Ecologica Montenegrina 44: 69-95 (2021) This journal is available online at: www.biotaxa.org/em http://dx.doi.org/10.37828/em.2021.44.10 Biodiversity, DNA barcoding data and ecological traits of caddisflies (Insecta, Trichoptera) in the catchment area of the Mediterranean karst River Cetina (Croatia) IVAN VUČKOVIĆ1*, MLADEN KUČINIĆ2**, ANĐELA ĆUKUŠIĆ3, MARIJANA VUKOVIĆ4, RENATA ĆUK5, SVJETLANA STANIĆ-KOŠTROMAN6, DARKO CERJANEC7 & MLADEN PLANTAK1 1Elektroprojekt d.d., Civil and Architectural Engineering Department, Section of Ecology, Alexandera von Humboldta 4, 10 000 Zagreb, Croatia. E-mails:[email protected]; [email protected] 2Department of Biology (Laboratory for Entomology), Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10 000 Zagreb, Croatia. E-mail: [email protected] 3Ministry of Economy and Sustainable Development, Radnička cesta 80/7, 10000 Zagreb, Croatia. E-mail: [email protected] 4Croatian Natural History Museum, Demetrova 1, 10 000 Zagreb, Croatia. E-mail: [email protected] 5Hrvatske vode, Central Water Management Laboratory, Ulica grada Vukovara 220, 10 000 Zagreb, Croatia. E-mail:[email protected] 6Faculty of Science and Education, University of Mostar, Matice hrvatske bb, 88000 Mostar, Bosnia and Herzegovina. E-mail: [email protected] 7Primary School Barilović, Barilović 96, 47252 Barilović and Primary School Netretić, Netretić 1, 47271 E-mail: [email protected] *Corresponding author: [email protected] **Equally contributing author Received 2 June 2021 │ Accepted by V. Pešić: 19 July 2021 │ Published online 2 August 2021. Abstract The environmental and faunistic research conducted included defining the composition and distribution of caddisflies collected using ultraviolet (UV) light trap at 11 stations along the Cetina River, from the spring to the mouth, and also along its tributaries the Ruda River and the Grab River with two sampling stations each, and the Rumin River with one station. The research was conducted in the period from August 2004 until August 2005 on the Cetina and the Ruda rivers, and from April 2007 to September 2007 on the Grab and the Rumin rivers. A total of 71 caddisfly species and 10,048 individuals were recorded in the study area. Results of the composition and structure of caddisflies were processed with statistical methods using the NMDS analysis. There were differences in the composition of caddisflies in the upper, middle and lower reaches. The paper also presents some ecological features of caddisflies, such as seasonal dynamics and diversity of caddisflies communities. Results of this work contribute to faunistic and ecological knowledge of Croatian caddisflies. Key words: aquatic insects/faunistic features/molecular data. Ecologica Montenegrina, 44, 2021, 69-95 CADDISFLIES OF RIVER CETINA Introduction Currently caddisfly fauna counts over 16,267 extant and ca. 521 fossil species (Holzenthal et al. 2007, Morse 2020), while in Europe there are over 1000 caddisfly species (Graf et al. 2015; Ibrahimi et al. 2016; Malicky 2004, 2005, 2020; Oláh 2010; Previšić et al. 2014; Valladolid et al. 2018, 2020; Vitecek et al. 2015). Along with aquatic Diptera, they represent one of the most complex and most numerous groups in aquatic ecosystems. Some caddisfly species are adapted to specific habitat types and to certain water quality, and are important bioindicators, thus representing one of the most important groups of aquatic organisms for biological methods of determining water quality based on benthic invertebrates (Graf et al. 2002, 2008b; Urbanič et al. 2005). Limnological research of caddisfly larvae in Croatia started in the second part of the 20th century (Habdija 1988; Habdija et al. 1994, 2004; Matoničkin 1959, 1987; Matoničkin & Pavletić 1961, 1967; Matoničkin et al. 1971), and are ongoing (Ćuk & Vučković, 2009, 2010, 2014; Ćuk et al. 2015; Graf et al. 2008a; Karaouzas et al. 2015; Kučinić et al. 2007, 2008; Previšić et al. 2012, 2014; Waringer et al. 2009). Records of adult specimens are required for safe determination to the species level and for a detailed insight into caddisfly comunities, because adults can generally be identified to species level (Malicky 2004), and larvae do not always provide correct and reliable identification (Waringer & Graf 2011). DNA barcoding was proposed in 2003 as a universal system for assignment of specimens to a particular species but also as a tool for recognition of yet undescribed, morphologically indistinguishable cryptic species (Hebert et al. 2003). DNA barcodes enable simple, fast and accurate identification for a great number of species, provide insights in geographical distribution of genetic diversity, and flag certain groups for further investigation (Ratnasingham & Hebert 2007). This method and other molecular methods are proven to be successful in the association of the adult and larval stages of caddisflies (e.g. Burington 2011; Graf et al. 2015; Ruiter et al. 2013; Shackleton & Webb 2013, Torii & Nakamura 2016; Zhou 2009; Zhou et al. 2007). So far, a reliable association has been made between the adult and larval stages of caddisfly fauna in Croatia using other molecular markers (e. g. use of the 3’-region of the COI gene) (Previšić et al. 2014; Ćukušić 2019; Valladolid et al. 2020). Detailed biodiversity, taxonomy, distribution, life cycles, seasonal dynamics and other ecological features were systematically studied in the area of Plitvice Lakes NP (Kučinić 2002; Kučinić & Malicky 2002; Kučinić et al. 2017; Marinković-Gospodnetić 1971, 1979; Previšić et al. 2007a; Šemnički et al. 2012), the Cetina River (Graf et al. 2008a; Previšić et al. 2014; Vučković et al. 2016; Waringer et al. 2009), the Krka River (Kučinić et al. 2011; Ridl et al. 2015); Drava River (Previšić et al. 2007b), the Gorski kotar region (Cerjanec et al. 2020; Ibrahimi et al. 2012; Previšić & Popijač 2010) and the Banovina region (Kučinić et al. 2010, 2013, 2020b). In these investigations several first records for the Croatian fauna of caddisflies were registered (e.g. Ćuk & Vučković 2010, 2014; Ćukušić et al. 2017; Kučinić et al. 2014, 2015), as well as some new species for science (Malicky et al. 2007; Oláh 2010; Previšić et al. 2014). So far in the Croatian fauna ca. 200 caddisfly species have been recorded (Ćuk & Vučković 2009, 2010, 2014; Graf et al. 2008a; Kučinić 2002; Kučinić & Ilić 1993a, 1993b; Kučinić & Malicky 2002; Kučinić et al. 1999, 2000, 2011; Malicky 2009; Malicky & Krušnik 1988; Malicky et al. 2007; Previšić & Popijač 2010; Previšić et al. 2007a, 2007b, 2010, 2014; Urbanič et al. 2000; Waringer et al. 2009). In the last few years the DNA barcoding method has been included in many studies of the caddisfly fauna of Croatia (Ćukušić, 2019; Ćukušić et al. 2017; Kučinić et al. 2013, 2019a, 2019b, 2019c, 2020a, 2020b) The objectives of this paper were to determine faunistic features of caddisflies (1), caddisfly ecology and distribution in the study area (2) and give a note on results of the DNA barcoding of some species (3). Material and Methods Study area. The catchment area of the Cetina River includes the Cetina River and the left tributaries the Šilova, Veliki and Mali Rumin (Rumin - one location), Kosinac, Velika and Mala Ruda, and Grab rivers (Fig. 1). This research encompassed the Cetina River from its spring to its mouth, the Veliki Rumin River (length of watercourse 1.4 km), the Velika Ruda River (length of watercourse 7.8 km) and the Grab River (length of watercourse 2.8 km) (Vučković 2011). 70 VUČKOVIĆ ET AL. Figure 1. Map of the study area with sampling stations. Names and corresponding abbreviations of the stations are listed in Table 1. The Cetina River is the longest river in Central Dalmatia, with a length of 105 km. It rises on the northern edge of Cetinjsko Polje, and its spring consists of several springs, predominantly of the Vauclusian type. From the spring all the way to Zadvarje (the area dowenstream from Trilj), the Cetina flows mostly parallel to the mountain ranges and geological structures, i.e. from northwest to southeast. From Zadvarje it flows westward to its mouth into the Adriatic Sea, near the town of Omiš. It is a regulated river, with three reservoirs/accumulation lakes along its course (Peruča, Đale and Prančević) constructed for the operation of four hydroelectric power plants (Peruča, Zakučac, Đale and Kraljevac) (Magdalenić 1971). In contrast to the majority of karstic rivers in France, Italy and Spain that dry out in the summer period, the rivers draining into the Eastern Adriatic coast are mainly permanent throughout the year, including the Cetina River. Ecologica Montenegrina, 44, 2021, 69-95 71 CADDISFLIES OF RIVER CETINA The most abundant rocks in the Cetina River drainage basin are limestones. These are in general well-permeable rocks, so precipitations falling on such ground mostly sinks into the underground and flows through fissures and joints. Karst poljes are mostly built of impervious Neogene clastic deposits that form a hydrogeological barrier over which groundwater reaches the surface, flowing through the fields along the surface. After a short surface flow through the field, at the renewed contact with the permeable carbonate rocks, the water flows underground to the next barrier, which consists of impermeable layers of the Karst polje at a lower altitude. The separate parts of the Cetina River are very different from each other in hydrological terms. In its upper parts the river flows through rocky fields, while in the lower course it passes through a canyon, all due to differences in geology (permeable / impermeable layers) (Bačani 2006; Magdalenić 1971). The climate in this region is continental and subcontinental in the upper and middle reaches, respectively. The lower reach has a maritime influence, with some aspects of the Mediterranean climate. Mean annual air temperature in the upper reach region is between 5 and 8°C and between 11 and 13°C in the lower reach region of the river (Bonacci & Roje-Bonacci 2003).