Biodiversity Data Journal 11: e97484 OO) doi: 10.3897/BDJ.11.e97484 open access Data Paper The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, documenting biodiversity for freshwater biomonitoring in a Mediterranean hotspot Joana Pauperio*S:!, Luis Martin Gonzalez", Jesus Martinez!, Marcos A Gonzalez", Filipa MS Martins+§, Joana Verissimo?$*, Pamela Puppo”, Joana Pinto*§, Catia Chaves*§, Catarina J. Pinho*$*, José Manuel Grosso-Silva‘, Lorenzo Quaglietta”$, Teresa Luisa L Silva?§, Pedro Sousat§, Paulo Celio Alves#8:’, Nuno Fonseca’, Pedro Bejat$:”, Sonia Ferreira? $.” + CIBIO, Centro de Investigagao em Biodiversidade e Recursos Genéticos, InBIO Laboratério Associado, Campus de Vairado, Universidade do Porto, 4485-661 Vairao, Vila do Conde, Portugal § BIOPOLIS Program in Genomics, Biodiversity and Land Planning, CIBIO, Campus de Vairao, 4485-661 Vairao, Vila do Conde, Portugal | European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge, United Kingdom 4] Departamento de Zoologia, Genética y Antropologia Fisica, Facultad de Biologia. Universidad de Santiago de Compostela, Santiago de Compostela, Spain # Departamento de Biologia, Faculdade de Ciéncias, Universidade do Porto, 4169-007, Porto, Portugal = Marshall University, Department of Biological Sciences, Huntington, West Virginia, United States of America « Museu de Historia Natural e da Ciéncia da Universidade do Porto, Porto, Portugal » CIBIO, Centro de Investigagaéo em Biodiversidade e Recursos Genéticos, InBIO Laboratério Associado, Instituto Superior de Agronomia, Universidade de Lisboa, Lisboa, Portugal “ EBM, Estacao Bioldgica de Mértola, Praca Luis de Camées, Meértola, Portugal Corresponding author: Sonia Ferreira (hiporame@gmail.com) Academic editor: Henrique Paprocki Received: 12 Nov 2022 | Accepted: 28 Dec 2022 | Published: 19 Jan 2023 Citation: Pauperio J, Gonzalez LM, Martinez J, Gonzalez MA, Martins FM, Verissimo J, Puppo P, Pinto J, Chaves C, Pinho CJ, Grosso-Silva JM, Quaglietta L, Silva TLL, Sousa P, Alves PC, Fonseca N, Beja P, Ferreira S (2023) The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, documenting biodiversity for freshwater biomonitoring in a Mediterranean hotspot. Biodiversity Data Journal 11: e97484. https://doi.org/10.3897/BDJ.11.e97484 © Pauperio J et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 2 Pauperio J et al Abstract Background The Trichoptera are an important component of freshwater ecosystems. In the Iberian Peninsula, 380 taxa of caddisflies are known, with nearly 1/3 of the total species being endemic in the region. A reference collection of morphologically identified Trichoptera specimens, representing 142 Iberian taxa, was constructed. The InBIO Barcoding Initiative (IBI) Trichoptera 01 dataset contains records of 438 sequenced specimens. The species of this dataset correspond to about 37% of Iberian Trichoptera species diversity. Specimens were collected between 1975 and 2018 and are deposited in the IBI collection at the CIBIO (Research Center in Biodiversity and Genetic Resources, Portugal) or in the collection Marcos A. Gonzalez at the University of Santiago de Compostela (Spain). New information Twenty-nine species, from nine different families, were new additions to the Barcode of Life Data System (BOLD). A success identification rate of over 80% was achieved when comparing morphological identifications and DNA barcodes for the species analysed. This encouraging step advances incorporation of informed Environmental DNA tools in biomonitoring schemes, given the shortcomings of morphological identifications of larvae and adult Caddisflies in such studies. DNA barcoding was not successful in identifying species in six Trichoptera genera: Hydropsyche (Hydropsychidae), Athripsodes (Leptoceridae), Wormaldia (Philopotamidae), Polycentropus_ (Polycentropodidae) Rhyacophila (Rhyacophilidae) and Sericostoma (Sericostomatidae). The high levels of intraspecific genetic variability found, combined with a lack of a barcode gap and a challenging morphological identification, rendered these species as needing additional studies to resolve their taxonomy. Keywords Trichoptera, occurrence records, species distributions, continental Portugal, continental Spain, DNA barcode, cytochrome c oxidase subunit | (COl) Introduction DNA barcoding is a molecular biology method for species identification that was proposed almost twenty years ago (Hebert et al. 2003). DNA barcoding relies on the comparison of a short mitochondrial DNA sequence of interest, usually a 658 bp fragment of the cytochrome c oxidase subunit | (COI) of the mitochondrial genome, known as the “Folmer region” (Folmer et al. 1994), although other regions and genes can also be used, including ones with different systematic scopes (e.g. Woese and Fox (1977)). For DNA barcoding to The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 3 work, the sequence of interest must be compared to a library containing sequences with known species identification (Hebert et al. 2003, Hebert et al. 2004). As such, the construction of comprehensive reference libraries is essential and these require the morphological identification of vouchers by an expert taxonomist (Baird and Sweeney 2011 , Ferreira et al. 2018, Kress et al. 2015). DNA barcoding applications have since expanded beyond single organism and species identification studies. Development of DNA metabarcoding (Taberlet et al. 2012) was made possible with the advances in PCR technologies and high-throughput sequencing (HTS) (Liu et al. 2019). Multiple DNA barcodes are sequenced in a single sample, allowing the study of complex samples as bulk samples and environmental DNA. DNA metabarcoding has broadened the use of the two. DNA barcodes are now a ubiquitous tool in ecological and biological conservation studies, as well as, for example, in forensic applications (DeSalle and Goldstein 2019, Fiser Pecnikar and Buzan 2013, Kress et al. 2015). Aquatic ecosystems are suffering high losses in biodiversity due to degradation and habitat destruction (Blancher et al. 2022). These ecosystems can be logistically challenging and time-consuming to monitor, as the current methodology is based on inventories and taxonomical diversity, based on morphology (Blancher et al. 2022). DNA metabarcoding has great potential for conservation and monitoring of aquatic ecosystems studies as it allows efficient, non-invasive and standardised sampling, without a priori knowledge of the existing biodiversity in an area (Thomsen and Willerslev 2015, Valentini et al. 2016). The choice of DNA markers and the biomass of the communities to monitor are important factors that can influence successful use of DNA metabarcoding (Thomsen and Wvillerslev 2015, Valentini et al. 2016, Casey et al. 2021). The Trichoptera, or caddisflies, is an order of holometabolous insects that rank seventh overall amongst insect orders regarding species number, with 16,267 described species (Morse 2022) and is the most speciose of the primarily aquatic insect orders. Species of this order can be found in all continents, except Antarctica (Morse et al. 2019). While adults are mostly terrestrial and capable of flight, most species’ eggs, larvae and pupae are found in freshwater habitats (Morse et al. 2019). Adult caddisflies are moth-like insects having their bodies covered with setae or hairs (Holzenthal et al. 2007, Morse et al. 2019, Thomas et al. 2020). Their larvae are known for their ability to use silk to construct shelters and retreats, but some species can also be free-living (Casey et al. 2021, Gonzalez and Cobo 2006, Holzenthal et al. 2015, Martin 2017, Martinez 2014, Morse et al. 2019, Thomas et al. 2020, Zhou et al. 2016). Caddisfly larvae provide several important ecological services, including their crucial role in the trophic dynamics and energy flow in the lakes, rivers and streams freshwater food webs (Holzenthal et al. 2015, Morse et al. 2019, Zhou et al. 2016). They show differential sensitivity to pollution and their diversity and abundance are widely used in biological freshwater monitoring (Resh and Rosenberg 1984). However, these programmes rely on larval morphological identification, which is much more challenging than adult determination and still impossible in the many species, whose larvae have not yet been described (Morse et al. 2019). 4 Pauperio J et al Environmental DNA has the potential to be used as a complement or as an alternative to the hurdles of current morphology-based identification in the scope of freshwater monitoring schemes (Lefrangois et al. 2020). However, successful application of eDNA in Europe will necessitate comprehensive reference collections of DNA sequences, representing existing European aquatic biodiversity (Baird and Sweeney 2011, Ferreira et al. 2018, Kress et al. 2015). Several studies have used barcodes to advance the knowledge on Trichoptera, either expanding the knowledge on their phylogeny or improving the DNA barcodes of Trichoptera species (e.g. Moriniere et al. (2017), Zhou et al. (2016)). In the Iberian Peninsula, approximately 380 Trichoptera taxa, from 23 families are known (Coppa et al. 2022, Gonzalez et al. 1992, Gonzalez and Martinez 2011, Malicky 2005, Martin 2017, Martinez 2014, Olah et al. 2019a, Olah et al. 2019b, Olah et al. 2020, Valladolid et al. 2018, Titos et al. 2018). Of these, 374 are known in Spain and 190 in Portugal. The rate of endemicity of Iberian caddisflies is very high, with around one third of the taxa known to occur in the region being endemic (Gonzalez et al. 1987, Martinez 2014, Martin 2017). In this work, we present a contribution to the DNA barcode library of the Iberian Peninsula species of Trichoptera representing 37% (n = 142) of the Caddisflies known in the region and 38% (n = 57) of the Known endemic Iberian taxa. This work was conducted within the framework of the InBIO Barcoding Initiative. General description Purpose: This dataset aims to provide a first contribution to an authoritative DNA barcode sequences library for Iberian Trichoptera, documenting biodiversity for freshwater biomonitoring in a Mediterranean hotspot. Such a library aims to enable DNA-based identification of species for both traditional molecular studies and DNA-metabarcoding studies. Furthermore, it constitutes a relevant resource for taxonomic research on Iberian Trichoptera and its distribution. Additional information: A total of 438 Trichoptera specimens were sequenced (Suppl. material 1). A full-length barcode of 658 bp was obtained for 400 specimens (91.3%) (Table 1, Suppl. material 2). These specimens represent 142 (37%) of the approximately 380 Caddisflies species known to occur in the Iberian Peninsula (Gonzalez and Martinez 2011, Martinez 2014, Martin 2017). Furthermore, 57 taxa are Iberian endemics, representing 38% of the total endemic Iberian taxa (Gonzalez and Martinez 2011, Martinez 2014, Martin 2017). The dataset includes 22 of the 23 families known to occur in the Iberian Peninsula (Table 1). These data contribute with 29 new taxa, 26 new species and three new subspecies of Trichoptera to the BOLD database (Table 1). For five additional species, the dataset contributes for the first time a full-length barcode. The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... Table 1. List of species that were collected and DNA barcoded within this project. Family Apataniidae Beraeidae Brachycentridae Calamoceratidae Ecnomidae Glossosomatidae Taxa Apatania theischingerorum Malicky, 1981 Beraea alva Malicky, 1975 Beraea malatebrera Schmid, 1952 Micrasema cenerentola Schmid, 1952 Micrasema longulum McLachlan, 1876 Micrasema minimum McLachlan, 1876 Micrasema moestum (Hagen, 1868) Micrasema servatum (Navas, 1918) Calamoceras marsupus Brauer, 1865 Ecnomus deceptor McLachlan, 1884 Agapetus delicatulus McLachlan, 1884 Agapetus fuscipes Curtis, 1834 Agapetus incertulus McLachlan, 1884 Agapetus nimbulus McLachlan, 1879 IBI code INVO05962 INV05488 INV04753 INV04267 INV05952 INV06484 INV05973 INV05974 INV00475 INV00476 INV00477 INV04731 INV04732 INV02470 INV05476 INV03539 INV03546 INVO3605 INV05502 INV05812 INV05813 INV05814 INV0O5815 INV05816 INV02468 INV05817 INV04759 INV04762 BOLD code IBITR421-20 IBITR348-20 IBITR267-20 IBITR173-20 IBITR414-20 IBITR435-20 IBITR430-20 IBITR431-20 IBITRO54-20 IBITRO55-20 IBITRO56-20 IBITR252-20 IBITR253-20 IBITR101-20 IBITR336-20 IBITR140-20 IBITR141-20 IBITR146-20 IBITR360-20 IBITR388-20 IBITR389-20 IBITR390-20 IBITR391-20 IBITR392-20 IBITRO99-20 IBITR393-20 IBITR273-20 IBITR274-20 BOLD BIN BOLD:ADL7734 BOLD:AAJ8091 BOLD:AAO02491 BOLD:AAQ3157 BOLD:AAK7456 BOLD:AAH6898 BOLD:AAO1660 BOLD:AAH3018 BOLD:AAO02482 BOLD:ABU6618 BOLD:AAE6313 BOLD:AAE6313 BOLD:AAJ7120 BOLD:AEC9758 BOLD:AAJ7120 BOLD:AEC9946 BOLD:AEM2297 6 Pauperio J et al Family Taxa Agapetus ochripes Curtis, 1834 Agapetus segovicus Schmid, 1952 Agapetus theischingeri Malicky, 1980 Catagapetus maclachlani Malicky, 1975 Glossosoma privatum McLachlan, 1884 Synagapetus diversus (McLachlan, 1884) Synagapetus lusitanicus Malicky, 1980 Goeridae Larcasia partita Navas, 1917 Silo graellsii Pictet, 1865 Helicopsychidae = Helicopsyche lusitanica McLachlan, 1884 IBI code INV04763 INV05818 INV05819 INV01163 INV04819 INV05823 INV05826 INV02477 INVO2929 INV03936 INV05824 INV05825 INV05831 INV00461 INV00468 INV04688 INV04689 INV05830 INV05491 INV05833 INV00320 INV00327 INV00329 INV00341 INV00451 INV02473 INV02474 INV04733 INV0O5951 INVO0005 BOLD code IBITR275-20 IBITR394-20 IBITR395-20 IBITRO75-20 IBITR317-20 IBITR396-20 IBITR399-20 IBITR108-20 IBITR127-20 IBITR169-20 IBITR397-20 IBITR398-20 IBITR401-20 IBITRO50-20 IBITRO52-20 IBITR219-20 IBITR220-20 IBITR400-20 IBITR351-20 IBITR402-20 IBITRO27-20 IBITRO29-20 IBITRO30-20 IBITRO34-20 IBITRO47-20 IBITR104-20 IBITR105-20 IBITR254-20 IBITR413-20 IBITRO12-20 BOLD BIN BOLD:AAB3823 BOLD:AEC7102 BOLD:AEC7102 BOLD:AEL9298 BOLD:ABA7173 BOLD:AAM0930 BOLD:ABX9025 BOLD:AA04326 BOLD:AEC6981 BOLD:AEC7954 BOLD:AEC8414 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... Family Hydropsychidae Taxa Helicopsyche sp. Cheumatopsyche lepida (Pictet, 1834) Diplectrona felix McLachlan, 1878 Hydropsyche ambigua Schmid, 1973 Hydropsyche brevis Mosely, 1930 Hydropsyche bulbifera McLachlan, 1878 Hydropsyche dinarica Marinkovic-Gospodnetic, 1979 Hydropsyche exocellata Dufour, 1841 IBI code INV04823 INV04824 INV04825 INV04778 INV04779 INV06149 INVO6590 INV04718 INV04719 INV05479 INV05957 INV03635 INV05480 INVO5960 INV04720 INV04721 INV05493 INV04780 INV04781 INV04783 INVO0809 INV04503 INV04782 INVO5956 INV02678 INV04785 INV00433 INV00434 INV02920 INVO2922 BOLD code IBITR321-20 IBITR322-20 IBITR323-20 IBITR283-20 IBITR284-20 IBITR432-20 IBITR436-20 IBITR243-20 IBITR244-20 IBITR339-20 IBITR418-20 IBITR151-20 IBITR340-20 IBITR420-20 IBITR245-20 IBITR246-20 IBITR353-20 IBITR285-20 IBITR286-20 IBITR288-20 IBITRO71-20 IBITR190-20 IBITR287-20 IBITR417-20 IBITR111-20 IBITR289-20 IBITRO45-20 IBITRO46-20 IBITR121-20 IBITR123-20 BOLD BIN BOLD:AED0915 BOLD:AEC8747 BOLD:AAD1893 BOLD:AAO02443 BOLD:AAB5092 BOLD:AAB9587 BOLD:AEC9027 BOLD:AAO01831 BOLD:AAE5138 BOLD:AAF0933 8 Pauperio J et al Taxa Family Hydropsyche iberomaroccana Gonzalez & Malicky, 1999 Hydropsyche infernalis Schmid, 1952 Hydropsyche instabilis (Curtis, 1834) Hydropsyche lobata McLachlan, 1884 Hydropsyche pictetorum Botosaneanu & Schmid, 1973 Hydropsyche siltalai Doehler, 1963 Hydropsyche tenuis Navas, 1932 Hydropsyche tibialis McLachlan, 1884 Hydroptilidae Agraylea sexmaculata Curtis, 1834 IBI code INV02979 INV04788 INV04789 INV04722 INV04723 INVO5959 INVO4501 INV04787 INV00561 INVO2669 INV03591 INV03592 INV04786 INV00421 INVO2962 INV04790 INVO5505 INV04269 INV00186 INV00460 INV04724 INVO3680 INV05481 INV05482 INV05494 INV00318 INV04725 INV04726 INV06211 INV04524 BOLD code IBITR135-20 IBITR292-20 IBITR293-20 IBITR247-20 IBITR248-20 IBITR419-20 IBITR189-20 IBITR291-20 IBITRO69-20 IBITR110-20 IBITR144-20 IBITR145-20 IBITR290-20 IBITRO37-20 IBITR132-20 IBITR294-20 IBITR363-20 IBITR175-20 IBITRO25-20 IBITRO49-20 IBITR249-20 IBITR160-20 IBITR341-20 IBITR342-20 IBITR354-20 IBITRO26-20 IBITR250-20 IBITR251-20 IBITR433-20 IBITR192-20 BOLD BIN BOLD:AED0538 BOLD:AAB5092 BOLD:AAB1966 BOLD:ABZ1867 BOLD:AAB1966 BOLD:AEC7586 BOLD:AAQ2260 BOLD:AAB5092 BOLD:AAQ2260 BOLD:AAB5092 BOLD:AAB9587 BOLD:AAB5092 BOLD:AAB9587 BOLD:AED0962 BOLD:AAE7232 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... Family Taxa Hydroptila fuentaldeala Schmid, 1952 Ithytrichia clavata Morton, 1905 Oxyethira frici Klapalek, 1891 Lepidostomatidae Lepidostoma hirtum (Fabricius, 1775) Leptoceridae Adicella meridionalis Morton, 1906 Adicella reducta (McLachlan, 1865) Athripsodes alentexanus Martin, Gonzalez & Martinez, 2016 Athripsodes braueri (Pictet, 1865) Athripsodes inaequalis (McLachlan, 1884) IBI code INV02924 INV02927 INV02928 INV03549 INV05477 INV00520 INV00839 INV05503 INVO0009 INV00010 INV00011 INV04579 INV04584 INV05510 INV00422 INV00426 INV00012 INV00013 INV00014 INV00470 INV00482 INV02475 INV04856 INV06592 INV06593 INV04268 INV05485 INV0O2463 INV02919 INV03273 BOLD code IBITR124-20 IBITR125-20 IBITR126-20 IBITR142-20 IBITR337-20 IBITRO66-20 IBITRO73-20 IBITR361-20 IBITRO02-16 IBITRO03-16 IBITRO04-16 IBITR202-20 IBITR204-20 IBITR367-20 IBITRO38-20 IBITRO41-20 IBITROO5-16 IBITROO06-16 IBITROO7-16 IBITRO53-20 IBITRO58-20 IBITR106-20 IBITR325-20 IBITR437-20 IBITR438-20 IBITR174-20 IBITR345-20 IBITRO94-20 IBITR120-20 IBITR137-20 BOLD BIN BOLD:AEC8395 BOLD:AEC8346 BOLD:ABY 2898 BOLD:AAB4052 BOLD:AEM0162 BOLD:AAJ1835 BOLD:AAI7978 BOLD:AED0841 10 Family Limnephilidae Pauperio J et al Taxa Athripsodes tavaresi (Navas, 1916) Ceraclea albimacula (Rambur, 1842) Ceraclea sobradieli (Navas, 1917) Leptocerus tineiformis Curtis, 1834 Mystacides azureus (Linnaeus, 1761) Oecetis testacea (Curtis, 1834) Setodes argentipunctellus McLachlan, 1877 Triaenodes ochreellus McLachlan, 1877 Allogamus laureatus (Navas, 1918) Allogamus ligonifer (McLachlan, 1876) IBI code INVO2764 INV03612 INV04754 INV00184 INV02233 INV04556 INVO2950 INV02948 INV04554 INV04510 INV05474 INV05484 INV00812 INV00846 INV04287 INV02239 INV03563 INV04818 INV05473 INV05352 INV05353 INV00549 INV04817 INV02467 INV0O2246 INV00321 INVO2462 INVO2466 INV04748 INV03724 BOLD code IBITR116-20 IBITR147-20 IBITR268-20 IBITRO24-20 IBITRO84-20 IBITR197-20 IBITRO01-16 IBITR128-20 IBITR196-20 IBITR191-20 IBITR334-20 IBITR344-20 IBITRO72-20 IBITRO74-20 IBITR177-20 IBITRO85-20 IBITR143-20 IBITR316-20 IBITR333-20 IBITR327-20 IBITR328-20 IBITRO68-20 IBITR315-20 IBITRO98-20 IBITRO86-20 IBITRO28-20 IBITRO93-20 IBITRO97-20 IBITR264-20 IBITR164-20 BOLD BIN BOLD:AEC8026 BOLD:AAN2950 BOLD:AAD8966 BOLD:AAD8965 BOLD:AAJ1160 BOLD:AAB1494 BOLD:AAD7208 BOLD:ACB2223 BOLD:AAJ8708 BOLD:AEC7060 BOLD:AAQ2353 Family The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 11 Taxa Allogamus mortoni (Navas, 1907) Annitella esparraguera (Schmid, 1952) Chaetopteryx atlantica Malicky, 1975 Drusus berthelemyi Sipahiler, 1992 Drusus bolivari (McLachlan, 1880) Enoicyla pusilla (Burmeister, 1839) Grammotaulius submaculatus (Rambur, 1842) Halesus radiatus (Curtis, 1834) Limnephilus bipunctatus Curtis, 1834 Limnephilus guadarramicus Schmid, 1955 Limnephilus hirsutus (Pictet, 1834) Limnephilus sparsus Curtis, 1834 Limnephilus vittatus (Fabricius, 1798) IBI code INV03727 INV04793 INV04794 INV04795 INVO5963 INVO5965 INVO05964 INV04791 INV04792 INV04796 INV02799 INV04740 INV01836 INV02469 INV04743 INV03722 INVO2609 INV0O3661 INVO03664 INV03946 INVO3655 INVO3685 INVO1281 INV04258 INV01284 INV03651 INV03653 INV04738 INV02256 INV04739 BOLD code IBITR167-20 IBITR297-20 IBITR298-20 IBITR299-20 IBITR422-20 IBITR424-20 IBITR423-20 IBITR295-20 IBITR296-20 IBITR300-20 IBITR117-20 IBITR257-20 IBITRO83-20 IBITR100-20 IBITR260-20 IBITR162-20 IBITR109-20 IBITR156-20 IBITR159-20 IBITR170-20 IBITR155-20 IBITR161-20 IBITRO78-20 IBITR171-20 IBITRO79-20 IBITR153-20 IBITR154-20 IBITR255-20 IBITRO89-20 IBITR256-20 BOLD BIN BOLD:AAM3837 BOLD:AAM4103 BOLD:AEC7901 BOLD:ACO5446 BOLD:ACO5618 BOLD:AAQ2902 BOLD:AEC8384 BOLD:AAF7718 BOLD:AAA4844 BOLD:AEC8200 BOLD:AAE6322 BOLD:AAB6375 BOLD:AAK8602 12 Pauperio J et al Family Taxa Mesophylax aspersus (Rambur, 1842) Potamophylax cingulatus (Stephens, 1837) Potamophylax latipennis (Curtis, 1834) Stenophylax fissus (McLachlan, 1875) Stenophylax mucronatus McLachlan, 1880 Stenophylax permistus McLachlan, 1895 Stenophylax sequax (McLachlan, 1875) Stenophylax vibex (Curtis, 1834) Odontoceridae Odontocerum albicorne (Scopoli, 1763) IBI code INV05478 INV04573 INV04662 INV04672 INV04530 INV01300 INV04746 INVO3662 INV05388 INV01299 INV02247 INV02253 INV0O2257 INV02472 INVO2800 INV04741 INV03616 INV03624 INV03642 INV04742 INV04744 INVO2900 INVO2951 INVO2964 INV04655 INV04656 INV04745 INV02957 INV00020 INVO0021 BOLD code IBITR338-20 IBITR199-20 IBITR207-20 IBITR208-20 IBITR193-20 IBITRO81-20 IBITR263-20 IBITR157-20 IBITR329-20 IBITRO80-20 IBITRO87-20 IBITRO88-20 IBITRO90-20 IBITR103-20 IBITR118-20 IBITR258-20 IBITR148-20 IBITR149-20 IBITR152-20 IBITR259-20 IBITR261-20 IBITR119-20 IBITR130-20 IBITR133-20 IBITR205-20 IBITR206-20 IBITR262-20 IBITR131-20 IBITRO13-20 IBITRO08-16 BOLD BIN BOLD:AAGS761 BOLD:AAC4985 BOLD:ABU7930 BOLD:AEC6836 BOLD:ABY 2452 BOLD:AED0879 BOLD:AAI0072 BOLD:AAE8973 BOLD:AAB5626 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 13 Family Philopotamidae Taxa Odontocerum lusitanicum Malicky, 1975 Chimarra marginata (Linnaeus, 1767) Philopotamus amphilectus McLachlan, 1884 Philopotamus montanus caurelensis Gonzalez & Terra, 1979 Philopotamus perversus McLachlan, 1884 IBI code INVO5968 INV05970 INV05971 INV05972 INV05508 INV0O5501 INV02459 INV00417 INV00419 INV00424 INV00425 INV00431 INV00432 INV00486 INV00506 INV02461 INV03259 INV05457 INV04696 INV02471 INV00334 INV00336 INVO2465 INV02476 INV04694 INV04695 INV00022 INV00023 INVO0024 INV00025 BOLD code IBITR426-20 IBITR427-20 IBITR428-20 IBITR429-20 IBITR365-20 IBITR359-20 IBITRO91-20 IBITRO35-20 IBITRO36-20 IBITRO39-20 IBITRO40-20 IBITRO43-20 IBITRO44-20 IBITRO60-20 IBITRO64-20 IBITRO92-20 IBITR136-20 IBITR331-20 IBITR225-20 IBITR102-20 IBITRO32-20 IBITRO33-20 IBITRO96-20 IBITR107-20 IBITR223-20 IBITR224-20 IBITRO14-20 IBITRO15-20 IBITRO16-20 IBITRO17-20 BOLD BIN BOLD:AEC9755 BOLD:AAO1593 BOLD:AED0394 BOLD:AAO1570 BOLD:AEC7824 BOLD:AAO1569 14 Family Phryganeidae Polycentropodidae Pauperio J et al Taxa Philopotamus variegatus (Scopoli, 1763) Wormaldia beaumonti Schmid, 1952 Wormaldia corvina (McLachlan, 1884) Wormaldia lusitanica Gonzalez & Botosaneanu, 1983 Wormaldia occipitalis (Pictet, 1834) Wormaldia triangulifera McLachlan, 1878 Wormaldia variegata mattheyi Schmid, 1952 Agrypnia varia (Fabricius, 1793) Cyrnus cintranus McLachlan, 1884 Plectrocnemia geniculata McLachlan, 1871 Plectrocnemia laetabilis McLachlan, 1880 Polycentropus corniger McLachlan, 1884 IBI code INV05487 INV05835 INV00458 INV00465 INV01598 INV03725 INV04698 INV04699 INV05840 INV05841 INV05836 INV05838 INV05837 INV05839 INV04765 INV04703 INV04702 INV03723 INV03726 INVO03663 INV05340 INVO5500 INV05953 INV04295 INV02464 INV04704 INV04705 INV05389 INV04773 INV04772 BOLD code IBITR347-20 IBITR403-20 IBITRO48-20 IBITRO51-20 IBITRO82-20 IBITR165-20 IBITR226-20 IBITR227-20 IBITR408-20 IBITR409-20 IBITR404-20 IBITR406-20 IBITR405-20 IBITR407-20 IBITR276-20 IBITR229-20 IBITR228-20 IBITR163-20 IBITR166-20 IBITR158-20 IBITR326-20 IBITR358-20 IBITR415-20 IBITR184-20 IBITRO95-20 IBITR230-20 IBITR231-20 IBITR330-20 IBITR278-20 IBITR277-20 BOLD BIN BOLD:AEC7364 BOLD:AAO2217 BOLD:AAQ2216 BOLD:ABU5927 BOLD:AAOQ2217 BOLD:AED0699 BOLD:AAH9306 BOLD:AED0151 BOLD:AAE4334 BOLD:AAL4393 BOLD:AAL0051 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 15 Family Psychomyiidae Taxa Polycentropus flavomaculatus (Pictet, 1834) Polycentropus intricatus Morton, 1910 Polycentropus kingi McLachlan, 1881 Polycentropus telifer McLachlan, 1884 Lype auripilis McLachlan, 1884 Lype phaeopa (Stephens, 1836) Paduniella vandeli Decamps, 1965 Psychomyia fragilis (Pictet, 1834) Psychomyia pusilla (Fabricius, 1781) Tinodes assimilis McLachlan, 1865 Tinodes foedellus McLachlan, 1884 IBI code INV05475 INV05483 INV00503 INV00504 INV04706 INV04707 INV05489 INV04709 INV05498 INV00478 INV04708 INV04417 INV04713 INV00485 INV04774 INV04775 INVO2695 INV04710 INV00427 INVO0806 INV02749 INV04711 INV05490 INVO5495 INV00521 INV01260 INVO2921 INV04716 INV04717 INV04714 BOLD code IBITR335-20 IBITR343-20 IBITRO62-20 IBITRO63-20 IBITR232-20 IBITR233-20 IBITR349-20 IBITR235-20 IBITR357-20 IBITRO57-20 IBITR234-20 IBITR185-20 IBITR238-20 IBITRO59-20 IBITR279-20 IBITR280-20 IBITR112-20 IBITR236-20 IBITRO42-20 IBITRO70-20 IBITR115-20 IBITR237-20 IBITR350-20 IBITR355-20 IBITRO67-20 IBITRO76-20 IBITR122-20 IBITR241-20 IBITR242-20 IBITR239-20 BOLD BIN BOLD:AACO0971 BOLD:ACR2507 BOLD:AAL0054 BOLD:AALO060 BOLD:AAMO0001 BOLD:AAQ2229 BOLD:AAC4581 BOLD:AAK7667 BOLD:AEC7914 BOLD:AAO1607 BOLD:AEC8086 BOLD:AAO1607 BOLD:AEC8086 BOLD:AAF 7459 BOLD:AAL9978 16 Family Ptilocolepidae Rhyacophilidae Pauperio J et al Taxa Tinodes maculicornis (Pictet, 1834) Tinodes waeneri (Linnaeus, 1758) Ptilocolepus extensus McLachlan, 1884 Ptilocolepus granulatus (Pictet, 1834) Rhyacophila adjuncta McLachlan, 1884 Rhyacophila dorsalis (Curtis, 1834) Rhyacophila dorsalis albarracina Malicky, 2002 Rhyacophila evoluta McLachlan, 1879 Rhyacophila intermedia McLachlan, 1868 Rhyacophila laevis Pictet, 1834 Rhyacophila laufferi Navas, 1918 IBI code INV04715 INVO05954 INV04777 INV00491 INV01280 INV04576 INV04581 INV04423 INV04500 INV04776 INV04266 INV04690 INV04691 INV05967 INV00035 INV00330 INV04677 INV04678 INV04811 INV05793 INV05794 INV05795 INV0O5790 INVO5791 INV04807 INV04680 INV05507 INV04810 INV04809 INV05800 BOLD code IBITR240-20 IBITR416-20 IBITR282-20 IBITRO61-20 IBITRO77-20 IBITR200-20 IBITR203-20 IBITR187-20 IBITR188-20 IBITR281-20 IBITR172-20 IBITR221-20 IBITR222-20 IBITR425-20 IBITRO18-20 IBITRO31-20 IBITR209-20 IBITR210-20 IBITR314-20 IBITR371-20 IBITR372-20 IBITR373-20 IBITR368-20 IBITR369-20 IBITR310-20 IBITR211-20 IBITR364-20 IBITR313-20 IBITR312-20 IBITR378-20 BOLD BIN BOLD:AAF 7446 BOLD:AAB9068 BOLD:AAL2306 BOLD:AAD5575 BOLD:AAC4103 BOLD:AAX8713 BOLD:AAF7929 BOLD:AAF8011 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... Family Taxa Rhyacophila lusitanica McLachlan, 1884 Rhyacophila martynovi Mosely, 1930 Rhyacophila melpomene Malicky, 1976 Rhyacophila meridionalis Pictet, 1865 Rhyacophila mocsaryi tredosensis Schmid, 1952 Rhyacophila munda McLachlan, 1862 Rhyacophila nevada Schmid, 1952 Rhyacophila obelix Malicky, 1979 Rhyacophila obliterata McLachlan, 1863 Rhyacophila occidentalis McLachlan, 1879 IBI code INVO2967 INV00039 INV03379 INV03633 INVO5504 INV05799 INV04808 INV04681 INV04682 INV04683 INV04684 INV05796 INV05803 INV05804 INVO5805 INV02949 INV03535 INV04572 INV04577 INVO3900 INV04420 INV05792 INV00044 INV00045 INV05947 INV05949 INV05797 INV05798 INV04685 INV04686 BOLD code IBITR134-20 IBITRO19-20 IBITR138-20 IBITR150-20 IBITR362-20 IBITR377-20 IBITR311-20 IBITR212-20 IBITR213-20 IBITR214-20 IBITR215-20 IBITR374-20 IBITR379-20 IBITR380-20 IBITR381-20 IBITR129-20 IBITR139-20 IBITR198-20 IBITR201-20 IBITR168-20 IBITR186-20 IBITR370-20 IBITRO20-20 IBITRO21-20 IBITR410-20 IBITR411-20 IBITR375-20 IBITR376-20 IBITR216-20 IBITR217-20 17 BOLD BIN BOLD:AEC8059 BOLD:AEC7148 BOLD:AEM0544 BOLD:AEC9268 BOLD:AEC7310 BOLD:AAM4449 BOLD:AEC7678 BOLD:AAM4448 BOLD:AEC7678 BOLD:AEC8711 BOLD:AEC8521 BOLD:AEC8711 BOLD:AAJ3548 18 Family Sericostomatidae Pauperio J et al Taxa Rhyacophila pascoei McLachlan, 1879 Rhyacophila pulchra Schmid, 1952 Rhyacophila relicta McLachlan, 1879 Rhyacophila sociata Navas 1916 Rhyacophila terpsichore Malicky, 1976 Rhyacophila terrai Gonzalez & Martinez, 2010 Rhyacophila tristis Pictet, 1834 Schizopelex festiva (Rambur, 1842) Sericostoma pyrenaicum Pictet, 1865 IBI code INV04755 INV04687 INV05806 INV04532 INV04531 INV05807 INV06215 INV05808 INVO5809 INV04756 INV04757 INV04758 INV05810 INV05811 INV00053 INVO0054 INV00513 INV02747 INVO2748 INV04292 INV04749 INV04803 INV05509 INV04288 INV04289 INV04290 INV04291 INV04293 INV04804 INV04805 BOLD code IBITR269-20 IBITR218-20 IBITR382-20 IBITR195-20 IBITR194-20 IBITR383-20 IBITR434-20 IBITR384-20 IBITR385-20 IBITR270-20 IBITR271-20 IBITR272-20 IBITR386-20 IBITR387-20 IBITRO22-20 IBITRO23-20 IBITRO65-20 IBITR113-20 IBITR114-20 IBITR182-20 IBITR265-20 IBITR306-20 IBITR366-20 IBITR178-20 IBITR179-20 IBITR180-20 IBITR181-20 IBITR183-20 IBITR307-20 IBITR308-20 BOLD BIN BOLD:AEC7530 BOLD:AEC8544 BOLD:AAI0887 BOLD:AAD5575 BOLD:AEC8427 BOLD:AEM3903 BOLD:ABA2486 BOLD:AAI0810 BOLD:AAJ7690 BOLD:AEC8551 BOLD:AAJ7690 BOLD:AEC8551 BOLD:ABZ0751 BOLD:AAJ7690 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 19 Family Thremmatidae Taxa Sericostoma vittatum Rambur, 1842 Thremma gallicum McLachlan, 1880 Thremma tellae Gonzalez, 1978 IBI code INV04806 INV05472 INV04270 INV04797 INV04822 INV05486 INV05492 INV04752 INV04798 INV04799 INV04800 INV04801 INV04820 INV04821 INV05950 INVO0056 INV00057 INV00058 INV04833 INV05497 BOLD code IBITR309-20 IBITR332-20 IBITR176-20 IBITR301-20 IBITR320-20 IBITR346-20 IBITR352-20 IBITR266-20 IBITR302-20 IBITR303-20 IBITR304-20 IBITR305-20 IBITR318-20 IBITR319-20 IBITR412-20 IBITROO9-16 IBITRO10-16 IBITRO11-16 IBITR324-20 IBITR356-20 BOLD BIN BOLD:AEC8551 BOLD:ABZ0751 BOLD:AAM4952 BOLD:AAJ7690 BOLD:ABZ0751 BOLD:AAJ7690 BOLD:AAM4952 BOLD:AEC9666 BOLD:AAJ7690 BOLD:AAF7946 BOLD:AAL9956 Average nucleotide composition of the Trichoptera sequences is 37.7% thymine (T), 17.9% cytosine (C), 30.5% adenine (A) and 13.9% guanine (G), for a total GC content of 31.8% for the COI barcode fragment analysed. Genetic p-distances ranged from 0.00% between Athripsodes alentexanus Martin, Gonzalez and Martinez, 2016 (n = 2) and A. braueri (Pictet, 1865) to 33.97% between Ptilocolepus granulatus (Pictet, 1834) (n = 1) and Potamophylax latipennis (Curtis, 1834) (n = 4) (Suppl. material 3). Intraspecifc genetic p- distances ranged from 0.00% in 12 species, including several species of Athripsodes, Hydropsyche and Rhyacophila (average n = 3.16 specimens per species), to 6.16% in Hydropsyche pictetorum Botosaneanu and Schmid, 1973 (n = 4), 6.22% in Psychomyia pusilla (Fabricius, 1781) (n = 7), 6.65% in Rhyacophila munda McLachlan, 1862 (n = 9) and 7.45% in Helicopsyche lusitanica McLachlan, 1884 (n = 2). Forty-seven species were represented by a single specimen in the dataset and, for this reason, no intraspecifc distance is calculated. 20 Pauperio J et al The BOLD BIN system uses algorithms to cluster sequences into operational taxonomic units (OTUs) that closely correspond to species (Ratnasingham and Hebert 2013). A total of 146 BINs were retrieved by BOLD (Ratnasingham and Hebert 2007). Seven specimens have not been BIN attributed as their sequence is only 418 bp and no other specimens have been sequenced (Suppl. material 1). Two specimens, identified to the genus level only as Helicopsyche sp., clustered together in a separate BIN, “BOLD:AEC8747”. Of the 146 BINs, 45 BINs are unique to our dataset (Table 1, Suppl. material 1). Using the criteria followed by Ratnasingham and Hebert (2013), there were 83.6% of matches, 3.7% of merges, 6.7% of splits and 6.0% of mixtures when comparing BINs to the morphological identifications (Fig. 1). The BINs generated by BOLD clustered together sequences that closely agree with the morphological identifications of the specimens, with only a few exceptions in nine of the 22 Trichoptera families analysed. BINs OTUs =MATCH ~MERGE «SPLIT ms MIXTURE Figure 1. EES] Comparison in OTU assignment performance between BOLD’s BIN and RESL stand-alone algorithms. The BIN dataset comprised 135 taxa (134 species) and the RESL stand-alone run comprised the entire 142 taxa (141 species) dataset. The four categories: MATCH, MERGE, SPLIT and MIXTURE into which the OTUs were divided, follow the criteria used by Ratnasingham and Hebert (2013). The independent RESL run (Ratnasingham and Hebert 2013, Ratnasingham and Hebert 2007) retrieved 153 OTUs, plus one OTU for the Helicopsyche sp. specimens (Suppl. material 4). The differences found between the RESL OTUs and the morphological identifications were similar to those found between the latter and BOLD’s BINs, with 81.7% of matches, 4.2% of merges, 7.7% of splits and 6.3% of mixtures when comparing OTUs to the morphological identifications (Fig. 1). Nevertheless, some differences existed between the RESL OTU clustering and the BINs created by BOLD (Suppl. materials 1, 4). In the family Hydropsychidae, sequences identified as Hydropsyche instabilis (Curtis, 1834) clustered into a single OTU, but were The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 21 split into two BINs. In the family Leptoceridae, sequences of specimens identified as Athripsodes alentexanus and A. brauer clustered in a single BIN. In the family Philopotamidae, sequences identified as Philopotamus perversus McLachlan, 1884 clustered into two OTUs, but were represented by a single BIN. In the family Polycentropodidae, sequences identified as Polycentropus flavomaculatus clustered into a single OTU, but were split into two BINs. In the family Rhyacophilidae, sequences identified as R. dorsalis (Curtis, 1834) and its subspecies, R. a. albarracina Malicky, 2002 clustered into a single OTU, but other sequences identified as R. dorsalis clustered into a different OTU. All R. dorsalis sequences share a single BIN, but the subspecies’ sequences have not been BIN attributed as their sequences are only 418 bp. Sequences identified as R. intermedia McLachlan, 1868 clustered into three OTUs, but were represented by a single BIN. Additionally, sequences identified as R. martynovi Mosely, 1930 clustered into two OTUs, but were represented by a single BIN. Furthermore, sequences identified as Rhyacophila munda clustered into two OTUs, but were split into three BINs. In the family Sericostomatidae, there was no separation of the species Sericostoma pyrenaicum and S. vittatum. These species clustered together into two different BINs, but sequences of S. pyrenaicum and S. vittatum also clustered in additional BINs (Suppl. materials 1, 4). This work provided new DNA barcode sequences and distributional data for 436 specimens of Iberian Trichoptera, plus two French specimens. The dataset represents 37% of the Caddisflies known to occur in Iberia and the work added 29 taxa previously not represented in the BOLD database. To our knowledge, this is the first study to focus on DNA barcoding of the Trichoptera order for the Iberian Peninsula. This study showed that DNA barcode sequences, based on the COI mitochondrial gene fragment, can be useful in identifying Iberian Trichoptera samples to species level. We achieved more than 80% success in matching the sequences generated to the morphological identification of the specimens. This is similar to the success rate achieved in 2017 (Moriniere et al. 2017) for German Caddisflies (79.8%). A DNA barcode library is an essential tool for incorporating Environmental DNA techniques in monitoring schemes of aquatic ecosystems that use Iberian Caddisflies (Lefrangois et al. 2020). Our results constitute a first step in the construction of a DNA barcode database of a curated reference collection of Iberian Trichoptera species, which could be used to overcome the difficulties in identifying many of the Trichoptera larval specimens of traditional biological freshwater monitoring studies. Incongruences were found in nine families. In six of them, Glossosomatidae, Helicopsychidae, Polycentropodidae, Limnephilidae, Rhyacophilidae and Psychomyiidae, the barcode analysis identified no species boundaries, with high levels of intraspecific genetic diversity (Suppl. material 3). It is possible that such levels of genetic diversity point to undescribed, distinct species. This hypothesis requires further morphological studies to search for diagnostic morphological traits that might separate these species. In the family Hydropsychidae, nine species of the genus Hydropsyche could be identified through their barcodes and their genetic distances ranged between 13.4% and 23%. 22 Pauperio J et al However, five other species could not be identified through DNA barcodes. These species, H. ambigua, H. infernalis, H. pictetorum, H. siltalai and H. tenuis were spliced between different BINS and OTUs, shared by some, but not all of the same species, further complicating their relationships. For the species with enough sequenced specimens, all were found to have moderate to high levels of intraspecific genetic diversity (Suppl. material 3). These species are difficult to identify morphologically and this study emphasises the need for further work towards a better understanding of the taxonomy of the genus in the Iberian Peninsula (Zamora-Munoz et al. 2017). In the family Leptoceridae, sequences identified as Athripsodes alentexanus and A. brauer clustered in a single BIN. All four sequences were identical. As such, DNA barcodes, based on COI, might not differentiate between these two species. This can be the result of an introgression event, if they had split very recently or alternatively, if their taxonomic identity needs revision. In the family Philopotamidae, two Wormaldia beaumonti and one W. /usitanica sequences were in the dataset. Two BINs are present in BOLD with both species represented in each (from previous data, but also with the new data). This genus is very difficult to identify morphologically and is likely that the morphological characters used are not able to separate both taxa. In the family Sericostomatidae, there were problems separating two species of the genus Sericostoma, S. pyrenaicum and S. vittatum. These species clustered together into two different BINs, but sequences of S. pyrenaicum and S. vittatum also clustered in additional BINs. Intraspecific genetic diversity is relatively high in both species (2.49% and 2.89%, respectively). Gonzalez et al. (1992) and Martinez (2014) already pointed out that, under these two names, a complex of species is actually hidden, some of them quite variable morphologically. A detailed morphological-molecular study may help to solve one of the most difficult taxonomic problems of our fauna. These findings suggest that both species need a taxonomic revision. Our results did not corroborate the findings of Valladolid et al. (2018) and suggest further work is necessary regarding the identity of Rhyacophila adjuncta and R. sociata. These authors restored the species R. sociata, previously considered a junior synonym of R. denticulata McLachlan, 1879. However, both BOLD clustering algorithms merged our samples, identified as R. adjuncta (2 specimens) and R. sociata (2 specimens), into a single BIN “BOLD:AAD5575”. Furthermore, this BIN includes all publicly available sequences in BOLD identified as R. adjuncta and R. sociata, including all sequences generated by Valladolid et al. (2018). In their paper, the authors did not investigate a possible relationship between these two species, nor was that relationship assessed in a subsequent study on the European species of the R. fasciata group (Valladolid et al. 2021). Finally, the BIN mentioned above also includes other sequences identified as R. tristis Pictet, 1834. and R. fasciata Hagen, 1859, although these are probably misidentifications. We also identified several cases that require further study by taxonomists. Other possibilities for the incongruence found amongst the results include the existence of The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 23 hybridisation, introgression or incomplete lineage sorting in these species, especially if they result from recent speciation events (e.g. Behrens-Chapuis et al. (2021), Moriniere et al. (2017), Zhou et al. (2016)). These hypotheses require the combination of nuclear and mitochondrial markers to be resolved, preferably in an integrative taxonomic approach. Project description Title: The InBIO Barcoding Initiative Database: DNA Barcodes of Iberian Trichoptera 01 Personnel: Luis Martin (taxonomist), Jesus Martinez (taxonomist), Marcos A. Gonzalez (taxonomist), affiliated to Universidad de Santiago de Compostela; Pedro Beja (project coordinator), Joana Paupeério (IBI manager), Sonia Ferreira (taxonomist and IB] manager), Fililpa M.S. Martins (molecular biologist), Joana Verissimo (molecular biologist), Pamela Puppo (molecular biologist), Joana C. Pinto (project technician), Catia Chaves (project technician), Catarina J. Pinho (project technician), Pedro Sousa (project technician), Lorenzo Quaglietta (ecologist), Teresa Silva (molecular biologist), Paulo Célio Alves, (molecular biologist), Nuno Fonseca (bioinformatician), all affiliated to CIBIO-InBIO, University of Porto and José Manuel Grosso-Silva (taxonomist), affiliated to the MHNC-UP, University of Porto. Study area description: Iberian Peninsula (Fig. 2). Pee Qe cme cs o7 ; a, ° bene x pote 3 7 a eo 4 - ‘s a or : p / on } e : nih & . a ¥ ‘ cs Pp “ ZS *; y/ ( ht / . ‘ f ¢ a AS “ a wn g re ) 2 : ian f ee ane sal ai el ~y tooo ae 0 100 200 km _. a is —k= 6 oe of rae nat me (ihe ma Figure 2. EES] Sampling localities of the Trichoptera specimens analysed in this study. Nine localities could not be mapped because geographic coordinates were not available. Design description: Specimens were collected during field expeditions in the Iberian Peninsula, from 1975 to 2018 (n = 434 Fig. 2, Suppl. material 1), with more than 60% of 24 Pauperio J et al specimens collected in the period between 2015 and 2017 (274 out of 434). Two additional specimens were collected in the French Pyrenees. Specimens kept at the InBIO Barcoding Initiative (IBI) reference collection (Vairao, Portugal), 230 in total, were stored in 96% ethanol. Specimens kept at the Coleccion Marcos A. Gonzalez (Universidad de Santiago de Compostela, Spain), 206 in total, were stored in either 70% or 96% ethanol. For each species, we selected, on average, three specimens for DNA sequencing, based on their location of capture, attempting to maximise the geographical coverage of the study. DNA was extracted using two different kits: EasySpin Genomic DNA Microplate Tissue Kit (Citomed, Odivelas, Portugal) or QlAmp DNA Micro Kit (Qiagen, Hilden, Germany). QlAmp DNA Micro Kit is designed to extract higher concentrations of genetic material from samples with small amounts of DNA. DNA amplification was performed using three different primer pairs, that amplify three overlapping fragments of the same 658 bp region of the COI mitochondrial gene. In the beginning of the project (2015), we used two primer pairs, LCO1490 (Folmer et al. 1994) + Il|__C_R (Shokralla et al. 2015) and III_B_F (Shokralla et al. 2015) + HCO2198 (Folmer et al. 1994) (henceforth referred to as LC and BH, respectively) to amplify two overlapping fragments of 325 bp and 418 bp, respectively. After the publication of the third primer pair, BF2 + BR2 (422 bp fragment), by (Elbrecht and Leese 2017), this started to be used instead of the second primer pair (IILB OF + HCO2198) due to higher amplification efficiency. PCRs were performed in 10 ul reactions, containing 5 ul of Multiplex PCR Master Mix (Qiagen, Germany), 0.3 (BF2-BR2) — 0.4 mM of each primer, and 1-2 ul of DNA, with the remaining volume in water. The thermocycling for PCR reactions was performed in T100 Thermal Cycler (Bio-Rad, California, USA) and carried out with an initial denaturation at 95°C for 15 min, followed by 5 cycles at 95°C for 30 sec, 47°C for 45 sec, 72°C for 45 sec (only for LC and BH); then 40 cycles at 95°C for 30 sec, 51°C for 45 sec (48°C for 60 sec for BF2 + BR2), 72°C for 45 sec; and a final elongation step at 60°C for 10 min. All PCR products were analysed by agarose gel electrophoresis and samples selected for sequencing were then organised for assignment of sequencing ‘indexes’. One of two types of index was used for each run. For Illumina indexes, samples were pooled into one plate, as described in Shokralla et al. (2015). When using custom indexes, designed, based on Meyer and Kircher (2010), no pooling was required. The latter allow for a maximum of 1920 unique index combinations. A second PCR was then performed where the ‘indexes’ and Illumina sequencing adapters were attached to the PCR product. The index PCR was performed in a volume of 10 ul, including 5 yl of Phusion® High-Fidelity PCR Kit (New England Biolabs, U.S.A.) or KAPA HiFi PCR Kit (KAPA Biosystems, U.S.A.), 0.5 ul of each ‘index’ and 2 ul of diluted PCR product (usually 1:4). This PCR reaction runs for 10 cycles at an annealing temperature of 55°C. The amplicons were purified using AMPure XP beads (Beckman Coulter Genomics, Massachusetts, United States) before quantification using NanoDrop 1000 (Thermo Fisher Scientific, Massachusetts, USA). Concentrations between samples were then normalised and samples were pooled, based on used primer sets. The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 25 Quantification of final pools was assessed through qPCR using the KAPA Library Quantification Kit IIlumina® Platforms (Kapa Biosystems) and the 2200 Tapestation System (Agilent Technologies, California, USA) was used for fragment length analysis as described by Paupeério et al. (2018). Sequencing was conducted in an Illumina MiSeq benchtop system, using a V2 MiSeq sequencing kit (2x 250 bp) to perform sequencing at CIBIO facilities. Sequences were filtered and processed with OBITools (Boyer et al. 2015) and the fragments were assembled into their consensus 658 bp-long sequences using Geneious 6.1.8 (https:/www.geneious.com). The obtained DNA sequences were then compared against the Barcode of Life Data Systems (BOLD) database (Ratnasingham and Hebert 2007) using the built-in identification engine, based on the BLAST algorithm. Sequences were submitted to the BOLD database and the Barcode Index Numbers (BIN) for every sequence were retrieved and analysed (Suppl. materials 1, 2). As not all our sequences matched the criteria used in BOLD (sequence length) to be clustered in a BIN, we ran the Refined Single Linkage algorithm (RESL, Ratnasingham and Hebert (2013)) on our dataset in the BOLD system (Ratnasingham and Hebert 2007) in an independent run (Suppl. material 4). This process clusters sequences independent of their BIN registry, generating OTUs that can be analysed independently. All DNA barcode sequences were aligned in Geneious 6.1.8 with MUSCLE (Edgar 2004) plug-in. Nucleotide composition of all sequences, as well as intra and interspecific p- distances,were calculated in MEGA11 (Tamura et al. 2021). Funding: InBIO Barcoding Initiative is funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 668981 and by the project PORBIOTA — Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The fieldwork benefited from EDP Biodiversity Chair, including research conducted at the Long Term Research Site of Baixo Sabor (LTER_EU_PT_002), the project “Promogao dos servigos de ecossistemas no Parque Natural Regional do Vale do Tua: Controlo de Pragas Agricolas e Florestais por Morcegos” funded by the Agéncia de Desenvolvimento Regional do Vale do Tua. SF was supported by individual research contract (2020.03526.CEECIND) and CUP, JV and FMSM by a PhD grant (SFRH/BD/145851/2019; SFRH/BD/133159/201 7; SFRH/BD/104703/2014) funded by FCT. Sampling methods Description: Iberian Peninsula. Sampling description: Specimens were captured during direct searches of the environment, using mainly hand-held sweep-nets or lured by light trapping, the latter with UV (black-light) LEDs. Morphological identification was done, based on Malicky (2004) 26 Pauperio J et al using a stereoscopic microscope for the study of genitalia. In some cases, genitalia were cleared in 10% potassium hydroxide (KOH) at room temperature for 4—8 hours, rinsed in water and placed in a drop of glycerine or resin (ODMHF) on a clean slide for further study. From each specimen, one tissue sample (a leg) was removed and stored in 96% ethanol for DNA extraction at the IBI collection. Quality control: All DNA barcode sequences were compared against the BOLD database and the 99 top results were inspected in order to detect possible problems due to contaminations or misidentifications. Prior to GBIF submission, data were checked for errors and inconsistencies with OpenRefine 3.3 (http://openrefine.org). Step description: Specimens were collected in 66 different localities in Portugal and 74 localities in Spain. Collections were carried out between 1975 and 2018. Specimens were collected during fieldwork by direct search of specimens, by sweeping the vegetation with a hand-net and by using light traps and were preserved in 96% alcohol. Captured specimens were deposited in the IBI reference collection at CIBIO (Research Center in Biodiversity and Genetic Resources) or in the collection Marcos A. Gonzalez at the University of Santiago de Compostela (Spain). Specimens were morphologically identified with the assistance of stereoscopic microscopes (Leica MZ12, 8x to 100x; Olympus SZX16, 7x to 115x). DNA barcodes were sequenced from all specimens. For this, one leg was removed from each individual, DNA was then extracted and a 658 bp CO! DNA barcode fragment was amplified and sequenced. All obtained sequences were submitted to BOLD and GenBank databases and, to each sequenced specimen, the morphological identification, when available, was contrasted with the results of the BLAST of the newly-generated DNA barcodes in the BOLD Identification Engine. Prior to submission to GBIF, data were checked for errors and inconsistencies with OpenRefine 3.3 (http://openrefine.org/). Geographic coverage Description: Specimens were collected in the Iberian Peninsula, 229 from 66 localities in Portugal and 207 from 74 localities in Spain (Fig. 2, Suppl. material 5 for further details). Two additional specimens were collected in two French localities. The Rhyacophila laevis Pictet, 1834 specimen represented in the dataset was collected in the French Pyrenees. Coordinates: -8.94 and -0.22 Latitude; 42.89 and 37.50 Longitude. Taxonomic coverage Description: This dataset is composed of data relating to 438 Trichoptera specimens. All specimens were determined to species level, with 14 specimens further identifed to subspecies level. Overall, 141 species are represented in the dataset. These species belong to 22 families. The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... Taxa included: Rank kingdom subkingdom phylum Class family family family family family family family family family family family family family family family family family family family family family family Scientific Name Common Name Animalia Animals Eumetazoa Arthropoda Insecta Apataniidae Beraeidae Brachycentridae Calamoceratidae Ecnomidae Glossosomatidae Goeridae Helicopsychidae Hydropsychidae Hydroptilidae Lepidostomatidae Leptoceridae Limnephilidae Odontoceridae Philopotamidae Phryganeidae Polycentropodidae Psychomyiidae Ptilocolepidae Rhyacophilidae Sericostomatidae Uenoidae 27 28 Pauperio J et al Temporal coverage Data range: 1975-5-03 - 2018-5-16. Collection data Collection name: InBIO Barcoding Initiative Collection identifier: 4ec2b246-f5fa-4b90-9a8d-ddafc2a3f970 Specimen preservation method: “Alcohol” Curatorial unit: DNA extractions - 1 to 438 Usage licence Usage licence: Creative Commons Public Domain Waiver (CC-Zero) Data resources Data package title: The InBIO Barcoding Initiative Database: DNA Barcodes of Iberian Trichoptera Resource link: http://dx.doi.org/10.5883/DS-IBITRO1 Number of data sets: 1 Data set name: DS-IBITRO1 IBI-Trichoptera 01 Download URL: http:/Awww.boldsystems.org/index.php/Public_ SearchTerms? query=DS-IBITRO1 Data format: dwc, xml, tsv, fasta Description: The InBIO Barcoding Initiative Database: DNA Barcodes of Iberian Trichoptera dataset can be downloaded from the PublicData Portal of BOLD (http:// www.boldsystems.org/index.php/Public_ SearchTerms?query=DS-IBITRO1) in different formats (data as dwc, xml or tsv and sequences as fasta files). Alternatively, BOLD users can log-in and access the dataset via the Workbench platform of BOLD. All records are also searchable within BOLD, using the research function of the database. The InBIO Barcoding Initiative will continue sequencing Iberian Trichoptera for the BOLD database, with the ultimate goal of comprehensive coverage. The version of the dataset, at the time of writing the manuscript, is included as in the form of one text file for record information as downloaded from BOLD, one text file with the collection and identification data in Darwin Core Standard format (downloaded from GBIF, Martin et al. (2022)) and of a fasta file containing all sequences as downloaded from BOLD. It The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 29 should be noted that, as the BOLD database is not compliant with the Darwin Core Standard format, the Darwin Core formatted file (dwc) that can be downloaded from BOLD is not strictly Darwin Core formatted. For a proper Darwin Core formatted file, see http://ipt.gbif.pt/ipt/resource?r=ibi_trichoptera _01&; v = 1.1 (Suppl. material 5). All data are available in the BioStudies database (http://www.ebi.ac.uk/biostudies) under accession number S-BSST920. Column label Column description processid Unique identifier for the sample. sampleid Identifier for the sample being sequenced, i.e. |B| catalogue number at Cibio-InBIO, Porto University. Often identical to the "Field ID" or "Museum ID". recordID Identifier for specimen assigned in the field. catalognum Catalogue number. fieldnum Field number. institution_storing The full name of the institution that has physical possession of the voucher specimen. bin_uri Barcode Index Number system identifier. phylum_taxID Phylum taxonomic numeric code. phylum_name Phylum name. class_taxID Class taxonomic numeric code. class_name Class name. order_taxID Order taxonomic numeric code. order_name Order name. family_taxID Family taxonomic numeric code. family_name Family name. subfamily_taxID subfamily_name genus_taxID genus_name species_taxID species_name Subfamily taxonomic numeric code. Subfamily name. Genus taxonomic numeric code. Genus name. Species taxonomic numeric code. Species name. identification_provided_by Full name of primary individual who assigned the specimen to a taxonomic group. identification_method The method used to identify the specimen. voucher_status Status of the specimen in an accessioning process (BOLD controlled vocabulary). tissue_type A brief description of the type of tissue or material analysed. 30 Pauperio J et al collectors The full or abbreviated names of the individuals or team responsible for collecting the sample in the field. lifestage The age class or life stage of the specimen at the time of sampling. sex The sex of the specimen. lat The geographical latitude (in decimal degrees) of the geographic centre of a location. 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. Acknowledgements The InBlO Barcoding Initiative was funded by the European Union’s Horizon 2020 Research and Innovation Programme under grant agreement No 668981 and by the project PORBIOTA - Portuguese E-Infrastructure for Information and Research on Biodiversity (POCI-01-0145-FEDER-022127), supported by Operational Thematic Program for Competitiveness and Internationalization (POCI), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). The fieldwork benefited from EDP Biodiversity Chair, the project “Promogao dos servicgos de ecossistemas no Parque Natural Regional do Vale do Tua: Controlo de Pragas Agricolas e Florestais por Morcegos” funded by the Agéncia de Desenvolvimento Regional do Vale do Tua and includes research conducted at the Long Term Research Site of Baixo Sabor (LTER_EU_PT_002). SF was supported by an_ individual research contract (2020.03526.CEECIND) and CJP, JV and FMSM by a PhD grant (SFRH/BD/145851/2019; SFRH/BD/133159/2017; SFRH/BD/104703/2014) funded by FCT. Work co-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). References ° Baird DJ, Sweeney BW (2011) Applying DNA barcoding in benthology: the state of the science. Journal of the North American Benthological Society 30 (1): 122-124. https:// doi.org/10.1899/10-154.1 The InBIO barcoding initiative database: DNA barcodes of Iberian Trichoptera, ... 31 Behrens-Chapuis S, Herder F, Geiger MF (2021) Adding DNA barcoding to stream monitoring protocols - What's the additional value and congruence between morphological and molecular identification approaches? 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Gonzalez, Martin Corley, José Manuel Grosso-Silva, Lorenzo Quaglietta, Pedro Sousa, Pedro Beja Data type: Record information - specimen data Brief description: The file includes information about all records in BOLD for the IBI-Trichoptera 01 library. It contains collection and identification data. The data are as downloaded from BOLD, without further processing. Download file (180.30 kb) Suppl. material 2: IBI-Trichoptera 01 library - DNA sequences EE) Authors: Joana Paupério, Luis Martin, Sonia Ferreira, Jesus Martinez, Marcus A. Gonzalez, Fililpa M.S. Martins, Joana Verissimo, Pamela Puppo, Joana C. Pinto, Catia Chaves, Catarina Pinho, Pedro Sousa, Pedro Beja Data type: Genomic data, DNA sequences Brief description: COl sequences in fasta format. Each sequence is identified by the BOLD ProcessID, species name, marker and GenBank accession number, separated by pipe. The data are as downloaded from BOLD. Download file (298.19 kb) Suppl. material 3: Genetic Distances EE) Authors: Joana Paupério, Luis Martin, Sonia Ferreira, Jesus Martinez, Marcus A. Gonzalez, Filipa M.S. Martins, Joana Verissimo, Pamela Puppo, Joana C. Pinto, Catia Chaves, Catarina Pinho, José Manuel Grosso-Silva, Lorenzo Quaglietta, Pedro Sousa, Paulo Célio Alves, Nuno Fonseca, Pedro Beja Data type: Genetic distances between analysed specimens Brief description: Brief description: Estimates of average genetic divergence (uncorrected p- distances) for species of Trichoptera. Values under the diagonal refer to interspecifc divergence, while values in the diagonal represent intraspecifc divergence. Download file (127.58 kb) Suppl. material 4: OTUs generated by the Refined Single Linkage algorithm (RESL,) EES) Authors: Joana Paupério, Luis Martin, Sonia Ferreira, Jesus Martinez, Marcus A. Gonzalez, Filipa M.S. Martins, Joana Verissimo, Pamela Puppo, Joana C. Pinto, Catia Chaves, Catarina Pinho, José Manuel Grosso-Silva, Lorenzo Quaglietta, Pedro Sousa, Paulo Célio Alves, Nuno Fonseca, Pedro Beja Data type: OTUs generated by the RESL algorithm and respective sequence composition Brief description: OTUs generated by the RESL algorithm (Ratnasingham and Hebert, 2013) in the BOLD system (Ratnasingham and Hebert, 2007), respective sequence composition and Nearest Neighbour genetic distance. The data are downloaded from BOLD, without further processing. Download file (43.36 kb) 36 Pauperio J et al Suppl. material 5: IBI-Trichoptera 01 library - Specimen details - Darwin Core Standard EG) Authors: Luis Martin, Sonia Ferreira, Jesus Martinez, Marcus A. Gonzalez, Martin Corley, José Manuel Grosso-Silva, Lorenzo Quaglietta, Pedro Sousa, Pedro Beja Data type: Record information - specimen data in Darwin Core Standard format Brief description: The file includes information about all records in BOLD for the IBI-Trichoptera 01 library. It contains collection and identification data. The data are downloaded from GBIF, without further processing. Download file (442.98 kb)