Psenulus, Kohl, 1897

Frey, David, Gayubo, Severiano Fernández, Mokrousov, Mikhail, Zanetta, Andrea, Řiha, Martin, Moretti, Marco & Cornejo, Carolina, 2019, Phylogenetic notes on the rare Mediterranean digger wasp Psenulus fulvicornis (Schenck, 1857) (Hymenoptera: Crabronidae) new to Switzerland, Revue suisse de Zoologie 126 (1), pp. 27-42 : 32-34

Psenulus sp.

1 female; La Sterpaia, Parco San Rossore ( IT: Pisa); 31.05.2005 - 09.06.2005; L. Strumina leg.; S. F. Gayubo det.; DNA-ID; PSC-7; GenBank accession Nr . MG 872069 .

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 S 1 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 S 1 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. S 1 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. fulvicornis - Group 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 South- Western 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.

Position of segregation sites A G G T T G A A A T A A G T A A T A C T A G - - - - - - - - - C - - - - - - - - - - - - I - - - - - A - - - C - - - - - - - - - - - - - - - - A - - - - C - G - - - - A - T - - - - - - - A - - - - C - G - - - - A - T - - - II - - - - A - - - - C - G - C - - A - T - - - - - - C A - - - - - - - - - - - A - T - G T VI - - - - A - G - G - - - - - - - A - - C - - - - - - A - G - G - - - - - - - A - - C - - - - - - A - G - G - - - - - - - A - T C - - - - - - A - G - G - - - - - - - A - T C - - III - - - - A - G - G C - - - - - - A - - C - - - - - - A - G - G C - - - - - - A - - C - - - - - - A - G - G C - - - - - - A - - C - - - - - - A - G - G C - - - - - - A - - C - - - - A - A A - G - C G - A - - G A G T - - A IV T A - - A - - G - - G - - - T - A - T - - A V * * 12 12 45 45 108 108 111 111 117 144 144 168 168 * 178 178 192 192 213 213 243 243 270 270 285 285 321 321 330 330 456 456 * 472 472 474 474 486 486 496 496 516 516 543 543

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.

Figures

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. 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).

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).

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.

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.