Corresponding author: Sónia Ferreira (
Academic editor: Henrique Paprocki
The
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
DNA barcoding is a molecular biology method for species identification that was proposed almost twenty years ago (
Development of DNA metabarcoding (
Aquatic ecosystems are suffering high losses in biodiversity due to degradation and habitat destruction (
The
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 (
In the Iberian Peninsula, approximately 380
In this work, we present a contribution to the DNA barcode library of the Iberian Peninsula species of
This dataset aims to provide a first contribution to an authoritative DNA barcode sequences library for Iberian
A total of 438
Average nucleotide composition of the
The BOLD BIN system uses algorithms to cluster sequences into operational taxonomic units (OTUs) that closely correspond to species (
The independent RESL run (
Nevertheless, some differences existed between the RESL OTU clustering and the BINs created by BOLD (Suppl. materials
This work provided new DNA barcode sequences and distributional data for 436 specimens of Iberian
This study showed that DNA barcode sequences, based on the COI mitochondrial gene fragment, can be useful in identifying Iberian
Incongruences were found in nine families. In six of them,
In the family
In the family
In the family
In the family
Our results did not corroborate the findings of
We also identified several cases that require further study by taxonomists. Other possibilities for the incongruence found amongst the results include the existence of hybridisation, introgression or incomplete lineage sorting in these species, especially if they result from recent speciation events (e.g.
The InBIO Barcoding Initiative Database: DNA Barcodes of Iberian
Luis Martín (taxonomist), Jesús Martínez (taxonomist), Marcos A. González (taxonomist), affiliated to Universidad de Santiago de Compostela; Pedro Beja (project coordinator), Joana Paupério (IBI manager), Sónia Ferreira (taxonomist and IBI manager), Filipa M.S. Martins (molecular biologist), Joana Veríssimo (molecular biologist), Pamela Puppo (molecular biologist), Joana C. Pinto (project technician), Cátia 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.
Iberian Peninsula (Fig.
Specimens were collected during field expeditions in the Iberian Peninsula, from 1975 to 2018 (n = 434 Fig.
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 QIAmp DNA Micro Kit (Qiagen, Hilden, Germany). QIAmp 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 (
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
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 (
All DNA barcode sequences were aligned in Geneious 6.1.8 with MUSCLE (
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 “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 do Tua. SF was supported by 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.
Iberian Peninsula.
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) 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 (DMHF) 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.
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 (
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. González 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 COI 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 (
Specimens were collected in the Iberian Peninsula, 229 from 66 localities in Portugal and 207 from 74 localities in Spain (Fig.
-8.94 and -0.22 Latitude; 42.89 and 37.50 Longitude.
This dataset is composed of data relating to 438
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InBIO Barcoding Initiative
4ec2b246-f5fa-4b90-9a8d-ddafc2a3f970
“Alcohol”
DNA extractions - 1 to 438
Creative Commons Public Domain Waiver (CC-Zero)
1
DS-IBITR01 IBI-
dwc, xml, tsv, fasta
The InBIO Barcoding Initiative Database: DNA Barcodes of Iberian
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. |
The InBIO 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 “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 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).
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
Sampling localities of the
List of species that were collected and DNA barcoded within this project.
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IBI-
Record information - specimen data
The file includes information about all records in BOLD for the IBI-
File: oo_767197.txt
IBI-
Genomic data, DNA sequences
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.
File: oo_767198.fas
Genetic Distances
Genetic distances between analysed specimens
Brief description: Estimates of average genetic divergence (uncorrected p- distances) for species of
File: oo_760686.xlsx
OTUs generated by the Refined Single Linkage algorithm (RESL,)
OTUs generated by the RESL algorithm and respective sequence composition
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.
File: oo_760687.xlsx
IBI-
Record information - specimen data in Darwin Core Standard format
The file includes information about all records in BOLD for the IBI-
File: oo_760685.txt