DOI: 10.2478/frp-2019-0021 Leśne Prace Badawcze / Forest Research Papers Available online: www.lesne-prace-badawcze.pl Wrzesień / September 2019, Vol. 80 (3): 227–232 REVIEW ARTICLE e-ISSN 2082-8926 DNA barcoding: A practical tool for the taxonomy and species identification of entomofauna Iwona Szyp-Borowska1*, Katarzyna Sikora2 Forest Research Institute, 1Department of Silviculture and Genetics of Forest Trees, 2Department of Forest Protection; Sękocin Stary, ul. Braci Leśnej 3, 05−090 Raszyn, Poland *Tel. +48 22 7150481, e-mail: [email protected] Abstract. DNA barcoding is an innovative system designed to provide rapid, accurate, and automatable species identification by using short, standardized gene regions as internal species codes. The mitochondrial cytochrome C oxidase I (COI) gene was proposed by Paul Hebert as an official marker for animals, because of its small intraspecific but large interspecific variation. Since the launch of the project Barcode of Life, this simple technique has caught the interest of taxonomists, ecologists and plant- quarantine officers charged with the control of pests and invasive species. The great diversity of insects and their importance have made this group a major target for DNA barcoding. In most cases, the identification of insect species by traditional methods based on morphological features requires specialist knowledge and is labor-intensive. DNA barcoding aims at meeting the challenge of monitoring and documenting the biodiversity of insects. The utility of DNA barcoding for identifying small insects, cryptic taxa or rare species, as well as many species of forest entomofau- na that are impossible to discriminate morphologically throughout all of their life stages, is a subject discussed in this review. Due to its usefulness, also in Poland in the Forestry Research Institute, a method for identifying selected species of saproxylic beetles based on the sequence of the COI region was developed. In the future, this method will be used to assess the state of biodiversity and the naturalness of forest ecosystems. Therefore, this and other future implications of this promising new technique are also discussed here. Keywords: DNA barcodes, entomofauna, species identification, taxonomy 1. Introduction features, the DNA barcoding method provides the possibi- lity of identifying organisms quickly and precisely (Hebert Insects are the most diverse group of animals, with over et al. 2003, 2004). The use of DNA sequences to obtain the a million species described and another million pending de- information on the taxonomic similarities of an unknown scription or simply undiscovered (Grimaldi, Engel 2005). In specimen consists of sequencing a short fragment of the acquiring useful knowledge about any group of organisms, mitochondrial sequence encoding the subunit of the cyto- the basic requirement is the ability to describe, classify and chrome oxidase I gene (cox1 CO1) and comparing this with then identify individual taxa of this group. The identification the reference library of the barcodes of known species. This of numerous insect species by traditional methods based on method is effective, especially in identifying small, cryptic morphological features requires specialist knowledge and is insects (Burns et al. 2008; Huemer et al. 2014) or rare spe- time consuming. Moreover, this diagnosis is often based on cies (Hebert et al. 2004; Sinclair, Gresens 2008; Sweeney the morphological differences of adult insect forms, which et al. 2011; Anderson et al. 2013; Jackson et al. 2014) and severely restricts the identification of specimens in juveni- attempting to link different stages of insect development le phases (Balakrishnan 2005). In many cases, due to the (Carew et al. 2005; Ekrem et al. 2007, 2010; Zhou et al. lack of diagnostic features or the large variability of these 2007, 2009; Stur, Ekrem 2011; Webb et al. 2012). Received: 12.06.2019 r., accepted after revision: 9.09.2019 r. © 2019 I. Szyp-Borowska, K. Sikora 228 I. Szyp-Borowska, K. Sikora / Leśne Prace Badawcze, 2019, Vol. 80 (3): 227–232 2. The taxonomic identification of insects The effectiveness of the DNA sequencing method in a selected, standard region of the genome to distinguish spe- The development of molecular phylogenetics has revo- cies (Hebert et al. 2003, 2004) was initiated by international lutionised taxonomy. Numerous research studies show how research programmes that resulted in the barcode libraries the analysis of the mitochondrial cytochrome C oxidase I available in the international GenBank databases (Clark (COI) locus can challenge existing taxonomic boundaries. et al. 2016) and the BOLDSystems (Barcode of Life Data For the stag beetle family, DNA barcoding confirmed the System) (Ratnasingham, Hebert 2007). In the first years of differences between several Lucanus species and Lucanus operation, 430,000 insect barcodes were collected, represen- cervus L. subspecies (Cox et al. 2013). Similarly, COI analy- ting approximately 50,000 species (30% of all known spe- sis enabled the species of two groups of beetles from the Hy- cies) (Silva-Brandão et al. 2009; International Barcode of drophilidae and Scarabaeidae families to be distinguished Life 2010b). Webb (2012) provided the largest set of DNA (Monaghan et al. 2005). Based on mtDNA analyses, Jordal barcode data for water insects. He collected 4,165 sequen- and Kambestad (2014) performed a taxonomic revision of ces from the mitochondrial region of the COI subunit gene, two species of bark beetles, Pityophthorus micrographus L. representing 354 species of mayflies (Ephemeroptera) from and Pityophthorus pityographus Ratz. DNA barcodes have Canada, Mexico and the United States. This allowed pre- also been used to identify tropical butterflies of the genus viously misidentified species to be corrected and extended Perichares (Lepidoptera, Hesperiidae). Since their first knowledge about the levels of variation of the COI locus, description in 1775, this group often included specimens both within and between species. Thanks to a project using that only superficially matched the described species. After DNA barcoding to prepare genetic and morphological keys mtDNA analysis, it turned out that some species are incor- to identify species of the Tanytarsini tribe of flies on Spits- rectly assigned, and many of them belong to a complex of bergen and Bear Island (Stur, Ekrem 2011), DNA barcode cryptic species (Burns et al. 2008). The correct assignment libraries have been developed for all Arctic species. Tany- of specimens to a species, especially in the case of cryptic tarsini is a large and diverse group of lake flies belonging species, has important implications for taxonomic, evolutio- to the Chironomidae family. The developed keys allow the nary and biodiversity studies, and their presence amongst identification of the Tanytarsini species’ larvae, pupae and pests also has economic consequences. finally metamorphosed specimens on Svalbard. DNA barcode analysis also allows ambiguities to be removed in determining species, resulting from the mor- 3. Protection of endangered species phological differences between representatives of different castes in the case of social insects, such as ants (Smith et In the context of protecting endangered species, it is worth al. 2008), or differences in the morphology of male and fe- mentioning that the DNA barcode method has the additional male species with high sexual dimorphism (Janzen 2005). advantage of allowing the use of only small anatomical frag- mtDNA analysis is an important tool in nature inventorying ments instead of whole individuals (Svensson et al. 2009). and biodiversity protection, facilitating both the identifica- This eliminates the need to obtain whole specimens, which tion of specimens living today as well as the description of is particularly important in the case of small populations. The taxa from museum collections, and, most importantly, it al- flagship taxon of significance for the protection of inverte- lows these samples to be linked to reconstruct the history of brates in Europe is the genus Osmoderma (hermit beetles), species. The inventory project of the geometer moth family comprising a complex of four species, including Osmoderma (Geometridae) in Costa Rica demonstrates the possibility of barnabita Motsch. – strictly associated with old, hollow trees, using DNA barcodes to describe new species (Chacon et al. which was intensively studied by Landvik et al. (2017). In 2012). Caterpillars of five butterfly species – Opsiphanes the study cited above, the authors focused on explaining the quiteria Stoll, Opsiphanes tamarindi Felder, Opsiphanes phylogeographic structure of the east European population of bogotanus Dist., Opsiphanes invirae Hübner and Opsipha- O. barnabita, based on specimens from Latvia and Finland, nes fabricii Boisduval – are very often encountered. A sixth as well as on previously published sequences and museum species in the genus Opsiphanes, whose adult males are very specimens. A sequence analysis of the mitochondrial COI similar to male specimens of the common O. fabricii, were gene has identified 26 closely related haplotypes whose diver- identified as Opsiphanes cassina fabricii (DeVries 1987; sity decreases from south to north. The observed distribution Bristow 1991). An analysis of the DNA barcode of these of O. barnabita also pointed to the recent expansion of this species showed that O. cassina fabricii is a new species for species in eastern Europe. This knowledge has a