Biologia 72/7: 796—806, 2017 Section Zoology DOI: 10.1515/biolog-2017-0087 DNA barcoding and first records of two rare Adicella species (Trichoptera: Leptoceridae) in Croatia Anđela Cukuši´ c´1,RenataCuk´ 2,AnaPrevišic´3,MartinaPodnar4,AntunDelic´5 &MladenKučinic´3* 1Geonatura Ltd. Consultancy in Nature Protection, Fallerovo šetalište 22, 10000 Zagreb, Croatia 2Hrvatske vode, Central Water Management Laboratory, Ulica grada Vukovara 220, 10000 Zagreb, Croatia 3Department of Biology, Laboratory for Entomology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia; e-mail: [email protected] 4Croatian Natural History Museum, Demetrova 1, 10 000 Zagreb, Croatia 5Faculty of Ecudation, Department in Petrinja, University of Zagreb, Matice Hrvatske 12, 44250, Petrinja, Croatia Abstract: Two species of the genus Adicella, A. cremisa Malicky, 1972 and A. balcanica Botosaneanu & Novak, 1965, were recorded in the summer period of 2014 which represent the first records of these species in Croatia. The former was collected at two relatively distant sites, the Krapina and the Zrmanja rivers, while the later was collected at the Krupa River. Both species were identified by morphological characteristics as well as DNA barcoding method. We calculated the uncorrected pairwise distances within Adicella and used molecular phylogenetic approach to delimit species.A.cremisafrom Croatia showed no significant difference in the mtCOI region and they are highly similar to A. cremisa from Italy. Additionally, the ecological preferences and distribution of Adicella species are presented. Our findings represent a significant contribution to the aquatic biodiversity of the Western Balkans. Key words: molecular identification; aquatic insects; caddisfly Introduction The Croatian caddisfly fauna was encompassed in several papers using the mtCOI gene in similar con- DNA barcoding method is used to identify species of text (e.g., Previši´c et al. 2009, 2014; Kučini´cetal. different groups of organisms (animals, plants, fungi) 2010), however, not the “barcode region” (mtDNA and is based on sequencing of the standardized segment COI-5P). The barcode region was used only to sup- of the mitochondrial (mt) cytochrome c oxidase subunit port the description of the new species Chaetopteryx 1 (COI) gene (Hebert et al. 2003). Advantages of us- bucari Kučini´c, Szivak & Deli´c, 2013 (Kučini´cetal. ing DNA barcodes in species identification compared 2013) and the new record of Tinodes antonioi Boto- to other parts of the genome are the lack of introns saneanu & Taticchi-Vigan`o, 1974 (Kučini´c et al. 2016). (unlike in nuclear genes), simple sequence alignment This paper presents the first finding of the two (compared to mitochondrial ribosomal genes, e.g., 12S, caddisfly species from the genus Adicella: A. cremisa 16S), a great range of phylogenetic signal, and widely Malicky, 1972 and A. balcanica Botosaneanu & No- used robust primers able to successfully amplificate the vak, 1965 in Croatia. The genus Adicella MacLachlan, barcode region in variety of taxa (Hebert et al. 2003). 1877 belongs to the tribe Triaenodini Morse, 1981 of DNA barcoding of caddisflies has overall wide ap- the long-horned caddisfly family Leptoceridae. Family plication in association of previously unknown larval Leptoceridae has a cosmopolitan distribution and it is stages with adults (e.g., Zhou et al. 2007; Zhou 2009; the second largest family of caddisflies in the world with Graf et al. 2009; Ruiter et al. 2013; Gill et al. 2014). a total of 1567 described species (Morse 2003; Holzen- Similarly, in European caddisflies, the barcode region thal & Pes 2004). This family has two subfamilies, the was used in studies mainly to support description of subfamily Triplectidinae distributed in southern hemi- new species and to associate previously unknown lar- sphere and the more cosmopolitan subfamily Leptoceri- val stages (e.g., Gíslason et al. 2015; Graf et al. 2015; nae, containing 14 and 30 genera, respectively (Morse Waringer et al. 2015; Vitecek et al. 2015b) and to per- & Holzenthal 1987; Holzenthal & Pes 2004). form phylogenetic analyses (Kučini´c et al 2013; Vitecek The genus Adicella is distributed in the West and et al. 2015a, 2017). East Palaearctic biogeographic region, the Afrotropical * Corresponding author c 2017 Institute of Zoology, Slovak Academy of Sciences Authenticated | [email protected] author's copy Download Date | 8/20/17 7:53 AM DNA barcoding and first records of two Adicella species in Croatia 797 Fig. 1. The study sites: A – the Krapina River at Krapina Selo; B – the Zrmanja River at Palanka; C – the Krupa River at Manastir. region, and the Oriental region (Huisman & Andersen 168 m a.s.l.) (Fig. 1A). According to the Croatian typol- 1997). According to Graf et al. (2008, 2016) there are 14 ogy, this part of the Krapina River is classified as “small species of the genus Adicella in Europe, two of which lowland rivers with gravel and pebble substrate” (Narodne A. filicornis (Pictet, 1834) and A. reducta (McLach- novine 2013, 2014). lan, 1865), are widely distributed, including Croatia The Zrmanja River is 69 km long and situated in the (Kučini´c et al. 2012; Robert 2015). Some ecological Dinaric ecoregion (ER5) (Illies 1978) (Mediterranean part of Croatia according to Berti´c et al. 2001), it originates un- preferences are known for A. cremisa, unlike A. bal- derneath Poštak peek and flows into the Adriatic Sea close canica whose both larva and ecological preferences are to the town Obrovac. Its catchment area is mostly built of unknown so far. To confirm the morphological identifi- karstic carbonate rocks and it is a part of the Dinaric karst. cation of collected specimens, we utilised sequence data Due to permeable carbonate rocks in some parts of the flow, of the mtCOI gene (i.e., the barcode region, mtDNA the Zrmanja spring and some other parts have a temporary COI-5P). As one of the first examples of the use of the flow (Šafarek & Šoli´c 2011). The study site at the Zrmanja ◦ ◦ barcode methodology in Croatian caddisflies, we further River was at the settlement Palanka (44 8.81 N, 16 4.27 E, highlight the importance of a simple and fast molecu- 264 m a.s.l.) (Fig. 1B). According to the Croatian typology, larmethodinidentificationof freshwater invertebrate this part of the course of the Zrmanja River is classified as “medium and large upland rivers” (Narodne novine 2013, taxa. Additionally, we give environmental data on their 2014). habitats, and discuss the ecology and the distribution The Krupa River is the longest right tributary of the of these invastegated species, both insufficiently inves- Zrmanja River and it is situated in the Dinaric ecoregion tigated not only taxonomically (unknown morphology (ER5). The Krupa River is 11.5 km long with all characteris- of larvae; Waringer & Graf 2011) but also ecologically tics of karst river (karst spring, river channel made of karstic (Graf et al. 2008). Finally, we present data of caddis- carbonate rocks with subterranean channels through which flies species syntopically collected with the two Adicella groundwater flows in conduits) (Šafarek & Šoli´c 2011). species. The study site at the Krupa River was at Krupa Mana- stir (Monastery) (44◦11.37 N, 15◦53.22 E, 105 m a.s.l.) (Fig. 1C). According to national typology the Krupa River Material and methods is classified as “short-flowing lowland rivers with a channel > Research area drop 5‰” (Narodne novine 2013, 2014). The Krapina River is situated in the Pannonian ecoregion (ER11) (Illies 1978) (Pannonian-peripanonian part of Croa- Sampling and laboratory methods tiaaccordingtoBerti´c et al. 2001) and is about 70 km Samples of adult caddisflies were collected using entomo- long. It springs at Ivanščica Mt., flows through the Hrvatsko logical net and UV light traps. The samples were stored zagorje region and then into the Sava River near Zapreši´c in 96% ethyl alcohol. Seven specimens of A. cremisa and (Šafarek & Šoli´c 2011). The study site at the Krapina River one specimen of A. balcanica have been deposited in the was in the village Krapina Selo (46◦4.34 N, 16◦12.00 E, NIP Trichoptera collection (collection formed as a part of Authenticated | [email protected] author's copy Download Date | 8/20/17 7:53 AM 798 A. Cukuši´ c´ et al. Table 1. Details of the specimens used in analysis with assigned species name, geographic origin, sample ID, BOLD Sequence ID number and GenBank Accession number. No. Species name Country Sample ID BOLD Sequence ID GenBank Accession number 1 A. balcanica Croatia TABAL 1 NIP010-16 BankIt1936914 TABAL 1 KX555470 2 A. cremisa Croatia TACRE 1 NIP009-16 BankIt1936914 TACRE 1 KX555471 3 A. cremisa Croatia TASYR 1 NIP008-16 BankIt1936914 TASYR 1 KX555472 4 A. cremisa KKCAD-0425 KKCAD417-07 5 A. cremisa Italy 08HMCAD-149 HMTRI149-08 6 A. cremisa Italy 08HMCAD-176 HMTRI176-08 7 A. cremisa Italy 08HMCAD-153 HMTRI153-08 8 A. cremisa Italy 08HMCAD-152 HMTRI152-08 9 A. cremisa Italy 08HMCAD-151 HMTRI151-08 10 A. cremisa Italy 08HMCAD-150 HMTRI150-08 11 A. filicornis Austria 12HMCAD-059 BHMKK222-12 12 A. filicornis France 10OFSI-0187 OFTRI186-10 13 A. filicornis Germany BCZSMAQU00856 FBAQU1141-12 14 A. reducta Germany BCZSMAQU00857 FBAQU1142-12 15 A. reducta Portugal HMCAD0810-3 HMKKT630-10 16 A. reducta Spain 09MNKK0411 KKUMN419-10 17 A. reducta Belgium UA-SG-TRICH-C18 TFLAN061-11 18 A. syriaca Hungary 10OFSI-0188 OFTRI187-10 HQ967420 the project: “EU Natura 2000 Integration Project – NIP”) deposited in Croatian Natural History Museum in Zagreb. All locations were visited three times: in spring, sum- mer and autumn period in 2014. For identification of col- lected specimens standard literature was used (Malicky 2004a). Systematic presentation follows Morse (2017). All physico-chemical parameters were analysed accord- ing to standard analytical methods for assessment of surface water quality (ISO norms), with Krapina River being sam- pled monthly while Zrmanja and Krupa rivers being sam- pled five (January, February, May, September and Novem- ber) and six times (January, February, May, July, Septem- ber and November), respectively, by staff of Hrvatske vode, Central Water Management Laboratory.