Biodiversity Data Journal 11: e98743 OO) doi: 10.3897/BDJ.11.e98743 open access Taxonomy & Inventories 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) Paolo Rosa?, Thomas Wood?, Teresa Luisa L. Silva$!, Joana Verissimo®!, Vanessa A. Mata®!!, Denis Michez?, Pedro BejaS1, Sénia Ferreira®:! + University of Mons, Research Institute for Biosciences, Laboratory of Zoology, Place du parc 20, 7000, Mons, Belgium § CIBIO, Centro de Investigagao em Biodiversidade e Recursos Genéticos, InBIO Laboratorio Associado, Campus de Vairao, Universidade do Porto, 4485-661 Vairao, Vila do Conde, Portugal | BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairao, 4485-661 Vairao, Vila do Conde, Portugal 4 CIBIO, Centro de Investigagao em Biodiversidade e Recursos Genéticos, InBIO Laboratdério Associado, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal Corresponding author: Sonia Ferreira (hiporame@gmail.com) Academic editor: Dominique Zimmermann Received: 12 Dec 2022 | Accepted: 25 Jan 2023 | Published: 01 Mar 2023 Citation: Rosa P, Wood T, Silva TLL, Verissimo J, Mata VA, Michez D, Beja P, Ferreira S (2023) 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). Biodiversity Data Journal 11: e€98743. https://doi.org/10.3897/BDJ.11.e98743 ZooBank: urn:lsid:zoobank.org:pub:79FFF43D-DF9F-4139-9BB8-85DE59F5BADD 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 © Rosa P et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Rosa P etal approximately 44% of the Iberian and 21% of the European fauna. The InBIO Barcoding Initiative (IB| - 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: Chrysis crossi Rosa, sp. nov. from southern Portugal and Hedychridium calcarium Rosa, sp. nov. from eastern Spain. Several taxonomic changes are proposed and Hedychrum rutilans Dahlbom, 1845 is found to consist of two different taxa that can be found in sympatry, Hedychrum rutilans s. str. and Hedychrum viridaureum Tournier, 1877 stat. nov. Stilbum westermanni Dahlbom, 1845 stat. nov. is confirmed as distinct from Stilbum calens (Fabricius, 1781), with the latter species not confirmed as present in Iberia; barcoded Stilbum material from Australia is distinct and represents Stilbum amethystium (Fabricius, 1775) sp. resurr.; Portuguese material identified as Hedychridium chloropygum Buysson, 1888 actually belongs to Hedychridium caputaureum Trautmann & Trautmann, 1919, the first confirmed record of this species from Iberia. Philoctetes parvulus (Dahlbom, 1845) is confirmed to be a synonym of Philoctetes punctulatus (Dahlbom, 1845). Chrysis lusitanica Bischoff, 1910 is confirmed as a valid species. Chrysis hebraeica Linsenmaier, 1959 stat. nov. is raised to species status. Keywords Portugal, Spain, Italy, DNA barcode, mitochondrial DNA, Cytochrome c oxidase subunit | (COl) 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 InBIO Barcoding Initiative Database: contribution to the knowledge ... 3 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. Gonzalez et al. 1999, Gonzalez 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 Vardal 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 Wagele 2004, Soon and Saarma 2011, Soon et al. 2014, Paukkunen et al. 2015, Orlovskyte 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, |. 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 (lan 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). Table 1. Rosa P etal List of species that were collected and DNA barcoded within this project. # Indicates species with new BINs. Genus Chrysidea Chrysis Species Chrysidea disclusa pumilionis (Linsenmaier, 1987)# Chrysis andradei Linsenmaier, 1959# Chrysis berlandi Linsenmaier, 1959# Chrysis blanchardi Lucas, 1849# Chrysis caeruliventris Abeille de Perrin, 1878 Chrysis castillana Du Buysson, 1894 Chrysis cerastes Abeille de Perrin, 1877# Chrysis rutilans Olivier, 1790# Chrysis chrysoprasina Forster, 1853 Chrysis chrysoscutella Linsenmaier, 1959# Chrysis comparata Lepeletier, 1806 Chrysis consanguinea Mocsary, 1889 Chrysis cortii Linsenmaier, 1951 Chrysis elegans Lepeletier, 1806# Chrysis emarginatula Spinola, 1808 Chrysis fugax Abeille de Perrin, 1878 Chrysis germari Wesmael, 1839# Chrysis gracillima aurofacies (Trautmann, 1926)# Chrysis grohmanni Dahlbom, 1854 Chrysis hydropica Abeille de Perrin, 1878# IBI code INV12702 INV12728 INV12729 INV12682 INV12683 INV12734 INV12732 INV12676 INV12731 INV12733 INV12689 INV12677 INV12743 INV12679 INV12687 INV12670 INV12673 INV12672 INV12661 INV12669 INV12663 INV12666 INV12735 BOLD code IBIHM1103-22 IBIHM1129-22 IBIHM1130-22 IBIHM1083-22 IBIHM1084-22 IBIHM1135-22 IBIHM1133-22 IBIHM1077-22 IBIHM1132-22 IBIHM1134-22 IBIHM1090-22 IBIHM1078-22 IBIHM1144-22 IBIHM1080-22 IBIHM1088-22 IBIHM1071-22 IBIHM1074-22 IBIHM1073-22 IBIHM1062-22 IBIHM1070-22 IBIHM1064-22 IBIHM1067-22 IBIHM1136-22 BOLD BIN BOLD:AES3051 BOLD:AES8383 BOLD:AES5679 BOLD:AEU3313 BOLD:AED3523 BOLD:AED2289 BOLD:AET4960 BOLD:AET4959 BOLD:AET4958 BOLD:AES1459 BOLD:AAU1528 BOLD:AED0671 BOLD:AAR9816 BOLD:AES1460 BOLD:AED6786 BOLD:AED3372 BOLD:AET6935 BOLD:AES2863 BOLD:AED6294 BOLD:AET2381 GenBank OP347205 OP347228 OP347302 OP347200 OP347265 OP347250 OP347222 OP347173 OP347274 OP347293 OP347268 OP347283 OP347305 OP347241 OP347212 OP347211 OP347219 OP347309 OP347230 OP347229 OP347234 OP347210 OP347243 Genus Chrysura The InBIO Barcoding Initiative Database: contribution to the knowledge ... Species Chrysis insperata Chevrier, 1870# Chrysis integra Fabricius, 1787# Chrysis irreperta Linsenmaier, 1959# Chrysis lusitanica Bischoff, 1910 Chrysis merceti (Trautmann, 1926)# Chrysis mixta Dahlbom, 1854# Chrysis monticola Linsenmaier, 1999# Chrysis mysticalis Linsenmaier, 1959# Chrysis peninsularis du Buysson, 1887# Chrysis crossi Rosa sp. nov.# Chrysis pulchella Spinola, 1808 Chrysis pulcherrima Lepeletier, 1806# Chrysis pyrophana Dahlbom, 1854# Chrysis ramburi Dahlbom, 1854 Chrysis sculpturata Mocsary, 1912 Chrysis scutellaris marteni Linsenmaier, 1951 Chrysis sexdentata Christ, 1791 Chrysis splendidula Rossi, 1790# Chrysis subsinuata Marquet, 1879# Chrysis varidens Abeille de Perrin, 1878 Chrysis zonata Dahlbom, 1854# Chrysura austriaca (Fabricius, 1804) Chrysura cuprea (Rossi, 1790) Chrysura dichroa (Dahlbom, 1854)# Chrysura hybrida (Lepeletier, 1806) IBI code INV12774 INV12741 INV12671 INV12747 INV12744 INV12664 INV12681 INV12730 INV12678 INV12736 INV12667 INV12727 INV12665 INV12688 INV12775 INV12674 INV12684 INV12738 INV12739 INV12783 INV12740 INV12662 INV12668 INV12742 INV12690 INV12692 INV12696 INV12776 INV12726 BOLD code IBIHM1175-22 IBIHM1142-22 IBIHM1072-22 IBIHM1148-22 IBIHM1145-22 IBIHM1065-22 IBIHM1082-22 IBIHM1131-22 IBIHM1079-22 IBIHM1137-22 IBIHM1068-22 IBIHM1128-22 IBIHM1066-22 IBIHM1089-22 IBIHM1176-22 IBIHM1075-22 IBIHM1085-22 IBIHM1139-22 IBIHM1140-22 IBIHM1184-22 IBIHM1141-22 IBIHM1063-22 IBIHM1069-22 IBIHM1143-22 IBIHM1091-22 IBIHM1093-22 IBIHM1097-22 IBIHM1177-22 IBIHM1127-22 BOLD BIN BOLD:AET2383 BOLD:AET2382 BOLD:AER8828 BOLD:ACQ6955 BOLD:AET3720 BOLD:AET3717 BOLD:AET3719 BOLD:AET3718 BOLD:AES0122 BOLD:AES0121 BOLD:AED0619 BOLD:AET0271 BOLD:AET0272 BOLD:AED5814 BOLD:ABU6373 BOLD:ACM0910 BOLD:ABU6376 BOLD:AES6413 BOLD:AES4620 BOLD:AEE0312 BOLD:AET8274 BOLD:AAJ3472 BOLD:AAP1055 BOLD:AET1511 BOLD:AAY6924 GenBank OP347258 OP347285 OP347244 OP347197 OP347260 OP347207 OP347289 OP347304 OP347245 OP347271 OP347226 OP347295 OP347198 OP347172 OP347213 OP347263 OP347310 OP347269 OP347185 OP347217 OP347180 OP347214 OP347174 OP347199 OP347193 OP347176 OP347273 OP347215 OP347177 Genus Hedychridium Rosa P etal Species Chrysura purpureifrons (Abeille de Perrin, 1878)# Chrysura radians (Harris, 1776) Chrysura refulgens (Spinola, 1806) Chrysura rufiventris (Dahlbom, 1854) Chrysura simplex (Dahlbom, 1854) Chrysura sulcata (Dahlbom, 1845) Chrysura varicornis (Spinola, 1838)# Hedychridium aereolum du Buysson, 1892 Hedychridium anale (Dahlbom, 1854) Hedychridium ardens (Coquebert, 1801) Headychridium buyssoni Abeille de Perrin, 1887# Hedychridium caputaureum Trautmann & Trautmann, 1919 Hedychridium valesiense Linsenmaier, 1959# Hedychridium chloropygum du Buysson, 1888 Hedychridium cupratum (Dahlbom, 1854) Headychridium cupritibiale Linsenmaier, 1987# Headychridium incrassatum (Dahlbom, 1854) IBI code INV12694 INV12693 INV12695 INV12849 INV12850 INV12851 INV12697 INV12777 INV12699 INV12691 INV12700 INV12698 INV12701 INV12809 INV12717 INV12786 INV12790 INV12760 INV12804 INV12801 INV12802 INV12806 INV12807 INV12769 INV12718 BOLD code IBIHM1095-22 IBIHM1094-22 IBIHM1096-22 IBIHM1250-22 IBIHM1251-22 IBIHM1252-22 IBIHM1098-22 IBIHM1178-22 IBIHM1100-22 IBIHM1092-22 IBIHM1101-22 IBIHM1099-22 IBIHM1102-22 IBIHM1210-22 IBIHM1118-22 IBIHM1187-22 IBIHM1191-22 IBIHM1161-22 IBIHM1205-22 IBIHM1202-22 IBIHM1203-22 IBIHM1207-22 IBIHM1208-22 IBIHM1170-22 IBIHM1119-22 BOLD BIN BOLD:AEU2029 BOLD:AED1166 BOLD:ABA8702 BOLD:ABA7395 BOLD:AEC6882 BOLD:AAY6923 BOLD:ABA7396 BOLD:AET0222 BOLD:AAY6930 BOLD:AED4749 BOLD:AAK4640 BOLD:AES9011 BOLD:AAU0775 BOLD:AET6828 BOLD:AAE3258 BOLD:AAY6946 BOLD:AES9012 BOLD:AEE0029 GenBank OP347231 OP347286 OP347257 OP347296 OP347297 OP347221 OP347225 OP347303 OP347290 OP347189 OP347249 OP347261 OP347192 OP347195 OP347270 OP347236 OP347196 OP347183 OP347275 OP347187 OP347240 OP347255 OP347252 OP347239 OP347267 Genus Hedychrum The InBIO Barcoding Initiative Database: contribution to the knowledge ... Species Hedychridium infans Abeille de Perrin, 1878# Hedychridium jucundum Mocsary, 1889# Headychridium krajniki Balthasar, 1946 Hedychridium mediocrum Linsenmaier, 1987 Hedychridium monochroum du Buysson, 1888 Hedychridium reticulatum Abeille de Perrin, 1878# Hedychridium roseum (Rossi, 1790) Hedychridium sculpturatum Abeille de Perrin, 1877# Hedychridium scutellare (Tournier, 1878)# Headychridium sevillanum Linsenmaier, 1968# Hedychridium calcarium Rosa sp. nov.# Hedychridium subroseum prochloropygum Linsenmaier, 1959# Hedychridium vachali Mercet, 1915# Hedychrum longicolle Abeille de Perrin, 1877 IBI code INV12768 INV12781 INV12784 INV12785 INV12810 INV12811 INV12764 INV12716 INV12803 INV12800 INV12763 INV12791 INV12792 INV12793 INV12805 INV12812 INV12813 INV12761 INV12788 INV12765 INV12767 INV12789 INV12794 INV12795 INV12796 INV12715 INV12766 INV12723 BOLD code IBIHM1169-22 IBIHM1182-22 IBIHM1185-22 IBIHM1186-22 IBIHM1211-22 IBIHM1212-22 IBIHM1165-22 IBIHM1117-22 IBIHM1204-22 IBIHM1201-22 IBIHM1164-22 IBIHM1192-22 IBIHM1193-22 IBIHM1194-22 IBIHM1206-22 IBIHM1213-22 IBIHM1214-22 IBIHM1162-22 IBIHM1189-22 IBIHM1166-22 IBIHM1168-22 IBIHM1190-22 IBIHM1195-22 IBIHM1196-22 IBIHM1197-22 IBIHM1116-22 IBIHM1167-22 IBIHM1124-22 BOLD BIN BOLD:AES6837 BOLD:AES6836 BOLD:AES6835 BOLD:AAZ0056 BOLD:AAE3260 BOLD:AAY1978 BOLD:AER9655 BOLD:AAE3259 BOLD:AET6828 BOLD:AAE3258 BOLD:AES0428 BOLD:AET6827 BOLD:AES0429 BOLD:AET9698 BOLD:AES2437 BOLD:AET6826 BOLD:AED0972 GenBank OP347254 OP347191 OP347262 OP347232 OP347170 OP347256 OP347237 OP347279 OP347308 OP347206 OP347248 OP347203 OP347282 OP347301 OP347281 OP347181 OP347276 OP347291 OP347175 OP347242 OP347259 OP347223 OP347204 OP347307 OP347169 OP347209 OP347280 OP347298 Genus Holopyga Parnopes Philoctetes Species Hedychrum micans europaeum Linsenmaier, 1959 Rosa P et al IBI code INV12724 Hedychrum niemelai Linsenmaier, 1959 INV12721 Hedychrum nobile (Scopoli, 1763) Hedychrum rutilans Dahlbom, 1854 INV12722 INV12771 INV12720 Hedychrum viridiaureum Tournier, 1877 INV12719 Holopyga calida Linsenmaier, 1951# Holopyga lucida (Lepeletier, 1806)# Holopyga fastuosa (Lucas, 1849) Holopyga fervida (Fabricius, 1781) Holopyga generosa (Forster, 1853) Holopyga inflammata (Férster, 1853)# Holopyga jurinei Chevrier, 1862# Holopyga similis Mocsary, 1889 Holopyga merceti Kimsey, 1991# Parnopes sp.# Philoctetes abeillei du Buysson, 1892# Philoctetes punctulatus (Dahlbom, 1854)# INV12770 INV12782 INV12705 INV12754 INV12708 INV12709 INV12710 INV12780 INV12753 INV12706 INV12757 INV12712 INV12713 INV12759 INV12714 INV12756 INV12787 INV12755 INV12779 INV12704 INV12703 INV12749 INV12750 BOLD code IBIHM1125-22 IBIHM1122-22 IBIHM1123-22 IBIHM1172-22 IBIHM1121-22 IBIHM1120-22 IBIHM1171-22 IBIHM1183-22 IBIHM1106-22 IBIHM1155-22 IBIHM1109-22 IBIHM1110-22 IBIHM1111-22 IBIHM1181-22 IBIHM1154-22 IBIHM1107-22 IBIHM1158-22 IBIHM1113-22 IBIHM1114-22 IBIHM1160-22 IBIHM1115-22 IBIHM1157-22 IBIHM1188-22 IBIHM1156-22 IBIHM1180-22 IBIHM1105-22 IBIHM1104-22 IBIHM1150-22 IBIHM1151-22 BOLD BIN BOLD:AAK4644 BOLD:AAU1294 BOLD:AAK4644 BOLD:AAM3491 BOLD:AAM3491 BOLD:AAK4643 BOLD:AAM3491 BOLD:AES0090 BOLD:AES0091 BOLD:AAZ6194 BOLD:AAY6928 BOLD:ACV6331 BOLD:AAY9735 BOLD:AAZ6194 BOLD:AET1451 BOLD:AET1450 BOLD:AED0274 BOLD:AES1216 BOLD:AET2814 BOLD:AEU5026 BOLD:AEU5027 GenBank OP347194 OP347300 OP347266 OP347168 OP347220 OP347294 OP347247 OP347174 OP347202 OP347227 OP347182 OP347292 OP347190 OP347253 OP347216 OP347208 OP347287 OP347288 OP347201 OP347284 OP347233 OP347235 OP347178 OP347186 OP347218 OP347179 OP347264 OP347272 OP347238 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 9 Genus Species IB| code BOLD code BOLD BIN GenBank Pseudochrysis Pseudochrysis humbolati (Dahlbom, INV12659 IBIHM1060-22 BOLD:AEU3425 OP347246 1845)# Pseudochrysis incrassata (Spinola, INV12660 IBIHM1061-22 BOLD:AEU3426 OP347278 1838)# Pseudomalus auratus (Linnaeus, 1758) =INV12751 IBIHM1152-22 BOLD:AAH8217 OP347251 Pseudomalus violaceus (Scopoli, 1763) INV12752 IBIHM1153-22 BOLD:ABX9998 OP347299 Spintharina Spintharina cuprata (Dahlbom, 1854)# INV12657 IBIHM1058-22 BOLD:AET6497 OP347277 Spintharina versicolor (Spinola, 1808) INV12656 IBIHM1057-22 BOLD:AAJ3630 OP347224 Stiloum Stiloum westermanni Dahlbom, 1845# INV12654 IBIHM1055-22 BOLD:AES3895 OP347311 INV12655 IBIHM1056-22 OP347306 Stiloum cyanurum (Forster, 1771) INV12725 IBIHM1126-22 BOLD:AAJ4180 OP347184 Trichrysis Trichrysis cyanea (Linnaeus, 1758) INV12658 IBIHM1059-22 BOLD:AAH7935 OP347188 A : : wee to 3 -e es, Figure 1. EES] Map of the localities where cuckoo wasps samples were collected. 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. 10 Rosa P etal Percentage species per genus @ Chrysis @ Hedychridium @ Chrysura @ Holopyga @ Hedychrum @ Philoctetes @ Stilbum @ Other Figure 2. EES Distribution of species (%) for each Chrysididae genus present in the dataset. Genera represented by less than 2% of species were lumped together. DNA was extracted using a Ql[Amp 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 ul reactions, containing 5 ul of Multiplex PCR Master Mix (Qiagen, Germany), 0.3 ul of each 10 mM primer and 1-2 ul of DNA, with the remaining volume in water. PCR cycling conditions consisted in an initial denaturation at 95°C for 15 min, followed by 45 cycles of denaturation at 95°C for 30 sec, annealing at 45°C for 45 sec and extension at 72°C for 45 sec, with a final elongation step at 60°C 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 ul, including 5 yl of KAPA HiFi PCR Kit (KAPA Biosystems, U.S.A.), 0.5 ul of each 10 mM indexing primer and 2 ul 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 55°C. 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® The InBIO Barcoding Initiative Database: contribution to the knowledge ... 11 Platforms (KAPA Biosystems, U.S.A.) and the 2200 Tapestation System (Agilent Technologies, California, USA) was used for fragment length analysis prior to sequencing (Pauperio 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). Acronyms and abbreviations NMLU = Natur-Museum, Lucerne, Switzerland PRC = Paolo Rosa Collection, Bernareggio, Italy TWC = Thomas J. Wood Collection, Mons, Belgium ICC = lan Cross Collection, Briantspuddle, Dorset, United Kingdom In the text, the following abbreviations are used for morphological terms: ° F1, F2, F3 = flagellomeres 1, 2, 3 ° MOD = anterior ocellar diameter ° MS = malar space, the shortest distance between base of mandible and margin of compound eye ° OOL = oculo-ocellar line, the shortest distance between lateral ocellus and compound eye ° P = pedicel ° PD = puncture diameter ° POL = the shortest distance between posterior ocelli ° T1—T3 = metasomal terga 1 to 3 ° vs. = versus 12 Rosa P etal Photographs were taken with a Camera Olympus E-M1 Mark II with the Olympus Zuiko 60 mm objective and stacked with the software Helicon Focus (ver. 7.6). Further image processing was completed with Adobe Photoshop CS6.0. 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). Taxon treatments Hedychridium calcarium Rosa, sp. n. * ZooBank 3F4762B4-7EAB-4B8F-9B63-F18EA15255C4 Materials Holotype: a. scientificName: Hedychridium sp.; phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Hedychndium; country: Spain; locality: Sierra de Baza, Prados del Rey; decimalLatitude: 37.375; decimalLongitude: -2.854; year: 2021; month: 6; day: 25; individuallD: INV12794; individualCount: 1; lifeStage: Adult; catalogNumber: INV12794; recordNumber: INV12794; recordedBy: Thomas Wood; otherCatalogNumbers: IBIHM1195-22; identifiedBy: Paolo Rosa; institutionCode: Universite de Mons; occurrence!D: 0D71E997-8420-5092-B584-2E6619F2EA61 Paratypes: a. scientificName: Hedychridium sp.; phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Hedychridium; country: Spain; locality: Noguera de Albarracin, Barranco de la Olmeda; decimalLatitude: 40.462; decimalLongitude: -1.614; year: 2021; month: 6; day: 27; individuallD: INV12795; individualCount: 1; lifeStage: Adult; catalogNumber: INV12795; recordNumber: INV12795; recordedBy: Thomas Wood; otherCatalogNumbers: IBIHM1196-22; identifiedBy: Paolo Rosa; institutionCode: Universite de Mons; occurrence!D: DF 256B6E-111E-51B6-8356-0E121E5033F4 b. scientificName: Hedychridium sp.; phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Hedychridium; country: Spain; locality: Villar del Cobo, Barranco de los Oncenachos; decimalLatitude: 40.397; decimalLongitude: The InBIO Barcoding Initiative Database: contribution to the knowledge ... 13 -1.674; year: 2021; month: 6; day: 19; individuallD: INV12796; individualCount: 1; lifeStage: Adult; catalogNumber: INV12796; recordNumber: INV12796; recordedBy: Thomas Wood; otherCatalogNumbers: IBIHM1197-22; identifiedBy: Paolo Rosa; institutionCode: Universite de Mons; occurrence!D: D746B3CE-8112-585E-B5AA- FBB5866002CB Description Female. Body length 5.0—5.4 mm (holotype 5.4 mm (Fig. 3)). Forewing length 3.0—-3.5 mm. Figure 3. EES] Hedychridium calcarium sp. n., A-F holotype, female. A habitus, dorsal view; B habitus, lateral view; C head, frontal view; D head and mesosoma, fronto-lateral view; E metasoma, postero- lateral view; F metasoma, ventral view; G parataype male, habitus, lateral view; H genital capsule. Scale bar: 1 mm. Head. Brow with medium, contiguous punctures (ca. 0.4 x MOD), suddenly decreasing diameter from frontal declivity to malar spaces and clypeus (Fig. 3C); face, in frontal view, micropunctate along inner eye margin; scapal basin with polished intervals; medial line complete from anterior ocellus to clypeus; clypeus finely punctate with wide polished intervals; clypeal apical margin thickened, triangularly-shaped, non-metallic brown; ocellar area with small punctures, without line connecting posterior ocelli; temples regularly rounded, double punctate. OOL = 1.9 x MOD; POL = 1.5 x MOD; MS = 0.6 x MOD; relative length of P:F1:F2:F3 = 1:1.1:0.8:0.8. Malar space as long as antennal thickness. 14 Rosa P etal Mesosoma. Pronotum with punctation irregularly sized, mostly contiguous and large, umbelicate punctures up to 0.6 MOD; intervals between large punctures densely micropunctate. Mesoscutum with similar punctuation, yet punctures relatively smaller and micropunctures sparser compared to intervals on pronotum. Scutellum with polished intervals and sparse micropunctures. Metascutellum with reticulate-foveate punctures (0.8 x MOD). Metapectal-propodeal complex with metapostnotum wider than in other species (Fig. 4A) and posterior propodeal projection [= propodeal teeth] triangular, with thickened, blunt apex, slightly pointing backwards. Forewing medial vein 1.5 times as long as RS stub, medially gently arched; Rs stub as along as pterostigma. Hind leg unmodified, metatibia entirely black, without visible spots or depressions. Figure 4. EES] Scutellum, metanotum, metapectal-propodeal complex A Hedychridium calcarium sp. nov., holotype, female; B Hedychridium jucundum, female, from Italy (PRC). Metasoma. Punctation on terga minute, even, sparse, regularly spaced, 1—2 PD apart. Third tergum laterally with denser, deeper punctures (Fig. 3E); posterior margin with hyaline rim (2 PD). 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. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 15 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 and H. 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 al. 2022) and modifications proposed by Pauli et al. (2019), based on multigene molecular analyses. anale group: Hedychridium anale (Dahlbom, 1854); H. dubium Mercet, 1904; ardens group: H. adventicium Zimmermann, 1961, H. aereo/um du Buysson, 1891, H. ardens (Coquebert, 1801), H. buyssoni Abeille de Perrin, 1887, H. cupritibiale Linsenmaier, 1987, H. ibericum Linsenmaier, 1959, H. infans Abeille de Perrin, 1879, H. infans santschii Trautmann, 1927, H. infantum Linsenmaier, 1987, H. jucundum (Mocsary, 1889), H. marteni Linsenmaier, 1951; H. reticulatum Abeille de Perrin, 1878, H. sevillanum Linsenmaier, 1968; coriaceum group: H. coriaceum (Dahlbom, 1854), H. krajniki Balthasar, 1953; cupratum group: H. cupratum (Dahlbom, 1854); femoratum group: H. elegantulum du Buysson, 1887, H. femoratum (Dahlbom, 1854), H. gratiosum Abeille de Perrin, 1878; heliophilum group: H. heliophilum du Buysson, 1887, H. vachali Mercet, 1915; incrassatum group: H. incrassatum (Dahlbom, 1854); monochroum group: H. balearicum Strumia, 2013, H. carmelitanum Mercet, 1915, H. minutussimum Mercet, 1915, H. monochroum du Buysson, 1888; plagiatum group: H. andalusicum Trautmann, 1920, H. franciscanum Linsenmaier, 1987; roseum group: H. chloropygum du Buysson, 1888, H. mediocrum Linsenmaier, 1987, H. roseum (Rossi, 1790), H. scutellare (Tournier, 1878), H. subroseum Linsenmaier, 1959, H. subroseum prochloropygum Linsenmaier, 1959. The following three species were moved in the genus Colpopyga, supported by morphological and molecular evidence (Rosa 2017, Pauli et al. 2019): C. auriventris 16 Rosa P etal (Mercet, 1904), C. flavipes (Eversmann, 1858) and C. temperata (Linsenmaier, 1959). Three species are also known for the Canary slands and are considered endemic: Hedychridium extraneum Linsenmaier, 1993, H. tricavatum Linsenmaier, 1993 and H. viridicupreum Linsenmaier, 1993. Another species, Hedychridium suave (Tournier, 1878), was described from Spain (Andalucia) 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 (Leman 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 Andalucia. 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. Tournier is well-known for confusing European and Moroccan localities, as has already happened in other insect families as well as in Chrysididae (see the case of Chrysis superba Tournier, 1879 in Linsenmaier (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 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 17 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. jJucundum, 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. ca/carium 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. Genetics: Hedychridium calcarium sp. nov. is very distinct genetically (Fig. 5), showing a low average intraspecific genetic distance of 0.30%. It is strongly separated from the nearest relative in the phylogenetic tree H. reticulatum by an average genetic distance of 16.45% (range 16.11-16.72%). By direct genetic distance, it is closest to H. jJucundum specimens from Italy, separated by an average of 16.13% (range 15.96-16.41%). As a note, H. jucundum specimens from Italy are separated from H. jJucundum specimens from Menorca by an average of 4.94% (range 4.71-5.17%). This requires further investigation. Etymology The epithet ca/carium derives from the Latin adjective ca/carius related to the limestone habitat of the species. 18 Rosa P etal 1BIHM1122-22_Hedychrum_niemelai_Spain_Granada IBIHM1119-22_Hedychridium_incrassatum_Spain_Granada IBIHM1186-22_Hedychridium_jucundum_Spain_Menorca 1BIHM1185-22_Hedychridium_jucundum_Spain_Menorca jucundum s.l. 1BIHM1212-22_Hedychridium_jucundum_Italy_Aosta IBIHM1211-22_Hedychridium_jucundum_Italy_Aosta 1BIHM1170-22_Hedychridium_cupritibiale_Portugal_Baixo_Alentejo IBIHM1187-22_Hedychridium_ardens_Belgium_Namur 1BIHM1210-22_Hedychridium_aereolum_Italy_Aosta 1BIHM1191-22_Hedychridium_buyssoni_Spain_Granada IBIHM1194-22_Hedychridium_reticulatum_Spain_Granada IBIHM1193-22_Hedychridium_reticulatum_Spain_Madrid reticulatum IBIHM1192-22_Hedychridium_reticulatum_Spain_Madrid IBIHM1197-22_Hedychridium_calcarium_spec_nov_Spain_Teruel_Oncenachos calcarium IBIHM1196-22_Hedychridium_calcarium_spec_nov_Spain_Teruel_Noguera sp. nov. IBIHM1195-22_Hedychridium_calcarium_spec_nov_Spain_Sierra_Baza 0.02 Figure 5. EES 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. 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). Figure 6. EES Villar del Cobo, Barranco de los Oncenachos, province of Teruel, Spain. Collecting site for Hedychridium calcarium Rosa sp. nov. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 19 Ecology The host is unknown, but is likely to be a small apoid wasp, in line with other members of the Hedychridium ardens group. Chrysis crossi Rosa, sp. n. ZooBank 759C3BE4-F 1BE-47B2-9C43-0FFD17612E43 Materials Holotype: a. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross/; country: Portugal; locality: Salema; decimalLatitude: 37.06; decimalLongitude: -8.83; year: 2017; month: 4; day: 16; individuallD: INV12727; individualCount: 1; sex: F; lifeStage: Adult; catalogNumber: INV12727; recordNumber: INV12727; recordedBy: I. C. Cross; otherCatalogNumbers: IBIHM1128-22; identifiedBy: Paolo Rosa; institutionCode: Universite de Mons; occurrence!lD: 6CF2BF38-8154-53D2- AB88-640F8CDD5821 Paratypes: a. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; country: Portugal; locality: 3 km N Mexilhoeira Grande, Poio; decimalLatitude: 37.2; decimalLongitude: -8.6; year: 2005; month: 4; day: 30; individualCount: 1; sex: M; lifeStage: Adult; recordedBy: J. Smit (JSC); identifiedBy: Paolo Rosa; occurrence!D: 9849F20C-7561-5544-B4E2-430469C8A89F phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross, country: Portugal; locality: Carrapateira, Praia da Bordeira; decimalLatitude: 37.2; decimalLongitude: -8.9; year: 2006; month: 4; day: 16; individualCount: 2; sex: M; lifeStage: Adult; recordedBy: M. & E. Howe (PRC); identifiedBy: Paolo Rosa; occurrence!lD: 01CC81B6-FEC3-5EBD-B89E-B7195661A5AD phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross, country: Portugal; locality: Carrapateira; decimalLatitude: 37.2; decimalLongitude: -8.9; year: 2016; month: 4; day: 26; individualCount: 1; sex: M; lifeStage: Adult; recordedBy: I.C. Cross (ICC); identifiedBy: Paolo Rosa; occurrencelD: 92FDDO0A6-54DE-5166-B4DF-372F 55037E84 phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross/; country: Portugal; locality: Estoi; decimalLatitude: 37.1; decimalLongitude: -7.9; year: 2019; month: 5; day: 4; individualCount: 1; sex: M; lifeStage: Adult; recordedBy: P. Rosa & M. Jacobs; identifiedBy: Paolo Rosa; occurrence!D: 6BBAAAE02-1293-52EB-8267-4DBC53119F00 phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross/; country: Portugal; municipality: Faro; locality: Montenegro; decimalLatitude: 37.0; decimalLongitude: -7.9; year: 2019; month: 5; day: 9; individualCount: 2; sex: M; lifeStage: Adult; recordedBy: P. Rosa & M. Jacobs (MJC); identifiedBy: Paolo Rosa; occurrence!D: 23B773FC-C44D-5C19-AE88-3CB26CCF3BA4 phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: cross/; country: Portugal; locality: Rocha; decimalLatitude: 37.1; decimalLongitude: -8.5; year: 2021; month: 5; day: 26; individualCount: 6; sex: 5 M, 1 F; 20 Rosa P etal lifeStage: Adult; recordedBy: M. Jacobs (MJC); identifiedBy: Paolo Rosa; occurrence!D: 4A481E5E-5CD0-5D06-A882-CA7D5B027CEC Description Female. Body length (holotype) 5.0 mm. Forewing length 3.5 mm. Head. Vertex and frons with small, contiguous punctures (from 0.2x to 0.3x MOD) and polished interspaces below brow; transverse frontal carina faint; scapal basin medially transversally microridged, laterally with small punctures increasing diameter towards eye (Fig. 7D); malar spaces densely punctate, elongate (1.7x MOD), shorter than first flagellomere (2.0x MOD) and with short dense, silver setae; genal carina fully developed to mandibular insertion; clypeus mostly polished, sparsely punctate along anterior margin; clypeus elongate, subantennal area 1.7 MOD; medially notched and apically thickened. First flagellomere elongate, Iw = 4 (width taken at base of flagellomere). OOL 1.4x MOD; POL 2.0x MOD; MS 1.7x MOD; relative length of P:F1:F2:F3 = 1.0:1.6:0.9:0.7. Figure 7. EESl Chrysis crossi Rosa, sp. nov., female, holotype. A habitus, lateral view; B habitus, dorsal view; C head and mesosoma, dorsal view; D head, frontal view; E metasoma, postero-lateral view; F metasoma, dorsal view. Scale bar: 1 mm. 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- The InBIO Barcoding Initiative Database: contribution to the knowledge ... 21 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. Figure 8. EESI Chrysis crossi Rosa, sp. nov. A male (Ph.: © M. Jacobs); B female (Ph.: © M. Jacobs); C collecting site of Chrysis crossi with Maarten Jacobs; D Male genital capsule; E Male genital capsule of Chrysis phryne from Italy (Emilia-Romagna, Oriano). 22 Rosa P etal 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. Figure 9. EES] A-B metasoma, dorsal view: A Chrysis crossi Rosa, sp. nov., male, paratype; B Chrysis phryne male, from Italy (Emilia-Romagna, Oriano); C-D metasoma, ventral view; C Chrysis crossi Rosa, sp., nov., male, paratype; D Chrysis phryne male, from Italy (Emilia-Romagna, Oriano). Genetics: Chrysis crossi is very distinct genetically, being separated from an Italian specimen of C. phryne by 8.51%. There are no other specimens separated by less than a genetic distance of 10.0%. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 23 Etymology The specific epithet crossi (masculine) is dedicated to lan 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. Distribution Portugal (Algarve). Ecology Label information from lan Cross reports that a male specimen was collected at an aggregation of Melitturga caudata Pérez, 1879 (Andrenidae), on the sand near empty snail shells. Chrysis phryne has been reported to attack Osmia (Allosmia) melanura Morawitz, 1871 (see Pauli 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 (Muller 2022). It is likely that a different snail shell- nesting O. (Allosmia) is used instead, all known species of O. (A/llosmia) 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 ( Muller 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. Notes According to Linsenmaier (1959), the phryne group includes only two species: Chrysis circe Mocsary, 1889 and C. phryne Abeille de Perrin, 1878, with three subspecies C. phryne s.str., C. phryne hebraeica Linsenmaier, 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. 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, Mocsary (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 (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) identified a specimen 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) and Mocsary 24 Rosa P etal (1889) and, in particular, on the detail on the punctation of the second tergum “réguliére 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. Analysis Hedychridium caputaureum Trautmann & Trautmann, 1919 and Hedychridium chloropygum du Buysson, 1888 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. chloropygum densum Linsenmaier, 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). 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 south- western Europe. Additional genetic samples from Spain and France are likely to fall between the Portuguese and central/northern European sequences. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 25 Figure 10. EES Hedychridium caputaureum Trautmann & Trautmann, 1919, from Algarve A habitus, dorsal view; B habitus, postero-lateral view; C genital capsule, ventral view; D genital capsule, dorsal view. IBIHM1187-22_Hedychridium_ardens_Belgium_Namur KY430845_Hedychrum_caputaureum_Germany_Mauchenheim MZ626517_Hedychridium_caputaureum_Finland_Regio_Aboensis MZ627137_Hedychridium_caputaureum_Finland_Nylandia capu ta ureum BACHR024-21_Hedychridium_caputauretum_listed_roseum_Austria 1BIHM1161-22_Hedychridium_caputaureum_Algarve_Lagos IBIHM1116-22_Hedychridium_subroseum_prochloropygum_Spain_Madrid_Campo_Real IBIHM1203-22_Hedychridium_chloropygum_lItaly_Aosta IBIHM1202-22_Hedychridium_chloropygum_Italy_Aosta (i chloropygum 1BIHM1213-22_Hedychridium_sculpturatum_group_ltaly_Parma IBIHM1205-22_Hedychridium_cf._valesiense_Italy_Parma | t t IBIHM1214-22_Hedychridium_cf._sculpturatum_Italy_Parma scu Pp ura um ag g . GMBUE782-14_Hedychridium_roseum_Bulgaria_Sofia IBIHM1206-22_Hedychridium_roseum_Italy_Parma GBAH0528-06_Hedychridium_roseum_Germany_Waldleiningen AJ514372_Hedychridium_roseum_ltaly_Pondel roseum KY430844_Hedychridium_roseum_Germany_Altleiningen MZ626798_Hedychridium_roseum_Finland_Karelia MZ623530_Hedychridium_roseum_Finland_Nylandia IBIHM1204-22_Hedychridium_mediocrum_lItaly_Aosta IBIHM1117-22_Hedychridium_mediocrum_Spain_Madrid_Campo_Real IBIHM1189-22_Hedychridium_scutellare_Algarve_Tavira 1 00 IBIHM1162-22_Hedychridium_scutellare_Portugal_Freimoninho Figure 11. EEN 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. 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 26 Rosa P etal moment, we consider it to be the northern African subspecies of H. chloropygum, based on its shallow and sparse punctation. Hedychrum rutilans Dahlbom, 1854 and Hedychrum viridiaureum Tournier, 1877 sp. resurr. 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 Mocsary, 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. viridijaureum (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. viridjaureum can be found in Iberia (such as IBIHM-1183-22) where they are associated with Philanthus pulchellus Spinola, 1842 that is smaller than P. trangulum. Equally, in central Spain, the large-bodied Philanthus dufourii Lucas, 1849 is much more frequently encountered than P The InBIO Barcoding Initiative Database: contribution to the knowledge ... 27 coronatus and, hence, this is likely the principal host of H. rutilans s. str. in this region. The correct placement of H. rutilans viridiauratum Mocsary, 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. 1BIHM1117-22_Hedychridium_mediocrum_Spain_Madrid KY430857_Hedychrum_longicolle_France_Lieuche IBIHM1124-22_Hedychridium_longicolle_Spain_Segovia_Buiteras KY430856_Hedychrum_gerstaeckeri_Rhineland-Palatinate MZ623931_Hedychrum_gerstaeckeri_Finland_Tavastia MZ629003_Hedychrum_gerstaeckeri_Finland_Savonia MZ624334_Hedychrum_gerstaeckeri_Finland_Nylandia HQ563042_Hedychrum_spp_Germany_Berlin CCDB-09785-D05_Hedychrum_spp_Bulgaria CCDB-09785-C12_Hedychrum_spp_Bulgaria MZ628991_Hedychrum_rutilans_Finland_Nylandia MZ626931_Hedychrum_rutilans_Finland_Nylandia MZ625850_Hedychrum_rutilans_Finland_Nylandia MZ626418_Hedychrum_rutilans_Finland_Nylandia ru ti i} ans GBACU707-12_Hedychrum_spp_Germany_Brandenburg 1BIHM1121-22_Hedychrum_rutilans_Segovia_Bernuy_de_Porreros e. st a GBACU2994-13_Hedychrum_spp_lItaly_Aosta GBACU2993-13_Hedychrum_spp_italy_Aosta CCDB-09785-D04_Hedychrum_spp_Bulgaria HQ563043_Hedychrum_spp_Germany_Saxony-Anhalt HQ563040_Hedychrum_spp_Germany_Brandenburg KY430858_Hedychrum_rutilans_Germany_Armsheim rica: IBIHM1183-22_Hedychrum_rutilans_viridauratum_Algarve_Boca_do_Rio vi ridia ure um 1BIHM1171-22_Hedychrum_rutilans_viridauratum_Portugal_Vila_Real S p. re Ss Uu rr. 1BIHM1120-22_Hedychrum_rutilans_viridauratum_Segovia_Bernuy_de_Porreros MZ623104_Hedychrum_nobile_Finland_Nylandia MZ628570_Hedychrum_nobile_Finland_Nylandia IBIHM1172-22_Hedychrum_nobile_Portugal_Loivos_de_Monte IBIHM1123-22_Hedychrum_nobile_Spain_Segovia_Riofrio KY430860_Hedychrum_nobile_Germany_Altleiningen IBIHM1125-22_Hedychrum_micans_europaeum_Segovia_Buiteras IBIHM1122-22_Hedychrum_niemelai_Spain_Granada MZ624023_Hedychrum_niemelai_Finland_Savonia MZ624501_Hedychrum_niemelai_Finland_Nylandia Figure 12. EEN 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 13. EEN Hedychrum rutilans Dahlbom, 1854, habitus, dorsal view A male from Austria, Hainburg (NMLU); B female from Austria, Hainburg (NMLU); C female from Spain, Segovia; D male genital capsule, Austria (NMLU). 28 Rosa P etal Figure 14. EES Hedychrum viridiaureum Tournier, 1877, habitus, dorsal view A male from Switzerland, Wallis (NMLU); B female from Spain, Soria (NMLU); C male from Spain, Segovia; D male genital capsule, Spain, Segovia. Philoctetes punctulatus (Dahlbom, 1845) and Philoctetes parvulus (Dahlbom, 1845) 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 species- specific and are probably caused or exaggerated by the smaller body size. Philoctetes parvulus is, therefore, considered to be a synonym of P. punctulatus. Figure 15. EEN Philoctetes punctulatus (Dahlbom, 1854), habitus, lateral view A female from Spain, Segovia; B female from Portugal, Tavira; C male from Portugal, Tavira. 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 Mocsary, 1912 with C. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 29 lusitanica, but the examination of both types revealed the occurrence of two distinct species (Rosa et al. 2017, PR, unpublished data). Soon et al. (2014) DNA barcoded and revalidated C. sculpturata in the clade of C. ignita (Linnaeus, 1758), whereas the placement of C. /usitanica remained unknown. IBIHM1106-22_Holopyga_calida_Spain_Cenes_de_la_Vega IBIHM1153-22_Pseudomalus_violaceus_Portugal_Carrapateira IBIHM1152-22_Pseudomalus_auratus_Portugal_Aljezur IBIHM1105-22_Philoctetes_abeillei_Spain_Granada_Sierra_Nevada KY430825_Philoctetes_putoni_France_Beuil IBIHM1151-22_Philoctetes_parvulus_Portugal_Tavira IBIHM1104-22_Philoctetes_punctulatus_Spain_Segovia pu n fs tu. la tus IBIHM1150-22_Philoctetes_parvulus_Portugal_Tavira Figure 16. EES) 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 17. EEN Chrysis lusitanica Bischoff, 1910, female, holotype (MfN) A habitus, lateral view; B head, frontal view; C head and mesosoma, dorsal view; D metasoma, dorsal view; E metasoma, postero-lateral view; F metasoma, ventral view. 30 Rosa P etal The DNA barcode of a recently-collected specimen places C. /usitanica in the clade of C. brevitarsis Thomson, 1870, a well-studied group after the molecular works of Soon et al. (2014) and Orlovskyte et al. (2016). In the framework of these previous projects, we can conclude that C. /usitanica 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, 1951 by 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 tergqum 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. /usitanica is considered to be a consistently differentiated species. JX292218_Chrysis_parabrevitarsis. _Germany_Messel ON052 MT888194_Chrysis_parabrevitarsis_Germany_Baden-Wuerttemberg_ON4369 MT888190_Chrysis_parabrevitarsis_Germany_Hesse_ON4668 95 KJ398927_Chrysis_parabrevitarsis_Estonia_Vehendi_VS002798 MT888211_Chrysis_parabrevitarsis_Germany_Hesse_ON4673 93 1 00 MZ628432_Chrysis_brevitarsis_Finland_Tavastia JX292241_Chrysis_brevitarsis_Sweden KJ396933_Chrysis_‘pseudobrevitarsis'_Italy_Sardinia lusitanica IBIHM1148-22 Chrysis_lusitanica_Portugal Algarve 100 MT888216_Chrysis_pseudobrevitarsis_Germany_Bavaria_ON7777 1 00 MZ623895_Chrysis_pseudobrevitarsis_Finland_Karelia pseudobrevitarsis MZ624866_Chrysis_pseudobrevitarsis_Finland_Savonia KJ398900_Chrysis_pseudobrevitarsis_Belarus_Giry IBIHM1058-22_Spintharina_cuprata_Spain_Segovia Figure 18. EES 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. 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 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 31 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. Chrysis splendidula group The splendidula group currently includes 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 sp/endidula group, we identify Chrysis maroccana Mocsary, 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. continentalis Linsenmaier, 1959. Specimen IBIHM1141-22 from southern Portugal (Algarve, Praia do Barril) is strongly separated from two sequences of C. sp/endidula from Italy by an average of 7.67% (range 7.49-7.84%, Fig. 19). We suspect that the Portuguese specimen represents C. maroccana, but additional sampling and genetic sequences from Morocco are required for confident determination, given the complexity within this species group Stilbum westermanni Dahlbom, 1845 sp. resurr. Linsenmaier (1959) listed six subspecies of Sti/lbum calens (Fabricius, 1781) distributed from Europe to China and he separated the two subspecies S. calens zimmermanni Linsenmaier, 1959 and S. calens subcalens Linsenmaier, 1959 (nec Mader 1933, an unavailable name) (Linsenmaier 1959, Linsenmaier 1997a, Linsenmaier 1997b). This latter taxon was later identified as S. calens wesmaeli Dahlbom, 1845 (Linsenmaier 1997a: 134, Linsenmaier 1997b: 287, Linsenmaier 1999: 254). However, Rosa and Vardal (2015) discovered that the type of Stilbum wesmaeli is actually related to S. cyanurum (Forster, 1771) and the first available name for this taxon is Stilbum westermanni Dahlbom, 1845. Rosa and Vardal (2015) considered this to be a subspecies of Stilbum calens, following Linsenmaier’s subspecific interpretation (Linsenmaier 1959, Linsenmaier 1997a, Linsenmaier 1997b). 32 Rosa P etal 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. IBIHM1103-22_Chrysidea_disclusa_pumilionis_Spain_Segovia IBIHM1090-22_Chrysis_cf.rutilans_Spain_Granada ? Sp Nov. IBIHM1175-22_Chrysis_insperata_Portugal_Tras-os-Montes MZ628465_Chrysis_rutilans_Finland_Tavastia MZ628641_Chrysis_rutilans_Finland_Savonia MZ627735_Chrysis_rutilans_Finland_Savonia ru tilans Ss. str. MZ610334_Chrysis_rutilans_Finland_Nylandia MZ607241_Chrysis_rutilans_Finland_Nylandia IBIHM1141-22_Chrysis_splendidula_Portugal_Algarve ? mMmaroccana AJ514390_Chrysis_splendidula_ltaly_Ozein 100 | splendidula s. str. KY430744_Chrysis_splendidula_ltaly_Aosta Figure 19. EEN 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. The specimens DNA barcoded and identified in BOLD as Stilbum cyanurum from Madagascar (M\VV983778 and M\VV983223) are clearly distinct and actually belong to the species Stilbum viride Guérin-Meéneville, 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. sp/endidium 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 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 33 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. AJ514401_Parnopes_grandior_Italy_Pondel HYQTB200-12_Stilbum_superbum_Australia_Queensland amethystinum HYQT111-08_Stilbum_superbum_Australia_Queensland 1 sp. resurr. HYQTBO045-11_Stilbum_superbum_Australia_Queensland AJ514399_Stilbum_cyanurum_cyanurum_Tunisia KMPMO016-19_Stilbum_cyanurum_South_Africa_Limpopo KY430803_Stilbum_cyanurum_Morocco_OQuarzazate 1BIHM1126-22_Stilbum_cyanurum_Portugal_Odemira ° IBIHM1056-22_Stilbum_westermanni_Spain_Almeria wes termannl sp. resurr, IBIHM1055-22_Stilbum_westermanni_Spain_Malaga KY430802_Stilbum_calens_zimmermanni_Italy_Piemonte calens AJ514398_Stilbum_calens_zimmermanni_ltaly_Pondel MW983778_'Stilbum_cyanurum'_Madagascar_Fianarantsoa 100 MW984224 'Stilbum_cyanurum'_Madagascar_Fianarantsoa 0.02 Figure 20. EES 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 21. | doi | Stilbum species, dorsal view. A Stilbum cyanurum (Forster, 1771), male from Portugal, Odemira; B Stilbum westermanni Dahlbom, 1845, female from Spain, Malaga. 34 Rosa P et al 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%. IBIHM1152-22_Pseudomalus_auratus_Portugal_Amoreira AJ514401_Parnopes_gran 2 ior_ltaly_Pondel grandior s. Str. KY430816_Parnopes_gran« a lior_Italy_Aosta KY430817_Parnopes_unicolor_Morocco_Ouarzazarte 1BIHM1180-22_Parnopes_aff_grandior_Boca_do_Rio G Sp. MW984245_Cephaloparnops_vareillesi_Egypt_El_Faiyum KY430818_Cephaloparnops_vareillesi_Algeria_El_Menia Figure 22. EEN 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 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. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 35 New additions to the Portuguese fauna Thanks to this barcoding project, we analysed and added for the first time the following taxa to the list of the Portuguese species: Hedychridium caputaureum Trautmann & Trautmann, 1919 Hedychridium cupritibiale Linsenmaier, 1987 Hedychridium sevillanum Linsenmaier, 1968 Holopyga fastuosa Lucas, 1849 Holopyga jurinei sensu Linsenmaier 1959 Chrysis castillana du Buysson, 1894 Chrysis cerastes Abeille de Perrin, 1877 Chrysis insperata Chevrier, 1870 Chrysis crossi Rosa, sp. nov. Stilbum westermanni Dahlbom, 1845 Acknowledgements The present work was funded by the project NORTE-01-0246-FEDER-000063, supported by Norte Portugal Regional Operational Programme (NORTE2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). InBIO Barcoding Initiative is co-funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 668981 and the project PORBIOTA —Portuguese_ E-Infrastructure for Information and Research on_ Biodiversity (POCI-01-0145- FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER) and by Horizon Europe under the Biodiversity, Circular Economy and Environment call (REA.B.3); co- funded by the Swiss State Secretariat for Education, Research and Innovation (SERI) under contract number 22.00173; and by the UK Research and Innovation under the Department for Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme. SF and VM were funded by the FCT through the programme ‘Stimulus of Scientific Employment, Individual Support—3rd Edition’ 2020.03526.CEECIND; 2020.02547.CEECIND). 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Download file (50.35 kb) Suppl. material 2: IBI - Hymenoptera 02 Chrysididae library - Specimen details doi Authors: Paolo Rosa, Thomas Wood, Sonia Ferreira Data type: Specimen data records Brief description: The file includes information about all records in BOLD for the IBI - Hymenoptera 02 library. It contains collecting and identification data. The data are as downloaded from BOLD in the DWC format, without further processing. Download file (50.35 kb) Suppl. material 3: IBI - Hymenoptera 02 Chrysididae library - DNA sequences EE) Authors: Paolo Rosa, Thomas Wood, Sonia Ferreira Data type: Genomic data, DNA sequences Brief description: COl sequences in fasta format. Each sequence is identified by the BOLD ProcessID, species name, marker and GenBank accession number, separated by pipe. The data are as downloaded from BOLD. Download file (99.27 kb)