This dataset aims to provide a contribution to the knowledge on DNA barcodes of Portuguese Orthoptera. Such a library should facilitate DNA-based identification of species for both traditional molecular studies and DNA metabarcoding studies. Furthermore, it constitutes a valuable resource for taxonomic research on Iberian Orthoptera and their distribution.

A total of 420 specimens of orthopterans were collected and DNA barcoded (Table 1, Suppl. material 2) corresponding to 119 species, about 77% of all the orthopteran species known from continental Portugal (Aires and Menano 1915, Pina et al. 2017, GBIF 2023, IUCN 2023). Figs 1, 2 illustrate examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera. The dataset includes 39 Iberian endemic species, of which six occur only in continental Portugal: Ephippigerida rosae, Lluciapomaresius anapaulae, Neocallicrania barrosi, Neocallicrania serrata, Pterolepis lusitanica and Pycnogaster cucullatus (Table 1). Additionally, the species Stenobothrus lineatus is recorded for the first time for Portugal in this dataset. A full-length barcode of 658 bp was obtained for all 420 specimens. This dataset contributes significantly to the representation of both species and genetic diversity of Orthoptera in public libraries. Of the 120 taxa barcoded, DNA barcodes of 54 are made public for the first time (marked with # in Taxa field of Table 1). The submitted sequences were found to cluster in 129 BINs, 35 of which were new to BOLD (unique BINs, marked with " in Taxa field of Table 1).

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Figure 1.

Examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera 01.

a: Neocallicrania barrosi Barat, 2013. Tettigoniidae. Photo by Francisco Barros. BIN URI BOLD:AEO7827;  
b: Tylopsis lilifolia (Fabricius, 1793). Tettigoniidae. Photo by Sónia Ferreira. BIN URI BOLD:AER2541;  
c: Eugryllodes escalerae (Bolívar, 1894). Gryllidae. Photo by Sónia Ferreira. BIN URI BOLD:AEP5287;  
d: Gryllotalpa vineae Bennet-Clark, 1970. Gryllotalpidae. Photo by Sílvia Pina. BIN URI BOLD:AEP5099; BOLD:AEP5100;  
e: Paramogoplistes ortini Llucià Pomares, 2015. Mogoplistidae. Photo by Sílvia Pina. BIN URI BOLD:AEO7942;  
f: Oecanthus dulcisonans Gorochov, 1993. Oecanthidae. Photo by Sílvia Pina. BIN URI BOLD:ACG7286.  

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Figure 2.

Examples of the diversity of species that are part of the dataset of distribution data and DNA barcodes of Portuguese Orthoptera.

a: Trigonidium cicindeloides Rambur, 1838. Trigonidiidae. Photo by Sílvia Pina. BIN URI BOLD:ACY3774;  
b: Sphingonotus lusitanicus Ebner, 1941. Acrididae. Photo by Sílvia Pina. BIN URI BOLD:AEI2886;  
c: Acinipe ignatii Llorente del Moral & Presa, 1983. Pamphagidae. Photo by Francisco Barros. BIN URI BOLD:AER2325;  
d: Pyrgomorpha conica (Olivier, 1791). Pyrgomorphidae. Photo by Sílvia Pina. BIN URI BOLD:AEO6938;  
e: Paratettix meridionalis (Rambur, 1838). Tetrigidae. Photo by Sílvia Pina. BIN URI BOLD:BOLD:AAP5007;  
f: Xya iberica Günther, 1990. Tridactylidae. Photo by Sónia Ferreira. BIN URI BOLD:AER2238.  

The results show multiple BINs for a recognised species in several cases. Namely, three BINs were obtained in each of the recognised species given: Antaxius spinibrachius, Nemobius sylvestris, Tessellana tessellata and Thyreonotus bidens. Two BINs were obtained in each of the following taxa: Gryllomorpha longicauda, Gryllotalpa vineae, Lluciapomaresius asturiensis, Neocallicrania lusitanica, Pezotettix giornae, Pycnogaster cucullatus, Platycleis sabulosa and for the subspecies Neocallicrania selligera meridionalis. Our dataset includes three BINs obtained for the species Antaxius spinibrachius, including the first sequences of the BIN BOLD:AER0568 from specimens collected in the Castelo Branco and Guarda Districts. In a previous study, aimed at the phylogeography of this species in the Iberian Peninsula, two different lineages of this species were identified, one of which occurs along the Cordillera Oretana and the other includes all other populations (Gutiérrez-Rodríguez et al. 2014). The genetic diversity found in DNA barcodes in this species highlights the necessity of a taxonomic revision. Additionally, in the family Tettigoniidae, the specimen of the Neocallicrania genus collected in the Setúbal Peninsula was morphologically identified as Neocallicrania lusitanica using the available literature (Barat 2007) and the unique BIN BOLD:AEO6978 was obtained. However, the revision conducted by Barat (2013) pointed out uncertainties related to specimens collected in this geographic area. Thus, our results emphasise the need for further work towards a better understanding of the taxonomy of this genus in the Iberian Peninsula.

Of the 119 species, 104 have direct correspondence between morphologic identification and BINs, leaving 15 species involved in BIN sharing. In the subfamily Tettigoniinae, two BINs were found to be shared by more than one species, the BIN BOLD:AEO6325 and the BIN BOLD:AEO7101 was shared by Platycleis albopunctata and Platycleis sabulosa and by Pterolepis lusitanica and Pterolepis spoliata, respectively. In the subfamily Gomphocerinae, the generated sequence of Pseudochorthippus parallelus shared BIN BOLD:AAC3399 with sequences of Pseudochorthippus montanus from Germany, Ukraine and Norway. Introgression caused by hybridisation is a well-studied phenomenon in areas where both species occur in sympatry in Germany (Hochkirch and Lemke 2011, Rohde et al. 2015). These species are morphologically very similar, but have different ecological requirements and both species occur in Spain, although Pseudochorthippus montanus is restricted to the northeast (Llucià-Pomares 2002). Additionally, in this subfamily, there are other Gomphocerinae groups of species that cannot be identified using the DNA barcode COI fragment. Sequences obtained from our specimens of Chorthippus binotatus, Chorthippus jacobsi and Chorthippus yersini shared the same BIN BOLD:AAC5779. Moreover and in line with the results by Hawlitschek et al. (2017), sequences obtained as Omocestus rufipes and Omocestus viridulus shared the same BIN. Likewise, in the subfamily Oedipodinae, two BINs were shared by more than one species, namely the BIN BOLD:AAJ3344 was shared by Sphingonotus azurescens and Sphingonotus nodulosus and the BIN BOLD:AEI2886 was shared by Sphingonotus lluciapomaresi and Sphingonotus lusitanicus. The success rate of DNA barcoding in the genus Sphingonotus was previously studied by Dey et al. (2020). Their results showed intensive sharing of identical or closely-related DNA barcodes indicating that the single analysis of the COI fragment is not sufficient enough to differentiate many of the Sphingonotus species.

This study shows that DNA barcode sequences, based on the COI mitochondrial gene fragment, can be useful in identifying Portuguese Orthoptera to species level. To our knowledge, this is the first study to focus on DNA barcoding of the Orthoptera order for the Iberian Peninsula. It also highlights several taxonomic challenges related to the rich fauna of this group and suggests intricated phylogeographic processes in the region that lead to the diversification of several taxa and high endemism levels in line with what previous studies have found (Barranco 2004, Solé et al. 2018). Our results constitute a first step in the construction of a DNA barcode database of a curated reference collection of Iberian (Portuguese) Orthoptera species that can be used in studies where it is necessary to identify specimens either by DNA barcoding or DNA metabarcoding.