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: e98743 doi: 10.3897/BDJ.11.e98743 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 Luísa L. Silva , Joana Veríssimo , Vanessa A. Mata , Denis Michez , Pedro Beja , Sónia Ferreira ‡ University of Mons, Research Institute for Biosciences, Laboratory of Zoology, Place du parc 20, 7000, Mons, Belgium § CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Campus de Vairão, Universidade do Porto, 4485-661 Vairão, Vila do Conde, Portugal | BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairão, 4485-661 Vairão, Vila do Conde, Portugal ¶ CIBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, InBIO Laboratório Associado, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal Corresponding author: Sónia Ferreira ([email protected]) Academic editor: Dominique Zimmermann Received: 12 Dec 2022 | Accepted: 25 Jan 2023 | Published: 01 Mar 2023 Citation: Rosa P, Wood T, Silva TLL, Veríssimo 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: e98743. 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. 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 NaturMuseum 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 I (COI) 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). 2Rosa P et al 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). The InBIO Barcoding Initiative Database: contribution to the knowledge ... 3 Genus Species IBI code BOLD code BOLD BIN GenBank Chrysidea Chrysidea disclusa pumilionis (Linsenmaier, 1987)# INV12702 IBIHM1103-22 BOLD:AES3051 OP347205 Chrysis Chrysis andradei Linsenmaier, 1959# INV12728 IBIHM1129-22 BOLD:AES8383 OP347228 INV12729 IBIHM1130-22 OP347302 Chrysis berlandi Linsenmaier, 1959# INV12682 IBIHM1083-22 BOLD:AES5679 OP347200 INV12683 IBIHM1084-22 OP347265 INV12734 IBIHM1135-22 OP347250 Chrysis blanchardi Lucas, 1849# INV12732 IBIHM1133-22 BOLD:AEU3313 OP347222 Chrysis caeruliventris Abeille de Perrin, 1878 INV12676 IBIHM1077-22 BOLD:AED3523 OP347173 Chrysis castillana Du Buysson, 1894 INV12731 IBIHM1132-22 BOLD:AED2289 OP347274 Chrysis cerastes Abeille de Perrin, 1877# INV12733 IBIHM1134-22 BOLD:AET4960 OP347293 Chrysis rutilans Olivier, 1790# INV12689 IBIHM1090-22 BOLD:AET4959 OP347268 Chrysis chrysoprasina Forster, 1853 INV12677 IBIHM1078-22 BOLD:AET4958 OP347283 Chrysis chrysoscutella Linsenmaier, 1959# INV12743 IBIHM1144-22 BOLD:AES1459 OP347305 Chrysis comparata Lepeletier, 1806 INV12679 IBIHM1080-22 BOLD:AAU1528 OP347241 Chrysis consanguinea Mocsáry, 1889 INV12687 IBIHM1088-22 BOLD:AED0671 OP347212 Chrysis cortii Linsenmaier, 1951 INV12670 IBIHM1071-22 BOLD:AAR9816 OP347211 Chrysis elegans Lepeletier, 1806# INV12673 IBIHM1074-22 BOLD:AES1460 OP347219 Chrysis emarginatula Spinola, 1808 INV12672 IBIHM1073-22 BOLD:AED6786 OP347309 Chrysis fugax Abeille de Perrin, 1878 INV12661 IBIHM1062-22 BOLD:AED3372 OP347230 Chrysis germari Wesmael, 1839# INV12669 IBIHM1070-22 BOLD:AET6935 OP347229 Chrysis gracillima aurofacies (Trautmann, 1926)# INV12663 IBIHM1064-22 BOLD:AES2863 OP347234 Chrysis grohmanni Dahlbom, 1854 INV12666 IBIHM1067-22 BOLD:AED6294 OP347210 Chrysis hydropica Abeille de Perrin, 1878# INV12735 IBIHM1136-22 BOLD:AET2381 OP347243 Table 1. List of species that were collected and DNA barcoded within this project. # Indicates species with new BINs. 4Rosa P et al Genus Species IBI code BOLD code BOLD BIN GenBank Chrysis insperata Chevrier, 1870# INV12774 IBIHM1175-22 BOLD:AET2383 OP347258 Chrysis integra Fabricius, 1787# INV12741 IBIHM1142-22 BOLD:AET2382 OP347285 Chrysis irreperta Linsenmaier, 1959# INV12671 IBIHM1072-22 BOLD:AER8828 OP347244 Chrysis lusitanica Bischoff, 1910 INV12747 IBIHM1148-22 BOLD:ACQ6955 OP347197 Chrysis merceti (Trautmann, 1926)# INV12744 IBIHM1145-22 BOLD:AET3720 OP347260 Chrysis mixta Dahlbom, 1854# INV12664 IBIHM1065-22 BOLD:AET3717 OP347207 Chrysis monticola Linsenmaier, 1999# INV12681 IBIHM1082-22 BOLD:AET3719 OP347289 INV12730 IBIHM1131-22 OP347304 Chrysis mysticalis Linsenmaier, 1959# INV12678 IBIHM1079-22 BOLD:AET3718 OP347245 INV12736 IBIHM1137-22 OP347271 Chrysis peninsularis du Buysson, 1887# INV12667 IBIHM1068-22 BOLD:AES0122 OP347226 Chrysis crossi Rosa sp. nov.# INV12727 IBIHM1128-22 BOLD:AES0121 OP347295 Chrysis pulchella Spinola, 1808 INV12665 IBIHM1066-22 BOLD:AED0619 OP347198 Chrysis pulcherrima Lepeletier, 1806# INV12688 IBIHM1089-22 BOLD:AET0271 OP347172 Chrysis pyrophana Dahlbom, 1854# INV12775 IBIHM1176-22 BOLD:AET0272 OP347213 Chrysis ramburi Dahlbom, 1854 INV12674 IBIHM1075-22 BOLD:AED5814 OP347263 Chrysis sculpturata Mocsáry, 1912 INV12684 IBIHM1085-22 BOLD:ABU6373 OP347310 Chrysis scutellaris marteni Linsenmaier, 1951 INV12738 IBIHM1139-22 BOLD:ACM0910 OP347269 INV12739 IBIHM1140-22 OP347185 Chrysis sexdentata Christ, 1791 INV12783 IBIHM1184-22 BOLD:ABU6376 OP347217 Chrysis splendidula Rossi, 1790# INV12740 IBIHM1141-22 BOLD:AES6413 OP347180 Chrysis subsinuata Marquet, 1879# INV12662 IBIHM1063-22 BOLD:AES4620 OP347214 Chrysis varidens Abeille de Perrin, 1878 INV12668 IBIHM1069-22 BOLD:AEE0312 OP347171 Chrysis zonata Dahlbom, 1854# INV12742 IBIHM1143-22 BOLD:AET8274 OP347199 Chrysura Chrysura austriaca (Fabricius, 1804) INV12690 IBIHM1091-22 BOLD:AAJ3472 OP347193 Chrysura cuprea (Rossi, 1790) INV12692 IBIHM1093-22 BOLD:AAP1055 OP347176 Chrysura dichroa (Dahlbom, 1854)# INV12696 IBIHM1097-22 BOLD:AET1511 OP347273 INV12776 IBIHM1177-22 OP347215 Chrysura hybrida (Lepeletier, 1806) INV12726 IBIHM1127-22 BOLD:AAY6924 OP347177 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 5 Genus Species IBI code BOLD code BOLD BIN GenBank Chrysura purpureifrons (Abeille de Perrin, 1878)# INV12694 IBIHM1095-22 BOLD:AEU2029 OP347231 INV12693 IBIHM1094-22 BOLD:AED1166 OP347286 INV12695 IBIHM1096-22 OP347257 INV12849 IBIHM1250-22 OP347296 INV12850 IBIHM1251-22 OP347297 INV12851 IBIHM1252-22 OP347221 Chrysura radians (Harris, 1776) INV12697 IBIHM1098-22 BOLD:ABA8702 OP347225 Chrysura refulgens (Spinola, 1806) INV12777 IBIHM1178-22 BOLD:ABA7395 OP347303 Chrysura rufiventris (Dahlbom, 1854) INV12699 IBIHM1100-22 BOLD:AEC6882 OP347290 Chrysura simplex (Dahlbom, 1854) INV12691 IBIHM1092-22 BOLD:AAY6923 OP347189 Chrysura sulcata (Dahlbom, 1845) INV12700 IBIHM1101-22 BOLD:ABA7396 OP347249 Chrysura varicornis (Spinola, 1838)# INV12698 IBIHM1099-22 BOLD:AET0222 OP347261 INV12701 IBIHM1102-22 OP347192 Hedychridium Hedychridium aereolum du Buysson, 1892 INV12809 IBIHM1210-22 BOLD:AAY6930 OP347195 Hedychridium anale (Dahlbom, 1854) INV12717 IBIHM1118-22 BOLD:AED4749 OP347270 Hedychridium ardens (Coquebert, 1801) INV12786 IBIHM1187-22 BOLD:AAK4640 OP347236 Hedychridium buyssoni Abeille de Perrin, 1887# INV12790 IBIHM1191-22 BOLD:AES9011 OP347196 Hedychridium caputaureum Trautmann & Trautmann, 1919 INV12760 IBIHM1161-22 BOLD:AAU0775 OP347183 Hedychridium valesiense Linsenmaier, 1959# INV12804 IBIHM1205-22 BOLD:AET6828 OP347275 Hedychridium chloropygum du Buysson, 1888 INV12801 IBIHM1202-22 BOLD:AAE3258 OP347187 INV12802 IBIHM1203-22 OP347240 Hedychridium cupratum (Dahlbom, 1854) INV12806 IBIHM1207-22 BOLD:AAY6946 OP347255 INV12807 IBIHM1208-22 OP347252 Hedychridium cupritibiale Linsenmaier, 1987# INV12769 IBIHM1170-22 BOLD:AES9012 OP347239 Hedychridium incrassatum (Dahlbom, 1854) INV12718 IBIHM1119-22 BOLD:AEE0029 OP347267 6Rosa P et al Genus Species IBI code BOLD code BOLD BIN GenBank Hedychridium infans Abeille de Perrin, 1878# INV12768 IBIHM1169-22 BOLD:AES6837 OP347254 INV12781 IBIHM1182-22 OP347191 Hedychridium jucundum Mocsáry, 1889# INV12784 IBIHM1185-22 BOLD:AES6836 OP347262 INV12785 IBIHM1186-22 OP347232 INV12810 IBIHM1211-22 BOLD:AES6835 OP347170 INV12811 IBIHM1212-22 OP347256 Hedychridium krajniki Balthasar, 1946 INV12764 IBIHM1165-22 BOLD:AAZ0056 OP347237 Hedychridium mediocrum Linsenmaier, 1987 INV12716 IBIHM1117-22 BOLD:AAE3260 OP347279 INV12803 IBIHM1204-22 OP347308 Hedychridium monochroum du Buysson, 1888 INV12800 IBIHM1201-22 BOLD:AAY1978 OP347206 Hedychridium reticulatum Abeille de Perrin, 1878# INV12763 IBIHM1164-22 BOLD:AER9655 OP347248 INV12791 IBIHM1192-22 OP347203 INV12792 IBIHM1193-22 OP347282 INV12793 IBIHM1194-22 OP347301 Hedychridium roseum (Rossi, 1790) INV12805 IBIHM1206-22 BOLD:AAE3259 OP347281 Hedychridium sculpturatum Abeille de Perrin, 1877# INV12812 IBIHM1213-22 BOLD:AET6828 OP347181 INV12813 IBIHM1214-22 BOLD:AAE3258 OP347276 Hedychridium scutellare (Tournier, 1878)# INV12761 IBIHM1162-22 BOLD:AES0428 OP347291 INV12788 IBIHM1189-22 OP347175 Hedychridium sevillanum Linsenmaier, 1968# INV12765 IBIHM1166-22 BOLD:AET6827 OP347242 INV12767 IBIHM1168-22 BOLD:AES0429 OP347259 INV12789 IBIHM1190-22 OP347223 Hedychridium calcarium Rosa sp. nov.# INV12794 IBIHM1195-22 BOLD:AET9698 OP347204 INV12795 IBIHM1196-22 OP347307 INV12796 IBIHM1197-22 OP347169 Hedychridium subroseum prochloropygum Linsenmaier, 1959# INV12715 IBIHM1116-22 BOLD:AES2437 OP347209 Hedychridium vachali Mercet, 1915# INV12766 IBIHM1167-22 BOLD:AET6826 OP347280 Hedychrum Hedychrum longicolle Abeille de Perrin, 1877 INV12723 IBIHM1124-22 BOLD:AED0972 OP347298 The InBIO Barcoding Initiative Database: contribution to the knowledge ... 7 Genus Species IBI code BOLD code BOLD BIN GenBank Hedychrum micans europaeum Linsenmaier, 1959 INV12724 IBIHM1125-22 BOLD:AAK4644 OP347194 Hedychrum niemelai Linsenmaier, 1959 INV12721 IBIHM1122-22 BOLD:AAU1294 OP347300 Hedychrum nobile (Scopoli, 1763) INV12722 IBIHM1123-22 BOLD:AAK4644 OP347266 INV12771 IBIHM1172-22 OP347168 Hedychrum rutilans Dahlbom, 1854 INV12720 IBIHM1121-22 BOLD:AAM3491 OP347220 Hedychrum viridiaureum Tournier, 1877 INV12719 IBIHM1120-22 BOLD:AAM3491 OP347294 INV12770 IBIHM1171-22 BOLD:AAK4643 OP347247 INV12782 IBIHM1183-22 BOLD:AAM3491 OP347174 Holopyga Holopyga calida Linsenmaier, 1951# INV12705 IBIHM1106-22 OP347202 INV12754 IBIHM1155-22 BOLD:AES0090 OP347227 Holopyga lucida (Lepeletier, 1806)# INV12708 IBIHM1109-22 BOLD:AES0091 OP347182 Holopyga fastuosa (Lucas, 1849) INV12709 IBIHM1110-22 BOLD:AAZ6194 OP347292 INV12710 IBIHM1111-22 OP347190 INV12780 IBIHM1181-22 OP347253 INV12753 IBIHM1154-22 BOLD:AAY6928 OP347216 Holopyga fervida (Fabricius, 1781) INV12706 IBIHM1107-22 BOLD:ACV6331 OP347208 INV12757 IBIHM1158-22 BOLD:AAY9735 OP347287 Holopyga generosa (Förster, 1853) INV12712 IBIHM1113-22 BOLD:AAZ6194 OP347288 INV12713 IBIHM1114-22 OP347201 Holopyga inflammata (Förster, 1853)# INV12759 IBIHM1160-22 BOLD:AET1451 OP347284 Holopyga jurinei Chevrier, 1862# INV12714 IBIHM1115-22 OP347233 INV12756 IBIHM1157-22 BOLD:AET1450 OP347235 Holopyga similis Mocsáry, 1889 INV12787 IBIHM1188-22 BOLD:AED0274 OP347178 Holopyga merceti Kimsey, 1991# INV12755 IBIHM1156-22 BOLD:AES1216 OP347186 Parnopes Parnopes sp.# INV12779 IBIHM1180-22 BOLD:AET2814 OP347218 Philoctetes Philoctetes abeillei du Buysson, 1892# INV12704 IBIHM1105-22 BOLD:AEU5026 OP347179 Philoctetes punctulatus (Dahlbom, 1854)# INV12703 IBIHM1104-22 BOLD:AEU5027 OP347264 INV12749 IBIHM1150-22 OP347272 INV12750 IBIHM1151-22 OP347238 8Rosa P et al Genus Species IBI code BOLD code BOLD BIN GenBank Pseudochrysis Pseudochrysis humboldti (Dahlbom, 1845)# INV12659 IBIHM1060-22 BOLD:AEU3425 OP347246 Pseudochrysis incrassata (Spinola, 1838)# INV12660 IBIHM1061-22 BOLD:AEU3426 OP347278 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 Stilbum Stilbum westermanni Dahlbom, 1845# INV12654 IBIHM1055-22 BOLD:AES3895 OP347311 INV12655 IBIHM1056-22 OP347306 Stilbum cyanurum (Forster, 1771) INV12725 IBIHM1126-22 BOLD:AAJ4180 OP347184 Trichrysis Trichrysis cyanea (Linnaeus, 1758) INV12658 IBIHM1059-22 BOLD:AAH7935 OP347188 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. Figure 1. Map of the localities where cuckoo wasps samples were collected. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 9 (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 (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. 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 16 Rosa P et al 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. 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. jucundum 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. jucundum specimens from Menorca by an average of 4.94% (range 4.71-5.17%). This requires further investigation. Etymology The epithet calcarium derives from the Latin adjective calcarius related to the limestone habitat of the species. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 17 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 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 6. Villar del Cobo, Barranco de los Oncenachos, province of Teruel, Spain. Collecting site for Hedychridium calcarium Rosa sp. nov. 18 Rosa P et al 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-F1BE-47B2-9C43-0FFD17612E43 Materials Holotype: a. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; country: Portugal; locality: Salema; decimalLatitude: 37.06; decimalLongitude: -8.83; year: 2017; month: 4; day: 16; individualID: 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; occurrenceID: 6CF2BF38-8154-53D2AB88-640F8CDD5821 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; occurrenceID: 9849F20C-7561-5544-B4E2-430469C8A89F b. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; 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; occurrenceID: 01CC81B6-FEC3-5EBD-B89E-B7195661A5AD c. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; 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; occurrenceID: 92FDD0A6-54DE-5166-B4DF-372F55037E84 d. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; 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; occurrenceID: 66AAAE02-1293-52EB-8267-4DBC53119F00 e. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; 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; occurrenceID: 23B773FC-C44D-5C19-AE88-3CB26CCF3BA4 f. phylum: Arthropoda; class: Insecta; order: Hymenoptera; family: Chrysididae; genus: Chrysis; specificEpithet: crossi; country: Portugal; locality: Rocha; decimalLatitude: 37.1; decimalLongitude: -8.5; year: 2021; month: 5; day: 26; individualCount: 6; sex: 5 M, 1 F; The InBIO Barcoding Initiative Database: contribution to the knowledge ... 19 lifeStage: Adult; recordedBy: M. Jacobs (MJC); identifiedBy: Paolo Rosa; occurrenceID: 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 0.2× to 0.3× 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 (1.7× MOD), shorter than first flagellomere (2.0× 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, l/w = 4 (width taken at base of flagellomere). OOL 1.4× MOD; POL 2.0× MOD; MS 1.7× MOD; relative length of P:F1:F2:F3 = 1.0:1.6:0.9:0.7. 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, anteroFigure 7. 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. 20 Rosa P et al 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. 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). The InBIO Barcoding Initiative Database: contribution to the knowledge ... 21 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. 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%. Figure 9. 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). 22 Rosa P et al 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. Distribution Portugal (Algarve). 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. 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 (Müller 2022). It is likely that a different snail shellnesting O. (Allosmia) 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. Notes According to Linsenmaier (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 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, 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 (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 Mocsáry The InBIO Barcoding Initiative Database: contribution to the knowledge ... 23 (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. 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 southwestern Europe. Additional genetic samples from Spain and France are likely to fall between the Portuguese and central/northern European sequences. 24 Rosa P et al 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 Figure 10. 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. 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. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 25 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 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. 32 Rosa P et al 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. 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 21. Stilbum species, dorsal view. A Stilbum cyanurum (Forster, 1771), male from Portugal, Odemira; B Stilbum westermanni Dahlbom, 1845, female from Spain, Malaga. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 33 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 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. 34 Rosa P et al 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-01-0145FEDER-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); cofunded 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). JV was funded by a PhD grant (SFRH/BD/133159/2017) from FCT. PR is supported by the EU Project ORBIT (DG Env 09.029901/2021/848268/SER/ ENV.D.2). TJW is supported by an F.R.S.-FNRS fellowship “Chargé de recherches”. We thank Villu Soon (Tartu, Estonia) and Juho Paukkunen (Helsinki, Finland) for reviewing the manuscript. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 35 References • Arens W (2010) Revision der Hedychridium roseum-Gruppe in Kleinasien (Hymenoptera: Chrysididae), mit Neubewertung zahlreicher europäischer Taxa und Beschreibung zweier neuer Arten. Linzer Biologische Beiträge 42 (1): 401‑458. • Baldock D, Wood T, Cross I, Smit J (2018) The bees of Portugal (Hymenoptera: Apoidea: Anthophila). Entomofauna Supplement 22: 1‑164. • Baldock D, Castro L, Cross I, Schmid-Egger C, Smit J, Wood T (2020) The vespiform wasps of Portugal (Hymenoptera: Scolioidea, Tiphioidea, Pompiloidea & Vespoidea). Monografias S.E.A. 14: 1‑74. • Baumgarten H (1995) Beobachtungen zum Verhalten von Hedychrum rutilans (Hymenoptera: Chrysididae) bei seinem Wirt, dem Bienenwolf Philanthus triangulum (Hymenoptera: Sphecidae). Bembix 5: 35‑37. • Boyer F, Mercier C, Bonin A, Le Bras Y, Taberlet P, Coissac E (2016) OBITOOLS: a UNIX-inspired software package for DNA metabarcoding. Molecular Ecology Resources 16 (1): 176‑182. https://doi.org/10.1111/1755-0998.12428 • Cross I, Baldock D, Wood T (2021) The spheciform wasps of Portugal (Hymenoptera: Apoidea: Ampulicidae, Sphecidae & Crabronidae). Monografias S.E.A. 15: 1‑84. • du Buysson R (1888) Descriptions de Chrysidides nouvelles. Revue d'Entomologie 7: 1‑13. • Elbrecht V, Leese F (2017) Validation and development of COI metabarcoding primers for freshwater macroinvertebrate mioassessment. Frontiers in Environmental Science 5 https://doi.org/10.3389/fenvs.2017.00011 • Elbrecht V, Braukmann TA, Ivanova N, Prosser SJ, Hajibabaei M, Wright M, Zakharov E, Hebert PN, Steinke D (2019) Validation of COI metabarcoding primers for terrestrial arthropods. PeerJ 7: e7745. https://doi.org/10.7717/peerj.7745 • Folmer O, Black M, Hoeh W, Lutz R, Vrijenhoek R (1994) DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Molecular Marine Biology and Biotechnology 3 (5): 294‑9. • González J, Gayubo S, Strumia F (1999) Biodiversità dei crisididi di un ambiente orticolo del territorio “Las Arribes del Duero” (Provincia di Salamanca, Spagna Occidentale) (Hymenoptera, Chrysididae). Frustula Entomologica 21: 26‑41. • González J, Gayubo S, Asís J, Tormos J (2009) Diversity and biogeographical significance of solitary wasps (Chrysididae, Eumeninae, and Spheciformes) at the Arribes del Duero Natural Park, Spain: Their importance for insect diversity conservation in the Mediterranean Region. Environmental Entomology 38 (3): 608‑626. https://doi.org/10.1603/022.038.0312 • Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution 27: 221‑224. https://doi.org/10.1093/molbev/msp259 • Huang X, Madan A (1999) CAP3: A DNA Sequence Assembly Program. Genome Research 9 (9): 868‑877. https://doi.org/10.1101/gr.9.9.868 • Kimsey L, Bohart R (1991) The chrysidid wasps of the World. Oxford University Press, New York, 652 pp. 36 Rosa P et al • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular Evolutionary Genetics Analysis across computing platforms. Molecular Biology and Evolution 35: 1547‑1549. https://doi.org/10.1093/molbev/msy096 • Linsenmaier W (1951) Die europäischen Chrysididen (Hymenoptera). Versuch einer natürlichen Ordnung mit Diagnosen. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 24: 1‑110. • Linsenmaier W (1959) Revision der Familie Chrysididae (Hymenoptera) mit besonderer Berücksichtigung der europäischen Spezies. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 32 (1): 1‑232. • Linsenmaier W (1968) Revision der Familie Chrysididae (Hymenoptera). Zweiter Nachtrag. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 41: 1‑144. • Linsenmaier W (1987) Revision der Familie Chrysididae. (Hymenoptera). 4 Teil. Mitteilungen der Schweizerischen Entomologischen Gesellschaft 60: 133‑158. • Linsenmaier W (1997a) Die Goldwespen der Schweiz. Veröffentlichungen aus dem Natur-Museum Luzern 9: 1‑140. • Linsenmaier W (1997b) Altes und Neues von den Chrysididen. (Hymenoptera, Chrysididae). Entomofauna 18: 245‑300. • Linsenmaier W (1999) Die Goldwespen Nordafrikas (Hymenoptera, Chrysididae). Entomofauna Supplement 10: 1‑210. • Michez D, Rasmont P, Terzo M, Vereecken N (2019) Bees of Europe. NAP Editions, Paris, 548 pp. • Mingo E, Gayubo S (1981) Contribución al conocimiento de los Crisídidos de la sierra de Béjar (Hym., Chrysididae). Eos 57: 153‑163. • Mingo E, Gayubo S (1986) Contribución al conocimiento de la crisididofauna de las sierras meridionales de la provincia de Badajoz (Hym. Chrysididae). Acta de las VIII Jornadas de la Asociación Española de Entomologia Sevilla1012‑1021. • Mingo E (1994) Hymenoptera Chrysididae. Fauna Iberica. Vol. 6. Museo Nacional de Ciencias Naturales, Consejo Superior de Investigaciones Científicas, Madrid, 256 pp. • Mitroiu M, Noyes J, Cetkovic A, Nonveiller G, Radchenko A, Polaszek A, Ronquist F, Forshage M, Pagliano G, Gusenleitner J, Bartalucci M, Olmi M, Fusu L, Madl M, Johnson N, Jansta P, Wahis R, Soon V, Rosa P, Osten T, Barbier Y, Jong Y (2015) Fauna Europaea: Hymenoptera-Apocrita (excl. Ichneumonoidea). Biodiversity Data Journal 3 (e4186): 1‑27. https://doi.org/10.3897/BDJ.3.e4186 • Mocsáry A (1889) Monographia Chrysididarum Orbis Terrarum Universi. Hungarian Academy of Science, Typis Societatis Franklinianæ, Budapest, 643 pp. • Morgan D (1984) Cuckoo-Wasps. (Hymenoptera, Chrysididae). Handbooks for the Identification of British Insects, 37 pp. • Müller A (2022) Palaearctic Osmia bees of the subgenera Allosmia and Neosmia (Megachilidae, Osmiini): biology, taxonomy and key to species. Zootaxa 5188 (3): 201‑232. https://doi.org/10.11646/zootaxa.5188.3.1 • Niehuis O, Wägele J (2004) Phylogenetic analysis of the mitochondrial genes LSU rRNA and COI suggests early adaptive differentiation of anal teeth in chrysidine cuckoo wasps (Hymenoptera: Chrysididae). Molecular Phylogenetics and Evolution 30: 615‑622. https://doi.org/10.1016/S1055-7903(03)00231-8 • Orlovskyté S, Budrys E, Budriene A, Radzevičiūte R, Soon V (2016) Sibling species in the Chrysis ignita complex: molecular, morphological and trophic differentiation of Baltic The InBIO Barcoding Initiative Database: contribution to the knowledge ... 37 species, with a description of two new cryptic species (Hymenoptera: Chrysididae. Systematic Entomology 41 (4): 771‑793. https://doi.org/10.1111/syen.12190 • Paukkunen J, Rosa P, Soon V, Johansson N, Ødegaard F (2014) Faunistic review of the cuckoo wasps of Fennoscandia, Denmark and the Baltic countries (Hymenoptera: Chrysididae. Zootaxa 3864 (1): 1‑67. https://doi.org/10.11646/zootaxa.3864.1.1 • Paukkunen J, Berg A, Soon V, Ødegaard F, Rosa P (2015) An illustrated key to the cuckoo wasps (Hymenoptera, Chrysididae) of the Nordic and Baltic countries, with description of a new species. ZooKeys 548: 1‑116. https://doi.org/10.3897/zookeys. 548.6164 • Pauli T, Castillo-Cajas RF, Rosa P, Kukowka S, Berg A, Berghe E, Fornoff F, Hopfenmü l, Niehuis M, Peters RS, Staab M, Strumia F, Tischendorf S, F S, Niehuis O (2019) Phylogenetic analysis of cuckoo wasps (Chrysididae) reveals the partially artificial nature of the current classification at the genus level in this family of Hymenoptera. Systematic Entomology 44 (2): 322‑335. https://doi.org/10.1111/syen. 12323 • Paupério J, Fonseca N, Egeter B, Galhardo M, Ferreira S, Oxelfelt F, Aresta S, Martins F, Mata V, Veríssimo J, Puppo P, Pinto JC, Chaves C, Garcia-Raventós A, Peixoto S, da Silva LP, Vasconcelos S, Gil P, Khalatbari L, Jarman S, Beja P (2018) Deliverable 4.4 (D4.4): Protocol for next-gen analysis of eDNA samples, EnMetaGen project (Grant Agreement No 668981). Zenodo https://doi.org/10.5281/zenodo.2586884 • Rognes T, Flouri T, Nichols B, Quince C, Mahé F (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4: e2584. https://doi.org/10.7717/peerj.2584 • Romano M (2006) La ricerca entomologica in Sicilia: protagonisti, cultori e collezioni a cavallo di tre secoli. Il Naturalista Siciliano 30 (2): 151‑226. • Rosa P, Soon V (2012) Hymenoptera: Chrysididae. Fauna Europaea version 2.5. http:// www.faunaeur.org. Accessed on: 2022-8-30. • Rosa P, Vårdal H (2015) An annotated catalogue of the types of Chrysididae (Hymenoptera)at the Swedish Museum of Natural History, Stockholm, with brief historical notes. ZooKeys 495: 79‑132. https://doi.org/10.3897/zookeys.495.9356 • Rosa P, Xu Z (2015) Annotated type catalogue of the Chrysididae (Insecta, Hymenoptera) deposited in the collection of Maximilian Spinola (1780–1857), Turin. ZooKeys 471: 1‑96. https://doi.org/10.3897/zookeys.471.6558 • Rosa P, Livory A, Baldock D (2015) Chrysis monticola Linsenmaier, 1999 (Hymenoptera, Chrysididae), a new species for the European fauna. Natural History Sciences 2 (2): 89‑94. https://doi.org/10.4081/nhs.2015.235 • Rosa P (2017) Review of the Palaearctic species of the genus Colpopyga (Hymenoptera: Chrysididae) with description of a new species. Zoosystematica Rossica 26 (2): 294‑306. https://doi.org/10.31610/zsr/2017.26.2.294 • Rosa P, Lelej A, Belokobylskij S, Loktionov V, Zaytseva L (2017) Family Chrysididae. In: Lelej A, Proshchalykin M, Loktionov V (Eds) Annotated catalogue of the Hymenoptera of Russia. Volume 1. Symphyta and Apocrita: Aculeata. Proceedings of the Zoological Institue RAS, 126-144. pp. https://doi.org/10.31610/trudyzin/2017.supl.6.5 • Rosa P, Heim R, Bernasconi MV (2022) The Palaearctic types of Chrysididae (Insecta, Hymenoptera) deposited in the Linsenmaier collection. Part 2. Elampini: genus Hedychridium Abeille de Perrin, 1878. Zootaxa 5090: 1‑94. https://doi.org/10.11646/ zootaxa.5090.1.1 38 Rosa P et al • Roslin T, Somervuo P, Pentinsaari M, Hebert PN, Agda J, Ahlroth P, Anttonen P, Aspi J, Blagoev G, Blanco S, Chan D, Clayhills T, deWaard J, deWaard S, Elliot T, Elo R, Haapala S, Helve E, Ilmonen J, Hirvonen P, Ho C, Itämies J, Ivanov V, Jakovlev J, Juslén A, Jussila R, Kahanpää J, Kaila L, Jari‐PekkaKaitila, Kakko A, Kakko I, Karhu A, Karjalainen S, Kjaerandsen J, Koskinen J, Laasonen E, Laasonen L, Laine E, Lampila P, Levesque‐Beaudin V, Lu L, Lähteenaro M, Majuri P, Malmberg S, Manjunath R, Martikainen P, Mattila J, McKeown J, Metsälä P, Miklasevskaja M, Miller M, Miskie R, Muinonen A, Veli‐MattiMukkala, Naik S, Nikolova N, Nupponen K, Ovaskainen O, Österblad I, Paasivirta L, Pajunen T, Parkko P, Paukkunen J, Penttinen R, Perez K, Pohjoismäki J, Prosser S, Raekunnas M, Rahulan M, Rannisto M, Ratnasingham S, Raukko P, Rinne A, Rintala T, Miranda Romo S, Salmela J, Salokannel J, Savolainen R, Schulman L, Sihvonen P, Soliman D, Sones J, Steinke C, Ståhls G, Tabell J, Tiusanen M, Várkonyi G, Vesterinen E, Viitanen E, Vikberg V, Viitasaari M, Vilen J, Warne C, Wei C, Winqvist K, Zakharov E, Mutanen M (2021) A molecular‐based identification resource for the arthropods of Finland. Molecular Ecology Resources 22 (2): 803‑822. https:// doi.org/10.1111/1755-0998.13510 • Shokralla S, Porter T, Gibson J, Dobosz R, Janzen D, Hallwachs W, Golding GB, Hajibabaei M (2015) Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform. Scientific Reports 5 (1). https://doi.org/ 10.1038/srep09687 • Soon V, Saarma U (2011) Mitochondrial phylogeny of the Chrysis ignita (Hymenoptera: Chrysididae) species group based on simultaneous Bayesian alignment and phylogeny reconstruction. Molecular Phylogenetics and Evolution 60: 13‑20. https://doi.org/ 10.1016/j.ympev.2011.04.005 • Soon V, Budrys E, Orlovskytė S, Paukkunen J, Ødegaard F, Ljubomirov T, Saarma U (2014) Testing the validity of Northern European species in the Chrysis ignita species group (Hymenoptera: Chrysididae) with DNA Barcoding. Zootaxa 3786 (3): 301‑330. https://doi.org/10.11646/zootaxa.3786.3.4 • Strumia F, Yildirim E (2009) Contribution to the knowledge of Chrysididae fauna of Turkey (Hymenoptera, Aculeata). Frustula Entomologica 30 (43): 55‑92. • Vamos E, Elbrecht V, Leese F (2017) Short COI markers for freshwater macroinvertebrate metabarcoding. Metabarcoding and Metagenomics 1: e14625. https://doi.org/10.3897/mbmg.1.14625 • Veenendaal R (1987) Het verborgen ei van Hedychrum rutilans (Hymenoptera: Chrysididae). Entomologische Berichten 47: 169‑171. The InBIO Barcoding Initiative Database: contribution to the knowledge ... 39 Supplementary materials Suppl. material 1: IBI - Hymenoptera 02 Chrysididae library - Specimen details Authors: Paolo Rosa, Thomas Wood, Sónia 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 tsv format, without further processing. Download file (50.35 kb) Suppl. material 2: IBI - Hymenoptera 02 Chrysididae library - Specimen details Authors: Paolo Rosa, Thomas Wood, Sónia 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 Authors: Paolo Rosa, Thomas Wood, Sónia Ferreira Data type: Genomic data, DNA sequences Brief description: COI 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) 40 Rosa P et al