DNA barcoding and first records of two rare Adicella species (Trichoptera: Leptoceridae) in Croatia

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 Ćukušić1,RenataĆuk2,AnaPrevišić 3,MartinaPodnar 4,AntunDelić 5 &MladenKučinić 3 * 1 Geonatura Ltd. Consultancy in Nature Protection, Fallerovo šetalište 22, 10000 Zagreb, Croatia 2 Hrvatske vode, Central Water Management Laboratory, Ulica grada Vukovara 220, 10000 Zagreb, Croatia 3 Department of Biology, Laboratory for Entomology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000 Zagreb, Croatia; e-mail: mladen.kucinic@biol.pmf.hr 4 Croatian Natural History Museum, Demetrova 1, 10 000 Zagreb, Croatia 5 Faculty 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 DNA barcoding method is used to identify species of different groups of organisms (animals, plants, fungi) and is based on sequencing of the standardized segment of the mitochondrial (mt) cytochrome c oxidase subunit 1 (COI) gene (Hebert et al. 2003). Advantages of using DNA barcodes in species identification compared to other parts of the genome are the lack of introns (unlike in nuclear genes), simple sequence alignment (compared to mitochondrial ribosomal genes, e.g., 12S, 16S), a great range of phylogenetic signal, and widely used robust primers able to successfully amplificate the barcode region in variety of taxa (Hebert et al. 2003). DNA barcoding of caddisflies has overall wide application in association of previously unknown larval stages with adults (e.g., Zhou et al. 2007; Zhou 2009; Graf et al. 2009; Ruiter et al. 2013; Gill et al. 2014). Similarly, in European caddisflies, the barcode region was used in studies mainly to support description of new species and to associate previously unknown larval stages (e.g., Gíslason et al. 2015; Graf et al. 2015; Waringer et al. 2015; Vitecek et al. 2015b) and to perform phylogenetic analyses (Kučinić et al 2013; Vitecek et al. 2015a, 2017). The Croatian caddisfly fauna was encompassed in several papers using the mtcoi gene in similar context (e.g., Previšić et al. 2009, 2014; Kučinić et al. 2010), however, not the barcode region (mtdna COI-5P). The barcode region was used only to support the description of the new species Chaetopteryx bucari Kučinić, Szivak & Delić, 2013 (Kučinić etal. 2013) and the new record of Tinodes antonioi Botosaneanu & Taticchi-Viganò, 1974 (Kučinić et al. 2016). This paper presents the first finding of the two caddisfly species from the genus Adicella: A. cremisa Malicky, 1972 and A. balcanica Botosaneanu & Novak, 1965 in Croatia. The genus Adicella MacLachlan, 1877 belongs to the tribe Triaenodini Morse, 1981 of the long-horned caddisfly family Leptoceridae. Family Leptoceridae has a cosmopolitan distribution and it is the second largest family of caddisflies in the world with a total of 1567 described species (Morse 2003; Holzenthal & Pes 2004). This family has two subfamilies, the subfamily Triplectidinae distributed in southern hemisphere and the more cosmopolitan subfamily Leptocerinae, containing 14 and 30 genera, respectively (Morse & Holzenthal 1987; Holzenthal & Pes 2004). The genus Adicella is distributed in the West and East Palaearctic biogeographic region, the Afrotropical * Corresponding author c 2017 Institute of Zoology, Slovak Academy of Sciences

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 1997). According to Graf et al. (2008, 2016) there are 14 species of the genus Adicella in Europe, two of which A. filicornis (Pictet, 1834) and A. reducta (McLachlan, 1865), are widely distributed, including Croatia (Kučini c et al. 2012; Robert 2015). Some ecological preferences are known for A. cremisa, unlike A. balcanica whose both larva and ecological preferences are unknown so far. To confirm the morphological identification of collected specimens, we utilised sequence data of the mtcoi gene (i.e., the barcode region, mtdna COI-5P). As one of the first examples of the use of the barcode methodology in Croatian caddisflies, we further highlight the importance of a simple and fast molecular method in identification of freshwater invertebrate taxa. Additionally, we give environmental data on their habitats, and discuss the ecology and the distribution of these invastegated species, both insufficiently investigated not only taxonomically (unknown morphology of larvae; Waringer & Graf 2011) but also ecologically (Graf et al. 2008). Finally, we present data of caddisflies species syntopically collected with the two Adicella species. Material and methods Research area The Krapina River is situated in the Pannonian ecoregion (ER11) (Illies 1978) (Pannonian-peripanonian part of Croatia according to Berti c et al. 2001) and is about 70 km long. It springs at Ivanščica Mt., flows through the Hrvatsko zagorje region and then into the Sava River near Zapreši c (Šafarek & Šoli c 2011). The study site at the Krapina River was in the village Krapina Selo (46 4.34 N, 16 12.00 E, 168 m a.s.l.) (Fig. 1A). According to the Croatian typology, this part of the Krapina River is classified as small lowland rivers with gravel and pebble substrate (Narodne novine 2013, 2014). The Zrmanja River is 69 km long and situated in the Dinaric ecoregion (ER5) (Illies 1978) (Mediterranean part of Croatia according to Berti c et al. 2001), it originates underneath Poštak peek and flows into the Adriatic Sea close to the town Obrovac. Its catchment area is mostly built of karstic carbonate rocks and it is a part of the Dinaric karst. Due to permeable carbonate rocks in some parts of the flow, the Zrmanja spring and some other parts have a temporary flow (Šafarek & Šoli c 2011). The study site at the Zrmanja River was at the settlement Palanka (44 8.81 N, 16 4.27 E, 264 m a.s.l.) (Fig. 1B). According to the Croatian typology, this part of the course of the Zrmanja River is classified as medium and large upland rivers (Narodne novine 2013, 2014). The Krupa River is the longest right tributary of the Zrmanja River and it is situated in the Dinaric ecoregion (ER5). The Krupa River is 11.5 km long with all characteristics of karst river (karst spring, river channel made of karstic carbonate rocks with subterranean channels through which groundwater flows in conduits) (Šafarek & Šoli c 2011). The study site at the Krupa River was at Krupa Manastir (Monastery) (44 11.37 N, 15 53.22 E, 105 m a.s.l.) (Fig. 1C). According to national typology the Krupa River is classified as short-flowing lowland rivers with a channel drop > 5 (Narodne novine 2013, 2014). Sampling and laboratory methods Samples of adult caddisflies were collected using entomological net and UV light traps. The samples were stored in 96% ethyl alcohol. Seven specimens of A. cremisa and one specimen of A. balcanica have been deposited in the NIP Trichoptera collection (collection formed as a part of

798 A. Ćukušić 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, summer and autumn period in 2014. For identification of collected specimens standard literature was used (Malicky 2004a). Systematic presentation follows Morse (2017). All physico-chemical parameters were analysed according to standard analytical methods for assessment of surface water quality (ISO norms), with Krapina River being sampled monthly while Zrmanja and Krupa rivers being sampled five (January, February, May, September and November) and six times (January, February, May, July, September and November), respectively, by staff of Hrvatske vode, Central Water Management Laboratory. Genomic DNA was extracted from legs of four specimens, two A. cremisa, one A. balcanica and one Rhyacophila vulgaris (Pictet, 1834), which are kept as a voucher in the Trichoptera DNA Barcode collection in Natural History Museum in Zagreb. Whole genomic DNA was extracted using GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, Germany) according to the manufacturer s specifications and eluted in 100 µl of elution buffer. Full-length COI-5P DNA barcodes were amplified using LCO1490/HCO2198 (Folmer et al. 1994) primer sets. The 50 µl polymerase chain reactions (PCR) mixture contained 1x Go Taq R Reaction Buffer (containing 1.5 mm MgCl2, Promega), 0.2 mm of each dntp, 0.4 µm of each primer, 1.25 units of Go Taq R DNA Polymerase (Promega) and 5 µl of DNA eluate. PCR cycling conditions comprised an initial denaturation step (94 C for 2 min) followed by 35 cycles of denaturation at 94 C for 30 s, annealing at 50 C for 30 s and elongation at 72 C for 90 s and a final extension step of 72 C for 7 min. Product purification and bidirectional sequencing was performed by Macrogen Inc. sequencing service (Seoul, South Korea) using the amplification primers. Sequences were edited manually and aligned using the program BioEdit (Hall 1999). DNA sequences were submitted to Barcode of Life Data Systems (BOLD, Ratnasingham & Hebert 2007) and GenBank. BOLD ID and accession number to GenBank are given in Table 1. Data set for phylogenetic analysis comprised the DNA barcodes amplified from A. cremisa adults from both locations and one adult of A. balcanica from the Krupa River as Fig. 2. A. cremisa, adult male. well as all available sequences from the BOLD database (Table 1). The sequence of R. vulgaris was included as outgroup. First we compared our DNA sequences with the ones available in the BOLD database using BOLD Identification Engine (accessed March 2016), second, the Neighbor-Joining (NJ) tree based on the Kimura-2-Parameter (K2P) distance model are built using MEGA 6.0. (Tamura et al. 2013) phylogenetic software. The robustness of branching was assessed by bootstrapping analysis (2000 replicates). Finally, we calculated the uncorrected pairwise distances between specimens (p-distances) based on the mtcoi barcode sequences using MEGA 6.0. (Tamura et al. 2013). Results Adult of A. cremisa (Fig. 2) were collected at two sites: the Krapina River at Krapina Selo on August 20 th 2014 (7 specimens: 5 males, 2 females) and the Zrmanja River at Palanka on August 11 th 2014 (2 specimens: 2 males). Figure 3 presents the male genitalia (Figs 3A,

DNA barcoding and first records of two Adicella species in Croatia 799 Fig. 4. A. balcanica: A, B female genitalia. Fig. 3. A. cremisa: A, B male genitalia; C, D female genitalia. B) and female genitalia (Figs 3C, D) of A. cremisa. Adult of A. balcanica were collected at the Krupa River near Krupa Manastir in 11 th August 2014 (2 females). Figure 4 presents the female genitalia of A. balcanica. Even though benthic macroinvertebrates are being sampled at all studied locations within the national monitoring of water quality, there are no larval records of A. balcanica and A. cremisa. According to BOLD Identification Tree, A. cremisa specimens from Croatia are highly similar to A. cremisa from Italy, from the Friuli-Venezia Giulia region in Italy (top similarity matches with A. cremisa in BOLD database is 100%). The NJ tree additionally supports all morphological identifications, i.e. A. cremisa specimens from both sites cluster within highly supported monophyletic A. cremisa clade. The sister taxa relationship of A. balcanica and A. cremisa is also highly supported (Fig. 5, electronic supplementary file Fig. 1S). The maximum value of the uncorrected pairwise distance (p-distance) between Adicella species is around 21% (Table 2). The p-distance between A. cremisa and A. balcanica is around 14% (Table 2).

800 A. Ćukušić et al. Fig. 5. Neighbour-Joining tree of the mtdna sequences of Adicella species from Croatia and BOLD database based on the Kimura- 2-Parameter (K2P) distance model. Table 2. Inter- and intraspecific genetic p-distances of the mitochondrial cytochrome oxidase I (mtcoi) gene fragments recorded for Adicella species from Croatia and BOLD database. A.cremisa A.cremisa A.balcanica A.reducta A. filicornis A. syriaca Zrmanja Krapina Krupa Portugal France Hungary A. cremisa Zrmanja A. cremisa Krapina 0.0032 A. balcanica Krupa 0.1399 0.1459 A. reducta Portugal 0.1512 0.1541 0.2024 A. filiconis France 0.1400 0.1420 0.2068 0.1065 A. syriaca Hungary 0.1295 0.1275 0.1948 0.1559 0.1477 Table 3. Range and average values of physico-chemical water parameters at sites where A. cremisa and A. balcanica were collected in Croatia. Krapina Krapina Selo Zrmanja Palanka Krupa Manastir No. of samplings 12 5 6 Min Mean Max Min Mean Max Min Mean Max Water temperature ( C) 5.6 11.6 18 9.8 10.6 13.3 9.9 11 13.5 ph 8 8.1 8.2 8.1 8.2 8.3 8 8.1 8.2 Conductivity (µs cm 1 ) 579 626 711 335 354 370 350 382 420 Alkalinity (mgcaco3 L 1 ) 304 313.8 334 180 185.2 191 181 202 223 Dissolved oxygen (mgo2 L 1 ) 7.5 9.5 11.3 9.1 10.9 11.4 10.7 11.2 11.4 Oxygen saturation (%) 77.2 86.2 103.1 80.2 97.8 106.2 97.1 101.7 108.7 BOD5 (mgo2 L 1 ) 0.7 1.7 4 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 COD-Mn (mgo2 L 1 ) 2.1 3.6 7 < 0.6 < 0.6 0.6 < 0.6 < 0.6 0.7 Ammonia (mgn L 1 ) < 0.01 0.0854 0.19 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.011 Nitrites (mgn L 1 ) 0.004 0.0202 0.045 < 0.0015 0.0018 0.0025 < 0.0015 < 0.0015 < 0.0015 Nitrates (mgn L 1 ) 0.56 0.8583 1.23 0.294 0.3392 0.378 0.172 0.2097 0.239 Total nitrogen (mgn L 1 ) 0.9 1.1317 1.52 0.2956 0.3507 0.415 0.172 0.2378 0.341 Ortophosphates (mgp L 1 ) 0.008 0.032 0.065 < 0.006 < 0.006 0.007 < 0.006 0.009 0.015 Total phosphorus (mgp L 1 ) 0.051 0.0882 0.133 < 0.015 < 0.015 < 0.015 < 0.015 0.0205 0.043 The sites where A. cremisa was recorded belong to the upper courses of the investigated rivers, the Krapina and the Zrmanja rivers, while the record of A. balcanica belong to the middle to lower course of the Krupa River.

DNA barcoding and first records of two Adicella species in Croatia 801 Table 4. Sympatric caddisfly species with two Adicella species presented in the current study. Species name Abundance data Sampling method Sympatric species with A. cremisa at the Krapina River by Krapina Selo Sympatric species with A. cremisa at Palanka site of the Zrmanja River Sympatric species with A. balcanica at Krupa Monastery at the Krupa River Ithytrichia lamellaris Eaton, 1873 22 adult male 14 adult female UV light trap Mystacides nigra (L., 1758) 1 adult male entomological net Potamophylax rotundipennis (Brauer, 1857) 2 adult female UV light trap Cyrnus trimaculatus (Curtis, 1834) 1 adult male entomological net Lype reducta (Hagen, 1868) 14 adult male entomological net Hydropsyche instabilis (Curtis, 1834) 1 adult male UV light trap Psychomia klapaleki Malicky, 1995 5 adult male 1 adult female entomological net Rhyacophila fasciata Hagen, 1859 1 adult male UV light trap Athripsodes bilineatus (L., 1758) 1 adult male entomological net Wormaldia subnigra McLachlan, 1865 1 adult male entomological net Polycentropus irroratus Curtis, 1835 1 adult male UV light trap Lype reducta (Hagen, 1868) 5 adult male 2 adult female UV light trap Relatively high ph values (> 8) were recorded at all sampling sites and other physico-chemical parameters show relatively high variability (Table 3). Based on the measured physico-chemical parameters all tree sampling sites were classified into high or good water quality status (Table 3). In Table 4 we showed sympatric species of caddisflies collected at the locations on which we found A. balcanica or A. cremisa. Discussion So far three species of the genus Adicella have been recorded in Croatia: A. filicornis Pictet, 1834, A. reducta (McLachlan, 1865) and A. syriaca Ulmer, 1907 (Kučinić et al. 2012). Acidella cremisa and A. balcanica represent new records for the Croatian caddisfly fauna (e.g., Ćuk & Vučković 2010, 2014; Ćuk et al. 2015; Previšić et al. 2009, 2013a, b, 2014; Previšić &Popijač 2010; Kučinić et al. 2011, 2012, 2014, 2015, 2016). Genetic identification and interspecific distance of Adicella species No study examined the interspecific p-distances of COI sequences within the genus Adicella, but maximum value of the p-distances similar to ours were recorded in the genus Smicridea, Hydropsychidae, also for a barcode region (20%, Pauls et al. 2010). The p-distance between A. cremisa and A. balcanica are higher than minimum values of the interspecific variability usually observed in caddisflies for the barcode region (8% Pauls et al. 2010; 5.3% Zhou 2009; 8.2% Graf et al. 2015). Thus, results of the DNA barcode method presented here support the morphological identification of a caddisfly species new for the Croatian fauna. The ability of the DNA barcode to delimit closely related caddisfly species is shown in many studies where a single locus was used (mtdna COI-5P, e.g., Pauls et al. 2010; Jackson et al. 2014). Intraspecific distance of A. cremisa specimens indicates gene flow between South European populations High similarity of A. cremisa specimens from Croatia with A. cremisa population in Italy (as it shown in BOLD illustrated Identification Tree) implies the existence of the contemporary or recent gene flow between disjunctly distributed populations in the wider region, as recorded in other taxa, e.g., the stonefly Dinocras cephalotes (Curtis, 1827) (Elbrecht et al. 2014) and other caddisflies, e.g., Lectrides varians Mosely, 1953 (Wickson et al. 2014), Plectrocnemia conspersa (Curtis, 1834) (Wilcock et al. 2005). Further evidence for such pattern is provided when genetic distance of A. cremisa specimens from two populations in Croatia are compared, i.e., the p distance between specimens from Zrmanja and Krapina rivers is only 0.3 % (Table 2). Similarly as in A. cremisa, low intraspecific variability in the DNA barcode region was observed in the mayfly Rhithrogena braaschi Jacob, 1974 within the Western Dinaric Balkan ecoregion (Vilenica et al. 2016). On the contrary, high intraspecific variability (p-distances up to 6.3%) was recorded in Drusus endemics (Drusinae, Limnephilidae) in the Western Balkans (e.g., Previšić et al. 2009; Pauls et al. 2006). Although some of these studies use a different fragment of the mtcoi gene (mtcoi-3p and mtcoi-5p), Graf et al. (2015) showed that differentiation level of these two mtcoi regions is very similar in Limnephilidae. The ecology of particular species (e.g., dispersal behaviour, habitat preferences etc.) is often reflected in the existence/absence of the contemporary gene flow. For instance, in contrast to lowland Adicella species (Graf et al. 2008, 2016), Drusus species inhabit montane springs and streams andthusshowhighdegreeofendemismandgenetic isolation (e.g., Previšić et al. 2009; Pauls et al. 2006, 2009; Ibrahimi et al. 2015, 2016; Vitecek et al. 2015a, b; Waringer et al. 2015). Distribution of A. balcanica and A. cremisa The distribution of A. balcanica and A. cremisa, is insufficiently investigated, and data are still scarce. Adicella balcanica was described from specimens collected in Bosnia and Herzegovina (Botosaneanu & Novak 1965) and A. cremisa is described from its type locality in Austria (Malicky 1972). According to Graf et al. (2008, 2016) A. cremisa has been recorded in ecore-

802 A. Ćukušić et al. Fig. 6. Distribution of A. cremisa (dark green field) and A. balcanica (light green field) compiled from BOLD data, www.freshwaterecology.info (Robert 2015) and Fauna Europaea (Malicky 2013) available at web portal Fauna Europaea with new findings in Croatia (red points presents A. cremisa and blue point A. balcanica). gions ER3 (Italy, Corsica and Malta), ER5 (Dinaric Western Balkan) and ER9 (Central Highlands, sensu Illies 1978). Adicela balcanica has a wider distribution in the Balkans, in the ER5, ER4 (Alps), ER6 (Hellenic Western Balkan) and ER11 (Hungarian lowlands) (Graf et al. 2008, 2016). However, according to Robert (2015), both species have more localised distribution only in some parts of the mentioned ecoregions. The sites at the Zrmanja River and Krupa River are situated in the ER5 while the site at the Krapina River is situated on the border between two ecoregions, ER5 and ER11. In Fig. 6 we present distribution of A. cremisa and A. balcanica in Europe according to Fauna Europaea (Malicky 2013) BOLD data, freshwaterecology.info (Graf et al. 2016) with recordsofa. cremisa in Italy (Cianficconi 2002; Cianficconi & Moretti 1987), Austria (Graf 2002) and Slovenia (Krušnik & Urbanič 2002) and records of A. balcanica in Bosnia and Herzegovina (Botosaneanu and Novak 1965), Serbia (Živić et al. 2002), Hungary (Nógardi and Uherkovich 2002), Italy (Malicky 2004b), Greece and Bulgaria (Kumanski 2004; Malicky 2005). The findings of these two species in Croatia thus represent an important contribution to the knowledge on their distribution ranges. A systematic inventory of caddisflies in Croatia was conducted on more than 220 sites in different geographical parts of Croatia in the last 20 years in different periods (e.g., Cerjanec 2012; Kučinić 2002; Kučinić et al. 2011; Vučković 2011). In these investigations including this study, A. cremisa was recorded at only two sites and A. balcanica at only one. All these data indicate that these two species are very rare in the fauna of Croatia and show a disjunct distribution (Fig. 6). Relatively high ph value (> 8) recorded at all sampling sites could be one of the preferences determining the distribution of these two species. Ecology of A. balcanica and A. cremisa According to Graf et al. (2008, 2016) Adicella species have a wide ecological valence what contributes to wide distribution of A. reducta and A. filicornis in Europe. The physico-chemical parameters at sites included in the current study also show seasonal variability (e.g., water temperature at the Krapina River varied more than in the Zrmanja and Krupa rivers; lowest water temperature was recorded at the Krapina River, but also highest values of conductivity and alkalinity (Table 3). These data suggest that A. balcanica and A. cremisa also have wide ecological valence. No records of larvae at any investigated site could be related to potentially small populations and specific microhabitat preferences (both are organic habitat specialists, Graf et al. 2008, 2016), therefore, it can be hard to record them even at sites where they normally occur. Consequently, we can assume that both species have wider distribution than currently known according to the available records (Fig. 6).

DNA barcoding and first records of two Adicella species in Croatia 803 Graf et al. (2008, 2016) indicate that A. cremisa occurs mainly in plains (< 300 m), rarely at higher altitudes (up to 1000 m). In Croatia, we found it in accordance with its main distribution preference, i.e. at low altitude (around 200 m a.s.l.). Adicella balcanica occurs at higher altitudes (300 1900 m a.s.l., Graf et al. 2008, 2016) and it is mostly a eucrenal species, also occurring in hypocrenal and epirhithral region (Graf et al. 2008, 2016). We found A. balcanica at 105 m a.s.l. in the Krupa canyon in the middle to lower river sections. It is typical for the Mediterranean kasrt rivers in the Dinaric region which do not follow the River Continuum Concept presented by Vannote et al. (1980) (e.g., Pinna et al. 2003; Vučković 2011). On the other hand, there are several underground springs along the Krupa River bed (Šafarek & Šolić 2011) changing the conditions more favourable for crenal species. Furthermore, records of adults only do not provide certain evidence of stream zonation preference since larvae were not collected within this study. For A. cremisa stream zonation preference is unknown. However, current findings suggest that it possibly occurs in the epirhithral and metarhithral zone, similar to other Adicella species presented in Graf et al. (2008). This conclusion is based on results of the current study. Within the study we sampled at the Zrmanja River at eucrenal, hypocrenal and epipotamal part of the river, but there were no Adicella species. According our physico-chemical water parameters, we can assume that A. balcanica and A. cremisa prefer alkaline environment. Regarding preference to ph value of two widely distributed Adicella species, A. filicornis prefers neutral to alkaline environment, while A. reducta is indifferent (Graf et al. 2008, 2016). The larva of A. cremisa was roughly described by Cianficconi & Moretti (1987). However, the head colouration pattern and case design are very close to A. reducta (Waringer & Graf 2011). Therefore, redescription of the larva should probably be taken into consideration. Larvae of A. cremisa are usually found in slow flowing streams and lentic zones, preferably occurring in macrophytes, mosses and woody debris (Graf et al. 2008). The larva of A. balcanica is unknown. Moreover, larvae of all Adicella species, including A. cremisa and A. balcanica, are organic habitat specialists (Graf et al. 2008, 2016). Even though larvae were not collected within the current study, habitats preferred by these species are present at sampling sites where adults were collected, e.g., woody debris, macrophytes and mosses (Fig. 1). Most of Adicella species have short emergence/ flight period during summer (Graf et al. 2008, 2016) or spring and summer (Šemnički et al. 2011). Emergence/flight periods recorded for both species in the current study were therefore mainly in line with typical periods of Adicella species. Both species were recorded only in the summer period even though the sites were visited three times throughout 2014. Emergence/flight period for A. balcanica is, therefore, shorter for one period (spring) than presented by Graf et al. (2008, 2016). However, further studies need to be conducted due to the small number of collected specimens within this study. Conclusions In the current study we present the first record of two Adicella species in Croatia and contributed to the knowledge of the Croatian caddisfly fauna. We also accentuated the importance of the Western Balkan region as a hot-spot of freshwater diversity, as is also pointed out by numerous previous studies (Kučinić et al. 2014; Previšić et al. 2014; Ibrahimi et al. 2015; Vitecek et al. 2015a, 2017). We used DNA barcode method to confirm morphological identification. This enabled a further insight into the genetic connectivity of A. cremisa populations in Croatia and the region. Furthermore, we presented valuable information on distribution and ecology of insufficiently studied Adicella species. Acknowledgements We are very grateful to employees of the Central Water Management Laboratory of Hrvatske vode for providing the results of physico-chemical parameters and Marwan Zeitoun (Zagreb) who helped us in the field collections. This study was supported by the EU Natura 2000 Integration Project (NIP) funded by the Croatian Ministry of Environmental and Nature Protection, by Croatian Science Foundation under the project (IP-2016-06-9988, DNA Barcoding of Diversity of Croatian Fauna, by M. Kučinić) and University of Zagreb funding. For technical help thank you to prof. Halil Ibrahimi from University of Pristina. We are also very grateful to two anonymous reviewers. References Bertić I., Lampek Pavčnik I. & Radovinović R. 2001. 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