Thus, in the bidentata-group the following species occur from West to East: C. bidentata, C. helladica, C. insignis, C. amasina, C. mzymtae, C. cilicia and C. charpentieri. The species C. coronata is genetically distant from this group, although morphologically close to the charpentieri-complex (Figure 23).

Cordulegaster coronata-Group
The coronata-group formed an extra clade with all three programs for both genes and also in combination of these two genes. This is the reason why this taxon was sometimes treated as a subspecies of C. insignis in the past [2,14]. C. coronata is a Middle Asian taxon, which has its Western distribution limits in North-East Iran (Razavi Khorasan) as recently documented [46,47]. In North-Khorasan, C. coronata and C. charpentieri may meet, therefore, it would be of interest to investigate specimens from this region to see if hybridisation between them is possible.