Biodiversity Data Journal : Data Paper (Biosciences)
Data Paper (Biosciences)
The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera
expand article infoSonia Ferreira, José Manuel Tierno de Figueroa§, Filipa MS Martins‡,|, Joana Verissimo‡,|, Lorenzo Quaglietta, José Manuel Grosso-Silva#, Pedro B Lopes¤, Pedro Sousa, Joana Paupério, Nuno A Fonseca, Pedro Beja‡,
‡ CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Universidade do Porto, Vairão, Portugal
§ Universidad de Granada, Granada, Spain
| Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Porto, Portugal
¶ CIBIO-InBIO, Centro de Investigação em Biodiversidade e Recursos Genéticos, Instituto Superior de Agronomia, Lisboa, Portugal
# Museu de História Natural e da Ciência da Universidade do Porto, Porto, Portugal
¤ Rua do Torgal nº16, Trigais - Covilhã, 6215-295 Erada, Covilhã, Portugal
Open Access



The use of DNA barcoding allows unprecedented advances in biodiversity assessments and monitoring schemes of freshwater ecosystems; nevertheless, it requires the construction of comprehensive reference collections of DNA sequences that represent the existing biodiversity. Plecoptera are considered particularly good ecological indicators and one of the most endangered groups of insects, but very limited information on their DNA barcodes is available in public databases. Currently, less than 50% of the Iberian species are represented in BOLD.

New information

The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera dataset contains records of 71 specimens of Plecoptera. All specimens have been morphologically identified to species level and belong to 29 species in total. This dataset contributes to the knowledge on the DNA barcodes and distribution of Plecoptera from the Iberian Peninsula and it is one of the IBI database public releases that makes available genetic and distribution data for a series of taxa.

The species represented in this dataset correspond to an addition to public databases of 17 species and 21 BINs. Fifty-eight specimens were collected in Portugal and 18 in Spain during the period of 2004 to 2018. All specimens are deposited in the IBI collection at CIBIO, Research Center in Biodiversity and Genetic Resources and their DNA barcodes are publicly available in the Barcode of Life Data System (BOLD) online database. The distribution dataset can be freely accessed through the Global Biodiversity Information Facility (GBIF).


Plecoptera, occurrence records, species distributions, continental Portugal, continental Spain, DNA barcode, COI


In freshwater ecosystems, biodiversity assessments and monitoring schemes often require the identification of aquatic insect species (e.g. Pawlowski et al. 2018), an often challenging step, namely when only first instars are available in the sample or when studies are developed in regions poorly known from a faunistic perspective. In such cases, DNA barcoding provides a powerful tool to overcome these challenges by using a fragment of DNA to assign organisms to a species in a rapid and automated way (Hebert et al. 2003). Furthermore, environmental DNA (eDNA) is an emerging tool with great potential in conservation for monitoring past and present biodiversity, both in terrestrial and aquatic ecosystems (Thomsen and Willerslev 2015), especially when DNA barcode reference collections are used to link the obtained sequences to reliably identified organisms. The use of DNA barcoding requires the construction of comprehensive reference collections of DNA sequences that represent the existing biodiversity (Ferreira et al. 2018, Kress et al. 2005, Baird et al. 2011). In Europe, initiatives like the DNA barcoding projects, overseen by the Bavarian State Collection of Zoology in Munich (SNSB-ZSM—www.barcoding. through the “Barcoding Fauna Bavarica project” (BFB——Haszprunar, 2009), launched in 2009 and by the “German Barcode of Life project” (GBOL—, launched in 2012 (Geiger et al. 2016), has led to the public release of DNA barcode sequence data of over 300 species of Ephemeroptera, Plecoptera and Trichoptera (Morinière et al. 2017). As part of the Mediterranean Basin Biodiversity Hotspot, the Iberian Peninsula presents not only high numbers of species, as it also harbours species with limited distribution range, with many absent in central and northern Europe. The InBIO Barcoding Initiative (IBI) was established to overcome the striking scarcity of genetic data associated with the high biodiversity found in Portugal, focusing mainly on invertebrate taxa. Within the project, a special focus was afforded to aquatic insects, given their role as indicators in biodiversity assessments and monitoring schemes (e.g. Weisser and Siemann 2004, Weigand et al. 2019) and their relevance to food webs and ecosystem functioning. Furthermore, many insect species occurring in the Iberian Peninsula are not represented in public barcode databases (Ferreira et al. 2019, Ferreira et al. 2018, Weigand et al. 2019) and those that exist often show high evolutionary distances to the sequences obtained in this region which may indicate cryptic diversity (Corley et al. 2019b, Corley et al. 2019a, Ferreira et al. 2018). DNA barcoding can therefore be used as a first step in new species discovery and, as such, be used as a tool to help address the taxonomic impediment problem (e.g. Kekkonen and Hebert 2014).

Plecoptera is a neopteran exopterygote insect order characterised by a combination of mainly primitive characters, whose phylogenetic relationships with other insect orders are not completely resolved (Zwick 2000). Except in a few cases, they are amphibiotic animals, with eggs and nymphs occurring in freshwaters and adults inhabiting the terrestrial environment. The commonly called stoneflies are worldwide distributed, except in Antarctica and many islands and are usually associated with unpolluted and well-preserved waters, mainly rivers and streams, where they play important roles as part of their biota (Fochetti and Tierno de Figueroa 2008, Stewart 2009) contributing to important ecosystem services (DeWalt and Ower 2019). Their high vulnerability to environmental changes have driven stoneflies to be one of the most endangered groups of insects (Fochetti and Tierno de Figueroa 2008, Tierno de Figueroa et al. 2010).

A total of 3718 Plecoptera species have been described all over the world and 489 of them have been reported in Europe (DeWalt and Ower 2019). The European stonefly fauna, included in seven of the 16 existing families, is one of the best studied worldwide, but the degree of knowledge differs between countries. Of the Western European countries, Portugal is one of the less studied from a taxonomic and faunistic point of view. Furthermore, less than 50% of the Iberian Plecoptera have their DNA barcode sequenced. Although the first reports of stonefly species in Portugal date from the mid-XIXth century (Pictet 1841), only a few new records were added for this country during the following hundred years by authors such as Pictet A.E., Albarda, Kempny or Navás (in: Sánchez-Ortega et al. 2002). It was not until 1963 when the first exhaustive work on faunistic and chorology of stoneflies from Portugal, particularly for those of Serra da Estrela, was published as part of a wider study on the Iberian Peninsula (Aubert 1963). Afterwards, the main contributions to the knowledge of the taxonomy and/or faunistics of Plecoptera from Portugal were those of Zwick (1972), Whytton da Terra (1979), Berthélemy and Whytton da Terra (1980), Tierno de Figueroa et al. (1998), increasing the number of recorded species in the country from 25 to 53. More recently, the Portuguese fauna have been also studied in general publications for the Iberian Peninsula, such as those by Sánchez-Ortega et al. (2002) and Tierno de Figueroa et al. (2003), Tierno de Figueroa et al. (2015). According to Tierno de Figueroa et al. (2018), a total of 56 species were recorded in continental Portugal, without considering Isoperla luzoni Tierno de Figueroa & Vinçon, 2005, whose presence should be confirmed. No Plecoptera species has ever been collected in the Portuguese archipelagoes of Madeira and the Azores. Other areas from the Iberian Peninsula have been better studied regarding their Plecoptera fauna. Currently, 148 species of Plecoptera have been reported in the Iberian Peninsula and Balearic Islands (two of them endemic from the Balearic Islands), 144 species in Spain and 43 in Andorra (Tierno de Figueroa et al. 2018).

The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera dataset contains records of 71 specimens of Plecoptera collected in the Iberian Peninsula, all of which were morphologically identified to species level, for a total of 29 species. This is the first IBI dataset on freshwater insects available in the Global Biodiversity Information Facility (GBIF). All specimens have their DNA barcodes made publicly available in the Barcode of Life Data System (BOLD). Overall, this paper increases the available information on Iberian freshwater insects by sharing and publicly disseminating the distribution records and DNA barcodes of specimens from our reference collection.

General description


This dataset aims to provide a first contribution to an authoritative DNA barcode sequences library for Iberian Plecoptera. Such a library should facilitate DNA-based identification of species for both traditional molecular studies and DNA-metabarcoding studies, as well as freshwater biomonitoring programmes and constitute a valuable resource for taxonomic research on Iberian Plecoptera and its distribution.

Additional information: 

A total of 71 specimens of Plecoptera were collected and DNA barcoded (Suppl. materials 1, 2, 3). Sequences of cytochrome c oxidase I (COI) DNA barcodes are 658 bp long (Folmer region) with the exception of Leuctra cazorlana, from which a fragment of 325 bp was obtained. From the 29 species barcoded, 18 (62%) from seven families are new to the DNA barcode database BOLD at the moment of the release (marked with quotation mark ('') in the Species field of Table 1). Six additional BINs from these datasets are new to BOLD (marked with asterisk symbol (*) in BOLD BIN field of Table 1). Therefore, this dataset represents a significant contribution to enhance the species and genetic diversity of Iberian Plecoptera fauna represented in public libraries.

Table 1.

List of species that were collected and DNA barcoded within this project. " Indicate species new to BOLD database and * new BINs in BOLD database.

Family Species IBI code BOLD code BOLD BIN GenBank BOLD BIN
Capniidae Capnioneura libera" INV00770 IBIPP042-20 AEC8556 MT407216 AEC8556
Capnioneura mitis" INV02034 IBIPP045-20 AEC7867 MT407200 AEC7867
INV02035 IBIPP046-20 MT407211 AEC7867
INV02036 IBIPP047-20 MT407226 AEC7867
Capnioneura petitpierreae" INV03768 IBIPP079-20 AEC8557 MT407228 AEC8557
Capnopsis schilleri INV03770 IBIPP009-19 ADV3255* MT407199 ADV3255
Chloroperlidae Chloroperla acuta" INV06355 IBIPP099-20 AEC9580 MT407268 AEC9580
Siphonoperla torrentium INV00467 IBIPP002-19 ADT9540 MT407262 ADT9540
INV03933 IBIPP011-19 MT407247 ADT9540
INV03934 IBIPP012-19 MT407207 ADT9540
INV06330 IBIPP095-20 MT407206 ADT9540
INV06354 IBIPP098-20 MT407244 ADT9540
INV06359 IBIPP100-20 MT407208 ADT9540
INV06360 IBIPP101-20 MT407265 ADT9540
Leuctridae Leuctra andalusiaca" INV03771 IBIPP010-19 ACX4018 MT407238 ACX4018
Leuctra cazorlana" INV03775 IBIPP082-20 MT407235
Leuctra franzi" INV03718 IBIPP076-20 AEC8030 MT407220 AEC8030
Leuctra geniculata INV02033 IBIPP007-19 AAM4209 MT407255 AAM4209
INV03720 IBIPP008-19 MT407267 AAM4209
Leuctra iliberis" INV02025 IBIPP052-20 AEC6493 MT407209 AEC6493
INV02026 IBIPP044-20 MT407261 AEC6493
Leuctra major INV03719 IBIPP077-20 ACB2856 MT407242 ACB2856
Tyrrhenoleuctra lusohispanica" INV00350 IBIPP017-19 ACD6989 MT407256 ACD6989
INV00405 IBIPP023-19 MT407263 ACD6989
INV02875 IBIPP048-20 MT407257 ACD6989
INV02926 IBIPP074-20 MT407222 ACD6989
Nemouridae Amphinemura guadarramensis" INV06379 IBIPP102-20 AEC7918 MT407223 AEC7918
Amphinemura sulcicollis INV00773 IBIPP004-19 AAM5074 MT407202 AAM5074
Nemoura cinerea INV00367 IBIPP033-20 ADS8217* MT407251 ADS8217
INV00368 IBIPP001-19 MT407266 ADS8217
Nemoura lacustris INV00389 IBIPP020-19 AEB8934* MT407258 AEB8934
INV00392 IBIPP021-19 MT407215 AEB8934
INV00345 IBIPP016-19 AEB9369* MT407225 AEB9369
INV00382 IBIPP018-19 MT407224 AEB9369
INV00385 IBIPP019-19 MT407214 AEB9369
INV00454 IBIPP024-19 MT407212 AEB9369
INV04847 IBIPP037-20 MT407250 AEB9369
INV06416 IBIPP103-20 MT407264 AEB9369
INV00404 IBIPP022-19 AEC1305* MT407237 AEC1305
Protonemura alcazaba INV02028 IBIPP067-20 AEC7157* MT407227 AEC7157
Protonemura meyeri INV02031 IBIPP005-19 ADS3277 MT407253 ADS3277
INV02032 IBIPP006-19 MT407252 ADS3277
Perlidae Eoperla ochracea" INV02021 IBIPP043-20 AEC9593 MT407249 AEC9593
Perla madritensis" INV00640 IBIPP026-19 AEB9929 MT407260 AEB9929
INV00677 IBIPP027-19 MT407245 AEB9929
INV00688 IBIPP028-19 MT407205 AEB9929
INV00764 IBIPP039-20 MT407233 AEB9929
INV05226 IBIPP038-20 MT407232 AEB9929
Perla marginata INV04280 IBIPP013-19 AAL2357 MT407210 AAL2357
INV04281 IBIPP014-19 MT407221 AAL2357
Perlodidae Guadalgenus franzi" INV00344 IBIPP015-19 AEB8450 MT407248 AEB8450
INV00355 IBIPP055-20 MT407230 AEB8450
INV00358 IBIPP032-20 MT407269 AEB8450
Hemimelaena flaviventris" INV00507 IBIPP025-19 AEC1314 MT407236 AEC1314
INV00766 IBIPP040-20 MT407243 AEC1314
INV02925 IBIPP031-19 MT407218 AEC1314
Isoperla bipartita" INV00828 IBIPP029-19 AEC0487 MT407203 AEC0487
INV00850 IBIPP030-19 MT407231 AEC0487
Isoperla grammatica INV00183 IBIPP053-20 AEC9627 MT407254 AEC9627
INV00479 IBIPP034-20 MT407240 AEC9627
INV00500 IBIPP003-19 MT407213 AEC9627
INV00548 IBIPP035-20 MT407234 AEC9627
INV00767 IBIPP058-20 MT407204 AEC9627
INV00768 IBIPP059-20 MT407219 AEC9627
INV03931 IBIPP084-20 MT407201 AEC9627
INV03932 IBIPP085-20 MT407246 AEC9627
INV04831 IBIPP036-20 MT407217 AEC9627
INV07241 IBIPP104-20 MT407229 AEC9627
Isoperla pallida" INV03772 IBIPP050-20 AED0411 MT407259 AED0411
Taeniopterygidae Brachyptera auberti" INV04837 IBIPP092-20 AEC8527 MT407241 AEC8527
Rhabdiopteryx thienemanni" INV03773 IBIPP051-20 AEC7722 MT407239 AEC7722

Project description


The name “The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera” refers to the first data release of DNA barcodes and distribution data of stoneflies within the InBIO Barcoding Initiative.


Pedro Beja (project coordinator), Nuno Fonseca (project chair), Sónia Ferreira (taxonomist and IBI manager), Joana Paupério (IBI manager), Pedro Sousa (project technician), Filipa MS Martins (PhD student), Joana Veríssimo (PhD student), all affiliated to CIBIO-InBIO and Jose Manuel Tierno de Figueroa (taxonomist), Department of Zoology, University of Granada.

Study area description: 

Iberian Peninsula (Fig. 1)

Figure 1.  

Map of the localities where Plecoptera samples were collected in the Iberian Peninsula.

Design description: 

Plecoptera specimens were collected in the field, morphologically identified and DNA barcoded.

Sampling methods

Study extent: 

Iberian Peninsula.

Sampling description: 

The studied material was collected in 40 different localities from the Iberian Peninsula (Suppl. materials 1, 2). Sampling was conducted between 2004 and 2018 on a wide range of habitats, using mainly hand-held sweep-nets or direct search for specimens. Collected specimens were examined in ethanol using a binocular stereoscopic microscope and they were stored in 96% ethanol for downstream molecular analysis. Morphological identification was performed using keys and descriptions from literature (mainly Tierno de Figueroa et al. 2003 and Tierno de Figueroa et al. 2015)

DNA extraction and sequencing followed the general pipeline used in the InBIO Barcoding Initiative (Ferreira et al. 2018). Briefly, genomic DNA was extracted from leg tissue using EasySpin Genomic DNA Tissue Kit (Citomed) following the manufacturer’s protocol. The cytochrome c oxidase I (COI) barcoding fragment (Folmer region) was amplified as two overlapping fragments (LC and BH), using two sets of primers: LCO1490 (Folmer et al. 1994) + Ill_C_R (Shokralla et al. 2015) and Ill_B_F (Shokralla et al. 2015) + HCO2198 (Folmer et al. 1994), respectively. The partial COI mitochondrial gene (Folmer region) was then sequenced in a MiSeq benchtop system. OBITools ( was used to process the initial sequences which were then assembled into a single 658 bp fragment using Geneious 9.1.8. (

Quality control: 

All DNA barcodes sequences were compared against the BOLD database and the 99 top hits were inspected in order to detect possible issues due to contamination or misidentifications. Prior submission to GBIF, data were checked for errors and inconsistencies with OpenRefine 3.2 (

Step description: 

Specimens were collected in 40 different localities of the Iberian Peninsula. Sampling was conducted from 2004 to 2018 and consisted of direct search of specimens on rocks and vegetation of streams and river margins and in the use of entomological nets to intercept specimens in flight. Specimens collected were stored in 96% ethanol. A tissue sample (leg) was removed, from which DNA was extracted and the COI DNA barcode fragment was sequenced. Data generated were submitted to BOLD, GenBank and GBIF.

Geographic coverage


Iberian Peninsula


35.97 and 43.99 Latitude; 9.55 and 3.34 Longitude.

Taxonomic coverage


This dataset is composed of data relating to 71 Plecoptera specimens. All specimens were determined to species level. Overall, 29 species are represented in the dataset. These species belong to 16 genera and seven families.

Taxa included:
Rank Scientific Name Common Name
kingdom Animalia Animals
phylum Arthropoda Arthropods
class Insecta Insects
order Plecoptera Stoneflies
family Capniidae
family Chloroperlidae
family Leuctridae
family Nemouridae
family Perlidae
family Perlodidae
family Taeniopterygidae

Temporal coverage

Data range: 
2004-6-22 - 2018-5-19.

The sampled material was collected in the period from 22 June 2004 to 19 May 2018

Collection data

Collection name: 
InBIO Barcoding Initiative
Collection identifier: 
Specimen preservation method: 
Curatorial unit: 
Voucher tube - 1 to 71, DNA extractions - 1 to 71

Usage rights

Use license: 
Creative Commons Public Domain Waiver (CC-Zero)

Data resources

Data package title: 
The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera
Number of data sets: 
Data set name: 
DS-IBIPP01 IBI-Plecoptera 01
Data format: 
dwc, xml, tsv, fasta

The InBIO Barcoding Initiative Database: contribution to the knowledge on DNA barcodes of Iberian Plecoptera dataset can be downloaded from the Public Data Portal of BOLD ( in different formats (data as dwc, xml or tsv and sequences as fasta files). Alternatively, BOLD users can log-in and access the dataset via the Workbench platform of BOLD. All records are also searchable within BOLD, using the search function of the database.

The InBIO Barcoding Initiative will continue sequencing Iberian Plecoptera 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 Suppl. materials 1, 2, 3 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, Ferreira et al. 2020) and of a fasta file containing all sequences as downloaded from BOLD.

It 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 (Suppl. material 2).

All data are available in the BioStudies database ( under accession number S-BSST402.

Column label Column description
processid Unique identifier for the sample
sampleid Identifier for the sample being sequenced, i.e. IBI 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 taxonomic numeric code
subfamily_name Subfamily name
genus_taxID Genus taxonomic numeric code
genus_name Genus name
species_taxID Species taxonomic numeric code
species_name 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
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


This project has received funding from 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), by EDP Biodiversity Chair and is part of the research conducted at the Long Term Research Site of Baixo Sabor, (LTER_EU_PT_002). FMSM was supported by FCT PhD grant SFRH/BD/104703/2014 and JV by FCT PhD grant SFRH/BD/133159/2017.


Supplementary materials

Suppl. material 1: IBI-Plecoptera 01 library - Specimen details 
Authors:  Sonia Ferreira, Jose Manuel Tierno de Figueroa, Lorenzo Quaglietta, José Manuel Grosso-Silva, Pedro B Lopes, Pedro Sousa, Pedro Beja
Data type:  Record information - specimen data
Brief description: 

The file includes information about all records in BOLD for the IBI-Plecoptera 01 library. It contains collection and identification data. The data are as downloaded from BOLD, without further processing.

Suppl. material 2: IBI-Plecoptera 01 library - Specimen details - Darwin Core Standard  
Authors:  Sonia Ferreira, Jose Manuel Tierno de Figueroa, Filipa Martins, Joana Veríssimo, 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-Plecoptera 01 library. It contains collection and identification data. The data are downloaded from GBIF, without further processing.

Suppl. material 3: IBI-Plecoptera 01 library - DNA sequences 
Authors:  Sonia Ferreira, Jose Manuel Tierno de Figueroa, Filipa Martins, Joana Veríssimo, Joana Paupério, Pedro Sousa, Pedro Beja
Data type:  Genomic data, DNA sequences
Brief description: 

COI sequences in fasta format. Each sequence is identified by the BOLD ProcessID, species name, marker and GenBank accession number, separated by pipe. The data are as downloaded from BOLD.