Study species

The cosmopolitan genus Psenulus Kohl, 1897 comprises more than 160 species, with the Oriental biogeographic region being particularly species rich (Bohart & Menke, 1976; Pulawski, 2018). About 26 species occur in the Palearctic region; with eight taxa found in Central Europe and Switzerland (Bohart & Menke, 1976; Blösch, 2000; Herrmann, 2005; Artmann-Graf, 2006; Pulawski, 2018; Info Fauna, 2018).

Ecological information is available for a small number of taxa, of which all nest in hollow or pithy stems (e.g. Sambucus spp.) or beetle borings, and prey on adult or nymph arthropod herbivores such as aphids, leafhoppers, delphacids and psyllids (Bohart & Menke, 1976; Blösch, 2000). Some species, e.g. P. fuscipennis (Dahlbom, 1843), accept artificial cavities and can serve as indicators to assess the effect of land-use (change) on hymenopteran-based food-webs (e.g. Tscharntke et al., 1998; Fabian et al., 2013); a research approach for which DNA barcoding can be useful (Turčinavičienė et al., 2016).

Psenulus fulvicornis has been reported from Algeria, Andorra, Bulgaria, Croatia, France, Germany, Greece, Hungary, Italy (Aosta), Russia, Spain, Switzerland, Syria and Turkey (Gayubo et al., 2002; Schmid-Egger, 2002; Nieves-Aldrey et al., 2003; Boillat, 2012; Cruz-Sanchez et al., 2005; Gayubo et al., 2006; Zsolt, 2008; González et al., 2009; Standfuss & Standfuss, 2012; Reder & Niehuis, 2014; Yildirim et al., 2015; Mokrousov & Popov, 2016) (Fig. 1). Older observations are given from Austria (Tirol), Italy (Trentino – Alto Adige) and Poland (Pomeriana) (Brischke, 1864; von Aichinger, 1870; Kohl, 1880, 1888). Taken together, these occurrences indicate a geographic range spanning from the Mediterranean to the Anatolian region, with additional occurrences in central and southern alpine valleys, and continental and steppe zones of Europe. Schmid-Egger (2002) observed a slight morphological divergence in several taxonomically important characters (e.g. the texture of the propodeal surface) between females from Algeria, Turkey and Syria on the one hand, and females from Central Europe on the other hand; a pattern which may indicate phylogeographic structuring.

In Central Europe P. fulvicornis is found in warm lowland habitats such as abandoned vineyards in the Upper Rhine Plain in Germany, which is a region characterized by a relatively mild climate (Blösch, 2000). In the Mediterranean area, specimens have been captured in olive and oak (Quercus ilex L.) groves, and vineyards. In Spain and in the North Caucasus they also occur on (shrubby) coastal sand dunes (see Results).

In the past, the taxonomic status of P. fulvicornis as a separate species has been doubted due to its morphological similarity with other taxa, the low number of observations and the lack of males, which have been discovered only recently (Schenck, 1861; De Beaumont, 1937–1939; Schmidt, 1971; Schmid-Egger, 2002). Psenulus fulvicornis type material has been studied by the latter two authors, who highlighted the close morphological relationship with P. schencki (Tournier, 1889) and delivered a detailed species diagnosis. Due to the close morphological relationship between the two taxa, and since P. fulvicornis was described first, we here refer to them as the Psenulus fulvicornis-Group. Currently, the status of P. fulvicornis as a separate species is accepted, and the taxon is included in the standard literature (Bohart & Menke, 1976; Blösch, 2000; Jacobs, 2007; de Jong et al., 2014; Pulawski, 2018).

Fig. 1.

Sampling sites of Psenulus fulvicornis (Schenck, 1857) (large filled circles) and Psenulus schencki (Tournier, 1889) (squares) used for the DNA barcode sequencing of the mitochondrial cox1 locus. Small filled circles show georeferenced distribution records of P. fulvicornis with confirmed determinations by Schmid-Egger (2002) together with newer records by Gayubo et al. (2012), Mokrousov & Popov (2016), Reder & Niehuis (2014), Standfuss & Standfuss (2012) and Zsolt (2008). The triangle represents the undetermined specimen (PSC-7) from Pisa, Italy.

Psenulus fulvicornis is considered rare in Germany and red-listed under the category “Vulnerable” due to a predicted (moderate) long-term population decline (Schmid-Egger, 2010a). In contrast, the number of records has slightly increased in recent years (Schmid-Egger, 2010a, b, 2015; Frommer, 2012; Reder & Niehuis, 2014) but it is unclear whether this is due to temporary and local population size increases, for example because of exceptionally favourable weather conditions, an increased sampling effort, or whether this represents an ongoing population size increase and/or range expansion, e.g. favoured by climate warming (Schmid-Egger, 2010a).

Sampling

Flying arthropods were sampled in 85 urban gardens in the city of Zurich between May 18 and August 19 2015 with three bowl traps fixed on a triangular wooden pole. Each bowl was sprayed with either UV-bright blue, white or yellow paint (Sparvar Leuchtfarbe, Spray-Color GmbH, Merzenich, Germany) (Westphal et al., 2008) and three quarters filled with 0.2% Rocima solution (Acima, Buchs, Switzerland). Aculeate hymenopterans (Formicidae excluded) were identified by one of the authors (M. Řiha), and vouchers of all taxa were deposited in the reference collection of the Swiss Federal Research Institute for Forest Snow and Landscape (WSL). One female of each of P. fulvicornis and P. schencki was caught and identified during this survey. In addition, to complete the dataset of specimens found in Switzerland, we were kindly allowed to sample one hind leg of the P. fulvicornis female captured in 2005 in the Canton of Geneva at the Swiss-French border (Boillat, 2012). Finally, to increase the geographic scale of the genetic analysis, specimens of both P. fulvicornis and P. schencki from areas of sympatry across almost the entire known distribution range of the P. fulvicornis were added from the collections of two of the authors (S. F. Gayubo and M. Mokrousov). Note that one such sample (PSC-7) from Pisa, Italy, which was originally determined as P. schencki, was genetically so divergent from all other samples of the P. fulvicornis-Group (2.3% sequence divergence over 614 base pairs) that we considered it an unknown species (see Results). Genetic analyses were further completed with publicly available cox1 sequences of Psenulus taxa in GenBank and BOLD (Ratnasingham & Hebert, 2007) (supplementary Table S1 in the Appendix).

Photographs of specimens and their discriminating characters were made with a Leica Digital Microscope DVM6 by using image-stacking. Georeferenced sampling sites and distribution records were mapped with ArcGIS (version 10.2; ESRI).

DNA extraction and cox1 sequencing

The DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) was used for the extraction following the manufacturer's instructions. The partial cox1 locus (approx. 650 nu) was sampled using the primer pair LepF1 and LepR1 (Hebert et al., 2003) following the published thermal regime. The JumpStart REDTaq ReadyMix Reaction Mix (Sigma, St. Louis, Missouri, U.S.A) was used in the PCR, and its products were purified enzymatically using Illustra ExoProStar (GE, Chicago, Illinois, U.S.A.) following the manufacturer's instructions. PCR products were sequenced in both forward and reverse directions on an ABI 3130 sequencer (Applied Biosystems, Foster City, California, U.S.A.) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) and PCR primers, following the manufacturer's indications. The cycle sequencing products were purified using the BigDye XTerminator Purification Kit (Applied Biosystems). The corresponding forward and reverse electropherograms were assembled with CLC Main Workbench 7 (Qiagen) and visually corrected.

Genetic diversity

The cox1 polymorphism of the P. fulvicornis-Group was analysed using the software DnaSP v.5.10.01 (Librado & Rozas, 2009) under the option for haploid mitochondrial DNA. No position contained gaps or missing data. Three codon positions were included with a total of 187 codon positions in the final dataset. We calculated the haplotype diversity (Hd; the probability that two randomly chosen haplotypes are different within the sample), the nucleotide diversity per site (Pi; the probability that two randomly taken nucleotides from the same position are different, and the average nucleotide differences (K). As we found that the inferred haplotypes belonged to divergent clades (see Results), genetic diversity was analysed in the entire sample (i.e. clades I-VI) and within each of the statistically well supported clades I-II (P. fulvicornis) and III (P. schencki).

Phylogenetic tree

The model of nucleotide substitutions was computed with the software jModelTest 0.1.1 (Guindon & Gascuel, 2003; Posada, 2006, 2008) and the best-fit model was selected based on the Akaike information criterion (Akaike, 1973). A maximum likelihood (ML) analysis used the RAxML-HPC BlackBox (Stamatakis, 2006) and a Bayesian analysis was performed with MrBayes 3.2.6 on the CIPRES Science Gateway (Miller et al., 2010). The statistical support of branches in the ML analysis was calculated based on 1,000 bootstrap replicates (Felsenstein, 1985). Bayesian analysis was run with 5 million generations and sampled every 100th generation, following a discarded burn-in of 12,500 generations. Convergence and the consequent proportion of burn-in were assessed using Tracer v1.5 (available from  http://beast.bio.ed.ac.uk/). Output tree files, with each containing the best tree found and labelled with the statistical support for each branch, were graphically represented with TreeGraph 2 (Stöver & Müller, 2010).

Review of Swiss museum and private collections of P. schencki accessions

The (undetected) presence of P. fulvicornis in Switzerland was checked in all major Swiss museums as well as in the most important private collections. To achieve this, we controlled the correct identification of P. schencki specimens since older identification keys do not discriminate the taxa (e.g. de Beaumont, 1937–1939). We followed Schmidt (1971) and Schmid-Egger (2002) for taxon identification. The subsequent collections were visited or contacted: Bündner Naturmuseum, ETH Zürich Entomological Collection (ETHZ), Muséum d'histoire naturelle de Genève (MHNG), Muséum d'histoire naturelle de Neuchâtel, Musée de la nature de Sion, Musée de zoologie, Lausanne (MZL), Museo cantonale di storia naturale (LUG), Naturhistorisches Museum Basel (NMB), Naturhistorisches Museum Bern (NMBE), Naturmuseum Luzern and Naturmuseum St. Gallen. Only collections harbouring P. schencki were visited except for the Musée de la nature de Sion, where the Crabronidae of Maurice Paul (1835–1898) were reviewed. Additionally, the following private collectors were contacted: Neumeyer, Rainer; Artmann-Graf, Georg; Müller, Andreas; Herrmann, Mike; Salzmann, Irene. The material examined is given in the Appendix.

Successfully barcoded material:

Fig. 2.

(A) Female Psenulus fulvicornis (Schenck, 1857) from the City of Zurich, Switzerland (D. Frey & A. Zanetta leg.; M. Řiha det.; R. Neumeyer conf.; WSL coll; GenBank accession Nr. KY039438). (B) Female Psenulus schencki (Tournier, 1889) from the City of Zurich (25.05.2015; D. Frey & A. Zanetta leg.; M. Řiha det.; R. Neumeyer conf.; WSL coll; GenBank accession Nr. KY039439). Note the (barely visible) oblong spot at the lower end of the mid-tibia, which is a unique and shared character for both taxa among Central European Psenulus species (Jacobs, 2007).

Discriminating morphological characters

The main discriminating characters between female P. fulvicornis and P. schencki are depicted in Fig. 3. In brief, the lateral surface of the propodeum of P. fulvicornis has short crosswise carinas that give the texture the characteristic coarse appearance (Fig. 3A). This trait is unique among females of Central European Psenulus. In P. schencki crosswise carinas are lacking, and the texture of the propodeum is smoother (Fig. 3C). Moreover, the pygidal area is usually longer and broader in P. fulvicornis (Fig. 3B) than in P. schencki (Fig. 3D). Likewise, the colour of the foretibia tends to be lighter in P. fulvicornis than in P. schencki (Fig. 2). More characters and a description of males are given by Schmid-Egger (2002) and Jacobs (2007).

cox1 sequence variation

The cox1 sequences of P. fulvicornis and P. schencki from Zurich were sampled successfully and sequences were deposited in GenBank (supplementary Table S1 in the Appendix). However, since the P. fulvicornis specimen from Geneva was collected several years ago, the extracted DNA was of poor quality and cox1 amplification failed in that sample, despite several amplification attempts with varying PCR parameters. All obtained sequences met barcode quality criteria with more than 500 bp sampled and no uncertain base calls (N’s).

We found 22 polymorphic sites within a 561 bp alignment of the P. fulvicornis-Group, which excluded gap positions on the flanking regions (Table 1). All variable sites were synonymous single-nucleotide polymorphisms (SNPs), except two nonsynonymous replacements found in the specimens from Krasnodar (Russia) and Salamanca (Spain) (Fig. 4). The overall haplotype diversity was relatively high (Table 1). The three samples from the Caucasus (clades IV, V, and VI) strongly contributed to the overall genetic diversity as each sample represented a unique haplotype and contained a large proportion of polymorphic sites (Table 1; Fig. 4).

cox1 phylogenetic tree

In total, eleven cox1 sequences were sampled (supplementary Table S1 in the Appendix). The dataset for the analysis of the genus Psenulus comprised 637 nucleotide sites and 42 sequences, including five sequences of two outgroup species, Diodontus minutus (Fabricius, 1793) and Pemphredon lethifer (Shuckard, 1837). Of the 637 sites, 204 were polymorphic. Both ML and Bayesian analyses resulted in highly similar topologies for Psenulus species (ML tree: Fig. 5; Bayesian tree: supplementary Fig. S1 in the Appendix), and the genus obtained maximal support under both analyses. Bootstrap values and posterior probabilities were high for most taxa, but while Bayesian analysis highly supported a closer relationship between P. pallipes, P. trisulcus, P. meridionalis and P. fuscipennis, RAxML calculated only a low bootstrap value of 63% for this branching. Additionally, the position of the accession JN934379 within the P. pallipes clade was poorly supported in both analyses. The unidentified specimen PSC-7 from Pisa formed a highly supported sister branch to all other P. fulvicornis and P. schencki samples in both analyses (RAxML: 94%; MrBayes: 0.99), confirming the unknown status of PSC-7 whose species identification failed with 2.3% dissimilarity from other Psenulus species in a BOLD System search ( http://www.boldsystems.org; July 10th, 2018). All samples of the P. fulvicornis-Group formed a highly supported monophyletic clade (RAxML: 81%; MrBayes: 0.99). The samples of the P. fulvicornisGroup were subdivided in six clades, all of which were congruent with taxonomic boundaries based on morphology (Fig. 5): On the one hand, P. fulvicornis was split in two well supported clades in Central and SouthWestern Europe, with clade I including the samples from Salamanca (RAxML: 88%; MrBayes: 0.99), and clade II including the samples from Zurich and Northern Spain (RAxML: 86%; MrBayes: 0.92). On the other hand, the seven P. schencki accessions from Canada, Germany and Zurich formed the well-supported monophyletic clade III (RAxML: 86%; MrBayes: 1.0). Finally, the three Caucasian specimens (one attributed morphologically to P. fulvicornis and the others to P. schencki) represented isolated branches (clades IV, V, and VI) whose relationships with one another and with Central European specimens remained unresolved.

Table 1.

Cox1 polymorphism in the Psenulus fulvicornis-Group and within each of the statistically well supported Central and SouthWestern European clades I-II (P. fulvicornis) and III (P. schencki) based on a 561 bp alignment.

Fig. 3.

Main discriminant characters between females of Psenulus fulvicornis (Schenck, 1857) and P. schencki (Tournier, 1889). (A) Propodeal surface and (B) pygidial area of P. fulvicornis from the City of Zurich, Switzerland (D. Frey & A. Zanetta leg.; M. Řiha det.; R. Neumeyer conf.; WSL coll; GenBank accession Nr. KY039438). (C) Propodeal surface and (D) pygidial area of P. schencki from the City of Zurich (25.05.2015; D. Frey & A. Zanetta leg.; M. Řiha det.; R. Neumeyer conf.; WSL coll; GenBank accession Nr. KY039439). In P. fulvicornis, the lateral surface of the propodeum (A) has short crosswise carinas which gives the texture coarse appearance. This character is unique among females of Central European Psenulus. In P. schencki such crosswise carinas are lacking (C), and the texture is finer. The pygidal area is longer and broader in P. fulvicornis (B) than in P. schencki (D), but this character can be difficult to observe in certain specimens.

Fig. 4.

Cox1 polymorphic sites within a 561 bp alignment of the P. fulvicornis-Group. Vertical numbers indicate the position of the segregation site in the alignment. Of 561 sites 539 were invariable, 10 singleton variable, and 12 parsimony informative sites. At position 543 of the alignment were three mutations, and replacements at the positions 178 and 472 were nonsynonymous (asterisks). Specimens are arranged according to their genetic distance. The Caucasian specimen PSC-11 was divergent from P. fulvicornis and P. schencki but it was similarly distant to both. Specimens are labelled with the specimen ID, the GenBank accession number or the Barcode Identification Number of the BOLD System (see supplementary Table S1). Rectangles include specimens belonging to the clades I–VI in the phylogenetic analysis (Fig. 5).

Review of museum and private collections

No P. fulvicornis were found among P. schencki accessions in Swiss museums and private collections. A total of 221 museum and eight specimens from private collections could be reviewed, of which 98% had an indication of the sampling site on the label (see Appendix). All major Swiss museum and private collections harbouring P. schencki could be visited apart from the Naturmuseum Luzern, which has three specimens that were not accessible for visitors in February 2018. The Bündner Naturmuseum, Muséum d'histoire naturelle Neuchâtel, Musée de la nature de Sion, Museo cantonale di storia naturale and Naturmuseum St. Gallen did not harbour P. schencki accessions.

Psenulus fulvicornis new to Switzerland

Surveys of urban arthropod communities in northern temperate cities frequently reveal alien species that originate from warmer climates (e.g. Germann et al., 2015; Frey et al., 2016; Zanetta et al., 2016). The urban heat island effect, which provides warmer temperatures in the city when compared to the rural surroundings, and, presumably, the availability of nesting sites (e.g. Sambucus and Rubus wood, and artificial nests) and feeding resources (e.g. aphids) in large allotment garden areas, could explain the finding of P. fulvicornis in an urban garden lot (e.g. Hall et al., 2017). Similarly, in Central Europe, P. schencki occurs in settlement areas and gardens (Blösch, 2000). Finally, discovering new cavity-nesting hymenopterans in anthropogenic habitats such as gardens may not be surprising since nests can be easily transported by humans over distances that are far beyond the dispersal capacity of adults. However, the naturalization status of P. fulvicornis in Switzerland remains unclear. This inconspicuous species is relatively rarely observed throughout its distribution range and it could have simply been overlooked. Yet our review of P. schencki accessions in Swiss museum and private collections indicates that P. fulvicornis could represent a recent colonizer, with to date, only two known observations: the two females that were caught in warm lowland habitats of the Cantons of Zurich and Geneva.

Taxon identification using cox1 sequence information

While at least females of P. fulvicornis and P. schencki can be readily identified morphologically (Fig. 3), we found that neither SNPs (Fig. 4) nor the phylogeny (Fig. 5) could consistently separate the two taxa at the large geographic scale investigated. Nevertheless, specimens sampled in Central Europe belonged either to the well supported clades II (P. fulvicornis) or III (P. schencki) and may also be separated by several diagnostic SNPs (Figs 4, 5). The existence of a such spatial pattern in cox1 sequence divergence within and among the taxa needs, however, further corroboration.

Phylogenetic relationships within the Psenulus fulvicornis-Group

We found that the P. fulvicornis -Group formed a highly supported monophyletic branch within the Central European Psenulus (note, however, that our phylogeny lacked P. chevrieri (Tournier, 1889) and the rare P. berlandi de Beaumont, 1937). This result confirms the morphology-based hypothesis of their close relationship (Schmidt, 1971; Schmid-Egger, 2002) and is in contrast to former authors, which grouped P. fulvicornis with P. fuscipennis (Schenck, 1861; de Beaumont, 1939). However, we found polyphyly within the P. fulvicornis -Group, which was split in at least three well supported clades in South-Western and Central Europe. All clades were coherent with the taxonomic classification of the samples, which excludes imperfect taxonomy as a cause for polyphyly. The unresolved phylogenetic relationship between the Caucasian specimens and the South-Western and Central European clades may be interpreted as a sampling gap (i.e. incomplete sampling) in the eastern part of the distribution range of P. fulvicornis (Fig. 1) (Cho et al., 2011; Wiens & Tiu, 2012).

Fig. 5.

Maximum likelihood tree showing the phylogenetic relationships within Psenulus taxa based on cox1 and the GTR+G substitution model. All bifurcations with bootstrap values below 60% were collapsed. The bold black lines indicate bootstrap of at least 70% and the thick grey bifurcations 60–70%. Both Diodontus minutus (Fabricius, 1793) and Pemphredon lethifer (Shuckard, 1837) sequences root this tree. Specimens are labelled with the sample ID, their GenBank accession number or the Barcode Identification Number of the BOLD System, and the collection site if known (see supplementary Table S1). Six lineages I–VI were found within the P. fulvicornis-Group.

Previous studies have shown that many taxa considered as valid species are polyphyletic at the cox1 locus, especially when sampled comprehensively across (large) distribution ranges, and intra- and interspecific variability (Funk & Omland, 2003; Meyer & Pauley, 2005). Specifically, it was also found in hymenopterans (e.g. Jansen et al., 2009; Nicholls et al., 2012; Eimanifar et al., 2018). The rather small morphological and genetic differences (Figs 2, 5) suggest that P. fulvicornis and P. schencki could be interpreted as incipient species, which are not yet reciprocally monophyletic at the cox1 locus (i.e. incomplete lineage sorting; Funk & Omland, 2003). Similar findings have also been made in other putatively closely related Central European hymenopterans (e.g. Schmidt et al., 2015). Other possible reasons for polyphyly include hybridisation and mitochondrial DNA introgression (Funk & Omland, 2003): the geographic range overlap and potentially similar habitat requirements between P. fulvicornis and P. schencki could set the stage for occasional hybridisation, and Gauss (1974) indeed observed a putative P. fulvicornis female in copula with a P. schencki male in South Western Germany. Yet we did not find a genetic sign of interspecific gene flow here, and, moreover, other (nuclear) loci may be more effective to detect hybrids (Petit & Excoffier, 2009; Nicholls et al., 2012; Patten et al., 2015; Beresford et al., 2017).