A new species of the paper wasp genus Polistes (Hymenoptera, Vespidae, Polistinae) in Europe revealed by morphometrics and molecular analyses

Abstract We combine multivariate ratio analysis (MRA) of body measurements and analyses of mitochondrial and nuclear data to examine the status of several species of European paper wasps (Polistes Latreille, 1802) closely related to P. gallicus. Our analyses unambiguously reveal the presence of a cryptic species in Europe, as two distinct species can be recognized in what has hitherto been considered Polistes bischoffi Weyrauch, 1937. One species is almost as light coloured as P. gallicus, and is mainly recorded from Southern Europe and Western Asia. The other species is darker and has a more northern distribution in Central Europe. Both species occur syntopically in Switzerland. Given that the lost lectotype of P. bischoffi originated from Sardinia, we selected a female of the southern species as a neotype. The northern species is described as P. helveticus sp. n. here. We also provide a redescription of P. bischoffi rev. stat. and an identification key including three more closely related species, P. biglumis, P. gallicus and P. hellenicus.


Introduction
The paper wasp genus Polistes Latreille, 1802 (Hymenoptera, Vespidae, Polistinae) is an important model group for behavioral and evolutionary studies. It includes a large number of eusocial species that exhibit varied forms of social organization (West-Eberhard 1969). Moreover, its comparatively small colony size and exposed nests facilitate both field observations and experiments (e.g., Cervo et al. 2008). More than 220 species are currently recognized worldwide (Arens 2011, Buck et al. 2012, Nugroho et al. 2012, ten of which occur in Europe (Arens 2011: 462, Carpenter 1997: 142, Castro and Dvořák 2009. Three of them, namely P. atrimandibularis Zimmermann, 1930, P. semenowi Morawitz, 1889, and P. sulcifer Zimmermann, 1930, are social parasites (Cervo 2006, and references therein) and were considered as members of a distinct genus (or subgenus) Sulcopolistes Blüthgen, 1938(Blüthgen 1961, Guiglia 1972, until Carpenter (1990) synonymized Sulcopolistes with Polistes. Later, phylogenetic analyses of one mitochondrial gene fragment showed that the three socially parasitic species formed a monophyletic group nested within other European Polistes (Choudhary et al. 1994: 33); the three social parasites constituted a monophyletic clade sister to a clade consisting of P. dominula (Christ, 1791) and P. nimpha (Christ, 1791). Blüthgen (1943) proposed the subgeneric name Leptopolistes for several non-parasitic European species, including P. associus (Kohl, 1898), the type species of Leptopolistes, as well as P. bischoffi Weyrauch, 1937 andP. gallicus (Linnaeus, 1767). Males of these taxa share non-convex, immediately narrowing genae, as seen in dorsal view (Blüthgen 1943: 99;Guiglia 1972: 49), giving the male head a characteristically slender aspect. Currently, all European Polistes species are assigned to the subgenus Polistes (Carpenter 1996b), although the species formerly included in Leptopolistes species are still considered to be closely related (Carpenter 1997).
In fen rotational fallows (Gigon et al. 2010) at the shore of Lake Greifen [Greifensee] in the Swiss midlands Neumeyer et al. (2011) found a population of paper wasps that could not be assigned to any described species. This taxon is colored almost as light as Polistes gallicus (Linnaeus, 1767) and was therefore tentatively called "Polistes cf. gallicus" by Neumeyer et al. (2011). Polistes gallicus is quite common in Southern Europe, but it does not usually occur in wetlands and is not known as far north in Switzerland. The unidentified taxon from the Swiss midlands, however, shares an important trait (a reduced epicnemial carina) with another taxon that has hitherto been referred to as Polistes bischoffi Weyrauch, 1937(e.g. Blüthgen 1961, Guiglia 1972, Mauss and Treiber 2004, Dvořák and Roberts 2006, Witt 2009), a common wetlanddweller in Switzerland and other countries of Central Europe.
To resolve the identity of the unidentified taxon from the Swiss wetlands, we examine its affinity to other European species using a combination of morphological, morphometric and molecular analyses. Recently, Buck et al. (2012) unraveled cryptic diversity in the Nearctic subgenus Fuscopolistes Richards, 1973 using multivariate morphometrics and DNA barcoding. In contrast to their study, we used a nuclear marker in addition to the mitochondrial marker and multivariate ratio analysis (MRA) instead of classic multivariate methods. MRA is a recently developed extension of principal component analysis (PCA) and linear discriminant analysis (LDA) that was specifically designed for the exploration of body measurements in a taxonomic context Leuenberger 2011, László et al. 2013).
Our analyses lead to the recognition of two distinct species within what has been hitherto referred to as P. bischoffi; we review the information on the type material of bischoffi, and designate a neotype to settle the status of this species. P. bischoffi turns out to be the valid name of the unidentified taxon ("cf. gallicus") found close to Zurich by ; a new name is required for the species referred to as bischoffi by some authors (Blüthgen 1961, Guiglia 1972, Mauss and Treiber 2004, Dvořák and Roberts 2006, Witt 2009): P. helveticus, which is described here. Lastly, we provide an identification key that, in combination with available keys (Mauss and Treiber 2004, Dvořák and Roberts 2006, Witt 2009), will facilitate the identification of the Central European species.

Material and methods
For the molecular and morphometric analyses we focus on the status of the two closely related morphs hitherto comprised under Polistes bischoffi (see introduction), as well as on the morphologically similar Polistes gallicus, and on their separation from other European Polistes. At this stage of the analyses, we deliberately avoid the concept of species and rather interprete them in the sense of operational taxonomic units, hereafter called "OTUs". The OTUs are labeled with their valid taxonomic names (Carpenter 1996b), except for the two taxa hitherto comprised under Polistes bischoffi which are labeled in a manner that already anticipates the outcome of our study and our neotype designation. Detailed information on the taxonomic status of these names will be provided after the presentation of the results from the molecular and morphometric analyses.

Molecular analyses a) Species included
Ninety-nine specimens were included in the molecular analysis, representing eleven OTUs. In addition, two specimens each of Vespula germanica (Fabricius, 1793) and V. vulgaris (Linnaeus, 1758) were used to root the trees; sequences for Polistes (Polistella) snelleni Saussure, 1862 and Polistes (Aphanilopterus) exclamans Viereck, 1906 were downloaded from Genbank and used with the two species of Vespula to root the trees in analyses of the mitochondrial sequences. Most specimens were collected in 80% ethanol in the field, but we also included some specimens that were killed with ethyl acetate. For specimens collected before 2012, DNA was extracted from the mesosoma, leaving the legs, wings, head and metasoma as vouchers; for specimens collected in 2012 and 2013, as well as specimens selected as type specimens, DNA was extracted from one single leg to preserve a nearly intact specimen. Most specimens were collected in Switzerland, but we also included specimens form Croatia, France, Greece, Italy and Portugal (Table 1). All DNA extractions are deposited in the DNA bank of the Swiss Barcode of Life initiative (Swissbol; www.swissbol.ch).

b) Lab protocols
Full lab protocols can be found in Praz et al. (2008). DNA was isolated using phenol-chloroform extractions; PCR reactions were performed with GoTaq polymerase (Promega) in a Biometra T1 thermocycler. PCR products were purified enzymatically using a mix of the enzymes exonuclease I (Fermentas) and FastAP thermosensitive alkaline phosphatase (Fermentas) and sequenced in both directions with the primers used in the original amplification using BigDye terminator technology (Applied Biosystems). Big Dye products were purified with Sephadex (GE Healthcare Life Sciences) and analyzed on a ABI-3500 DNA sequencer.

c) Markers and primers
We sequenced two fast-evolving genetic markers: the 600 bp fragment of the mitochondrial gene cytochrome oxidase 1 (COX1) used as an universal barcode (Hebert et al. 2003) and the nuclear marker ITS1; we chose ITS1 rather than ITS2 because preliminary analyses revealed that ITS2 was polymorphic in P. bischoffi and could not be sequenced directly.
For COX1 we used the universal primers LepF and LepR (Hebert et al. 2004) with the following conditions: an initial denaturation of 1 min at 94 °C, then six cycles of 1 min at 94 °C, 1.5 min at 45 °C, and 1.25 min at 72 °C, followed by 36 cycles of 1 min at 94 °C, 1.5 min at 51 °C, and 1.25 min at 72 °C, with a final step of 5 min at 72 °C. For specimens with degraded DNA, we used another universal forward primer, UAE3 (Zhang and Hewitt 1996) in combination with LepR to amplify a 400 bp fragment of the barcode. The conditions for this 400 bp fragment were as above, except that the extension time at 72 °C was 45 seconds in each cycle.
The presence of nuclear pseudogenes, or NUMTs, was carefully examined by visually detecting "ghost bands" on the agarose gel, and especially by detecting double peaks in the chromatograms. No indication of the presence of NUMTs was found in the specimens analyzed, with the exception of P. nimpha. For this OTU, double peaks were found in up to 20 nucleotide positions in every specimen, strongly suggesting the presence of NUMTs; no indels were found, and no stop codons were found in the translated amino acid sequence for these sequences, even when polymorphism was allowed, suggesting that the NUMTs were highly similar to the true mitochondrial sequences Table 1. Locality information, voucher numbers and GenBank accession numbers for sequences used in this study.
* sequenced with UAE3/LepR instead of LepF/LepR and thus of recent origin. The presence of NUMTs in P. nimpha was therefore unlikely to affect our results, especially given that P. nimpha was not the focus of our study, as it is not closely related to any of the main OTUs.
For ITS1, we used the primers CAS18sF1 and CAS5p8sB1d (Ji et al. 2003) to amplify a 700 bp fragment. For most specimens, the chromatograms were clean, without double peaks, indicating no within-specimen polymorphism in ITS1. In P. dominula, a few sites were polymorphic, and one insertion rendered the sequencing difficult in some specimens at position 550; in P. nimpha, several sites were polymorphic and insertions or deletions prevented direct sequencing in all specimens, except two (the numbers 57 and 65). Given that P. nimpha was not the focus of our study, we did not clone the PCR products to obtain clean sequences of the individual copies of ITS1, and merely included two specimens in our analysis.

d) Analyses
Genetic distances between each terminal were computed under the GTR model of nucleotide substitution in Paup 4.0b10 (Swofford 2002). We then performed maximum likelihood analyses of each marker separately using RAXML (Stamatakis et al. 2005), performing 1000 bootstrap replicates. For the mitochondrial marker, the first and second position were combined in one partition, while the third codon position constituted a second partition. For ITS1, we coded each insertion or deletion as an additional, binary character added as a separate partition, hereafter referred to as the "gap" partition; one insertion or deletion was considered as one character, regardless of the size of the indel. In total, the coding of the insertions and deletions resulted in 42 characters, 38 of which were parsimony informative and four of which were autapomorphic. We do not intend to unravel the phylogenetic relationships among the European species of Polistes, and therefore we do not present an analysis of a matrix combining both genes.
We applied a GTR + G model to each DNA partition; the gap partition was analyzed as a binary character with two states, with a gamma shape to accommodate rate heterogeneity. FigTree v1.3.1 (Rambaut 2009) was used to visualize the trees and produce the figures.

Morphometrics
We restricted the morphometric analyses to the five most morphologically similar OTUs, namely biglumis, bischoffi, gallicus, hellenicus, and helveticus. For convenience, we refer hereafter to this group as the gallicus-group. We stress that we consider this group to be neither monophyletic nor taxonomically relevant.

b) Morphometric analysis
We applied the multivariate ratio analysis (MRA) of Baur and Leuenberger (2011) to our data. MRA comprises a set of tools for analyzing size and shape of body measurements in a multivariate mathematical framework that is entirely consistent with the customary usage of body lengths and ratios in taxonomic works (e.g., in descriptions, diagnoses). In systematic and taxonomic studies, MRA offers several advantages over conventional explorative multivariate methods, such as principal component analysis (PCA) and linear discriminant analysis (LDA). MRA removes biases from spurious contradictions in the results due to different definitions of size and shape. Furthermore, the numeric output of MRA can be used directly in the descriptive part of a taxonomic study. László et al. (2013) reviewed these issues in an application to parasitic wasps. Following Baur and Leuenberger (2011), we first calculated isometric size (isosize), defined as the geometric mean of all variables. We then performed a shape PCA (i.e., a principal component analysis in the space of all ratios) for evaluating how the morphometric pattern corresponds to the OTUs revealed in the molecular analyses. In order to decide how many components to retain we inspected the scree graph (Rencher 2002: 398-399). We also plotted isosize against shape PCs, because the correlation of size with shape is a measure of the amount of allometry in the data. Two graphical tools, the PCA ratio spectrum and allometry ratio spectrum respectively, were also employed in some cases. Finally, we used the LDA ratio extractor to extract the best ratios, and calculated the standard distance as well as the measure δ. The R language and environment for statistical computing was used for data analysis (R Development Core Team 2013; version 3.0.1). For the above methods we employed slightly modified versions of the R-scripts provided by Baur and Leuenberger (2011, under "Supplementary material"). Scatterplots were generated with the package "ggplot2" (Wickham 2009).

Taxonomic treatment, voucher and type specimens
For taxonomy and classification we followed Carpenter (1996b). Abbreviations used for specimen depositories and other institutions or private collections cited in this study are given in Table 3. Stack-photographs of mounted specimens were taken with a Keyence VHX-2000 digital microscope at the NMBE. All known Polistes collections in Switzerland (CH), as well as several collections elsewhere (Table 3), have been examined by one of the authors (RN). We also examined the relevant type material. Table 3. Abbreviations of depositories (museums and private collections) and other institutions. "CH" means Switzerland.

Data resources
The morphometric data underpinning the analyses reported in this paper as well as a series of images showing the exact character definitions are deposited in the Dryad Digital Repository at http://doi.org/10.5061/dryad.9b8tt.

Molecular analyses a) Sequencing
Of the 99 ingroup specimens included, complete COX1 sequences were obtained for 96 specimens, and ITS sequences for 80 specimens (Table 1). This difference is due to 12 ITS sequences of P. nimpha that were polymorphic and excluded, as well as some specimens with degraded DNA, which could be sequenced for the shorter mitochondrial fragment but not for ITS1.

b) COX1
Analyses of the COX1-sequences ( Fig. 1) reveal that P. helveticus and P. bischoffi represent two distinct, well-supported clades (Bootstrap support, hereafter BS, of 100 and 94%, respectively). Sequences of all included specimens of P. bischoffi, including the 10 specimens from Switzerland and one specimen from Corsica, were absolutely identical (genetic distance of 0); similarly, sequences of the 20 specimens of P. helveticus were identical. The genetic distance between these two clades was 2.6%. The relationship between these two clades, as well as the relationships among the species of the gallicus-group, were not resolved.
More generally, most OTUs included in this study were recovered as monophyletic with high bootstrap support >90%, with the exception of P. dominula. Sequences for this OTU formed two well-supported clades (see below). The two specimens identified as Polistes gallicus by Arens (2011)   P. biglumis. Within OTU-distances were higher for P. nimpha (2.4%) and especially for P. dominula (up to 4.9%; see below). For P. nimpha, although two weakly supported clades are revealed within this OTU (Fig. 1), the ranges of distance within (0-0.6% and 0-0.7%) and between these clades (0.4-2.4%) overlapped. In contrast, sequences for P. dominula formed two distinct clades that did not overlap. All sequences within the first clade were identical, thus the distance within this clade was equal to 0. In the second clade, the distances ranged from 0 to 0.67%; the distances between these two clades were between 3.6 and 4.9%. These two clades were weakly associated with geographic location: specimens originating from western Switzerland (Geneva, Valais and one location in Vaud) and from one site close to Zurich formed one clade, whereas specimens originating from the Grisons, from one location in Vaud and from the southern parts of the canton of Zurich formed the other clade; specimens from one locality in Zurich were distributed in both clades. The minimal distance between two OTUs was 2.6%, observed between P. helveticus and P. bischoffi, as indicated above, as well as between the two included social parasites, P. semenowi and P. sulcifer. 19

c) ITS1
Analyses of ITS1 ( Fig. 2) again strongly suggest that P. helveticus and P. bischoffi represent two distinct, well supported clades (both with BS of 95%). Sequences for all of the eleven specimens of P. bischoffi, including one specimen from Corsica, were identical; within P. helveticus, the genetic distance was 0.17% due to one single polymorphic site. The genetic distances between both clades were between 2.23% and 2.37%. The relationship between these two species, as well as the relationships among the different species of the gallicus-group, were not resolved.
All other OTUs were recovered as well supported clades, with bootstrap supports > 85% (Fig. 2). No sequence of ITS1 could be obtained for the two specimens of P. sp. aff. gallicus from Greece. The two clades observed in analyses of the mitochondrial marker in P. dominula were not recovered in analyses of ITS1, although maximal within-OTU distances were comparatively high for this OTU (0.77%). However, no distance correlation between ITS1 and COX1 was observed; for example, some specimens exhibiting high mitochondrial distances (eg, numbers 5 and 43) had identical ITS1 sequences. Other within-OTU genetic distances were as follows: 0% for P. sulcifer, P. associus and P. gallicus; 0.24% for P. biglumis; 0.32% for P. hellenicus.
The smallest interspecific distance in ITS1 sequences was 0.8%, between P. biglumis and P. hellenicus; the maximal distance in our ingroup was 11.5%, observed between P. nimpha and P. biglumis. The minimum distance between P. bischoffi and any other OTU was 1.72%, between bischoffi and P. biglumis.

Multivariate ratio analysis (MRA) of the gallicus-group
As mentioned above in material and methods, we restricted the MRA to the five OTUs of the gallicus-group (s. Table 4 for an overview of measurements). We first performed a shape PCA to see how well the monophyletic OTUs recovered by molecular analyses (Figs 1 and 2) are supported by morphometric variation. A PCA is convenient because it does not require a priori assignment of OTUs to particular groups but assumes instead that all OTUs belong to one single group. A PCA thus avoids circular reasoning with respect to particular groupings (see Peters and Baur 2011). According to the scree graph (not shown), only the first and second shape PC were relevant, comprising more than 60% of the total variation. Scatterplots of the two axes gave a very similar result for both sexes (Figs 3a, b). P. biglumis was clearly separable from the other species along the first shape PC. The other OTUs were much closer, with P. bischoffi and P. helveticus still being rather distinct. The ranges of the two remaining OTUs, P. gallicus and P. hellenicus, were entirely overlapping. A scatterplot of isosize and the first shape PC revealed a strong correlation between size and shape (Figs 3c,d). This was mainly caused by the presence of P. biglumis, which was clearly the largest OTU in both sexes. The others were largely overlapping in their size ranges.
As mentioned in the introduction, two of the main target OTUs of our study, P. bischoffi and P. helveticus, are separated from the others by a reduced epicnemial carina. We therefore conducted a shape PCA including only these two OTUs for examining their morphometric differences. Only the first shape PC was informative and was plotted against isosize to evaluate the amount of allometric variation in the data (Fig. 4). Both sexes were well differentiated by the first shape PC. Furthermore, females of P. helveticus were very slightly larger than those of P. bischoffi (4a), whereas males were entirely overlapping in the size range (Fig. 4b).  To interpret the first shape PC, the PCA ratio spectrum was plotted (Fig. 5, graph with blue bars). In a PCA ratio spectrum, only ratios calculated with variables lying at the opposite ends of the spectrum are relevant for a particular shape PC (Baur and Leuenberger 2011). In a similar manner, the most allometric ratios are found in an allometry ratio spectrum (Fig. 5, graph with green bars). For females (Fig. 5a) the PCA ratio spectrum was dominated by ratios msp.l : eye.h, msp.l : tb3.l, and msp.l :  flgfirst.l; for males ( Fig. 5b) only a single ratio was most important, msp.l : flglast.l. The same ratio was also the most allometric (though both variables showed broad confidence intervals, see allometry ratio spectrum for males, Fig. 5b), whereas for females the dominating ratios were not among the most allometric ones (Fig. 5a). This result was in accordance with the general observation that allometric variation played a minor role in distinguishing the two groups, as they were of comparable size (compare Fig. 4).
The LDA ratio extractor is a tool for finding the best discriminating ratios for use in identification keys and diagnoses (see Baur and Leuenberger 2011). In contrast to a PCA, group membership must be specified beforehand. The results are compiled in Table 5 showing various contrasts, listed by sex. Generally, males were more distinct than females, as the groups were more widely separated in their ranges and the standard distances were on average higher, though overlapping (3.50-8.36 for males versus 4.07-6.19 for females). The ranges of two female comparisons (biglumis-rest, helveticus-bischoffi) were more or less distinct, for males a third one could be added (bischoffi-hellenicus). Ratios that separated the groups well were used for the key and diagnoses (see below). For both sexes, δ (a measure of how well shape discriminates in comparison with size) was always relatively close to zero (0.01-0.31), indicating that separation was mainly due to shape rather than size.

Taxonomic treatment a) Status of OTUs
Our molecular and morphometric analyses clearly revealed that all operational taxonomic units (hitherto called OTUs) formed well-supported taxonomic units (i.e., species). We can thus confidently conclude that the three species examined in this study, P. bischoffi, P. gallicus, and P. helveticus sp. n., represent valid species.

b) Diagnoses and descriptions of species
The following section provides information on all five species of the gallicus-group, as these can most easily be confused with each other, including the two main target taxa, P. bischoffi and P. helveticus sp. n. Males: Gena in dorsal view convex (Fig. 12l). Epicnemium and mesosternum yellow. Head breadth : head height 1.18-1.28; lower face : clypeus breadth 1.29-1.45; terminal flagellomere length : lateral ocelli distance 0.85-1.38; terminal flagellomere length : malar space 0.72-1.07; terminal flagellomere length : terminal flagellomere breadth 1.64-2.68.

Polistes biglumis
Comments. The holotype of Vespa biglumis Linnaeus, 1758, presently held at the Linnean Society of London, is not available for loan. We have, however, examined pictures online (http://linnean-online.org/16745/). Although no clear epicnemial carina is recognizable from the picture due to the condition of the specimen, the pubescence on the mesoscutum appears too long for P. helveticus sp. n. Therefore, we have no reason to question the current concept of P. biglumis.
Similarly, we have examined pictures (http://linnean-online.org/16772/) of the holotype (LINN 2807) of Vespa rupestris Linnaeus, 1758, also held at the Linnean Society of London and unavailable for loan. The genae of this male specimen are clearly convex in dorsal view (Fig. 12l), excluding any confusion with P. helveticus sp. n. or P. bischoffi.
The holotype of Vespa bimaculata Geoffroy in Fourcroy, 1785 is missing (Blüthgen 1961: 54), as are the syntypes of Polistes geoffroyi Lepeletier & Serville, 1825. According to the original descriptions both taxa seem to refer to dark individuals, but since no epicnemial carina is mentioned, a synonymy with P. helveticus sp. n. can neither be excluded nor proved.
The lectotype of Polistes dubius Kohl, 1898 was examined; we did not detect any characters allowing separation from P. biglumis. This view is also supported by our morphometric analyses (Fig. 3b, d [I]), which revealed that the lectotype of P. dubius does not plot far away from other males of P. biglumis. In any case it is a male with convex genae (Fig. 12l), making any confusion with the otherwise similarily colored male of P. helveticus sp. n. impossible.
We have seen three (ZSM-HYM-000006, ZSM-HYM-000007, ZSM-HYM-000009) of four syntypes of Polistes bimaculatus pamirensis Zirngiebl, 1955. Although they are dark females, occasionally with the entire mandible (ZSM-HYM-000007) or the apical part of the clypeus (ZSM-HYM-000006, ZSM-HYM-000007) black, the flagellum is not dark even on its dorsal side. The epicnemial carina is very pronounced in all three specimens, excluding confusion with P. helveticus sp. n. or P. bischoffi. However, morphology as well as morphometry (Fig. 3a, c [A]) cast doubt on whether this taxon is conspecific with P. biglumis. More material and further studies are needed to elucidate the status of this taxon.
The holotype (ZSM-HYM-000008) of Polistes bimaculatus nigrinotum Zirngiebl, 1955 is a very dark female; the apical part of the clypeus is entirely black and there is only a very small yellow spot on the mandible. The epicnemial carina is distinct, excluding confusion with P. helveticus sp. n. We see no trait distinguishing this specimen from P. biglumis, a view supported by our morphometric analysis (Fig. 3a, c [B]). Type study. Polistes bischoffi was described by Weyrauch (1937: 274) in a mere footnote indicating neither the type material nor the type locality. Later, Weyrauch (1938: 277 ff.) gave a key to the Palearctic species of Polistes, including P. bischoffi, but a more precise indication of the type material and the type locality is given only in Weyrauch (1939: 163), where a female from Macomer (Sardinia, Italy) is mentioned as the "type [Typus]". However, following article 74.5 of the ICZN (2012) this specimen is considered as a lectotype here. Unfortunately, this lectotype is lost (Blüthgen 1956: 85), as well as most paralectotypes from various localities (Italy, Malta, and Turkey; see Weyrauch 1939: 164), with the exception of two presumed paralectotypes that we were able to examine: a female (RN0287) from the Greek Island of Poros (see below, examined material), and a male (RN0325) from Glattbrugg in Switzerland. While the male from Glattbrugg clearly belongs to the dark (Fig. 10), northern (Fig. 11) taxon (Polistes helveticus sp. n.), the female from Poros belongs without any doubt to the southern (Fig. 11), bright ( Fig. 6) taxon (Polistes bischoffi). Consequently, Weyrauch (1939) most likely considered both taxa as geografically separated color morphs of the same species. Evidence for this statement can be found in his redescription of P. bischoffi (Weyrauch 1939: 163 ff.), where he writes that the antenna is "dorsally blackened in the northern part of the species range [Fühler im Norden des Verbreitungsgebietes oberseits geschwärzt]".
It must be stressed that both taxa (P. bischoffi, P. helveticus sp. n.) run to "bischoffi" in the keys of Weyrauch (1938: 277 ff.;1939: 195 ff.). In more recent keys (Blüthgen 1961, Dvořák and Roberts 2006, Guiglia 1972, Mauss and Treiber 2004, Witt 2009 for Central Europe however, Polistes helveticus sp. n. would run to "bischoffi", whereas Polistes bischoffi would run to "gallicus" due to the entirely bright flagellum. Unfortunately, the identity of the lost lectotype from Macomer (Sardinia, Italy) is unclear and can not be guessed from Weyrauch (1937Weyrauch ( , 1938Weyrauch ( , 1939. Therefore, the designation of a neotype is necessary for the clarification of the identity of Polistes bischoffi. Our attempts to locate the lectotype in all institutions likely to host some of Weyrauch's material were unsuccessful (e.g.: MFNB, Michael Ohl, pers. comm.; MHNL, Claus Rasmussen, pers. comm.; FMLT, Emilia Perez, pers. comm.), and so were our attempts to locate any specimen of Polistes bischoffi from Sardinia, including during a field trip to Macomer in 2013. Consequently, we designate a female from Galeria on the island of Corsica (France), north of Sardinia, as the neotype of Polistes bischoffi. Given that there is only a distance of 12 km between the two neighboring islands (Corsica, Sardinia), and that both of them share a similar fauna (Corti et al. 1999;Kwet 2005;Tolman and Lewington 1997), we are confident that this specimen matches the lost lectotype of Polistes bischoffi Weyrauch, 1937. In fact both, Corsica and Sardinia are probably located too far south to host the taxon called Polistes helveticus sp. n. here, since the southernmost individual (RN0378) of P. helveticus sp. n. that we are familiar with was found about 200 km north of the French Mediterranean coast (Fig. 11). Moreoever, the neotype is a well preserved female of the southern, light colored species (P. bischoffi) that appears at the center of the scatter of points in our morphometric analysis and clearly lies outside the area of overlap with P. gallicus (Fig. 3a, c [C]). Lastly, this specimen (RN0366) yielded high-quality DNA and could be included in our molecular analysis.
Diagnosis. Small and moderately bright species with flagellum on upper side bright yellow in both sexes (Figs 6a, 6c, 6d, 7a, 7b, 7d, 7e) or faintly darkened, especially in large females; pedicel and extreme base of flagellomere 1 always black on upper side.
Head: Clypeus yellow, with a black margin and a large central black spot usually isolated (Fig. 6a) but seldom shaped like a (rhomboid) crossband reaching lateral margin. Face with large, almost triangular yellow spot touching inner orbit (Fig. 6a).
Mesosoma: Change in sculpture between coarse mesepisternum and smooth epicnemium frequently gradual (Fig. 12b). Pronotum along posterior margin with pair of longitudinal yellow stripes not reaching yellow cross stripe on pronotal collar (Fig. 6e). Scutellum with pair of yellow, somewhat triangular spots, followed by rectangular pair of spots on metanotum and crescent-shaped pair of spots on dorsal propodeum (Fig. 6e). Mesopleuron with yellow spot (Figs 6c, 6d). Propodeal valve yellow (Fig. 6c). Tegula yellow anteriorly and posteriorly, with transparent area in between (Fig. 6e). Legs apically yellow and orange, black only on coxa, trochanter and most of femur (Figs 6d, 6e), including base.
Metasoma: Each tergum with continuous, but indented terminal yellow band (Figs 6d, 6e). Tergum 2 also with two large yellow spots (Fig. 6e). Tergum 1 occasionally with two small yellow spots. Sterna 2 and 3 with continuous terminal yellow bands, on sternum 3 occasionally centrally indented close to interruption. Sternum 4 with interrupted terminal yellow band. Sternum 5 with broadly interrupted terminal band, reduced to two lateral yellow spots.
Mesosoma: Pronotum with yellow cross stripe along collar, often extending down both sides to longitudinal pair of yellow stripes along pronotal side margin ( Fig. 7d; white arrow). Epicnemium and mesosternum yellow (Fig. 7d). Legs yellow and partially orange, except for superior side of coxa, trochanter and femur, which are black (Figs 7d, 7e). Rest of mesosoma colored as in females.
Metasoma: Tergum 2 with terminal yellow band laterally extending towards base ( Fig. 7d; red arrow), even if occasionally interrupted. Terga otherwise colored as in females. Sternum 2 with pair of large yellow spots mostly isolated ( Fig. 7d; black arrow), seldom fused. Sternum 3 with both terminal and basal yellow bands ( Fig. 7d; blue arrow). Sterna 4 and 5 both with continuous terminal yellow band, the latter interrupted on sternum 6 and absent on hypopygium.
Comments. This is one of the smallest Polistes species in Europe and besides P. helveticus sp. n., the only one with often absent epicnemial carina in the female sex.
The two locally syntopic species (Fig. 11;Neumeyer et al. 2011) are, however, easy to distinguish in both sexes due to differently colored antennae. Furthermore, the ratio metatibia length : malar space is an unambiguous separator for females, whereas the best separating ratio for males (P. bischoffi, P. helveticus sp. n.) is the ratio terminal flagellomere length : malar space ( Table 5). The same ratio weakly separates the sometimes similar females of P. bischoffi and P. gallicus. It is impossible to confuse the males of P. bischoffi with the males of P. hellenicus or P. biglumis due to the strikingly different color patterns and the diagnostic head shape of P. biglumis males within the gallicus-group.
Two morphs can be distinguished within P. bischoffi (rev. status), one with the flagellum entirely bright (yellow to orange) and the other with the flagellum dorsally faintly darkened. Often, the brighter morph (e.g. RN0137) has the clypeus with a central black spot (Fig. 6a), whereas in the darker morph the clypeus usually has a horizontal black band reaching the lateral margin. These two color morphs are probably the two extremes of an otherwise gradual continuum, but more individuals would have to be examined to verify this hypothesis. It would be even more important to examine whether such color variations are associated with geography or not. Limited evidence suggests that these variations are not associated with geographic location, since two nests were found (16 Aug 2013) in Zurich (Katzensee Allmend) with both morphs in each. In these colonies, the dark morph was more common among large females (presumably young queens), rather than among small females (presumably workers) or males. Also the neotype (RN0366) of bischoffi belongs to the darker morph and is presumably a queen, since it was collected on 19 April. More observations are needed to confirm this correlation between coloration and caste. Different color morphs within the same nest population are also reported in P. gallicus (Gusenleitner 1985: 105).
Distribution. Based on the material that we have examined, P. bischoffi occurs at least in Southern Europe and Turkey from the Atlantic coast of southern France to Turkish Kurdistan (Fig. 11). The northernmost confirmed locality is in the Pannonian region of Austria (Neusiedl am See), followed by several localities in Switzerland where the species occured already in 1927 at the river Versoix near Geneva (individuals RN0170, RN0171). In all other, more northern Swiss sites P. bischoffi occurs syntopically  with P. helveticus sp. n. and was not detected before 1992, suggesting a possible recent range expansion due to climate warming.
Ecology. According to our experience in Switzerland, P. bischoffi appears to be restricted to large wetlands, especially to fens on lake shores, more so than P. helveticus sp. n. The altitudinal records range from sea level for several beach records (see "Material examined"), including the neotype (RN0366), to 540 m a.s.l. for a female (RN0076) in Switzerland (Wetzikon, Canton of Zurich). However, the Turkish locality (road from Yüksekova to Şemdinli) where three females (RN0363, RN0364, RN0365) were found was probably higher than 540 m a.s.l., since Yüksekova is situated at 1950 m, Şemdinli at 1450 m a.s.l., but the precise elevation of the locality is neither indicated on the label nor in the publication (Madl 1997: 824). Most individuals were found in August or September. The earliest record in the season is the neotype female from Comments. The holotype of Vespa gallica Linnaeus, 1767 (LINN 2790), presently held at the Linnean Society of London, is not available for loan. We have, however, examined pictures (http://linnean-online.org/16757/). They clearly show the bright flagellum all around, excluding identity with P. helveticus sp. n. A careful examination of this specimen would be needed to confirm the identify of P. gallicus.
Except for Vespa gallica and for the lost lectotype of Polistula omissa Weyrauch, 1938 we have examined and measured type specimens of all taxa listed as synonymous with P. gallicus. Since each of them appears to be clearly distinct from P. bischoffi in our morphometric analyses (Fig. 3), we only compare them with P. helveticus sp. n. in the following section. Blüthgen (1941: 245) and Guiglia (1972: 49) claim that the upper side of the flagellum of P. foederatus Kohl, 1898 is "slightly blackened [leicht geschwärzt]" or "darkened [assombrie]", respectively, even in the male sex, unlike the flagellum of P. omissus (Weyrauch, 1938). However, the flagellum of the male lectotype of P. foederatus from Azerbaijan is bright yellow all around, as noted by Blüthgen (1943: 129), thus excluding any confusion with P. helveticus sp. n. Furthermore, the clypeus of this lectotype has not even a trace of a longitudinal furrow, although this trait has also been regarded as diagnostic for P. foederatus (Blüthgen 1941: 245). Currently, both P. foederatus and P. omissus are synonyms of gallicus (Day 1979: 63;Gusenleitner 1985: 105). This view is supported by our morphometric analysis (Fig. 3b, d [J]) for P. foederatus.
The lectotype of Polistes gallicus mongolicus Buysson, 1911 has its epicnemium and mesosternum largely black as in P. hellenicus, but is otherwise a large, very light colored male with an extremely broad head. Its flagellum is bright all around, excluding any synonymy with P. helveticus sp. n. The terminal flagellomere is in fact "very short" [très court], as Buysson (1911: 218) states. Morphologically it appears doubtful that the taxon mongolicus belongs to P. gallicus (C. van Achterberg, pers. comm.). This view is also supported by our morphometric analyses (Fig. 3b, d [K]).
We have seen two syntypes (RN0444, RN0445) of Polistes foederatus obscuricornis Mader, 1936. In contrast to the statement of Mader (1936: 263) the flagella of these two females are not "entirely black [ganz schwarz]" dorsally, but only grey. Only the scape, the pedicel, and the very base of the first flagellomere are entirely black dorsally. Both of these otherwise light colored individuals have a pronounced epicnemial carina, excluding confusion with P. helveticus sp. n. Although this taxon belongs without any doubt to the gallicus-group, our morphometric analyses (Fig. 3a, c [D]) do not entirely support their synonymy with P. gallicus.
The female holotype of Polistes omissus ordubadensis Zirngiebl, 1955 is colored very light with the clypeus unspotted, the flagellum colored light dorsally, the hypopygium (Blüthgen 1956: 85) and even the malar space largely yellow, and the epicnemial carina distinct, all of them excluding confusion with P. helveticus sp. n. This view is also supported by morphometry (Fig. 3a, c [E]). In our opinion this taxon (P. omissus ordubadensis) may not even belong to the gallicus-group.
The holotype of Polistes omissus kaszabi Giordani Soika, 1970 is a large, dark female with both malar space and mandibles entirely black. The epicnemial carina is distinct, excluding confusion with P. helveticus sp. n. Flagellum, clypeus and hypopygium are colored and patterned as in P. biglumis or P. nimpha. In fact, a synonymy with P. gallicus is not supported by our morphometric analyses (Fig. 3a, c [F]) and this taxon (P. omissus kaszabi) may not even belong to the gallicus-group (C. van Achterberg, pers. comm.). Giordani Soika, 1976 is currently on loan and could not be examined, but we have examined the only paratype (RN0326, MSNV-04702) mentioned in Giordani Soika (1976: 272). It is an extremely dark female with the flagellum dorsally black and the epicnemial carina unilaterally reduced. It is morphologically and even morphometrically (Fig. 3a, c [G]) similar to a very dark P. helveticus sp. n., except that the bright spots and stripes are not only reduced, but also of ivory-white rather than of yellow color. These striking characters make it unlikely that this taxon (albellus) belongs to a species reaching Europe (C. van Achterberg, pers. comm.).

The holotype of Polistes foederatus albellus
These observations indicate that the taxon gallicus should be carefully revised; our analysis of the mitochondrial marker further indicates that two specimens (P. sp. aff. gallicus; RN0126, RN0129) from Greece, both identified as P. gallicus by Arens (2011), may represent another species. The upper side of the flagellum in both of these individuals is slightly darkened. This trait, as well as others (e.g. a broad malar space), suggest that this taxon (P. sp. aff. gallicus) may be P. foederatus obscuricornis. More data are required to resolve this problem.
Head: Clypeus yellow with black margin and large central black spot; this spot either isolated (Fig. 9a) or more often extended as crossband reaching the lateral margins of clypeus (Fig. 12g). Face with nearly triangular yellow spot touching inner orbit (Fig. 9a). Upper gena with small, elongate spot (Fig. 9d). Frons with pair of horizontal yellow stripes seldom confluent (Fig. 9a).
Mesosoma: Change in sculpture between coarse mesepisternum and smooth epicnemium frequently gradual (Fig. 12b). Pronotum along posterior margin with pair of longitudinal stripes not reaching cross stripe on pronotal collar (Fig. 9e). Scutellum and metanotum each with pair of yellow bars (Fig. 9e). Propodeum dorsally usually  (c) is pointing to the rather reduced epicnemial carina, and the white arrow to the quite distinct mesopleural signum (sensu Carpenter 1996a), a structure also called a sternopleural groove (Richards 1973). with pair of crescent-shaped spots (Fig. 9e). Mesopleuron with yellow spot (Figs 9c,  9d). Propodeal valve yellow (Fig. 9d). Tegula yellow anteriorly and posteriorly, with more transparent area in between (Fig. 9e). Legs yellow and orange, black only on coxa, trochanter and most of femur, including entire base (Figs 9d, 9e).
Metasoma: Each tergum with continuous, but slightly indented terminal yellow band (Figs 9d, 9e). Tergum 2 also with two yellow spots (Fig. 9e). Tergum 1 seldom with two small yellow spots. Sterna 2 and 3 with terminal yellow bands usually inter- rupted, even though often only slightly so. Sterna 3, 4 and 5 with broadly interrupted terminal bands, manifested only as lateral terminal yellow spots.
Mesosoma: Pronotum with yellow cross stripe along collar, occasionally extending down to sharp angle of pronotum ( Fig. 10c; arrow). Legs yellow and orange, except for upper sides of coxa, trochanter and femur, which are black (Figs 10c, 10d); black area occasionally reaching (yellow) lower side of hind femur, yellow area occasionally reaching (black) upper side of pro-and mesocoxa. Rest of mesosoma colored as in females (Fig. 10d).
Metasoma: Tergum 2 with terminal yellow band extending laterally toward base, even if occasionally discontinuous. Other terga colored as in females. Sternum 2 most of the time with two yellow spots. Sterna 3, 4 and 5 usually with continuous terminal yellow band, the latter interrupted on sternum 6 and absent on hypopygium.
Comments. Except for P. bischoffi, Polistes helveticus sp. n. is the only European species with an epicnemial carina that is often absent in the female sex. These two species are easy to distinguish in both sexes due to their differing color patterns, mainly on the antennae. Furthermore, the ratio metatibia length : malar space separates females, whereas the best separating ratio for males (P. bischoffi, P. helveticus sp. n.) is terminal flagellomere length : malar space (Table 5). Confusion with P. gallicus or P. hellenicus is unlikely due to the very different color patterns in both sexes. Males are virtually impossible to confuse with P. biglumis; however, the very similarly colored females of P. helveticus sp. n. and P. biglumis are likely to be confused in specimens of P. helveticus sp. n. with an exceptionally developed epicnemial carina. For such cases, we provide the ratio malar space : lateral ocelli distance, which fully separates the two.
Since most collected specimens labeled as "Polistes bischoffi" are presumed to belong to P. helveticus sp. n., rather than to P. bischoffi Weyrauch, 1937 (revised status), at least in Central European museums, their identity must be checked. In fact, according to the CSCF (www.cscf.ch; in litt.) there are about 450 individuals of P. helveticus sp. n. from Switzerland deposited in Swiss museums, but only very few (< 10) individuals of P. bischoffi, at least before the material of the present study was deposited. A similar situation may apply to other Central European museums, especially in Austria and Germany. In contrast, the relatively few individuals labeled as "P. bischoffi" that we examined from Southern Europe (Greece, Italy, Southern France) are, in fact, determined correctly (mostly by Josef Gusenleitner).
The specimen that we have chosen as the holotype of P. helveticus sp. n. clearly belongs to P. helveticus sp. n. according to molecular and morphological analyses. Ac-cording to its body measurements, however, it lies in an area of overlap with P. gallicus (Fig. 3a,c [H]). Unfortunately, it is the only specimen that was both intact and suitable for molecular analyses.
Nevertheless, Fig. 11 indicates a geographical separation between P. helveticus sp. n. (in the north) and P. bischoffi (in the south), leaving only a small area of overlap. Real syntopy (habitat sharing) between the two species has thus far only been assessed in Switzerland but both species also occur sympatrically in Austria. Furthermore, the verified range of P. helveticus sp. n. (Fig. 11) is distinctly smaller than that of P. bischoffi, although P. helveticus sp. n. is considered to be in a period of expansion , Mauss 2001.
Ecology. In Switzerland, P. helveticus sp. n. is widespread (Fig. 11), usually occurring in wet habitats such as floodplains, fens, bogs, and pits (gravel, sand). The Syntopy of both species Figure 11. Distribution of examined specimens of Polistes bischoffi Weyrauch, 1937 and Polistes helveticus sp. n. While P. bischoffi mainly occurs from Southern Europe to Western Asia, P. helveticus appears to have a more northern distribution in Central Europe. Thus far, the only incidences of syntopy (P. bischoffi, P. helveticus) are from Switzerland. altitudinal records range from 200 m above sea level (Le Champ-près-Froges, France) for a female (individual RN0378) to 980 m a.s.l. (Muggio, Canton of Ticino, CH) for a male (RN0387). The seasonal records range from 02 April (Saint-Blaise, CH) for a female to 24 November (Gnadental, Germany) for a female (RN0283), but most individuals of both sexes are recorded in July and August (CSCF, in litt.). The earli-

c) Key to species of the Polistes gallicus-group
The following dichotomous key only applies to the described species of the gallicusgroup. The text denotes diagnostic traits. However, traits described after a hyphen (-) are those that apply in most cases to species in that half of the couplet, but that may also apply to some species in the alternative half of the same couplet. To determine all European species of Polistes including the dominula-group, we recommend the keys of Mauss and Treiber (2004), Dvořák and Roberts (2006), and Witt (2009: 129-132), whereby Mauss and Treiber (2004)  Flagellum black on upper side, bright yellow to orange on lower side (Fig.  12c). -Metacoxa black. Mesoscutum usually without yellow spot (Fig. 12k). Clypeus with central, black spot (Fig. 12h) or more often with black, horizontal bar (Fig. 12g) Epicnemial carina reduced (Fig. 12b) or absent. Change in sculpture between mesepisternum and epicnemium frequently gradual (Fig. 12b). Terminal flagellomere length : malar space 1.00-1.24. -Clypeus yellow, although almost never entirely so, frequently with central black spot (Fig. 12h), occasionally even with horizontal black band (Fig. 12g). Mesoscutum usually without yellow spot (Fig. 12k), only occasionally with pair of yellow spots (Fig. 12i) (Fig. 12a) or reduced, usually marking a sudden change in sculpture between coarse mesepisternum and smooth epicnemium (Fig. 12a). Terminal flagellomere length : malar space 0.74-1.11. -Clypeus yellow, with or without central, black spot (Fig. 12h) Hypopygium entirely black, only rarely spotted yellow at tip. Lateral part of propodeum with yellow spot usually more than half the size of mesopleural spot (Fig. 6c). Mesoscutum frequently with pair of yellow spots (Fig. 12i). Pronotum with paired longitudinal yellow stripes along posterior margin usually not reaching yellow cross stripe on pronotal collar (Fig. 12i)  Although it probably belongs to the gallicus-group too, the ambiguous taxon from Greece (and possibly elsewhere) referred to as "Polistes sp. aff. gallicus" is not included in this key because there was not enough material available to examine.

Discussion
Status of Polistes bischoffi and Polistes helveticus sp. n. Our study unambiguously demonstrates that two distinct species are included within what has been so far considered as Polistes bischoffi Weyrauch, 1937: a light colored species (Polistes bischoffi) with a Southern European to West Asian distribution, and a dark, Central European species described here as Polistes helveticus sp. n.
The distinctivness of these taxa (P. bischoffi, P. helveticus sp. n.) is revealed in analyses of two independent molecular markers (COX1, ITS1), as well as in our morphometric analyses. Moreover, P. helveticus sp. n. is probably closely related to P. bischoffi (as suggested by the reduced epicnemial carina and the association with wetlands) and occurs in the same habitats, sometimes syntopically, but appears not to interbreed . Taken together, these results suggest that three independent criteria are met to reveal the presence of a new species: molecules, morphology, and syntopy without interbreeding .
The unnoticed presence of a cryptic species in Europe is surprising and calls for an explanation. Interestingly, the first record for Polistes bischoffi Weyrauch, 1937 (rev. status) in Switzerland refers to two individuals (RN0170, RN0171) found in 1927 in Versoix near Geneva, in the extreme southwest of Switzerland where Polistes gallicus is known to have occurred before 1900 (cf. our examined individual RN0208). The second Swiss record (RN0156) of P. bischoffi is from Chabrey on Lake Neuchâtel in 1992, and the third (RN0169) from Regensdorf near Zurich in 1997, all together suggesting a recent range expansion from the southwestern to the northeastern part of the Swiss midlands, where P. gallicus still does not occur. We hypothesize that P. bischoffi was originally present but remained undetected within the range of the superficially similar P. gallicus, and became conspicuous only after it expanded beyond the range of P. gallicus, possibly due to global warming.
Morphometry. By applying multivariate ratio analysis (MRA) most taxa of the gallicus-group are rather well differentiated (Fig. 3), with the exception of P. gallicus and P. hellenicus. The use of further measurements may have resulted in better differentiation between these two species, as the addition of characters has indeed improved the separation of sibling species in some other Hymenoptera (e.g., Mills 1998, Villemant et al. 2007). However, such analyses are beyond the scope of this study, as P. bischoffi and P. helveticus sp. n. were clearly separated by the first shape PC (Fig. 4).
The latter two species were of comparable size, so allometric variation did not interfere with the interpretation of the data. This was also unlikely to bias the differentiation of P. biglumis, although this large species accounted for the rather strong correlative pattern between size and shape in the shape PCA of all five species of the gallicus-group (Figs 3c, d). Since P. biglumis could clearly be separated from the rest of the gallicus-group by qualitative morphological characters and molecular analysis, the morphometric separation was assumed to be based on "true" shape differences and not merely on an indirect size effect. Hence, we see no need to correct the best separating ratio (Tab. 5) for allometric size effects, although such a procedure has sometimes been suggested (Janzon 1986, Seifert 2008, Bartels et al. 2011. The LDA ratio extractor revealed ratios that separated some of the species with very little or no overlap (Table 5, species comparisons marked with *). It is noteworthy that these ratios were composed of measurements from widely separated body parts; for instance metatibia length (tib3.l) to malar space (msp.l) was the best ratio for separating the females of P. helveticus sp. n. from P. bischoffi. This is in contrast to more commonly used ratios that are calculated from measurements of the same or adjacent body parts, such as eye length to breadth or clypeus height to breadth (e.g., Arens 2011). In our study, such standard ratios are clearly less powerful for separating taxa (compare PCA ratio spectra in Fig. 5), an observation that was also made by László et al. (2013) in their application of MRA to parasitic wasps.
Utility of the molecular markers. An important question when using molecular markers to separate closely related species, is whether a clear gap (the barcoding gap) exists between "within-species" distances and "between-species" distances. Buck et al. (2012)'s detailed study of the Nearctic Fuscopolistes revealed no barcoding gap within this group for COX1. In fact, half of the species included in their study showed a "negative barcoding gap", i. e. a situation where "the maximum intraspecific divergence was greater than the distance to the nearest neighbour from another species" (Buck et al. 2012: 34). In our case, the evaluation of such a barcoding gap would strongly depend on our interpretation of the two clades found within P. dominula with the mitochondrial marker. Two hypotheses can be formulated: firstly, two cryptic species may be present in Central Europe; alternatively, two distinct mitochondrial haplotypes may exist within one single species. As P. dominula was not the focus of our study, we did not perform any morphometric analyses for this taxon. The nuclear marker ITS1 did not recover these two clades. As a nuclear DNA marker, ITS1 has a lower rate of mutation than the mitochondrial marker, as indicated by the overall smaller genetic distances between species for ITS1 than for COX1. It is therefore possible that ITS1 evolves too slowly to recover the recent divergence between the two clades observed within P. dominula. However, ITS1 appeared highly suitable for recovering differences between other closely related species. Therefore, we favor the hypothesis that two mitochondrial haplotypes may coexist in Central Europe within P. dominula, as demonstrated for other species (Avtzis et al. 2008, Arthofer et al. 2010. Possibly, the two haplotypes revealed in P. dominula reflect two distinct Pleistocene refugia that have facilitated sequence divergence in the mitochondrial marker; divergence time was presumably not long enough for preventing the populations from successfully interbreeding when they entered in contact again. Our example stresses the importance of using additional criteria (morphometry, nuclear DNA) in addition to one single mitochondrial marker (e.g., the universal barcode) to examine the status of populations in systematics. Deep within-species divergences in mitochondrial DNA sequences may be more widespread than hitherto assumed, especially when sampling is done over the entire range of a species (Bergsten et al. 2012, Buck et al. 2012. In conclusion, our study demonstrates the power of the combined use of morphometrics and molecular markers in unraveling cryptic diversity, as proposed under the framework of integrative taxonomy (Schlick-Steiner et al. 2010). It also stresses the importance of using multiple molecular markers to evaluate the status of unclear taxa.