Ptomaphagus thebeatles n. sp., a previously unrecognized beetle from Europe, with remarks on urban taxonomy and recent range expansion (Coleoptera: Leiodidae)

Anthropogenic environmental change is leading to changes in distribution for many organisms. While this is frequently discussed for prominent organisms of high conservation value, the same is true for the many cryptic species that rarely figure in debates on the human impact. One such cryptic taxon is the European Ptomaphagus sericatus (Chaudoir, 1845) and related forms. During a citizen science expedition in the Vondelpark, Amsterdam, the Netherlands, we obtained two forms of this species complex. We placed the examination of these specimens in the context of a re-analysis of the species group, and, using DNA barcoding and genital study on material collected thoughout Europe, found that the P. sericatus species complex consists of three distinct, partly sympatric species, one of which was previously undescribed. On the basis of collection data, at least two species, P. medius and P. thebeatles sp. n., show signs of having recently undergone (possibly anthropogenic) range changes, with P. medius even reaching North America. We describe P. thebeatles sp. n.; we raise two subspecies, viz. P. sericatus sericatus (Chaudoir, 1854) and P. sericatus medius (Rey, 1889) to the level of species, and designate a neotype for the former; we identify P. dacicus Jeannel, 1934 and P. pyrenaeus Jeannel, 1934 as junior synonyms of P. sericatus , and P. compressitarsus (Rey, 1889) as a junior synonym of P. subvillosus Goeze, 1777; we identify P. septentrionalis Jeannel, 1934 and P. miser (Rey, 1889) as junior synonyms of P. medius ; we designate lectotypes for P. medius and P. miser .


Introduction
Although the coleopteran fauna of western Europe has been studied intensively for over two centuries and, as it has always been a popular group of insects, their species-level taxonomy has been elucidated to a relatively high degree of accuracy, certain taxa remain poorly understood. One such group is the genus Ptomaphagus Hellwig, 1795 (Leiodidae: Cholevinae; Fig. 1), which consists of a large number of species, all belonging to the nominal subgenus, that are externally very similar and even in male and female genitalia display only subtle interspecific differences. For this reason, a revision of the subgenus Ptomaphagus s.str. is currently under way (Schilthuizen et al., unpubl.). One particularly controversial group within the genus is the species complex surrounding P. sericatus (Chaudoir, 1845). Multiple intraspecific taxa have been described (Jeannel, 1936), which have variously been interpreted as good species, subspecies, or taxonomically insignificant variations (Sokolowski, 1956;Szymczakowski, 1956Szymczakowski, , 1971Zwick, 1983;Schilthuizen, 1989).
In this paper, we present a separate taxonomic treatment of this species complex. The impetus for this paper was a recent 'taxon expedition' , a field course / field trip aimed at discovering and describing new species together with citizen scientists (Schilthuizen et al., 2017). Although taxon expeditions are normally held in remote, poorly studied locations, this particular expedition was to the Vondelpark of Amsterdam, and was meant to highlight the fact that even in the heart of the capital of one of the best studied countries in the world, new discoveries can be made. During this expedition, besides undescribed parasitic Hymenoptera ( Van Achterberg et al., 2020), an undescribed member of the P. sericatus complex was found. This species had already been recognised previously by one of us (M.S.), and this opportunity was taken to work out a revision of the controversial P. sericatus species complex. new beetle from an urban park in amsterdam Downloaded from Brill.com04/04/2022 02:44:28PM via free access

Molecular phylogenetic analysis
Two individuals of Ptomaphagus thebeatles n. sp., collected during the Vondelpark Taxon Expedition, were treated as follows. Total genomic DNA was isolated from the whole specimen using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Barcoding PCR was conducted by amplifying the mitochondrial gene encoding COI as previously described (Maestri et al., 2019). The following primers, Nancy and Ron (Simon et al., 1994), tailed with adaptors to allow indexing prior to MinION library preparation were used. Nancy: To incorporate index sequences and allow the sequencing of multiple samples in each MinION flow cell, a second round of PCR was carried out in a 100 μL reaction volume using 48 μL of the purified COI-PCR amplicons from the first round (0.5 nM), 2 μL of indexed primers provided in the EXP-PBC001 kit (ONT), 0.25 mM of each dNTP, 1× Herculase II reaction buffer, and 1 μL (20 U/μL) of Herculase II fusion DNA polymerase. The amplification profile consisted of an initial denaturation step (3 min at 95°C) followed by 15 cycles of 15 s at 95°C, 15 s at 62°C, 30 s at 72°C, and a final extension for 3 min at 72°C. Indexed PCR products were purified using 0.8X (80 μL) AMPureXP beads (Beckman Coulter), quantified as described above and pooled in equimolar concentrations.
Then, 1 μg of pooled amplicons was used to prepare sequencing libraries with the SQK-LSK109 DNA Sequencing kit (ONT) according to the manufacturer's instructions. The library was loaded on a FLO-MIN106 flow cell (R9.4.1 Figure 1 Ptomaphagus thebeatles n. sp., habitus. Drawing by Erik-Jan Bosch. All other samples were treated as follows. DNA extractions were performed on individual legs and/or abdomens using the NucleoMag 96 Tissue kit (Macherey-Nagel Gmbh & Co., Düren, Germany) on the KingFisher Flex magnetic particle processor (Thermo Scientific). The KingFisher Flex protocols were slightly adjusted from the manufacturer's protocol (http://www.thermoscientific.com) to include longer mixing of beads with lysed sample and a drying step before elution. A volume of 150 μl was used for elution. PCR reactions contained 18.75 μl ultrapure H2O, 2.5 μl 10x PCR buffer, 1.0 μl 10 mM of each primer, 0.5 μl 2.5 mM dNTPs and 0.25 μl 5U Qiagen Taq. We amplified part of the cytochrome oxidase I gene of mtDNA using the primers 'Ron' , 5'-GGAT-CACCTGATATAGCATTCCC-3' , and 'Nancy' , 5'-CCCGGTAAAATTAAAATATAAACTTC-3', of Simon et al. (1994). PCRs were performed using an initial denaturation step of 180 s at 94°C, followed by 40 cycles of 15 s at 94°C, 30 s at 50°C and 40 s at 72°C, and ended with a final extension of 300 s at 72°C and pause at 12 °C. Bi-directional Sanger sequencing was performed at either Macrogen (http://www. macrogen.com) or BaseClear (http://www. baseclear.com).
Sequences were edited manually (primers trimmed off and obvious reading errors corrected) using Sequencher 4.10.1 (Gene Codes Corporation).
In addition to the sequences we generated ourselves, we also obtained Ptomaphagus s. str. sequences available in GenBank and BOLD (Barcoding Of Life Database; www. boldsystems.org). An overview of all the specimens (and their details) for which we have DNA sequences is given in supplementary table S1.
Sequences were aligned in Geneious v.11.1.3, using the MUSCLE algorithm and default settings. Since we used a dataset consisting only of partial COI sequences and therefore not suitable for thorough phylogenetic analysis, we subjected this dataset to a relatively simple analysis, intended primarily for separating the species in the P. sericatus complex from each other and from the rest of the subgenus. The alignment (consisting of 137 sequences belonging to 12 putative taxa) was subjected to a maximum-likelihood analysis using PhyML as incorporated in Geneious v.11.1.3 (Guindon et al., 2010), with a GTR substitution model, fixed proportion of invariable sites, 4 substitution rate categories, estimated gamma distribution, and 100 bootstrap replicates. Trees were displayed and manipulated in FigTree v.1.4.2, and Ptomaphagus (Adelops) hatchi Jeannel, 1933 was used as outgroup to root the trees.

Systematic part
The group comprising Ptomaphagus sericatus (Chaudoir, 1845) and related forms is composed of several taxa that are widespread and common throughout Europe. Some of these have variously been overlooked or misinterpreted. A brief overview of their chequered taxonomic history is therefore warranted.
It is probably best to begin with Jeannel's (1934Jeannel's ( , 1936 (Chaudoir, 1845), (ii) the Swedish P. sericatus septentrionalis Jeannel, 1934, and (iii) the West European P. sericatus medius (Rey, 1889). He also presented one variety, miser (Rey, 1889) of southeastern France. Jeannel (1936), however, did not clearly specify to which subspecies he considered var. miser to belong, writing that "miser of eastern France is a small-sized variety, which is found mixed with medius, but which, curiously, has the characteristics of the typical sericatus" (our translation). Sokolowski (1949Sokolowski ( , 1956 and Szymczakowski (1971) claim that the differences between P. s. sericatus and P. s. medius are sufficient to raise medius to the status of separate species. For Central Europe, therefore, they distinguish just two species, P. sericatus (of the highlands) and P. medius (of the lowlands). Zwick (1983), however, casts serious doubt on the existence of multiple taxa, stating that he cannot recognize consistent differences in the aedeagus and therefore considers all Central European material as P. sericatus. Schilthuizen (1989Schilthuizen ( , 2010, by contrast, reports that the two species are easily distinguishable and sympatric in the Netherlands, and that, moreover, P. sericatus s.str. is a recent arrival in that country. The current consensus (Perreau, 2000), however, seems to be that P. sericatus consists of two subspecies, P. sericatus sericatus and P. sericatus medius, that P. sericatus septentrionalis has been ignored since Jeannel (1936), and that miser is a variety of medius with no taxonomic validity. As we show below, aided by molecular phylogenetics, the complex forms a monophyletic group of three genetically and morphologically distinct species, which are sympatric in parts of their range, and have recently undergone significant range shifts. The species are dealt with in detail below, but here follows a synopsis: -P. thebeatles n. sp. occurs across southern Europe, from the Caucasus to the Iberian peninsula. It is sympatric, in part of its range, with P. sericatus and P. medius. Its arrival in the Netherlands is, based on collection label dates, probably of relatively recent date. This new species has always been overlooked and usually interpreted as P. sericatus. -P. sericatus (Chaudoir, 1845) is a mainly central and east-European species. In part of its range, it is sympatric with P. thebeatles and with P. medius. Some records of P. sericatus from the literature probably apply to P. thebeatles. P. pyrenaeus Jeannel, 1934 is here considered a junior synonym of P. sericatus. -P. medius (Rey, 1889) is a good species from western and northern Europe, covering large parts of Scandinavia, the British Isles, France, the Netherlands, Spain, and Germany. In France and the Netherlands, it is sympatric with P. thebeatles. It has recently spread into Hungary, where it is sympatric with P. sericatus. It has also recently expanded westward into Canada. P. miser (Rey, 1889) and P. sericatus septentrionalis Jeannel, 1934 are junior synonyms of P. medius. Crucial for this resolution was the study of the type series of Claudius Rey in the Musée des Confluences in Lyon. Rey (1889) described three taxa from the P. sericatus complex, namely, P. medius, P. compressitarsis, and P. miser, the latter two having been considered junior synonyms of P. medius by Jeannel (1934Jeannel ( , 1936, and later authors. For clarity, we here provide the results of our study of all the specimens in Rey's type series: 1. Labelled as "C. compressus" ([sic]; probably an earlier, manuscript version of the published name Catops compressitarsus): -1 female without any labels (therefore, from "région Lyonnais sensu lato"; Harold Labrique, pers. comm.). Since Jeannel (1934Jeannel ( , 1936 stated, for P. compressitarsis (sic): "type: Lyon", we consider this single specimen schilthuizen et al.
the lectotype for P. compressitarsus and have labelled it as such. However, the specimen is, in fact, a P. subvillosus, which renders P. compressitarsus a junior synonym of P. subvillosus (syn. n.). 2. Labelled as "C. medius": -1 male with a pale blue round label, which, according to Rey's colour scheme, denotes it was collected in the Pyrénées Orientales (Harold Labrique, pers. comm.). Upon dissection, this specimen indeed has an aedeagus that places it within our interpretation of P. medius. Rey (1889) reports four collection localities for his medius, one of which (Collioure) is in the Pyrénées Orientales and probably concerns this specimen. Jeannel (1934Jeannel ( , 1936 states, however: "type: Lyon". Jeannel did not see Rey's material and we therefore consider Jeannel's statement not as a lectotypification, but only as a restriction of the type locality, which allows us to designate this specimen (the only "true" P. medius in the type series --see below) as lectotype for Catops medius Rey, 1889. We have labelled it as such. -1 male without any labels (i.e., from "région Lyonnais sensu lato"; Harold Labrique, pers. comm.). We dissected this specimen and based on its aedeagus it proved to be a P. 3. Labelled as "C. miser": -2 males without any labels (therefore, probably from "région Lyonnais sensu lato"; Harold Labrique, pers. comm.). We dissected these specimens and by their aedeagi identified them as P. medius. We selected one of these as lectotype of Catops miser Rey, 1889 and labelled it as such. -2 females without any labels (therefore, probably from "région Lyonnais sensu lato"; Harold Labrique, pers. comm.). These specimens belong to the sericatus-group. However, as they are females, they cannot be unambiguously identified to species level. -1 female with the label "Var". This specimen belongs to the sericatus-group. However, as it is a female, it cannot be unambiguously identified to species level. Given the external similarity of the three species, we first provide a general description for the P. sericatus complex as a whole, and then treat each of the three constituent species, giving cross diagnoses only. In the cross diagnoses, only those traits are mentioned for which we could detect interspecific differences, i.e., the aedeagus and the antennae only. We could not find species-specific differentiation in the spiculum gastrale, the spermatheca, or any other characters. 4. General description for the sericatus-group as a whole: Habitus: small (2.4-3.2 mm), long-oval to cuneiform, relatively flat; pronotum as wide as or slightly wider than the elytra; elytra in a gentle curve narrowed apically, their greatest width around one-sixth from the basis; the pronotum deep brown, the elytra often slightly brighter reddish brown. Eyes: relatively small, occupying c. 50% of the distance between the occipital carina and the antennal basis. Pronotum: rounded laterally, greatest width around one-fifth from the basis, new beetle from an urban park in amsterdam Antenna: slender, longer than the pronotum, last antennomere as dark as the previous ones, antennal club darker brown than the basis; 3rd antennomere shorter and narrower than the 2nd. Protarsus: dilated in the male, the first 4 tarsomeres jointly 2.5 times as long as broad. Metatibia: straight in the male. Spermatheca: shaped like a narrow "J". Aedeagus: slender, apically gradually narrowed into a symmetric, angular, pointed apex; in lateral view either gradually bent ventrad or with a subapical sinuosity. Genital tergite: Spiculum gastrale long, linear, more than ten times as long as in the middle wide. Caudally widened to a rounded hammer-head shape. While we were able to discern distinct interspecific differences in the shape of the aedeagus, we have failed to detect interspecific differences in the female spermatheca. However, we cannot exclude the possibility that future studies will reveal diagnostic characters in the spermatheca. Despite subtle differences in the antennal segments, it proved impossible to unambiguously recognise females morphologically. This, plus the fact that most sequenced individuals were deliberately chosen to be males, makes that we lack sufficient reliably identified female material to make pronouncements on the presence or absence of spermathecal differentiation.

Materials and methods
Material from the citizen science expedition From August 4th until August 9th 2019, a 'taxon expedition' (Schilthuizen et al., 2017) was organised in the 'Koeienweide' (52.3558°N, 4.8585°E), a restricted, ecologically managed part of the Vondelpark in the centre of Amsterdam. The area is surrounded by water, and the vegetation consists of reedland and grassland, and fringing strips of woodland consisting of willow, poplar, alder, and other characteristic species of Dutch peatbog areas. During this expedition, citizen scientists Where appropriate, we also included notes on material that we studied previously, from the Zoological Museum of Copenhagen (ZMUC), and the private collection of B.J. van Vondel, Hendrik-Ido-Ambacht (BJVV). We only list material for which the identity was confirmed by dissection of the genitalia and/or DNA-sequencing. Distribution maps were generated in SimpleMappr (Shorthouse, 2010).

Notes
The type, from Kiev, is presumed lost. Chaudoir's collection is held in Paris, but Jeannel (1934Jeannel ( , 1936 did not see the type, and subsequent authors (Sokolowksi, 1956;Perreau, 2000) have also been unable to locate it. Given that the identity of P. sericatus has been unclear, we therefore find it important to designate a neotype, from Kiev, the original type locality.

DNA barcodes
We have generated 11 DNA-barcodes for specimens from the south of France (Languedoc-Rousillon and Ariège; figs 6, 7). However, as these were generated with the internal 'Ron' and 'Nancy' primers (which avoid the amplification problems encountered with the 'Folmer' primers), they are less than 500 bp in length and therefore do not qualify for Barcode Identification Number (BIN). Genetic distance with P. thebeatles amounts to c. 3%. Unfortunately, we were unable to obtain DNA barcodes for material from eastern and central Europe.  Rey, 1889(Rey, 1889; syn. n.

Notes
The existence of this previously unrecognized species is probably one of the causes for much of the confusion regarding the sericatus-complex over the past century. The shape of the aedeagus of P. medius is strikingly different from that of P. thebeatles, and workers who had access to many P. thebeatles specimens would have considered these as P. sericatus and as different from P. medius at the species level. On the other hand, researchers whose image of P. sericatus was formed mostly by specimens of the true P. sericatus, of which the aedeagus shape is somewhat intermediate between P. thebeatles and P. medius, may have been more dubious about its specific status.

DNA barcodes
We have obtained 16 closely-related DNAbarcodes for specimens from France, the Netherlands, and Germany ( fig. 7), most of which were confirmed with genital dissection. These sequences were either obtained from GenBank or generated with the internal 'Ron' and 'Nancy' primers (which avoid the amplification problems encountered with the 'Folmer' primers), and less than 500 bp in length. In both situations, they do not qualify for a Barcode Identification Number (BIN) to be determined. Genetic distance with P. sericatus amounts to c. 3%.

Discussion
Our study, which was partly conducted by citizen scientists in an urban park in Amsterdam, shows that there is scope for urban taxonomy: primary taxonomic studies carried out in urbanised areas by their inhabitants. In fact, during the same field trip that yielded P. thebeatles, we also discovered and described a new species of braconid wasp (Van Achterberg et al., 2020a). Many invertebrate taxa are so poorly studied, even in densely populated areas and even in well-studied countries like the Netherlands, that new species remain to be discovered in streets, backyards, and city parks. Moreover, recent shifts in distribution, caused directly or indirectly by human action, will lead to urban taxa that may be new arrivals in the country in question ( Van Achterberg et al., 2020b). Indeed, the transatlantic expansion of P. medius was first detected in a private garden in Canada (S.B. Peck, pers. comm.). And, based on the distribution localities and dates, it seems likely that the new P. thebeatles has only arrived in the Netherlands in the past few decades, possibly as a result of climate change, without which it may have taken longer to have been noticed. Involving city inhabitants in these studies and highlighting them to the local authorities may serve several benefits (Schilthuizen et al., 2017). First, it may raise the conservation value of the urban greenspace where the new species is discovered. Second, it makes urbanites aware of the undiscovered biological richness of their immediate surroundings. Finally, it increases awareness about the field of taxonomy and the gaps in our knowledge for large parts of our local biodiversity.
with organising material from Naturalis. Dr. Jan van Tol advised on matters of typification. Some of the DNA sequences were generated by Roland Butôt, Jessy Dommershuijzen, Cindy Scholte, Iris Sipman, Devi van der Horst, and Renske van Wijk. Erik-Jan Bosch is kindly acknowledged for his beautiful drawing for Fig. 1. Remco Daalder, and other urban ecologists of the municipality of Amsterdam were helpful in assisting with the organisation of the Vondelpark expedition by Taxon Expeditions, which allowed the collecting of the holotype and the description of P. thebeatles n. sp. SEM-photos were prepared with the help of Roel van Elsas, Vrije Universiteit, Amsterdam. This research received support from the SYNTHESYS Project http://www.synthesys.info/ which is financed by European Community Research Infrastructure Action under the FP7 "Capacities" Program, and from the Uyttenboogaart-Eliasen Foundation. An earlier version of this paper was improved by comments from Jan Ružička and Javier Fresneda. We acknowledge two anonymous reviewers, whose comments helped improve the paper.