The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of cuckoo wasps, with the description of new species from the Iberian Peninsula (Hymenoptera, Chrysididae)

Abstract Background DNA barcoding technologies have provided a powerful tool for the fields of ecology and systematics. Here, we present a part of the InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of cuckoo wasps (Hymenoptera, Chrysididae) dataset representing 144 specimens and 103 species, covering approximately 44% of the Iberian and 21% of the European fauna. The InBIO Barcoding Initiative (IBI – DNA Barcoding Portuguese terrestrial invertebrate biodiversity) aims to fill the barcoding gap for the terrestrial invertebrate taxa. All DNA extractions are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and specimens are deposited in the University of Mons collection (Belgium) and in the Natur-Museum in Lucerne (Switzerland). New information This dataset increases the knowledge on the DNA barcodes and distribution of 102 species of cuckoo wasps. A total of 52 species, from 11 different genera, were new additions to the Barcode of Life Data System (BOLD), with DNA barcodes for another 44 species added from under-represented taxa in BOLD. All specimens have their DNA barcodes publicly accessible through the BOLD online database. Nine cuckoo wasp species are newly recorded for Portugal. Additionally, two new species for science are described: Chrysiscrossi Rosa, sp. nov. from southern Portugal and Hedychridiumcalcarium Rosa, sp. nov. from eastern Spain. Several taxonomic changes are proposed and Hedychrumrutilans Dahlbom, 1845 is found to consist of two different taxa that can be found in sympatry, Hedychrumrutilans s. str. and Hedychrumviridaureum Tournier, 1877 stat. nov. Stilbumwestermanni Dahlbom, 1845 stat. nov. is confirmed as distinct from Stilbumcalens (Fabricius, 1781), with the latter species not confirmed as present in Iberia; barcoded Stilbum material from Australia is distinct and represents Stilbumamethystium (Fabricius, 1775) sp. resurr.; Portuguese material identified as Hedychridiumchloropygum Buysson, 1888 actually belongs to Hedychridiumcaputaureum Trautmann & Trautmann, 1919, the first confirmed record of this species from Iberia. Philoctetesparvulus (Dahlbom, 1845) is confirmed to be a synonym of Philoctetespunctulatus (Dahlbom, 1845). Chrysislusitanica Bischoff, 1910 is confirmed as a valid species. Chrysishebraeica Linsenmaier, 1959 stat. nov. is raised to species status.


Introduction
In Europe, the diversity of cuckoo wasps is highest in the Mediterranean region, with relatively few species found in the north (Paukkunen et al. 2014, Paukkunen et al. 2015) and the British Isles (Morgan 1984).Chrysidids are more common in southern European countries in part due to their ecology, since most species are heliophilous and thermophilous, favouring warm and sunny habitats.Another reason is their reproductive biology, as the number of host species of bees and aculeate wasps is also greater in Mediterranean countries (Michez et al. 2019).
The total number of valid cuckoo wasp species is approximately 2,800 (Rosa 2017).Of this world total, about 480 have been recorded from Europe, plus 135 accepted subspecies, whose possible specific rank has yet to be evaluated (Mitroiu et al. 2015).In Portugal, a total of 130 species and four subspecies are known to date, but this number is likely to be far from the true total given the much larger number of species reported from neighbouring Spain (e.g.González et al. 1999, González et al. 2009, Mingo and Gayubo 1981, Mingo and Gayubo 1986).Mingo (1994) compiled the most recent faunistic survey for the Iberian Peninsula, including identification keys.In this important monograph, Portuguese species are not clearly separated from Spanish ones.This volume is a valuable guide for beginners, yet includes only 170 species for the Iberian Peninsula, overlooking many of the species described or published from Spain.The real number of species exceeds 230 known taxa.However, new field research on Portuguese bees and aculeate wasps (e.g.Rosa et al. 2015, Rosa et al. 2015, Rosa and Vårdal 2015, Rosa and Xu 2015, Baldock et al. 2018, Baldock et al. 2020, Cross et al. 2021) has reinvigorated work on the Iberian cuckoo wasp fauna and a new illustrated catalogue of the Portuguese fauna is in preparation, including new records for the country and for Europe.
Despite the fact that the Iberian Chrysididae fauna is one of the richest in Europe (Mingo 1994), this fauna has essentially never been investigated using molecular tools, with only a handful of DNA barcodes sequences available from a small selection of species (e.g.Pauli et al. (2019)).To a certain extent, this is a function of the limited number of studies that have presented barcode data for West Palaearctic Chrysididae in general (Niehuis and Wägele 2004, Soon and Saarma 2011, Soon et al. 2014, Paukkunen et al. 2015, Orlovskyté et al. 2016, Roslin et al. 2021).The present work represents the first attempt to generate DNA barcodes for Iberian Chrysididae at a faunal level and, thus, represents a major step in documenting the genetic diversity in the Mediterranean cuckoo wasp fauna.

Materials and methods
This dataset is composed of data relating to 144 Chrysididae specimens.Specimens were collected during field expeditions in the Iberian Peninsula, Belgium, Italy and Morocco from 2014 to 2022 by T.J. Wood, I. Cross (Dorchester, UK) and P. Rosa (Fig. 1, Table 1).Specimens were pinned and dried and are preserved in the collection of T.J. Wood at the University of Mons (Belgium); Italian specimens are preserved in ethanol (98%) and preserved in the collection of P. Rosa at the University of Mons (Belgium).Holotypes of the newly-described species are deposited in the Natur-Museum (Lucerne, Switzerland -NMLU) and paratypes are deposited in the following private collections: PRC (Paolo Rosa Collection, Bernareggio, Italy), TWC (Thomas J. Wood Collection, Mons, Belgium); ICC (Ian Cross Collection, Briantspuddle, Dorset, United Kingdom).The majority of specimens were determined to species level, though some specimens in challenging or unclear taxonomic groups were identified as 'cf.' or simply to the species group.Overall, 103 species are represented in the dataset.These species belong to 13 genera (Fig. 2).Specimens were captured with an entomological net, euthanised by exposure to ethyl acetate and pinned and dried within 24 hours to achieve maximum suitability for DNA extraction and amplification.Map of the localities where cuckoo wasps samples were collected.
The InBIO Barcoding Initiative Database: contribution to the knowledge ... DNA was extracted using a QIAmp DNA Micro Kit that is designed to extract higher concentrations of genetic material from samples with small amounts of DNA.DNA amplification was performed using two different primer pairs, that amplify partially overlapping fragments (LC + BH) of the 658 bp barcoding region of the COI mitochondrial gene (Folmer et al. 1994).We used the primers FwhF1 (Vamos et al. 2017) + C_R (Shokralla et al. 2015) for LC and BF3 (Elbrecht et al. 2019) + BR2 (Elbrecht and Leese 2017) for BH amplification, all modified with Illumina adaptors.PCRs were performed in 10 µl reactions, containing 5 µl of Multiplex PCR Master Mix (Qiagen, Germany), 0.3 µl of each 10 mM primer and 1-2 µl of DNA, with the remaining volume in water.PCR cycling conditions consisted in an initial denaturation at 95ºC for 15 min, followed by 45 cycles of denaturation at 95ºC for 30 sec, annealing at 45ºC for 45 sec and extension at 72ºC for 45 sec, with a final elongation step at 60ºC for 10 min.All DNA extracts were deposited in the IBI collection.
Successful amplification was validated through 2% agarose gel electrophoresis and samples selected for sequencing followed for a second PCR, where Illumina P5 and P7 adapters with custom 7 bp long barcodes were attached to each PCR product.The index PCR was performed in a volume of 10 µl, including 5 µl of KAPA HiFi PCR Kit (KAPA Biosystems, U.S.A.), 0.5 µl of each 10 mM indexing primer and 2 µl of diluted PCR product (usually 1:4).PCR cycling conditions were as before, except that only 10 cycles were performed and at an annealing temperature of 55ºC.The amplicons were purified using AMPure XP beads (New England Biolabs, U.S.A.) and quantified using NanoDrop 1000 (Thermo Scientific, U.S.A.).Clean PCR products were then pooled equimolarly per fragment.Each pool was quantified with KAPA Library Quantification Kit Illumina® Distribution of species (%) for each Chrysididae genus present in the dataset.Genera represented by less than 2% of species were lumped together.
Platforms (KAPA Biosystems, U.S.A.) and the 2200 Tapestation System (Agilent Technologies, California, USA) was used for fragment length analysis prior to sequencing (Paupério et al. 2018).DNA sequencing was done at CIBIO facilities on an Illumina MiSeq benchtop system, using a V2 MiSeq sequencing kit (2x 250 bp).
Illumina sequencing reads were processed using OBITools (Boyer et al. 2016) and VSEARCH (Rognes et al. 2016).Briefly, paired-end reads were aligned, collapsed into exact sequence variants, filtered by length, denoised and checked for chimeras.The resulting sequences from both LC and BH fragments of each sample were further assembled using CAP3 (Huang and Madan 1999) to produce a single 658 bp contig per sample.
All sequences in the dataset were submitted to BOLD and GenBank databases and, to each sequenced specimen, the morphological identification was contrasted with the results of the BLAST of the newly-generated DNA barcodes in the BOLDIdentification Engine.In order to clarify the taxonomic status of problematic groups, DNA barcodes generated here were analysed with other sequences from across the West Palaearctic downloaded from BOLD and GenBank.Sequences (658 bp) were aligned using SeaView (Gouy et al. 2010) and a neighbour-joining phylogeny was run with 10,000 bootstraps.Intra-and interspecific distances were calculated using MEGA-X (Kumar et al. 2018).

Data resources
The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of "European Chrysididae" dataset can be downloaded from the Public Data Portal of BOLD (http://dx.doi.org/10.5883/DS-IBIHY02) in different formats (data as dwc, xml or tsv and sequences as fasta files).Alternatively, BOLD users can log-in and access the dataset via the Workbench platform of BOLD.All records are also searchable within BOLD, using the search function of the database.
The version of the dataset, at the time of writing the manuscript, is included as Suppl.material 1 in the form of one text file with specimen data information, as Suppl.material 2 in the form of DWC file specimen data and one fasta file containing all sequences as downloaded from BOLD (Suppl.material 3).
Hind leg unmodified, metatibia entirely black, without visible spots or depressions.
Colouration.Head blue with two large golden-red spots on brow, between anterior ocellus and eyes; clypeus, malar space and base of mandible greenish; ocelli area blackish.Pronotum and mesonotum red, lateral and posterior margin of scutellum green; rest of mesosoma blue, with green mesopleuron and legs.Metasoma dorsally red, ventrally black with two large, oblique green to blue spots on second sternum (Fig. 3C).Mandible entirely dark brown.Scape black with slight metallic reflection, pedicel and flagellomeres black; tegula black.Wings slightly infuscate.
Male: Paratype from Teruel similar to female, with face laterally covered with appressed, silvery setae; antennae elongate, with slender flagellum and cylindrical articles.Paratype from Granada smaller (4.0 mm) with red colouration turned to green (Fig. 3G); genital capsule as in Fig. 3H, with slender cuspis, apically unmodified.

Diagnosis
The genus Hedychridium Abeille de Perrin, 1878 in Iberia includes 34 species and two subspecies (Rosa and Soon 2012), whereas three previous members were recently moved from the genus Hedychridium to the genus Colopopyga Semenov-Tian-Shanskji, 1954 (Rosa 2017).Twenty-five of these species are known from Portugal (Rosa et al., in preparation).Mingo (1994) listed only 26 species for Iberia, another nine were overlooked, but has previously been described or cited from Spain by Linsenmaier (Linsenmaier 1959, Linsenmaier 1968, Linsenmaier 1987) and two species, H. infantum Linsenmaier, 1997 andH. balearicum Strumia, 2013, were described later.
The list of Hedychridium species known from Iberia is given below, with species subdivided by species groups following Linsenmaier's classification (Rosa et (Linsenmaier, 1959).Three species are also known for the Canary slands and are considered endemic: Hedychridium extraneum Linsenmaier, 1993, H. tricavatum Linsenmaier, 1993and H. viridicupreum Linsenmaier, 1993.Another species, Hedychridium suave (Tournier, 1878), was described from Spain (Andalucía) and has been considered to be a synonym of H. roseum by Linsenmaier (1951), Mingo (1994) and Kimsey and Bohart (1991), who erroneously placed the type locality in Switzerland (Léman area).None of these authors examined the type deposited at the Museum in Geneva.According to the labels pinned with the type specimen, the type locality is Tangier in Morocco and not Andalucía.Hedychridium suave does not belong to the roseum group, but to the femoratum group; it is a valid species and, based on its aspect and colouration, this taxon should be a North African species and the Andalusian locality is an error.(1968).For the moment, we do not consider H. suave to be a member of the Iberian fauna.
Hedychridium calcarium sp.nov.belongs to the ardens species group due to the shape of the second metatarsomere which is longer than the third, the punctate scapal basin, the general habitus and the body colouration (Fig. 3).Hedychridium calcarium sp.nov.has small to medium dimensions, from 4.0 to 5.4 mm; head blue with two red patches on brow between anterior ocellus and compound eye; black ocellar area; red pronotum and mesonotum, rest of mesosoma blue with greenish reflections; metasoma dorsally red and ventrally black with two large and oblique green-bluish spots on the second sternum.Punctation dense, even and deep on vertex; the largest punctures deep and umbelicate on pronotum, with intervals densely micropunctate; mesoscutum with smaller, shallower and sparser punctures, intervals less densely micropunctate compared to pronotum; metanotum with sparse micropunctures on shining intervals; metapostnotum distinctly enlarged compared to the same morphological part of the closest species, H. jucundum (Fig. 4), in which it is triangular.Metasomal sculpture with even, dense and small punctures equally spaced; apical margin of the third tergum with wide hyaline margin (2-3 PD).
Besides different body sculpture and morphological characters, Hedychridium calcarium sp.nov.can be immediately separated from H. ardens, H. marteni and H. ibericum by its blue metanotum, contrasting with red scutellum (concolourous in the other species); from H. cupritibiale by the blue face, contrasting with the red head on vertex (entirely red in H. sevillanum); from H. sevillanum by the different body colour, which is green to bronze in the latter and by the metanotum bronze to green, slightly contrasting with the rest of the red body colour.For comparison, pictures of H. ardens can be found in Paukkunen et al. (2015) and pictures of all the remaining species can be found in the illustrated catalogue of Linsenmaier's types (Rosa et al. 2022).Finally, H. infans, H. adventicium and H. infantum can be immediately separated by their very small size (2-3 mm) and the different colouration, the first having metallic tegulae (a unique feature), the other two a green line along the posterior margin of the pronotum.
The species morphologically and chromatically closer to H. calcarium sp.nov.are H. jucundum, H. reticulatum sensu Linsenmaier (1959) and H. buyssoni.However, H. jucundum can be differentiated by a dark to black spot on discum of second tergum and by the vertex entirely golden to red; in case of doubt, the triangular shape of the metapostnotum is diagnostic (Fig. 4); H. reticulatum by the red mesopleuron and, finally, H. buyssoni by the green vertex, the stocky body, the first tergum shorter medially and angled on anterior margins, the metasoma with denser and deeper punctures.The male of H. calcarium sp.nov.has the same colouration of the female and can be separated from the similar male of H. jucundum by the colour of the head and the shape of the genital capsule with cuspis apically slender, unmodified (vs.apically enlarged and curved in H. jucundum (see Rosa 2017)).
Outside the Iberian Peninsula, only Hedychridium bytinskii Linsenmaier, 1959 can be confused with H. calcarium sp.nov.H. bytinskii was described from Palestine and is known from Greece and Turkey (Linsenmaier 1968, Linsenmaier 1999).Linsenmaier ( Linsenmaier 1968, Linsenmaier 1999) listed this species from Morocco, but the Moroccan specimens may actually belong to the western Mediterranean species H. calcarium.The latter can be immediately recognised by the dark metasomal sterna, with two small dark green spots on the second sternum, whereas H. bytinskii specimens from the east Mediterranean have the first sternum (largely) and second sternum (entirely) bright green (see pictures of the type in Rosa et al. (2022), Fig. 7E).
The second sternum of H. bytinskii is also characterised by only a few and sparse punctures bearing long setae, whereas H. calcarium has a denser punctation (Fig. 1F) with short setae that are approximately one third as long as those of H. bytinskii.The colour pattern of the head also differs between the two species, with the entire vertex in H. bytinskii coloured flame red, distinctly contrasting the blue head and the green declivity of the frons, with the ocelli area flame red; the red area on the vertex of H. calcarium is less strongly contrasting and the ocelli area is black.The scutellum is entirely flame red in H. bytinskii, whereas it is metallic green on its posterior margin in H. calcarium.Barcoding analyses of the eastern Mediterranean H. bytinskii are needed to evaluate the genetic distance between the two species.

Etymology
The epithet calcarium derives from the Latin adjective calcarius related to the limestone habitat of the species.

Distribution
Spain (provinces of Teruel and Granada).At each locality, the species was found in dry grassland on calcareous soil, such as at the Barranco de los Oncenachos (Fig. 6).Mesosoma.Medial pronotal line narrow and short, reaching half pronotal length; pronotum antero-laterally slightly bulging (Fig. 7); pronotal punctation double and interspaces polished with sparse minute dots; notaulus basally formed by small subrectangular foveae becoming smaller and rounded at apex; parapsidal signum as a linear depression; mesoscutellum dense puncture and irregular interspaces, antero- medially corrugated and becoming polished towards base; scrobal sulcus of mesopleuron formed by large foveae aligned, limited to upper half; episternal sulcus formed by large and irregular, subsquare foveae; punctation with dots on interspaces and larger punctures on mesepisterum; scutellar-metanotal suture deep and wide; metanotum with contiguous punctures, larger than other punctures on mesosoma; posterior propodeal projections slightly divergent; wing venation unmodified.
Metasoma.First tergum double punctate, with large punctures separated by small punctures on interspaces; second and third tergum double punctate, larger punctures smaller than those on first tergum; punctures on metasomal separated by polished interspaces (Fig. 7B, Fig. 7F); pit row composed by small, deep pits, apical margin of third tergum continuous, dark blue, medially arcuate; black spots of the second sternum large, covering almost all segment length, reaching median line.
Colouration.Head and mesosoma dark blue, pronotum and lateral areas of mesoscutum flame red, scutellum with light blue highlights; metasoma red to purplish, apical margin of third tergum blue.Scape, pedicel and first tergum black with weak greenish-metallic lustre, rest of flagellum black; tegula blue; metasomal venter black, with only a narrow blue thin line between the black spots and the apical margin of the second sternum.Legs blue, tarsi dark brown.
Male.Body length 5.0-6.0 mm.Similar to female in shape, sculpture and colouration.Malar space slightly shorter, scapal basin laterally covered by short, dense, appressed and silvery pubescence; blue segments of mesososoma with greenish reflection, propodeum and propodeal angles dorsally green to golden green; brown.Male genital capsule (Fig. 8A) with inner margin of the gonocoxa straight.The InBIO Barcoding Initiative Database: contribution to the knowledge ...

Diagnosis
Medium-sized, slender species (5-6 mm); head and mesosoma blue, pronotum and lateral areas of mesoscutum red; mesosoma dorsally red to purple, apical margin of third tergum blue; metasoma ventrally black, black spots on second sternum large, covering almost all surface and touching mid-line, without being clearly fused with each other; narrow stripe on apical margin of second tergum blue.Metasoma punctation double, dense, with polished interspaces between the large and small punctures.Chrysis crossi sp.nov. is chromatically and morphologically similar to C. phryne Abeille de Perrin, 1878, but it is clearly separated genetically (see below).The main diagnostic characters to separate both sexes from C. phryne is the punctation, which consists of distinct double punctures on the metasomal scutum, these being separated by polished interspaces (Fig. 7E, Fig. 9A), whereas in C. phryne, the punctation is even and dense, without polished spaces.The metasomal venter is black in both sexes; black spots on second sternum large, covering almost all surface and touching mid-line with a narrow blue line between the black spots and the apical margin of the segment; in C. phryne, the sternum is clearly metallic green to golden green, with black spots distinctly separate from mid-line.The male genital capsule of the two species is different (Fig. 8) being narrower and more slender in C. crossi sp.nov., with the inner margin of the gonocoxa straight.Male can be also recognised by their brown tarsi, which are pale to yellowish in C. phryne.

Etymology
The specific epithet crossi (masculine) is dedicated to Ian Cross (Dorchester, Dorset, UK) for his active research on Portuguese Hymenoptera, including cuckoo wasps, many specimens of which were used for the current InBIO Barcoding Initiative work.

Ecology
Label information from Ian Cross reports that a male specimen was collected at an aggregation of Melitturga caudata Pérez, 1879 (Andrenidae), on the sand near empty snail shells.(Romano 2006).Chrysis destefanii was considered to be a synonym of C. phryne by Linsenmaier (1959) and Kimsey and Bohart (1991).Strumia and Yildirim (2009)  The specimen IBIHM1161-22 collected in the Algarve (Lagos, Fig. 10) shows a rich golden colouration of the anterior part of the body and third tergum is entirely violet, with the metasoma densely punctate.DNA barcodes demonstrate there is moderate genetic differentiation between the Portuguese specimen and H. caputaureum from northern and central Europe (Fig. 11), being separated by an average of 2.50% (range 2.43-2.59%).The clade of H. caputaureum from Austria, Finland and Germany shows low average intraspecific distance of 0.19% (range 0.00-0.34%)and has bootstrap support of 98%.
However, the Portuguese specimen is much more strongly separated from two sequences of H. chloropygum from Italy, showing average genetic differentiation of 8.43% (range 8.36-8.51%).When including the Portuguese specimen within H. caputaureum, the two clades are separated by an average interspecific genetic distance of 8.56% (range 8.36-9.00%).We, therefore, consider H. caputaureum and H. chloropygum to be two different species and include Portuguese material within H. caputaureum, with the observed genetic distance considered to be variation, given the geographic distance between southern Portugal and Germany.The overall distribution of H. caputaureum must be revised, as this Portuguese specimen is the first reported record of this taxon in southwestern Europe.Additional genetic samples from Spain and France are likely to fall between the Portuguese and central/northern European sequences.
The subspecies H. chloropygum berberiacum Linsenmaier, 1959 from Algeria and Morocco shows a similar colouration to the Algarve specimen, though it has more extensively metallic violet colouration laterally on the metasoma, but also sparser punctation (Rosa et al. 2022).Genetic analyses are needed to clarify the placement of this taxon, but for the  The InBIO Barcoding Initiative Database: contribution to the knowledge ...
moment, we consider it to be the northern African subspecies of H. chloropygum, based on its shallow and sparse punctation.
Hedychrum rutilans Dahlbom, 1854 is one of the most common European cuckoo wasp species, known to be a cleptoparasite of Philanthus species ( Linsenmaier 1997a).In addition to typical cleptoparasitic behaviour, the female does not have to enter the host nest for ovipositing, but can oviposit directly on the prey (Apis mellifera Linnaeus) while it is being transported to the nest by the host (Veenendaal 1987, Baumgarten 1995).This species is also known in literature as H. intermedium sensu auctorum for an incorrect interpretation of the type materials (Rosa and Xu 2015).Linsenmaier (Linsenmaier 1959, Linsenmaier 1997a, Linsenmaier 1997b) considered three European subspecies, namely: rutilans s.str., ssp.viridiaureum Tournier, 1877 and ssp.viridiauratum Mocsáry, 1889.
Two specimens from the same sampling locality in central Spain (Segovia, Bernuy de Porreros, IBIHM1120-22 and IBIHM1121-22) were both identified as H. rutilans, but are separated by a genetic distance of 5.27%.Integrating all newly-acquired sequences and sequences from BOLD and GenBank (some without identifications beyond Hedychrum sp.) shows that H. rutilans s.l.comprises two taxa (Fig. 12).Sequences from Spain, Italy, Germany, Finland and Bulgaria belong to H. rutilans s. str.(Fig. 12).They show low average intraspecific distance of 0.55% (range 0.00-1.48)and form a clade with bootstrap support of 100.Sequences from Portugal, Spain and western Germany (Rhineland-Palatinate) also show a low average intraspecific distance of 0.16% (range 0.00-0.33)and have bootstrap support of 100%.The two clades are separated by an average genetic distance of 5.16% (range 4.78-5.77%).
Following Linsenmaier (1997a), the second clade is called Hedychrum viridiaureum Tournier, 1877 sp.resurr.Linsenmaier (Linsenmaier 1959, Linsenmaier 1997a) noted differences between these two taxa and employed a subspecific framework.Hedychrum rutilans s. str.(Fig. 13) is usually larger and Linsenmaier noted a host association with Philanthus coronatus (Thunberg, 1784), whereas H. viridiaureum (Fig. 14) is usually smaller and is associated with the respectively smaller host Philanthus triangulum (Fabricius, 1775).Based on the DNA barcodes presented here, combined with the distributional framework of Linsenmaier, in Europe, H. rutilans s. str.appears to be more widely distributed, from central Iberia across the continent, whereas H. viridiaureum appears to be restricted to Iberia and western Europe, to Switzerland, western Germany and north to Belgium and the Netherlands.Detailed revision is necessary to clarify these range limits and all host associations as, in some regions where H. rutilans s.str.occurs, only P. triangulum is present.Our observations would support the host associations noted by Linsenmaier, with additional points.Very small individuals of H. viridiaureum can be found in Iberia (such as IBIHM-1183-22) where they are associated with Philanthus pulchellus Spinola, 1842 that is smaller than P. triangulum.Equally, in central Spain, the large-bodied Philanthus dufourii Lucas, 1849 is much more frequently encountered than P. coronatus and, hence, this is likely the principal host of H. rutilans s. str. in this region.The correct placement of H. rutilans viridiauratum Mocsáry, 1889, described from Algeria (types examined) and cited by Linsenmaier (Linsenmaier 1959, Linsenmaier 1997a) from the Iberian Peninsula is unclear and must be evaluated by means of molecular analyses.It could be a synonym of H. viridiaureum Tournier or a separate northern African species.Philoctetes parvulus (Dahlbom, 1845) was considered to be a valid species by Rosa and Soon (2012) following type examination.Based on the consistently smaller size, dark colouration and different punctation of both sexes in comparison with P. punctulatus (Fig. 15), specimens of P. parvulus were considered to be distinct.However, DNA barcodes (Fig. 16) demonstrate that the small individuals displaying the typical morphology of P. parvulus (INV12750, INV12749) show almost no genetic differentiation from P. punctulatus (0.00% and 0.02%).The differences in morphology are, therefore, not considered to be speciesspecific and are probably caused or exaggerated by the smaller body size.Philoctetes parvulus is, therefore, considered to be a synonym of P. punctulatus.

Chrysis lusitanica Bischoff, 1910
The identity of Chrysis lusitanica Bischoff, 1910 (Fig. 17) has to date remained unclear.Kimsey and Bohart (1991) synonymised Chrysis sculpturata Mocsáry, 1912 with C.     (2014) and Orlovskyté et al. (2016).In the framework of these previous projects, we can conclude that C. lusitanica is a member of the brevitarsis clade and that it is also present in Sardinia (first record for Italy, previously published as C. pseudobrevitarsis Linsenmaier, 1951by Soon et al. (2014)).Chrysis lusitanica can be easily separated from the other species of this clade by the small, even and dense punctures on the second tergum, these punctures being smaller or similar to those on the first tergum and the mesosoma densely punctate, with uniform dark blue colouration.
Chrysis lusitanica (including the Sardinian specimen that differs from the Portuguese specimen by 0.46%) is strongly separated from C. pseudobrevitarsis by an average interspecific distance of 4.07% (range 3.80-4.41%,Fig. 18).It is less strongly separated from C. brevitarsis by an average interspecific distance of 2.28% (range 2.05-2.43%),but because intraspecific variation is low (0.46% and 0.16%, respectively), both the C. lusitanica and C. brevitarsis clades have bootstrap support of 100%.Genetic differentiation within the brevitarsis clade is generally low, with C. brevitarsis separated from C. parabrevitarsis by an average interspecific distance of 3.06% (range 2.46-3.50%).In this context, C. lusitanica is considered to be a consistently differentiated species.

Chrysis scutellaris marteni Linsenmaier, 1951
Linsenmaier (1951) described a Spanish subspecies of Chrysis scutellaris Fabricius, 1794, based on its larger size (9.0-10.5 mm) and stocky aspect, with the apical margin of the third tergum indistinctly undulate.This form can be collected in sympatry with the nominotypical species.The two specimens DNA barcoded here conform morphologically to C. scutellaris marteni and show a genetic distance of 6.26% and 6.42% from a sample of C. scutellaris from Eisenberg (Rhineland-Palatinate) in Germany ( KY430717).The subspecies may, therefore, be distinct, but additional samples are required, particularly of typical C. scutellaris from Iberia.
Genetic results unambiguously support a species-level difference between S. calens westermanni from Spain and Portugal and S. calens zimmermanni from Italy (Fig. 20).These taxa are separated by an average genetic distance of 6.62%, with bootstrap support of 100% for each clade.Both taxa are well-separated from S. cyanurum (Fig. 21A), by an average of 6.68% for S. calens westermanni and by 6.86% for S. calens zimmermanni.
Though collected over a large area from Portugal to South Africa, S. cyanurum shows low intraspecific variability, with average separation of 1.26% (range 0.47-1.89%).Stilbum westermanni stat.nov.is, therefore, restored to species status (Fig. 21B).For now, we follow the interpretation of Linsenmaier that material from Central Europe should be referred to as S. calens zimmermanni until genetic samples are available from Siberia, the locus typicus of S. calens s. str.
The specimens DNA barcoded and identified in BOLD as Stilbum cyanurum from Madagascar (MW983778 and MW983223) are clearly distinct and actually belong to the species Stilbum viride Guérin-Méneville, 1842, the sole and endemic Madagascan Stilbum (Kimsey and Bohart 1991).Additionally, samples from Australia identified as 'S.superbum' are also clearly distinct.However, the name 'Stilbum superbum' is unavailable and it is likely an incorrect spelling of Stilbum splendidum (Fabricius, 1775) that has recently been used on online sites.The Australian Stilbum species is clearly morphologically different from all other known species, but its taxonomic status has been confused.In literature, it has been commonly referred to as S. splendidium auct.or S. amethystinum auct.The type of the first taxon proved to be morphologically conspecific with S. cyanurum and considered to be a subjective synonym by Kimsey and Bohart (1991).The second was also considered to be a subjective synonym of S. cyanurum by Kimsey and Bohart (1991), but two syntypic specimens in London (Banks Collection, Natural History Museum) belong to a morphological separated species, characterised by smaller size and short malar spaces.Since this taxon is morphologically and genetically distinct, we here resurrect S. amethystinum (Fabricius, 1775) sp.resurr.from its previous synonymy with S. cyanurum.We also designate here the lectotype of Chrysis amethystina Fabricius, 1775 with one of the two specimens housed in the Banks Collection.

Parnopes sp.
The genetic sequence of this Parnopes specimen is strongly separated from the sequence of P. grandior from Italy by an average of 8.71% (Fig. 22).It is closer to a P. unicolor sequence from Morocco, but is still separated by 5.18%.Taken together, this taxon and Parnopes unicolor form a clade with bootstrap support of 89%, strongly separated from the P. grandior clade that has bootstrap support of 100%.
The discovery of another Parnopes species in the Iberian Peninsula is not so surprising as it seems, even though a name currently cannot be confidently assigned to this taxon.In recent years, a new species from Sardinia, Parnopes linsenmaieri Agnoli, 1995 (described as subspecies of Parnopes grandior) was described and another species was found through DNA barcoding Bulgarian specimens (BOLD, unpublished sequences).However, several new species of West Palearctic Parnopes will be described in an upcoming revision.These species have been overlooked because, classically, only three species were considered to be valid in the West Palearctic: Parnopes grandior (known from Europe to central Asia), P. unicolor (northern Africa) and P. glasunowi (western Asia to central Asia) and specimens were identified, in part, based on the collecting locality and, in part, on body colouration.In this sense, all the variations and subspecies of P. grandior were considered to be only colour variation (Kimsey and Bohart 1991).The Portuguese female barcoded may be related to Parnopes marokkanus Trautmann, 1927, a taxon not mentioned by Kimsey and Bohart (1991), Linsenmaier (1959), Linsenmaier (1968), Linsenmaier (1997b) and Linsenmaier (1999).In any case, many more genetic sequences and analyses are needed to understand the limits of variability within this genus.In fact, colouration is still seemingly very variable within populations, but could also represent the presence of valid sibling species.

Figure 5 .
Figure 5. Phylogenetic tree (neighbour-joining) of members of the Hedychridium ardens group based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 11 .
Figure 11.Phylogenetic tree (neighbour-joining) of members of the Hedychridium roseum group, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 12 .
Figure 12.Phylogenetic tree (neighbour-joining) of Hedychrum species, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 16 .
Figure 16.Phylogenetic tree (neighbour-joining) of Elampini species with a focus on Philoctetes, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 18 .
Figure 18.Phylogenetic tree (neighbour-joining) of members of the Chrysis ignita group with a focus on the species around Chrysis brevitarsis based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 19 .
Figure 19.Phylogenetic tree (neighbour-joining) of the Chrysis splendidula group, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 20 .
Figure 20.Phylogenetic tree (neighbour-joining) of Stilbum species, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.

Figure 22 .
Figure 22.Phylogenetic tree (neighbour-joining) of Parnopes species, based on the DNA barcoding mitochondrial COI gene fragment.Numbers adjacent to branches represent bootstrap support (values of < 0.75 are omitted).The scale-bar indicates the % of sequence divergence.
The InBIO Barcoding Initiative Database: contribution to the knowledge ...
(Müller 2022)(2019)been reported to attack Osmia (Allosmia) melanura Morawitz, 1871 (seePauli et al. (2019)), but this cannot be the typical host across much of its range as, in Europe, O. melanura is restricted to southern Italy, North Macedonia and southern Ukraine(Müller 2022).It is likely that a different snail shell-Comments on the specific status of C. phryne burgenlandia (known from Austria to Greece) should be postponed until genetic sequences are available, because the main diagnostic characters are based on body colouration only.Finally, Mocsáry (1889) described Chrysis destefanii, based on the description of a specimen collected in Sicily by De Stefani-Perez and identified as C. candens by du Buysson (1888).The type of Chrysis destefanii is currently considered to be lost, as is large part of De Stefani's collection Linsenmaier, 1959 and is used instead, all known species of O. (Allosmia) using this nesting substrate.The likely host is O. ( Allosmia) rufohirta Latreille, 1811 which is widely distributed across Europe and is the only O. (Allosmia) known from Portugal( Müller 2022), being common in the Algarve(Baldock et al. 2018).We suggest that O. rufohirta is the likely host of C. crossi, though this must be confirmed through direct observations.NotesAccording toLinsenmaier (1959), the phryne group includes only two species: Chrysis circe Mocsáry, 1889 and C. phryne Abeille de Perrin, 1878, with three subspecies C. phryne s.str., C. phryne hebraeicaLinsenmaier, 1959 and C. phryne burgenlandia  Linsenmaier, 1968.The types of these subspecies have been examined and C. hebraeica stat.nov.has to be considered to be a distinct species, based on morphological analyses, as it displays greater morphological differences from C. phryne s.str.than C. crossi.

Hedychridium caputaureum Trautmann & Trautmann, 1919 and Hedychridium chloropygum du Buysson, 1888
Mocsáry (1889)pecimen from Turkey as Chrysis destefanii, yet this record may be related to Chrysis hebraeica or to an undescribed species of the similar rubricata group that has already been observed in the Middle East (PR, unpublished data).Based on the descriptions by du Buysson (1888) andMocsáry (1889)and, in particular, on the detail on the punctation of the second tergum "régulière formée de points égaux, assez serrés", the synonymy between Chrysis destefanii and C. crossi is excluded and De Stefani's specimen would appear to be conspecific with C. phryne.Since the type of Chrysis destefanii is lost, we treat C. destefani as nomen dubium, until such a point that molecular data are available for Sicilian specimens.Arens (2010) considered Hedychridium caputaureum Trautmann & Trautmann, 1919 to be a subspecies of H. chloropygum du Buysson, 1888, based on morphological affinities and noticeable variation in surface sculpture.According to Arens (2010), H. chloropygum s.str. is limited to south-western Europe, whereas H. chloropygum caputaureum is distributed in northern, central and south-eastern Europe.The main difference between these two taxa is based on the colouration.In H. chloropygum s.str., the anterior part of the body is richly adorned with copper or gold and the metasoma is metallic blue to violet on the third tergum, to a varying extent and intensity.In H. chloropygum caputaureum, the anterior part of the body may have a similar colouration, although Nordic and central European specimens may be darker, with faint coppery or golden reflections (described as H. Linsenmaier, 1959 andsenmaier, 1959 and synonymised by Arens (2010)with H. chloropygum caputaureum); however, the metasoma is always without metallic reflections.The two taxa can additionally be separated by the denser and coarser punctation of the metasoma in H. caputaureum which is apparently locally variable (Arens 2010).

Stilbum westermanni Dahlbom, 1845 sp. resurr.
Linsenmaier, 1959.Linsenmaier (1959)twelve Palearctic species, yet the real number of the species in this group is unclear and requires detailed revision.Several morphospecies are found in the Mediterranean region, in particular those related to the subgroup of C. rutilans Olivier, 1790 and identified with the name Chrysis insperata Chevrier, 1870, including small and slender species.Other morphospecies closely related to C. rutilans are awaiting description (PR, unpublished data).The identity of specimen IBIHM1090-22 from the Sierra Nevada, tentatively identified as C. rutilans, is unclear, but it may represent another undescribed taxon within this group as it is clearly separated from C. rutilans sequences from Finland.Within the splendidula group, we identify Chrysis maroccana Mocsáry, 1883 as a species closely related to C. splendidula Rossi, 1790, which lacks the raised apical margin at the apex of the second tergum.This species was previously reported from Morocco, Sardinia and Corsica(Linsenmaier 1987).Mingo (1994)recorded C. maroccana from Portugal and Spain, but her identifications clearly refer to another species already known from Iberia which was not mentioned in the monograph, namely C. continentalisLinsenmaier, 1959.Linsenmaier (1959)listed six subspecies of Stilbum calens (Fabricius, 1781) distributed from Europe to China and he separated the two subspecies S. calens zimmermanniLinsenmaier, 1959 and S. calens subcalens Linsenmaier, 1959 ( nec Mader 1933, an  unavailable name)