Pteromalidae are known from all regions in Germany and collecting these tiny wasps with an average (body) size of roughly 2-3 mm can be done in many ways. There is already some helpful literature about collecting chalcidoid wasps, including, for example, Noyes (1982), Bouček (1988), Grissell and Schauff (1997), Noyes (2021), which also reference other resources for further reading. Here, only a short synopsis of sampling and preparation methods will be given. Due to their size, Pteromalidae can hardly be observed and sampled on sight; therefore, the most effective methods of sampling include active methods like sweep netting, as well as trapping and rearing. Sweep netting is an easy, widely applied method and if done mindfully, it can give some additional information on plants and habitats, from where specimens have been sampled for biological implications. It is generally advised to use a sturdy net, with strong fabric to withstand beating through vegetation like meadows and shrubs. Noyes (1982) designed a very suitable net for this purpose, being subtriangular in form and heavily reinforced. Its form allows it to be swept just above the ground, catching species, largely dwelling close to the ground. It is also fitted with a screen to avoid large amounts of vegetation and debris getting inside the net, making it easier to recover caught specimens. Collecting specimens out of the net sack is best done by using an aspirator with a long tube to precisely pick out individuals. Keeping the net opening away from the sun and upright forces specimens to walk to the top of the net, allowing for easier aspiration. As a euthanising agent, pure and highly concentrated (99.9%) ethanol is advised, as it preserves the specimens and guarantees DNA stability for subsequent molecular analysis. Other effective, but rather rare, active sampling methods for chalcidoids include suction samplers, vegetation beating, light tower collecting and advanced chemical techniques, like canopy fogging.

In contrast to active sampling methods, trapping techniques allow for a neutral assessment of biodiversity, free of collector bias, for example, for monitoring purposes, but with the caveat of catching non-target organisms, requiring additional sorting time to separate the groups of interest. The most widely used method of trapping flying insects is the malaise trap (Townes 1972). Correctly set up, this flight interception trap will sample autonomously, requiring little maintenance, but only regular changing of the collecting jar. Intervals of changing the jar depend on the scientific question, but it is advised to not let too much time pass, because evaporation, influence of the sun and temperature might damage collected material when left in the collecting liquid for too long. Again, pure and highly concentrated ethanol is advised as the euthanising agent, but it might be diluted to about 80% for the purpose of biomass assessment (Ssymank et al. 2018). Other flight interception traps, like window traps are also reliable methods to catch microhymenoptera. Colour pans are a more common way of collecting parasitoid wasps as well, although they need more attention and care when being deployed in the field and are more selective due to their attracting properties (Buffington et al. 2021). Light trapping is a rather unexplored method, but might also be useful to assess pteromalid diversity. Pitfall traps are rarely used, but can yield rarely collected, ground dwelling species. When trying to assess the complete diversity of parasitoid wasps, it would be recommended to use several sampling techniques.

In order to gain specific biological information about a species, rearing is an ideal method; however, it is fairly time consuming. Everything potentially harbouring parasitoids can be collected, such as plant galls, dead wood, insect pupae, egg masses, parts of plants, potential hosts and many more. Collected material should be kept in containers that allow for enough airflow to prevent mould, but preventing the emerging wasps from escaping. Tightly sealable plastic boxes fitted with fine mesh have proven to be an easy and cheap way to store material. In some cases, it can be useful to keep the material in the container slightly moist, to keep the insects inside from drying out and dying. Material should ideally be stored in a natural climate in order to allow parasitoids to develop normally. Containers need to be checked regularly to catch emerging specimens alive, in order to preserve them and avoid collapsing body parts when drying in air. If possible, it is advised to separate hosts to gain more biological information, because often several potential host taxa might be present.

Storing samples can easily be done by leaving them in pure and highly concentrated ethanol. If molecular analyses are to be performed, freezing the samples at -20°C is advised, although this might affect morphological studies negatively (Marquina et al. 2021).

Before drying and mounting, specimens often have to be presorted. This can be especially tedious with trapping samples containing many non-target individuals. Automatic sieving (Buffington and Gates 2008) has proven to be extremely useful and effective, presorting specimens by size. Pteromalidae will mainly be found in the smaller fraction and can be sorted out more easily, without the risk of damage while sieving, due to their strong sclerotisation.

It is uncommon to slide-mount Pteromalidae; instead, the most widely used method of mounting is card mounting. For this method, it is necessary to dry specimens gently before mounting, either by using critical point drying (Gordh and Hall 1978) or chemical drying (Heraty and Hawks 1998), preventing collapsing bodies and deformed extremities. There are several techniques for card mounting, depending on preference (Noyes 2021). Gluing specimens on the point of triangular cards or on top of rectangular cards appears to be best practice as it allows us to view the specimen from all sides. This is especially true for point mounting, although the specimens are more in danger of being damaged when handled without care. Deans (2018) provides an in-depth review of potential glues for entomotaxy; however, with no clear recommendation. Choice is therefore dependent on the needs of the user. Although not recommended by Deans (2018), shellac is used by the authors to great effect, as it has the ideal properties and mounting results are excellent. Mounted specimens should be stored in air-tight insect drawers under dark and climate stable conditions in order to preserve colours and avoid damage.

Species now part of Pteromalidae have already been described by Carl von Linné as early as 1758 in his “Systema naturae”, with many scientists following in the centuries after. Dalman, a Swedish naturalist, first coined the taxonomic term “Pteromalini” in 1820 as a family, including many taxa today being part of other chalcidoid families or other superfamilies (Dalman 1820). In comparison to other parasitoid hymenopteran families, Pteromalidae are rather well recorded from Germany, but actual species numbers can only be estimated. A review on the historic research of parasitoid wasps in Germany is given by Vidal (2005), including the pre-Linean era. Here, a brief history of work on Pteromalidae in Germany after Linné will be given in the following, although it has to be noted that larger taxonomic and systematic works on Pteromalidae were mostly conducted outside of Germany (Walker 1839a, Walker 1839b, Thomson 1876, Thomson 1878, Ashmead 1904, Graham 1969, Bouček 1988).

Johan Christian Fabricius, a Danish zoologist, mostly focused on studying arthropods and was probably the first to conduct work on Chalcidoidea and Pteromalidae in Germany. Like his mentor Carl von Linné, Fabricius dedicated much of his work to describing species in several large monographs (e.g. Fabricius 1775, Fabricius 1787, Fabricius 1793). This also included several species of Chalcidoidea, part of the German fauna, for example, the morphologically remarkable Leucospis dorsigera Fabricius, 1775, the striking pteromalid Cratomus megacephalus (Fabricius, 1793) or Cheiropachus quadrum (Fabricius, 1787), which was described from material collected in Halle, Germany. Fabricius curated several collections; therefore, specimens he described and worked with are dispersed between different institutions, with the largest parts housed in the Zoological Museum in Copenhagen, the Zoological Museum at Kiel University and the Natural History Museum in London (Tuxen 1967). Although his descriptions were not comprehensive and hardly informative by today’s standards, it can only be imagined how difficult it must have been to study organisms of few millimetres at that time. This achievement is also highlighted by contemporary artwork, as is shown in Panzers "Fauna insectorum Germaniae initia", a book series of German insects, published between 1796 and 1813.

One of the first to publish a comprehensive list of German parasitoid wasps, included in a record of insects in general, was Nikolaus Joseph Brahm (1790). Only 20 species of Ichneumon are listed in his work, a genus which, at the time, included many parasitoid wasp taxa, which belong to several different superfamilies today. Therein included is also Ichneumon puparum Linnaeus, 1758, known today as Pteromalus puparum (Linnaeus, 1758), one of the oldest formally described Pteromalidae by Linné. Brahm did not describe any species anew, but provided a rudimentary key and scarce notes on the biology of the Ichneumon wasps. In 1802, Franz von Paula Schrank published a regional list of Bavarian insects, also encompassing species of the Genus Ichneumon (Schrank 1802). Schrank also listed biological information for each species, where possible, but did not describe new species of Pteromalidae within his work.

In 1834, the remarkable German naturalist Christian Gottfried Daniel Nees von Esenbeck published his opus, the two volumes of “Hymenopterorum Ichneumonibus affinium monographiae”. In those monographs, Nees von Esenbeck provided descriptions and keys to a multitude of species, of which many, although synonymised over the years, are still part of the German fauna. Nees von Esenbeck’s important collection of Hymenoptera was reportedly severely damaged, but its remains are now part of the collection of the University Museum in Oxford, UK (Graham 1988). Seven years after Esenbecks publication in 1841, Arnold Foerster published the “Beiträge zur Monographie der Pteromalinen Nees.” adding to the work of Nees von Esenbeck, whom he praises"… as one of the greatest naturalists of our time…" (Foerster 1841). Within this, he gives a short historic overview of pteromalid research, lists known biological observations of different species and provides a key and descriptions to new species, regarded at that time to be part of Pteromalidae. Foerster followed this work up with several publications on the taxonomy and systematics of Chalcidoidea and Pteromalidae (Foerster 1856a, Foerster 1856b, Foerster 1868, Foerster 1878), describing many new species from Germany. His collection of parasitoid wasps is now part of the collection of the Natural History Museum in Vienna (Hörn 1928). Although the taxonomy and systematics of Pteromalidae and Chalcidoidea would change drastically since then, it is apparent that profound examinations were conducted even at that time, to shed light on those tiny wasps that "… allow us to take a deep look into the proceedings and liveliness of nature." (freely translated from Foerster 1841). Foerster’s work was later partly updated by the Austrian naturalist Karl Wilhelm von Dalla Torre, renaming some of his described species due to homonymy (Dalla Torre 1898).

At around the same time of Foerster’s workings, Julius Theodor Christian Ratzeburg also published on Chalcidoidea. Ratzeburg was a German naturalist mainly interested in forestry, but some of his work focused heavily on parasitoid wasps, especially with regard to forestry pests. Over the years, he published several volumes which included biological observations and species descriptions of chalcidoid wasps amongst other taxa (Ratzeburg 1844a, Ratzeburg 1844b, Ratzeburg 1848). Unfortunately, his collection, including type material, appears to have largely been destroyed during the Second World War, with only few remnants remaining in the Deutsches Entomologisches Institut (Königsmann 1964, Vidal 2005), now situated in Müncheberg.

Another noteworthy German hymenopterist partly working on Pteromalidae and Chalcidoidea at the beginning of the 20^th century was Otto Schmiedeknecht (e.g. Schmiedeknecht 1907, Schmiedeknecht 1909). His book chapter on the family Chalcididae in 1909 (Schmiedeknecht 1909) was critically received. It was viewed to be a faulty copy of Ashmeads’ comprehensive publication in 1904 on the superfamily of Chalcidoidea (Ashmead 1904) and was even being described as "… a tragedy and a comedy of errors" (Girault 1910). Nonetheless, Schmiedeknecht continued his work and, in 1930, provided an update of his extensive catalogue on the Hymenoptera of northern and central Europe, the first version of which he had published 16 years prior. Therein, he listed all occurring genera of Pteromalidae and provided comprehensive keys for their identification (Schmiedeknecht 1914, Schmiedeknecht 1930). His collection, although largely focused on Ichneumonidae and Anthophila, is severely torn apart, but an exhaustive account is given by Oehlke (1968) of its whereabouts.

In the middle of the 20^th century, the Italian field entomologist Vittorio Delucchi described many pteromalid species and genera from Western Europe including Germany. An encompassing account of his described species including literature is given by Baur (2001).Numerous of those species were described from holotypes collected in Germany, either by Delucchi himself or from historic collections, for example, Foerster or Schmiedeknecht. The whereabouts of Delucchis’ collection are also detailed in Baur (2001).

Marcus William Robert de Vere Graham should be named here as an entomologist who made the vastness of European Pteromalidae more widely accessible through his work. Especially noteworthy here is his monograph from 1969 on the Pteromalidae of north-western Europe (Graham 1969). Therein, he provides keys to subfamilies, genera and even species, with detailed information on type material, distributions and where available, biological information. His work is, to this date, mostly the first to consult when identifying Pteromalidae from Germany and Europe. Zdeněk Bouček was a contemporary of Graham, dedicating his research to the superfamily Chalcidoidea, with Pteromalidae being one of the main focuses of his work (Noyes 2005). A full list of his publications can be found in Noyes (2005), which, in great parts, is also relevant when studying the German pteromalid fauna. His and Jean-Yves Rasplus “Illustrated key to West-Palearctic Genera of Pteromalidae” has to be mentioned here specifically, as it is a beginner-friendly introduction to pteromalid identification, aided by line drawings and SEM images (Bouček and Rasplus 1991).

After the middle of the 20^th century, research focusing on and including Pteromalidae in Germany diversified, with some examples of different researchers and working groups listed in the fields of ecology (Abraham 1970, Kruess and Tscharntke 1994, Kruess 1996, Tscharntke et al. 2001), taxonomy (Werner and Peters 2018), morphology (Krogmann and Abraham 2003, Krogmann 2005, Vilhelmsen et al. 2010), phylogenetic analysis (Heraty et al. 2013, Peters et al. 2018), molecular studies (König et al. 2019) and autecology (Abraham and König 1977, Steidle and Schöller 1997, Ruther and Steidle 2000, Niehuis et al. 2013, König et al. 2015, Niedermayer et al. 2016, Tappert et al. 2017).

In 2001, the most recent species list of German Hymenoptera was published, listing 663 species of Pteromalidae (Vidal 2001). Since then, the number of species has been tracked by web-based databases, for example, the Chalcidoidea database (Noyes 2021) and the thereon-based Checklist of the German Chalcidoidea (Zoologische Staatssammlung München 2021), listing 734 pteromalid species or the German Barcode of Life taxon lists (German Barcode of Life Consortium et al. 2011), based on Fauna Europea (de Jong et al. 2014), listing 695 recorded species. The reasons for the discrepancy in species numbers are unknown, but it has to be speculated that the Chalcidoidea database was curated regularly until March 2019; therefore, it is potentially the best source for the most reliable species number. Only one species (Doczkal 2017) was not yet added, resulting in a total of 735 pteromalid species recorded from Germany to date.

In more recent history, the German Barcode of Life project (German Barcode of Life Consortium et al. 2011) was initiated, aiming to inventory the German fauna and flora in the form of a barcode database, also including Pteromalidae. With the advent of metabarcoding methods of insect material (e.g. Morinière et al. 2016, Toju and Baba 2018, Piper et al. 2019, Liu et al. 2020), barcoding databases will be heavily relied upon in the future, especially for the identification of morphologically diverse and difficult groups. This results in the necessity to fill the databases with correct and comprehensive data. Geiger et al. (2016) demonstrated that, in Germany, metabarcoding of malaise trap samples yielded an especially low identification success of about 40% for genus and species level in the two most abundant orders of insects, the Diptera and Hymenoptera. In Hymenoptera, especially, many of the alleged parasitoid barcodes could not be assigned to a genus or species identification. This, in combination with their ecological importance and potential use as indicator groups (Shaw and Hochberg 2001, Anderson et al. 2011), highlights the urgency to further advance the molecular databases and continue working on those elusive groups.

In a first step, we here present 41 newly-recorded species for the German fauna, collected and processed within the GBOL project. DNA barcodes are supplied to most of the presented species. In addition, more general information on the family Pteromalidae will be given to exemplify the importance to advance research efforts, further shedding light on this important taxon and parasitoid wasps in general.

The pteromalid specimens, used in this study, were collected in different localities throughout Germany (Fig. 2) between the years 2011 to 2021. The specimens treated here (n=122) were collected through various methods, mainly by malaise trapping (n=73), sweep netting (n=39) and others (n=10). Before laboratory procedures were conducted, all specimens were kept in 99.6% pure ethanol at -20°C.

Figure 2.  

Origin of material studied from Germany. © Bundesamt für Kartographie und Geodäsie, Frankfurt am Main, 2011; OpenStreetMap -contributors.

All molecular work was conducted in the State Museum of Natural History Stuttgart (SMNS), except for sequencing, which was done at Eurofins Genomics, formerly GATC Biotech AG (Germany, Ebersberg).

The DNA extraction protocol, including buffer recipes, was based on the protocol of Ivanova et al. (2006), but modified to suit the small size of the specimens and working with a Xiril Neon 100 pipetting robot. Differences to the original protocol of Ivanova et al. (2006) will be specified in the following. Due to the small size of the specimens, the whole body had to be used for DNA extraction. A semi-destructive approach was chosen to yield enough DNA for analysis, allowing for a morphological specimen voucher. By removing one of the hind legs, an opening was created for the lysis buffer to penetrate the heavily sclerotised body, otherwise hardly permeable for the buffer and the released DNA. Every specimen was submerged in 100 µl ProteinaseK and Insect Lysis Buffer (ILB) mixture within a 1.5 ml tube and incubated at 56°C for about 30 hours in a Thermoshaker, agitating the lysis mix at 200 rpm. Afterwards, the lysate was separated from the specimen and transferred to a 96 format deep well plate for DNA isolation. The isolation was performed by the pipetting robot, utilising a silica-based isolation system. Due to the initially larger amount of lysis buffer, 200 µl of binding mix was used to fix the DNA to the filter. Centrifugation steps from Ivanova et al. (2006) are not realisable in the robot and were substituted via a vacuum pump, removing the residue washing buffers from the membrane-bound DNA. The purified DNA was transferred to an elution plate, which was stored for a short time at -20°C until after the PCR had been conducted. Afterwards, long-term storage at -80°C is provided.

The PCR was conducted using different iterations of the FastGene® Optima Taq from Nippon Genetics. In total 25 µl were used for PCR reaction, including 4 µl eluate DNA and other components mixed to the manufacturers’ recommendation. Primers LCO1490 and HCO2198 (Folmer et al. 1994) were used with the PCR-protocol in the reaction, following the cycler protocol in Table 1. Purification and sequencing of the resulting PCR products was conducted by GATC Biotech AG, now Eurofins Genomics. Final sequences are deposited in the Barcode of Life Data (BOLD) Systems (Ratnasingham and Hebert 2007, Ratnasingham and Hebert 2013), as well as Genbank (Clark et al. 2015) and can be accessed via their respective IDs given in the material section of each specimen.

To match undescribed males to described females, the mean in between group p-distance was calculated with MEGA-X (Kumar et al. 2018). Standard errors were assessed by bootstrap (1000 replicates) and the rate variation amongst sites was modelled with a gamma distribution.

After lysis, specimens were washed in a soapy water solution to rid them of buffer residue. The solution was discarded and specimens were transferred to a 70% MEK-ethanol solution, which was substituted in steps by higher concentrations (90%, 95% and 99.6%) of MEK-ethanol over the course of several days. For drying and subsequent mounting, specimens were transferred to Hexamethyldisilazan (Heraty and Hawks 1998) and kept in open Eppendorf tubes until they were completely dry. The specimens were mounted on card points using shellac glue, labelled and stored in the entomological collection of the State Museum of Natural History Stuttgart (SMNS) for later identification. Each specimen was given a unique accession number of the format “SMNS_Hym_Pte_XXXXXX'' under which all collected information is stored in the SMNS collection Database “Diversity Workbench” (Triebel et al. 1999). Accession numbers are abbreviated in the text in the format “Pte_XXXXXX”

Morphological identification was conducted by multiple taxonomists, predominantly using a Leica M205 C stereomicroscope, with a measuring eyepiece. The main literature used for identification included Bouček and Rasplus (1991) for genus level identification and Graham (1969) for genus and species level identification. If available, more recent literature was used to identify specimens to species level. Information on the distribution outside of Germany and the biology of species was supplemented through consultation of the following references: Graham (1969), Bouček and Rasplus (1991), Noyes (2021). A list with all newly-recorded species is given in Table 2.

Our results were split into two sections within the manuscript. Firstly, within the taxon treatment section, we want to focus on the description of two previously-undescribed males of the species Rhicnocoelia impar (Walker, 1836) and Rohatina inermis Bouček, 1954. Both species are new records for Germany and are also treated in the faunistic part of our results. Our faunistic data are presented in the checklist section of the manuscript, where detailed information is given on the newly-recorded species and genera of Germany. The checklist is sorted by subfamily and Table 2 can serve as an overview.