The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal

Biodiversity Data Journal 12: e118010 doi: 10.3897/BDJ.12.e118010 Data Paper The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal Sílvia Pina , Joana Pauperio , Francisco Barros , Cátia Chaves , Filipa MS Martins , Joana Pinto , Joana Veríssimo , Vanessa A Mata , Pedro Beja , Sónia Ferreira ‡ 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 ¶ European Molecular Biology Laboratory, European Bioinformatics Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom # Rua da Eira 3, S. Salvador, 2550-251, Cercal, Portugal ¤ EBM, Estação Biológica de Mertola, Praça Luis de Camoes, Mertola, Mertola, Portugal Corresponding author: Sónia Ferreira ([email protected]) Academic editor: Edward Baker Received: 30 Dec 2023 | Accepted: 24 Feb 2024 | Published: 15 May 2024 Citation: Pina S, Pauperio J, Barros F, Chaves C, Martins FM, Pinto J, Veríssimo J, Mata V, Beja P, Ferreira S (2024) The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal. Biodiversity Data Journal 12: e118010. https://doi.org/10.3897/BDJ.12.e118010 Abstract Background The InBIO Barcoding Initiative (IBI) Orthoptera dataset contains records of 420 specimens covering all the eleven Orthoptera families occurring in Portugal. Specimens were collected in continental Portugal from 2005 to 2021 and were morphologically identified to species level by taxonomists. A total of 119 species were identified corresponding to about 77% of all the orthopteran species known from continental Portugal. ‡,§,| ‡,¶ # ‡,§ ‡,§ ‡,§ ‡,§ ‡,§ ‡,§,| ‡,§,¤ © Pina S 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. New information DNA barcodes of 54 taxa were made public for the first time at the Barcode of Life Data System (BOLD). Furthermore, the submitted sequences were found to cluster in 129 BINs (Barcode Index Numbers), 35 of which were new additions to the Barcode of Life Data System (BOLD). All specimens have their DNA barcodes publicly accessible through BOLD online database. Stenobothrus lineatus is recorded for the first time for continental Portugal. This dataset greatly increases the knowledge on the DNA barcodes and distribution of Orthoptera from Portugal. All DNA extractions and most specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources. Keywords Orthoptera, species distributions, continental Portugal, DNA barcode, cytochrome c oxidase subunit I (COI) Introduction Insects are very challenging to study due to their astonishing biological diversity, the lack of taxonomic expertise for several groups and traditional morphology-based species identification being often logistically or financially unsustainable. DNA metabarcoding is rapidly emerging to overcome these challenges (van Klink et al. 2022), but its application relies on the existence of comprehensive reference collections of DNA barcodes. This method identifies multiple species from a mixed sample, based on DNA barcoding using high-throughput sequencing (HTS) of a specific DNA marker, usually the mitochondrial cytochrome c oxidase I (COI) gene. DNA barcoding is a molecular biology method for species identification that relies on the comparison of a short mitochondrial DNA sequence of interest to a library of sequences with known species identity (Hebert et al. 2003). Hence, for these comparisons, it is important to guarantee that a good sequence library is being used, i.e. all DNA barcodes come from specimens identified by taxonomists (Chimeno et al. 2023). The Barcode of Life Data Systems (BOLD) is an international barcode reference database made available in 2007 (Ratnasingham and Herbert 2007) and large national barcoding initiatives worldwide have contributed to increasing this library (Janzen and Hallwachs 2011,Geiger et al. 2016, deWaard et al. 2019). In Portugal, the InBIO Barcoding Initiative (IBI) was the first project aimed to develop a reference collection of DNA barcoding sequences, having largely focused on Portuguese invertebrate taxa, particularly insects (Ferreira et al. 2018). Orthoptera are a diverse group of herbivorous insects and playing an important role in ecosystem functioning as both primary consumers in different habitats and significant food sources for higher trophic levels (e.g. birds, mammals) (Catry et al. 2018, Valdez and Cryan 2013). Due to the strong connection with plants, orthopterans are good indicators of 2Pina S et al habitat changes and, therefore, often used in environmental monitoring and assessment (Bieringer et al. 2013, Vasconcelos et al. 2019). However, despite their functional importance, they are still poorly studied in areas with high levels of endemic species, such as the Iberian Peninsula (Hochkirch et al. 2016). Studies on the Portuguese Orthoptera fauna have been published in a very scattered manner over time, generally lacking comprehensive inventories or focusing on certain Orthoptera taxa (Pina et al. 2017). Although some of these more recent studies report new findings for the country (e.g. Lemos et al. (2016), Monteiro et al. (2016)), the prime information, such as which species occur in Portugal and their distributions, remains very incomplete. In Europe, large DNA barcoding initiatives have been established mainly in the northern and central countries (Gaytán et al. 2020). In the study conducted by Hawlitschek et al. (2017), DNA barcodes of Orthoptera from four barcoding initiatives (Barcoding Fauna Bavarica (Germany), German Barcode of Life, Austrian Barcode of Life and Swiss Barcode of Life) comprising three central European countries (Austria, Germany and Switzerland) were made available. A total of 748 COI sequences were obtained for several central European Orthoptera taxa that also occur in other countries, namely in Portugal. This study also showed that barcoding studies can be successfully applied to Orthoptera revealing an overall congruence of 76.2% of the 127 Orthoptera taxa in the study. However, more work is necessary to create a library of barcodes of the European Orthoptera because the representation of certain groups (e.g. Tetrigidae, Gryllidae and Bradyporinae) (Kasalo et al. 2023) and regional areas, such as the southern European peninsulas, remains insufficient. The Iberian Peninsula is a known hotspot of endemic Orthoptera species, but many of its species remain unrepresented in the DNA barcoding reference collections. The IBI Orthoptera dataset contains records of 420 specimens of Orthoptera collected in continental Portugal, all morphologically identified to species level, for a total of 119 species. Our results constitute a first step in the construction of a DNA barcode database of a curated reference collection of Iberian Orthoptera species. General description Purpose: This dataset aims to provide a contribution to the knowledge on DNA barcodes of Portuguese Orthoptera. Such a library should facilitate DNA-based identification of species for both traditional molecular studies and DNA metabarcoding studies. Furthermore, it constitutes a valuable resource for taxonomic research on Iberian Orthoptera and their distribution. Additional information: A total of 420 specimens of orthopterans were collected and DNA barcoded (Table 1, Suppl. material 2) corresponding to 119 species, about 77% of all the orthopteran species known from continental Portugal (Aires and Menano 1915, Pina et al. 2017, GBIF 2023, IUCN 2023). Figs 1, 2 illustrate examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera. The dataset includes 39 Iberian endemic species, of which six occur only in continental Portugal: Ephippigerida rosae, Lluciapomaresius anapaulae, Neocallicrania barrosi, The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 3 Neocallicrania serrata, Pterolepis lusitanica and Pycnogaster cucullatus (Table 1). Additionally, the species Stenobothrus lineatus is recorded for the first time for Portugal in this dataset. A full-length barcode of 658 bp was obtained for all 420 specimens. This dataset contributes significantly to the representation of both species and genetic diversity of Orthoptera in public libraries. Of the 120 taxa barcoded, DNA barcodes of 54 are made public for the first time (marked with # in Taxa field of Table 1). The submitted sequences were found to cluster in 129 BINs, 35 of which were new to BOLD (unique BINs, marked with " in Taxa field of Table 1). Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Acrotylus insubricus (Scopoli, 1786) INV02543 IBIOR108-17 BOLD:AAP4452 OR974638 Acrididae Acrotylus insubricus (Scopoli, 1786) INV02544 IBIOR109-17 BOLD:AAP4452 OR974794 Acrididae Acrotylus insubricus (Scopoli, 1786) INV05545 IBIOR196-22 BOLD:AAP4452 OR974607 Acrididae Acrotylus insubricus (Scopoli, 1786) INV06642 IBIOR436-22 BOLD:AAP4452 OR974455 Acrididae Acrotylus patruelis (Herrich-Schäffer, 1838) INV00063 IBIOR002-17 BOLD:AAJ6293 OR974610 Acrididae Acrotylus patruelis (Herrich-Schäffer, 1838) INV03740 IBIOR326-22 BOLD:AAJ6293 OR974601 Acrididae Acrotylus patruelis (Herrich-Schäffer, 1838) INV05270 IBIOR392-22 BOLD:AAJ6293 OR974494 Acrididae Acrotylus patruelis (Herrich-Schäffer, 1838) INV10594 IBIOR535-22 BOLD:AAJ6293 OR974432 Acrididae Aiolopus puissanti Defaut, 2005 INV00065 IBIOR004-17 BOLD:AAZ1689 OR974633 Acrididae Aiolopus puissanti Defaut, 2005 INV02502 IBIOR067-17 BOLD:AAZ1689 OR974542 Acrididae Aiolopus strepens (Latreille, 1804) INV00066 IBIOR005-17 BOLD:AAP4386 OR974796 Acrididae Aiolopus strepens (Latreille, 1804) INV02503 IBIOR068-17 BOLD:AAP4386 OR974414 Acrididae Aiolopus strepens (Latreille, 1804) INV02539 IBIOR104-17 BOLD:AAP4386 OR974707 Acrididae Aiolopus strepens (Latreille, 1804) INV02540 IBIOR105-17 BOLD:AAP4386 OR974749 Acrididae Aiolopus strepens (Latreille, 1804) INV04053 IBIOR351-22 BOLD:AAP4386 OR974447 Acrididae Aiolopus strepens (Latreille, 1804) INV06052 IBIOR406-22 BOLD:AAP4386 OR974623 Acrididae Anacridium aegyptium (Linnaeus, 1764) INV02504 IBIOR069-17 BOLD:AAO1248 OR974491 Table 1. List of taxa that were collected and DNA barcoded within this project. In column Taxa: - indicates Iberian endemic species; - indicates continental Portugal endemic species; - indicates taxa without a public DNA barcode prior to this study; " - indicates unique BINs. 1 2 # 4Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Anacridium aegyptium (Linnaeus, 1764) INV02505 IBIOR070-17 BOLD:AAO1248 OR974554 Acrididae Anacridium aegyptium (Linnaeus, 1764) INV04117 IBIOR166-19 BOLD:AAO1248 OR974781 Acrididae Arcyptera tornosi Bolívar, 18841 INV02598 IBIOR188-22 BOLD:AER6128 OR974776 Acrididae Arcyptera tornosi Bolívar, 18841 INV02599 IBIOR189-22 BOLD:AER6128 OR974669 Acrididae Calephorus compressicornis (Latreille, 1804) INV02562 IBIOR120-17 BOLD:AEA6681 OR974480 Acrididae Calephorus compressicornis (Latreille, 1804) INV02563 IBIOR121-17 BOLD:AEA6681 OR974777 Acrididae Calliptamus barbarus (Costa, 1836) INV00067 IBIOR006-17 BOLD:ACQ0538 OR974431 Acrididae Calliptamus barbarus (Costa, 1836) INV00068 IBIOR007-17 BOLD:ACQ0538 OR974606 Acrididae Calliptamus barbarus (Costa, 1836) INV02550 IBIOR113-17 BOLD:ACQ0538 OR974538 Acrididae Calliptamus barbarus (Costa, 1836) INV03646 IBIOR314-22 BOLD:ACQ0538 OR974580 Acrididae Calliptamus barbarus (Costa, 1836) INV04028 IBIOR346-22 BOLD:ACQ0538 OR974686 Acrididae Calliptamus wattenwylianus Pantel, 1896 INV00069 IBIOR008-17 BOLD:AEZ7204 OR974823 Acrididae Calliptamus wattenwylianus Pantel, 1896 INV00070 IBIOR009-17 BOLD:AEZ7204 OR974681 Acrididae Calliptamus wattenwylianus Pantel, 1896 INV11426 IBIOR550-22 BOLD:AEZ7204 OR974745 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV00071 IBIOR010-17 BOLD:AED8559 OR974762 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV00072 IBIOR011-17 BOLD:AED8559 OR974600 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV02506 IBIOR071-17 BOLD:AED8559 OR974536 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV02507 IBIOR072-17 BOLD:AED8559 OR974696 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV02532 IBIOR097-17 BOLD:AED8559 OR974673 Acrididae Chorthippus apicalis (Herrich-Schäffer, 1840) INV02533 IBIOR098-17 BOLD:AED8559 OR974657 Acrididae Chorthippus binotatus (Charpentier, 1825) INV03941 IBIOR331-22 BOLD:AAC5779 OR974519 The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 5 Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Chorthippus binotatus (Charpentier, 1825) INV05546 IBIOR197-22 BOLD:AAC5779 OR974508 Acrididae Chorthippus binotatus (Charpentier, 1825) INV05547 IBIOR198-22 BOLD:AAC5779 OR974733 Acrididae Chorthippus jacobsi Harz, 19751 INV02535 IBIOR100-17 BOLD:AAC5779 OR974828 Acrididae Chorthippus jacobsi Harz, 19751 INV02536 IBIOR101-17 BOLD:AAC5779 OR974425 Acrididae Chorthippus jacobsi Harz, 19751 INV02537 IBIOR102-17 BOLD:AAC5779 OR974577 Acrididae Chorthippus jucundus (Fischer, 1853) INV03734 IBIOR323-22 BOLD:AER0035"OR974582 Acrididae Chorthippus vagans (Eversmann, 1848) INV00073 IBIOR012-17 BOLD:AAL3938 OR974714 Acrididae Chorthippus vagans (Eversmann, 1848) INV00074 IBIOR013-17 BOLD:AAL3938 OR974540 Acrididae Chorthippus vagans (Eversmann, 1848) INV02548 IBIOR111-17 BOLD:AAL3938 OR974820 Acrididae Chorthippus vagans (Eversmann, 1848) INV02704 IBIOR571-22 BOLD:AAL3938 OR974618 Acrididae Chorthippus yersini Harz, 19751 INV05556 IBIOR207-22 BOLD:AAC5779 OR974682 Acrididae Chorthippus yersini Harz, 19751 INV05557 IBIOR208-22 BOLD:AAC5779 OR974805 Acrididae Dociostaurus genei (Ocskay, 1832) INV00075 IBIOR014-17 BOLD:ADU3247 OR974636 Acrididae Dociostaurus genei (Ocskay, 1832) INV00076 IBIOR015-17 BOLD:ADU3247 OR974806 Acrididae Dociostaurus hispanicus Bolívar, 18981 INV00079 IBIOR018-17 BOLD:ADV6985 OR974787 Acrididae Dociostaurus jagoi Soltani, 1978 INV00077 IBIOR016-17 BOLD:ADT8663 OR974505 Acrididae Dociostaurus jagoi Soltani, 1978 INV00078 IBIOR017-17 BOLD:ADT8663 OR974567 Acrididae Dociostaurus jagoi Soltani, 1978 INV02549 IBIOR112-17 BOLD:ADT8663 OR974476 Acrididae Dociostaurus jagoi Soltani, 1978 INV03536 IBIOR308-22 BOLD:ADT8663 OR974516 Acrididae Dociostaurus jagoi Soltani, 1978 INV03540 IBIOR191-22 BOLD:ADT8663 OR974736 Acrididae Dociostaurus jagoi Soltani, 1978 INV03547 IBIOR309-22 BOLD:ADT8663 OR974585 Acrididae Dociostaurus jagoi Soltani, 1978 INV03556 IBIOR310-22 BOLD:ADT8663 OR974495 Acrididae Dociostaurus jagoi Soltani, 1978 INV03628 IBIOR313-22 BOLD:ADT8663 OR974808 Acrididae Dociostaurus jagoi Soltani, 1978 INV06726 IBIOR438-22 BOLD:ADT8663 OR974514 Acrididae Dociostaurus jagoi Soltani, 1978 INV07282 IBIOR464-22 BOLD:ADT8663 OR974807 6Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Dociostaurus jagoi Soltani, 1978 INV07542 IBIOR473-22 BOLD:ADT8663 OR974544 Acrididae Dociostaurus jagoi Soltani, 1978 INV07619 IBIOR480-22 BOLD:ADT8663 OR974532 Acrididae Dociostaurus jagoi Soltani, 1978 INV11390 IBIOR546-22 BOLD:ADT8663 OR974482 Acrididae Dociostaurus jagoi Soltani, 1978 INV11391 IBIOR547-22 BOLD:ADT8663 OR974635 Acrididae Dociostaurus jagoi Soltani, 1978 INV11393 IBIOR549-22 BOLD:ADT8663 OR974646 Acrididae Dociostaurus maroccanus (Thunberg, 1815) INV00080 IBIOR019-17 BOLD:AAX8673 OR974534 Acrididae Dociostaurus maroccanus (Thunberg, 1815) INV00081 IBIOR020-17 BOLD:AAX8673 OR974574 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00083 IBIOR021-17 BOLD:ADJ5172 OR974465 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00084 IBIOR022-17 BOLD:ADJ5172 OR974592 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00085 IBIOR023-17 BOLD:ADJ5172 OR974659 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00086 IBIOR024-17 BOLD:ADJ5172 OR974625 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00087 IBIOR025-17 BOLD:ADJ5172 OR974559 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV00088 IBIOR026-17 BOLD:ADJ5172 OR974666 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV02579 IBIOR135-17 BOLD:ADJ5172 OR974674 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV02580 IBIOR136-17 BOLD:ADJ5172 OR974803 Acrididae Euchorthippus elegantulus Zeuner, 1940# INV03557 IBIOR311-22 BOLD:ADJ5172 OR974428 Acrididae Eyprepocnemis plorans (Charpentier, 1825) INV05585 IBIOR236-22 BOLD:AAV4939 OR974729 Acrididae Eyprepocnemis plorans (Charpentier, 1825) INV05586 IBIOR237-22 BOLD:AAV4939 OR974676 Acrididae Locusta migratoria (Linnaeus, 1758) INV02510 IBIOR075-17 BOLD:AAD9526 OR974584 Acrididae Locusta migratoria (Linnaeus, 1758) INV02511 IBIOR076-17 BOLD:AAD9526 OR974821 Acrididae Locusta migratoria (Linnaeus, 1758) INV05513 IBIOR141-19 BOLD:AAD9526 OR974698 The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 7 Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Mioscirtus wagneri (Eversmann, 1859) INV05564 IBIOR215-22 BOLD:ABV1949 OR974436 Acrididae Mioscirtus wagneri (Eversmann, 1859) INV05565 IBIOR216-22 BOLD:ABV1949 OR974457 Acrididae Morphacris fasciata (Thunberg, 1815) INV02560 IBIOR118-17 BOLD:ACR8091 OR974668 Acrididae Morphacris fasciata (Thunberg, 1815) INV02569 IBIOR125-17 BOLD:ACR8091 OR974488 Acrididae Oedaleus decorus (Germar, 1825) INV00091 IBIOR027-17 BOLD:AAE5868 OR974713 Acrididae Oedaleus decorus (Germar, 1825) INV00092 IBIOR142-19 BOLD:AAE5868 OR974497 Acrididae Oedipoda caerulescens (Linnaeus, 1758) INV00093 IBIOR028-17 BOLD:AAC9181 OR974732 Acrididae Oedipoda caerulescens (Linnaeus, 1758) INV00094 IBIOR029-17 BOLD:AAC9181 OR974612 Acrididae Oedipoda caerulescens (Linnaeus, 1758) INV02551 IBIOR114-17 BOLD:AAC9181 OR974535 Acrididae Oedipoda caerulescens (Linnaeus, 1758) INV04033 IBIOR161-19 BOLD:AAC9181 OR974793 Acrididae Oedipoda charpentieri Fieber, 1853 INV00095 IBIOR030-17 BOLD:AAC9180 OR974452 Acrididae Oedipoda charpentieri Fieber, 1853 INV00096 IBIOR031-17 BOLD:AAC9180 OR974742 Acrididae Oedipoda charpentieri Fieber, 1853 INV02512 IBIOR077-17 BOLD:AAC9180 OR974548 Acrididae Oedipoda charpentieri Fieber, 1853 INV02513 IBIOR078-17 BOLD:AAC9180 OR974571 Acrididae Oedipoda coerulea Saussure, 1884# INV07299 IBIOR465-22 BOLD:AEO7756 OR974419 Acrididae Omocestus panteli (Bolívar, 1887) INV00097 IBIOR032-17 BOLD:AAX0669 OR974413 Acrididae Omocestus panteli (Bolívar, 1887) INV00098 IBIOR033-17 BOLD:AAX0669 OR974740 Acrididae Omocestus panteli (Bolívar, 1887) INV00099 IBIOR034-17 BOLD:AAX0669 OR974780 Acrididae Omocestus panteli (Bolívar, 1887) INV00100 IBIOR035-17 BOLD:AAX0669 OR974546 Acrididae Omocestus panteli (Bolívar, 1887) INV00101 IBIOR036-17 BOLD:AAX0669 OR974726 Acrididae Omocestus panteli (Bolívar, 1887) INV02531 IBIOR096-17 BOLD:AAX0669 OR974790 Acrididae Omocestus panteli (Bolívar, 1887) INV10730 IBIOR265-22 BOLD:AAX0669 OR974565 Acrididae Omocestus raymondi (Yersin, 1863) INV02588 IBIOR178-22 BOLD:AEO7761 OR974430 Acrididae Omocestus raymondi (Yersin, 1863) INV02589 IBIOR179-22 BOLD:AEO7761 OR974511 Acrididae Omocestus rufipes (Zetterstedt, 1821) INV02575 IBIOR131-17 BOLD:AAE1108 OR974755 Acrididae Omocestus rufipes (Zetterstedt, 1821) INV02576 IBIOR132-17 BOLD:AAE1108 OR974773 Acrididae Omocestus viridulus (Linnaeus, 1758) INV05555 IBIOR206-22 BOLD:AAE1108 OR974599 Acrididae Paracinema tricolor (Thunberg, 1815) INV06628 IBIOR432-22 BOLD:ACU5079 OR974730 1 1 1 1 1 1 1 8Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Acrididae Pezotettix giornae (Rossi, 1794) INV00102 IBIOR037-17 BOLD:AEO7437"OR974583 Acrididae Pezotettix giornae (Rossi, 1794) INV00103 IBIOR038-17 BOLD:AEO7437"OR974543 Acrididae Pezotettix giornae (Rossi, 1794) INV02538 IBIOR103-17 BOLD:AEO7437"OR974709 Acrididae Pezotettix giornae (Rossi, 1794) INV03278 IBIOR306-22 BOLD:AEO7436 OR974802 Acrididae Pseudochorthippus parallelus (Zetterstedt, 1821) INV03712 IBIOR319-22 BOLD:AAC3399 OR974527 Acrididae Pseudochorthippus parallelus (Zetterstedt, 1821) INV05554 IBIOR205-22 BOLD:AAC3399 OR974441 Acrididae Sphingonotus azurescens (Rambur, 1838) INV02556 IBIOR116-17 BOLD:AAJ3344 OR974768 Acrididae Sphingonotus azurescens (Rambur, 1838) INV02557 IBIOR172-22 BOLD:AAJ3344 OR974647 Acrididae Sphingonotus azurescens (Rambur, 1838) INV02594 IBIOR184-22 BOLD:AAJ3344 OR974570 Acrididae Sphingonotus azurescens (Rambur, 1838) INV06744 IBIOR439-22 BOLD:AAJ3344 OR974738 Acrididae Sphingonotus imitans Brunner von Wattenwyl, 1882 # INV02555 IBIOR171-22 BOLD:AER3084"OR974661 Acrididae Sphingonotus imitans Brunner von Wattenwyl, 1882 # INV08093 IBIOR244-22 BOLD:AER3084"OR974539 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV00105 IBIOR040-17 BOLD:AEI2886 OR974734 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV00106 IBIOR041-17 BOLD:AEI2886 OR974489 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV00107 IBIOR042-17 BOLD:AEI2886 OR974721 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV03709 IBIOR316-22 BOLD:AEI2886 OR974433 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV03711 IBIOR318-22 BOLD:AEI2886 OR974624 Acrididae Sphingonotus lluciapomaresi (Defaut, 2005) INV11392 IBIOR548-22 BOLD:AEI2886 OR974569 Acrididae Sphingonotus lusitanicus Ebner, 1941 # INV08094 IBIOR245-22 BOLD:AEI2886 OR974699 1 1 1 1 1, 1, 1 1 1 1 1 1 1, The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 9 Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Conocephalus conocephalus (Linnaeus, 1767) INV06925 IBIOR446-22 BOLD:AEQ8460 OR974573 Tettigoniidae Conocephalus conocephalus (Linnaeus, 1767) INV06944 IBIOR449-22 BOLD:AEQ8460 OR974561 Tettigoniidae Conocephalus conocephalus (Linnaeus, 1767) INV06948 IBIOR453-22 BOLD:AEQ8460 OR974493 Tettigoniidae Conocephalus fuscus (Fabricius, 1793) INV05583 IBIOR234-22 BOLD:AEQ8462 OR974609 Tettigoniidae Conocephalus fuscus (Fabricius, 1793) INV06634 IBIOR435-22 BOLD:AEQ8462 OR974829 Tettigoniidae Conocephalus fuscus (Fabricius, 1793) INV09078 IBIOR513-22 BOLD:AEQ8462 OR974579 Tettigoniidae Cyrtaspis scutata (Charpentier, 1825)# INV07949 IBIOR493-22 BOLD:AEQ8625 OR974541 Tettigoniidae Cyrtaspis scutata (Charpentier, 1825)# INV08776 IBIOR510-22 BOLD:AEQ8625 OR974639 Tettigoniidae Cyrtaspis scutata (Charpentier, 1825)# INV09030 IBIOR512-22 BOLD:AEQ8625 OR974656 Tettigoniidae Decticus albifrons (Fabricius, 1775)# INV00115 IBIOR049-17 BOLD:AEQ8654"OR974549 Tettigoniidae Decticus albifrons (Fabricius, 1775)# INV00116 IBIOR271-22 BOLD:AEQ8654"OR974550 Tettigoniidae Decticus albifrons (Fabricius, 1775)# INV02508 IBIOR073-17 BOLD:AEQ8654"OR974446 Tettigoniidae Ephippigerida diluta (Bolívar, 1878) # INV08107 IBIOR258-22 BOLD:AEP5780"OR974778 Tettigoniidae Ephippigerida diluta (Bolívar, 1878) # INV08108 IBIOR259-22 BOLD:AEP5780"OR974678 Tettigoniidae Ephippigerida rosae Barat & Correas, 2015 # INV08109 IBIOR260-22 BOLD:AEP5781"OR974415 Tettigoniidae Incertana decorata (Fieber, 1853)# INV02577 IBIOR133-17 BOLD:AEO9013 OR974700 Tettigoniidae Incertana decorata (Fieber, 1853)# INV02578 IBIOR134-17 BOLD:AEO9013 OR974759 Tettigoniidae Leptophyes punctatissima (Bosc, 1792) INV05562 IBIOR213-22 BOLD:ADT5783 OR974467 Tettigoniidae Lluciapomaresius anapaulae (Schmidt, 2009) # INV02573 IBIOR129-17 BOLD:AEO8290 OR974723 Tettigoniidae Lluciapomaresius asturiensis (Bolívar, 1898) # INV03388 IBIOR307-22 BOLD:AEO6324"OR974819 Tettigoniidae Lluciapomaresius asturiensis (Bolívar, 1898) # INV03952 IBIOR333-22 BOLD:AEO6324"OR974672 Tettigoniidae Lluciapomaresius asturiensis (Bolívar, 1898) # INV05560 IBIOR211-22 BOLD:AEO8296 OR974648 1, 1, 1,2, 1,2, 1, 1, 1, 16 Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Lluciapomaresius asturiensis (Bolívar, 1898) # INV05561 IBIOR212-22 BOLD:AEO8296 OR974697 Tettigoniidae Lluciapomaresius stalii (Bolívar, 1877) # INV08100 IBIOR251-22 BOLD:AEO8295 OR974783 Tettigoniidae Meconema thalassinum (De Geer, 1773) INV03732 IBIOR159-19 BOLD:AAK7060 OR974440 Tettigoniidae Meconema thalassinum (De Geer, 1773) INV03944 IBIOR160-19 BOLD:AAK7060 OR974750 Tettigoniidae Meconema thalassinum (De Geer, 1773) INV04094 IBIOR165-19 BOLD:AAK7060 OR974507 Tettigoniidae Metrioptera ambigua Pfau, 1986 # INV02600 IBIOR190-22 BOLD:AEO8174 OR974461 Tettigoniidae Metrioptera ambigua Pfau, 1986 # INV05542 IBIOR193-22 BOLD:AEO8174 OR974767 Tettigoniidae Neocallicrania barrosi Barat, 2013 # INV08101 IBIOR252-22 BOLD:AEO7827"OR974485 Tettigoniidae Neocallicrania barrosi Barat, 2013 # INV08102 IBIOR253-22 BOLD:AEO7827"OR974563 Tettigoniidae Neocallicrania lusitanica (Aires & Menano, 1916) # INV08110 IBIOR261-22 BOLD:AEO7822 OR974578 Tettigoniidae Neocallicrania lusitanica (Aires & Menano, 1916) # INV08111 IBIOR262-22 BOLD:AEO7822 OR974435 Tettigoniidae Neocallicrania lusitanica (Aires & Menano, 1916) # INV08112 IBIOR263-22 BOLD:AEO7826"OR974827 Tettigoniidae Neocallicrania miegii (Bolívar, 1873) # INV08103 IBIOR254-22 BOLD:AEO7823 OR974490 Tettigoniidae Neocallicrania miegii (Bolívar, 1873) # INV08104 IBIOR255-22 BOLD:AEO7823 OR974513 Tettigoniidae Neocallicrania selligera meridionalis (Pfau, 1996) # INV02574 IBIOR130-17 BOLD:AEO7820"OR974631 Tettigoniidae Neocallicrania selligera meridionalis (Pfau, 1996) # INV04091 IBIOR354-22 BOLD:AEO7825 OR974528 Tettigoniidae Neocallicrania selligera meridionalis (Pfau, 1996) # INV07436 IBIOR470-22 BOLD:AEO7825 OR974462 Tettigoniidae Neocallicrania selligera selligera (Charpentier, 1825) # INV06585 IBIOR429-22 BOLD:AEO7824"OR974724 Tettigoniidae Neocallicrania serrata (Bolívar, 1885) # INV08105 IBIOR256-22 BOLD:AEO7821"OR974605 Tettigoniidae Neocallicrania serrata (Bolívar, 1885) # INV08106 IBIOR257-22 BOLD:AEO7821"OR974589 1, 1, 1, 1, 1,2, 1,2, 1, 1, 1, 1, 1, 1, 1, 1, 1, 1,2, 1,2, The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 17 Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Odontura glabricauda (Charpentier, 1825)# INV00117 IBIOR050-17 BOLD:AEO7751 OR974651 Tettigoniidae Odontura glabricauda (Charpentier, 1825)# INV02515 IBIOR080-17 BOLD:AEO7751 OR974587 Tettigoniidae Odontura glabricauda (Charpentier, 1825)# INV06518 IBIOR419-22 BOLD:AEO7751 OR974459 Tettigoniidae Odontura glabricauda (Charpentier, 1825)# INV06520 IBIOR420-22 BOLD:AEO7751 OR974804 Tettigoniidae Odontura glabricauda (Charpentier, 1825)# INV08771 IBIOR509-22 BOLD:AEO7751 OR974664 Tettigoniidae Odontura macphersoni MoralesAgacino, 1943 # INV05587 IBIOR238-22 BOLD:AEO7752 OR974608 Tettigoniidae Odontura macphersoni MoralesAgacino, 1943 # INV05588 IBIOR239-22 BOLD:AEO7752 OR974456 Tettigoniidae Phaneroptera nana Fieber, 1853 INV02559 IBIOR117-17 BOLD:AEO7449 OR974643 Tettigoniidae Phaneroptera nana Fieber, 1853 INV03968 IBIOR334-22 BOLD:AEO7449 OR974614 Tettigoniidae Phaneroptera nana Fieber, 1853 INV03978 IBIOR335-22 BOLD:AEO7449 OR974650 Tettigoniidae Phaneroptera nana Fieber, 1853 INV04008 IBIOR341-22 BOLD:AEO7449 OR974667 Tettigoniidae Phaneroptera nana Fieber, 1853 INV04019 IBIOR344-22 BOLD:AEO7449 OR974798 Tettigoniidae Phaneroptera nana Fieber, 1853 INV04021 IBIOR345-22 BOLD:AEO7449 OR974799 Tettigoniidae Phaneroptera nana Fieber, 1853 INV04042 IBIOR349-22 BOLD:AEO7449 OR974444 Tettigoniidae Phaneroptera nana Fieber, 1853 INV06063 IBIOR408-22 BOLD:AEO7449 OR974764 Tettigoniidae Phaneroptera nana Fieber, 1853 INV06087 IBIOR412-22 BOLD:AEO7449 OR974416 Tettigoniidae Phaneroptera nana Fieber, 1853 INV06558 IBIOR424-22 BOLD:AEO7449 OR974510 Tettigoniidae Phaneroptera nana Fieber, 1853 INV06560 IBIOR425-22 BOLD:AEO7449 OR974758 Tettigoniidae Phaneroptera nana Fieber, 1853 INV06629 IBIOR433-22 BOLD:AEO7449 OR974655 Tettigoniidae Phaneroptera nana Fieber, 1853 INV09088 IBIOR515-22 BOLD:AEO7449 OR974469 Tettigoniidae Phaneroptera nana Fieber, 1853 INV10234 IBIOR520-22 BOLD:AEO7449 OR974481 Tettigoniidae Phaneroptera nana Fieber, 1853 INV10271 IBIOR522-22 BOLD:AEO7449 OR974586 Tettigoniidae Phaneroptera nana Fieber, 1853 INV10339 IBIOR528-22 BOLD:AEO7449 OR974813 Tettigoniidae Phaneroptera nana Fieber, 1853 INV11453 IBIOR555-22 BOLD:AEO7449 OR974449 Tettigoniidae Phaneroptera sparsa Stål, 1857# INV06918 IBIOR444-22 BOLD:AEO7448"OR974512 1, 1, 18 Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Phaneroptera sparsa Stål, 1857# INV06926 IBIOR447-22 BOLD:AEO7448"OR974518 Tettigoniidae Phaneroptera sparsa Stål, 1857# INV07540 IBIOR472-22 BOLD:AEO7448"OR974619 Tettigoniidae Phaneroptera sparsa Stål, 1857# INV07543 IBIOR474-22 BOLD:AEO7448"OR974771 Tettigoniidae Platycleis affinis Fieber, 1853 INV00122 IBIOR053-17 BOLD:AAH9443 OR974752 Tettigoniidae Platycleis affinis Fieber, 1853 INV02516 IBIOR081-17 BOLD:AAH9443 OR974816 Tettigoniidae Platycleis albopunctata (Goeze, 1778) INV03998 IBIOR338-22 BOLD:AEO6325 OR974784 Tettigoniidae Platycleis albopunctata (Goeze, 1778) INV05544 IBIOR195-22 BOLD:AEO6325 OR974442 Tettigoniidae Platycleis albopunctata (Goeze, 1778) INV06511 IBIOR417-22 BOLD:AEO6325 OR974815 Tettigoniidae Platycleis albopunctata (Goeze, 1778) INV07110 IBIOR458-22 BOLD:AEO6325 OR974626 Tettigoniidae Platycleis intermedia (Serville, 1838) INV02518 IBIOR083-17 BOLD:AAE4790 OR974615 Tettigoniidae Platycleis intermedia (Serville, 1838) INV10586 IBIOR532-22 BOLD:AAE4790 OR974728 Tettigoniidae Platycleis sabulosa Azam, 1901# INV00131 IBIOR058-17 BOLD:AEO6325 OR974479 Tettigoniidae Platycleis sabulosa Azam, 1901# INV00132 IBIOR059-17 BOLD:AEO6325 OR974756 Tettigoniidae Platycleis sabulosa Azam, 1901# INV00133 IBIOR060-17 BOLD:AEO6325 OR974450 Tettigoniidae Platycleis sabulosa Azam, 1901# INV00134 IBIOR061-17 BOLD:AEO6325 OR974690 Tettigoniidae Platycleis sabulosa Azam, 1901# INV02572 IBIOR128-17 BOLD:AEO6325 OR974581 Tettigoniidae Platycleis sabulosa Azam, 1901# INV05439 IBIOR399-22 BOLD:AAL4618 OR974658 Tettigoniidae Platycleis sabulosa Azam, 1901# INV05515 IBIOR403-22 BOLD:AEO6325 OR974429 Tettigoniidae Platycleis sabulosa Azam, 1901# INV05516 IBIOR404-22 BOLD:AAL4618 OR974716 Tettigoniidae Platycleis sabulosa Azam, 1901# INV07352 IBIOR468-22 BOLD:AEO6325 OR974679 Tettigoniidae Platycleis sabulosa Azam, 1901# INV07353 IBIOR469-22 BOLD:AAL4618 OR974671 Tettigoniidae Platycleis sabulosa Azam, 1901# INV10326 IBIOR527-22 BOLD:AEO6325 OR974468 Tettigoniidae Platycleis sabulosa Azam, 1901# INV10593 IBIOR534-22 BOLD:AAL4618 OR974814 Tettigoniidae Platycleis sabulosa Azam, 1901# INV11434 IBIOR551-22 BOLD:AEO6325 OR974630 Tettigoniidae Platycleis sabulosa Azam, 1901# INV11452 IBIOR554-22 BOLD:AEO6325 OR974596 Tettigoniidae Platystolus martinezii (Bolívar, 1873) # INV02526 IBIOR091-17 BOLD:AEO7253 OR974818 Tettigoniidae Platystolus martinezii (Bolívar, 1873) # INV02527 IBIOR092-17 BOLD:AEO7253 OR974545 Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV06068 IBIOR410-22 BOLD:AEO7102"OR974774 Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV06069 IBIOR411-22 BOLD:AEO7102"OR974761 1, 1, The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 19 Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV06609 IBIOR431-22 BOLD:AEO7102"OR974483 Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV06631 IBIOR434-22 BOLD:AEO7102"OR974566 Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV07175 IBIOR570-22 BOLD:AEO7102"OR974830 Tettigoniidae Pterolepis grallata (Pantel, 1886)# INV07351 IBIOR467-22 BOLD:AEO7102"OR974809 Tettigoniidae Pterolepis lusitanica (Bolívar, 1900) # INV05574 IBIOR225-22 BOLD:AEO7101 OR974551 Tettigoniidae Pterolepis lusitanica (Bolívar, 1900) # INV07563 IBIOR479-22 BOLD:AEO7101 OR974437 Tettigoniidae Pterolepis spoliata Rambur, 1838 # INV06945 IBIOR450-22 BOLD:AEO7101 OR974831 Tettigoniidae Pterolepis spoliata Rambur, 1838 # INV08090 IBIOR241-22 BOLD:AEO7101 OR974424 Tettigoniidae Pycnogaster cucullatus (Charpentier, 1825) # INV02590 IBIOR180-22 BOLD:AEO6978"OR974521 Tettigoniidae Pycnogaster cucullatus (Charpentier, 1825) # INV05577 IBIOR228-22 BOLD:AEO6980"OR974622 Tettigoniidae Pycnogaster jugicola Graells, 1851 # INV05563 IBIOR214-22 BOLD:AEO6979"OR974704 Tettigoniidae Ruspolia nitidula (Scopoli, 1786) INV02561 IBIOR119-17 BOLD:AAJ7497 OR974654 Tettigoniidae Ruspolia nitidula (Scopoli, 1786) INV04086 IBIOR163-19 BOLD:AAJ7497 OR974782 Tettigoniidae Ruspolia nitidula (Scopoli, 1786) INV04089 IBIOR164-19 BOLD:AAJ7497 OR974529 Tettigoniidae Steropleurus pseudolus (Bolívar, 1878) # INV02592 IBIOR182-22 BOLD:AEO6528"OR974515 Tettigoniidae Steropleurus pseudolus (Bolívar, 1878) # INV02593 IBIOR183-22 BOLD:AEO6528"OR974620 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV00136 IBIOR062-17 BOLD:AER1298 OR974473 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV00137 IBIOR063-17 BOLD:AAF2021 OR974526 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV03387 IBIOR154-19 BOLD:AAF2021 OR974616 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV06544 IBIOR422-22 BOLD:AER1298 OR974556 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV06545 IBIOR423-22 BOLD:AAF2021 OR974522 Tettigoniidae Tessellana tessellata (Charpentier, 1825) INV09723 IBIOR583-22 BOLD:AER6209"OR974718 1,2, 1,2, 1, 1, 1,2, 1,2, 1, 1, 1, 20 Pina S et al Family Taxa IBI code BOLD code BOLD BIN GenBank Tettigoniidae Tettigonia viridissima (Linnaeus, 1758) INV02524 IBIOR089-17 BOLD:AAF1423 OR974547 Tettigoniidae Tettigonia viridissima (Linnaeus, 1758) INV02525 IBIOR090-17 BOLD:AAF1423 OR974531 Tettigoniidae Tettigonia viridissima (Linnaeus, 1758) INV05451 IBIOR401-22 BOLD:AAF1423 OR974520 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV02581 IBIOR137-17 BOLD:AER1533"OR974766 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV04118 IBIOR359-22 BOLD:AER1532 OR974598 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV05307 IBIOR396-22 BOLD:AER1532 OR974597 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV06570 IBIOR427-22 BOLD:AER1532 OR974439 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV07109 IBIOR457-22 BOLD:AER1532 OR974499 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV07232 IBIOR462-22 BOLD:AER1532 OR974725 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV07796 IBIOR490-22 BOLD:AER1531"OR974500 Tettigoniidae Thyreonotus bidens (Bolívar, 1887) # INV07805 IBIOR491-22 BOLD:AER1533"OR974471 Tettigoniidae Tylopsis lilifolia (Fabricius, 1793)# INV00140 IBIOR064-17 BOLD:AER2541 OR974552 Tettigoniidae Tylopsis lilifolia (Fabricius, 1793)# INV00141 IBIOR065-17 BOLD:AER2541 OR974797 Tettigoniidae Tylopsis lilifolia (Fabricius, 1793)# INV02520 IBIOR085-17 BOLD:AER2541 OR974792 Tettigoniidae Tylopsis lilifolia (Fabricius, 1793)# INV02521 IBIOR086-17 BOLD:AER2541 OR974422 Tettigoniidae Tylopsis lilifolia (Fabricius, 1793)# INV10591 IBIOR533-22 BOLD:AER2541 OR974502 Tridactylidae Xya iberica Günther, 1990 # INV02567 IBIOR124-17 BOLD:AER2238 OR974754 Trigonidiidae Natula averni (Costa, 1855)# INV02565 IBIOR123-17 BOLD:ACP3886 OR974642 Trigonidiidae Natula averni (Costa, 1855)# INV02582 IBIOR138-17 BOLD:ACP3886 OR974795 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV02258 IBIOR144-19 BOLD:AAC3438 OR974524 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV02570 IBIOR126-17 BOLD:AER5488"OR974523 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV02571 IBIOR127-17 BOLD:AER5488"OR974691 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV03615 IBIOR157-19 BOLD:AAC3438 OR974826 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV03758 IBIOR328-22 BOLD:AAC3438 OR974675 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV03759 IBIOR329-22 BOLD:AAC3438 OR974769 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV07640 IBIOR484-22 BOLD:AEO7832"OR974684 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV07769 IBIOR489-22 BOLD:AEO7832"OR974746 Trigonidiidae Nemobius sylvestris (Bosc, 1792) INV08603 IBIOR504-22 BOLD:AER5488"OR974466 Trigonidiidae Pteronemobius lineolatus (Brullé, 1835)# INV03739 IBIOR325-22 BOLD:AEO6935 OR974731 1, 1, 1, 1, 1, 1, 1, 1, 1, The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 21 Family Taxa IBI code BOLD code BOLD BIN GenBank Trigonidiidae Trigonidium cicindeloides Rambur, 1838 INV02529 IBIOR094-17 BOLD:ACY3774 OR974533 Trigonidiidae Trigonidium cicindeloides Rambur, 1838 INV02530 IBIOR095-17 BOLD:ACY3774 OR974420 Trigonidiidae Trigonidium cicindeloides Rambur, 1838 INV07034 IBIOR456-22 BOLD:ACY3774 OR974412 Trigonidiidae Trigonidium cicindeloides Rambur, 1838 INV10193 IBIOR519-22 BOLD:ACY3774 OR974786 The results show multiple BINs for a recognised species in several cases. Namely, three BINs were obtained in each of the recognised species given: Antaxius spinibrachius, Nemobius sylvestris, Tessellana tessellata and Thyreonotus bidens. Two BINs were obtained in each of the following taxa: Gryllomorpha longicauda, Gryllotalpa vineae, Lluciapomaresius asturiensis, Neocallicrania lusitanica, Pezotettix giornae, Pycnogaster cucullatus, Platycleis sabulosa and for the subspecies Neocallicrania selligera meridionalis. Our dataset includes three BINs obtained for the species Antaxius spinibrachius, including the first sequences of the BIN BOLD:AER0568 from specimens collected in the Castelo Branco and Guarda Districts. In a previous study, aimed at the phylogeography of this species in the Iberian Peninsula, two different lineages of this species were identified, one of which occurs along the Cordillera Oretana and the other includes all other populations (Gutiérrez-Rodríguez et al. 2014). The genetic diversity found in DNA barcodes in this species highlights the necessity of a taxonomic revision. Additionally, in the family Tettigoniidae, the specimen of the Neocallicrania genus collected in the Setúbal Peninsula was morphologically identified as Neocallicrania lusitanica using the available literature (Barat 2007) and the unique BIN BOLD:AEO6978 was obtained. However, the revision conducted by Barat (2013) pointed out uncertainties related to specimens collected in this geographic area. Thus, our results emphasise the need for further work towards a better understanding of the taxonomy of this genus in the Iberian Peninsula. Of the 119 species, 104 have direct correspondence between morphologic identification and BINs, leaving 15 species involved in BIN sharing. In the subfamily Tettigoniinae, two BINs were found to be shared by more than one species, the BIN BOLD:AEO6325 and the BIN BOLD:AEO7101 was shared by Platycleis albopunctata and Platycleis sabulosa and by Pterolepis lusitanica and Pterolepis spoliata, respectively. In the subfamily Gomphocerinae, the generated sequence of Pseudochorthippus parallelus shared BIN BO LD:AAC3399 with sequences of Pseudochorthippus montanus from Germany, Ukraine and Norway. Introgression caused by hybridisation is a well-studied phenomenon in areas where both species occur in sympatry in Germany (Hochkirch and Lemke 2011, Rohde et al. 2015). These species are morphologically very similar, but have different ecological requirements and both species occur in Spain, although Pseudochorthippus montanus is restricted to the northeast (Llucià-Pomares 2002). Additionally, in this subfamily, there are other Gomphocerinae groups of species that cannot be identified using the DNA barcode 22 Pina S et al COI fragment. Sequences obtained from our specimens of Chorthippus binotatus, Chorthippus jacobsi and Chorthippus yersini shared the same BIN BOLD:AAC5779. Moreover and in line with the results by Hawlitschek et al. (2017), sequences obtained as Omocestus rufipes and Omocestus viridulus shared the same BIN. Likewise, in the subfamily Oedipodinae, two BINs were shared by more than one species, namely the BIN BOLD:AAJ3344 was shared by Sphingonotus azurescens and Sphingonotus nodulosus and the BIN BOLD:AEI2886 was shared by Sphingonotus lluciapomaresi and Sphingonotus lusitanicus. The success rate of DNA barcoding in the genus Sphingonotus was previously studied by Dey et al. (2020). Their results showed intensive sharing of identical or closely-related DNA barcodes indicating that the single analysis of the COI fragment is not sufficient enough to differentiate many of the Sphingonotus species. a b c d e f Figure 1. Examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera 01. a: Neocallicrania barrosi Barat, 2013. Tettigoniidae. Photo by Francisco Barros. BIN URI BOL D:AEO7827; b: Tylopsis lilifolia (Fabricius, 1793). Tettigoniidae. Photo by Sónia Ferreira. BIN URI BOLD:AE R2541; c: Eugryllodes escalerae (Bolívar, 1894). Gryllidae. Photo by Sónia Ferreira. BIN URI BOLD:A EP5287; d: Gryllotalpa vineae Bennet-Clark, 1970. Gryllotalpidae. Photo by Sílvia Pina. BIN URI BOLD: AEP5099; BOLD:AEP5100; e: Paramogoplistes ortini Llucià Pomares, 2015. Mogoplistidae. Photo by Sílvia Pina. BIN URI BOLD:AEO7942; f: Oecanthus dulcisonans Gorochov, 1993. Oecanthidae. Photo by Sílvia Pina. BIN URI BOLD :ACG7286. The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 23 This study shows that DNA barcode sequences, based on the COI mitochondrial gene fragment, can be useful in identifying Portuguese Orthoptera to species level. To our knowledge, this is the first study to focus on DNA barcoding of the Orthoptera order for the Iberian Peninsula. It also highlights several taxonomic challenges related to the rich fauna of this group and suggests intricated phylogeographic processes in the region that lead to the diversification of several taxa and high endemism levels in line with what previous a b c d e f Figure 2. Examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera. a: Trigonidium cicindeloides Rambur, 1838. Trigonidiidae. Photo by Sílvia Pina. BIN URI BOLD :ACY3774; b: Sphingonotus lusitanicus Ebner, 1941. Acrididae. Photo by Sílvia Pina. BIN URI BOLD:AEI2 886; c: Acinipe ignatii Llorente del Moral & Presa, 1983. Pamphagidae. Photo by Francisco Barros. BIN URI BOLD:AER2325; d: Pyrgomorpha conica (Olivier, 1791). Pyrgomorphidae. Photo by Sílvia Pina. BIN URI BOLD: AEO6938; e: Paratettix meridionalis (Rambur, 1838). Tetrigidae. Photo by Sílvia Pina. BIN URI BOLD:BO LD:AAP5007; f: Xya iberica Günther, 1990. Tridactylidae. Photo by Sónia Ferreira. BIN URI BOLD:AER2238 24 Pina S et al studies have found (Barranco 2004, Solé et al. 2018). Our results constitute a first step in the construction of a DNA barcode database of a curated reference collection of Iberian (Portuguese) Orthoptera species that can be used in studies where it is necessary to identify specimens either by DNA barcoding or DNA metabarcoding. Project description Title: The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from Portugal. Personnel: Pedro Beja (project coordinator), Sónia Ferreira (taxonomist and IBI manager), Sílvia Pina (taxonomist), Joana Paupério (IBI manager), Francisco Barros (taxonomist), Cátia Chaves (project technician), Filipa M.S. Martins (molecular biologist), Joana Pinto, Joana Veríssimo (molecular biologist), Vanessa Mata (molecular biologist). Study area description: Continental Portugal (Fig. 3). Design description: Orthoptera specimens were collected in the field, morphologically identified and DNA barcoded. Funding: The present work was funded by National Funds through FCT-Fundação para a Ciência e a Tecnologia in the scope of the project LA/P/0048/2020. InBIO Barcoding Initiative was 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 Figure 3. Map of the localities where Orthoptera samples were collected in continental Portugal. Portuguese districts are also represented. The InBIO Barcoding Initiative Database: DNA barcodes of Orthoptera from ... 25 lon The geographical longitude (in decimal degrees) of the geographic centre of a location. elev Elevation of sampling site (in metres above sea level). country The full, unabbreviated name of the country where the organism was collected. province_state The full, unabbreviated name of the province ("Distrito" in Portugal) where the organism was collected. region The full, unabbreviated name of the municipality ("Concelho" in Portugal) where the organism was collected. exactsite Additional name/text description regarding the exact location of the collection site relative to a geographic relevant landmark. subspecies_taxID Subspecies taxonomic numeric code. subspecies_name Subspecies name. Acknowledgements The present work was funded by National Funds through FCT-Fundação para a Ciência e a Tecnologia in the scope of the project LA/P/0048/2020. The authors are grateful to Ana Rita Gonçalves, José M. Grosso-Silva, Martin Corley, Pedro Brito Lopes, Pedro Sousa and Rui Andrade for collecting specimens. InBIO Barcoding Initiative was 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). Fieldwork benefitted from the project PTDC/ BIA-BIC/ 2203/2012-FCOMP-01-0124-FEDER-028289 by FEDER Funds through the Operational Programme for Competitiveness Factors–COMPETE and by National Funds, EDP Biodiversity Chair, the project “Promoção dos serviços de ecossistemas no Parque Natural Regional do Vale do Tua: Controlo de Pragas Agrícolas e Florestais por Morcegos” funded by the Agência de Desenvolvimento Regional do Vale doTua and includes research conducted at the Long Term Research Site of Baixo Sabor (LTER_EU_PT_002). The work was partially Funded 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. 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It contains collection and identification data. Download file (161.27 kb) Suppl. material 2: IBI – Orthoptera 01 library - Specimen details - Darwin Core Standard Authors: Sílvia Pina, Joana Paupério, Francisco Barros, Cátia Chaves, Filipa M.S. Martins, Joana Pinto, Joana Veríssimo, Vanessa Mata, Pedro Beja, Sónia Ferreira Data type: Specimen data records in the Darwin Core Standard format. Brief description: The file includes information about all records in BOLD for the DS-IBIOR01 InBIO Barcoding Initiative - Orthoptera 01. It contains collection and identification data. Download file (191.09 kb) Suppl. material 3: IBI - Orthoptera 01 library - DNA sequences Authors: Sílvia Pina, Joana Paupério, Francisco Barros, Cátia Chaves, Filipa M.S. Martins, Joana Pinto, Joana Veríssimo, Vanessa Mata, Pedro Beja, Sónia Ferreira Data type: Specimen genomic data, DNA sequences. Brief description: COI sequences in fasta format. Each sequence is identified by the BOLD ProcessID, species name, genetic marker name and GenBank accession number, all separated by a vertical bar. The data are as downloaded from BOLD. Download file (307.09 kb) 36 Pina S et al