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: ) in

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 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 ; 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 (, 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

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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 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 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

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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. Genomic DNA was extracted from legs of four spec- imens, two A. cremisa,oneA. balcanica and one Rhya- cophila vulgaris (Pictet, 1834), which are kept as a voucher in the Trichoptera DNA Barcode collection in Natural His- tory Museum in Zagreb. Whole genomic DNA was extracted Fig. 2. A. cremisa, adult male. 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 well as all available sequences from the BOLD database (Ta- LCO1490/HCO2198 (Folmer et al. 1994) primer sets. The ble 1). The sequence of R. vulgaris was included as outgroup. 50 µl polymerase chain reactions (PCR) mixture contained First we compared our DNA sequences with the ones avail-  1x Go Taq Reaction Buffer (containing 1.5 mM MgCl2, able in the BOLD database using BOLD Identification En- Promega), 0.2 mM of each dNTP, 0.4 µM of each primer, gine (accessed March 2016), second, the Neighbor-Joining  1.25 units of Go Taq DNA Polymerase (Promega) and 5 µl (NJ) tree based on the Kimura-2-Parameter (K2P) distance of DNA eluate. PCR cycling conditions comprised an initial model are built using MEGA 6.0. (Tamura et al. 2013) phy- ◦ denaturation step (94 C for 2 min) followed by 35 cycles of logenetic software. The robustness of branching was assessed ◦ ◦ denaturation at 94 C for 30 s, annealing at 50 C for 30 s by bootstrapping analysis (2000 replicates). Finally, we cal- ◦ and elongation at 72 C for 90 s and a final extension step of culated the uncorrected pairwise distances between speci- ◦ 72 C for 7 min. Product purification and bidirectional se- mens (p-distances) based on the mtCOI barcode sequences quencing was performed by Macrogen Inc. sequencing ser- using MEGA 6.0. (Tamura et al. 2013). vice (Seoul, South Korea) using the amplification primers. Sequences were edited manually and aligned using the pro- Results gram BioEdit (Hall 1999). DNA sequences were submitted to Barcode of Life Data Systems (BOLD, Ratnasingham & Adult of A. cremisa (Fig. 2) were collected at two sites: Hebert 2007) and GenBank. BOLD ID and accession num- th ber to GenBank are given in Table 1. the Krapina River at Krapina Selo on August 20 2014 Data set for phylogenetic analysis comprised the DNA (7 specimens: 5 males, 2 females) and the Zrmanja th barcodes amplified from A. cremisa adults from both loca- River at Palanka on August 11 2014 (2 specimens: tions and one adult of A. balcanica from the Krupa River as 2 males). Figure 3 presents the male genitalia (Figs 3A,

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Fig. 4. A. balcanica: A, B – 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 11th August 2014 (2 females). Figure 4 presents the female genitalia of A. balcanica. Even though benthic macroinvertebrates are being sam- pled at all studied locations within the national mon- itoring 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 sup- ports all morphological identifications, i.e. A. cremisa specimens from both sites cluster within highly sup- ported 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 Fig. 3. A. cremisa: A, B – male genitalia; C, D – female genitalia. A. cremisa and A. balcanica is around 14% (Table 2).

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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 (µScm−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)0.71.74< 1.5 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 COD-Mn (mgO2 L−1)2.13.67< 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 and the Zrmanja rivers, while the record of A. balcanica the upper courses of the investigated rivers, the Krapina belong to the middle to lower course of the Krupa River.

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Table 4. Sympatric caddisfly species with two Adicella species presented in the current study.

Species name Abundance data Sampling method

Sympatric species with Ithytrichia lamellaris Eaton, 1873 22 adult male 14 adult female UV light trap A. cremisa at the Krapina nigra (L., 1758) 1 adult male entomological net River by Krapina Selo 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

Sympatric species with Hydropsyche instabilis (Curtis, 1834) 1 adult male UV light trap A. cremisa at Palanka site Psychomia klapaleki Malicky, 1995 5 adult male 1 adult female entomological net of the Zrmanja River Rhyacophila fasciata Hagen, 1859 1 adult male UV light trap

Sympatric species with bilineatus (L., 1758) 1 adult male entomological net A. balcanica at Krupa Wormaldia subnigra McLachlan, 1865 1 adult male entomological net Monastery at the Krupa Polycentropus irroratus Curtis, 1835 1 adult male UV light trap River Lype reducta (Hagen, 1868) 5 adult male 2 adult female UV light trap

Relatively high pH values (> 8) were recorded at with A. cremisa population in Italy (as it shown in all sampling sites and other physico-chemical param- BOLD illustrated Identification Tree) implies the exis- eters show relatively high variability (Table 3). Based tence of the contemporary or recent gene flow between on the measured physico-chemical parameters all tree disjunctly distributed populations in the wider region, sampling sites were classified into high or good water as recorded in other taxa, e.g., the stonefly Dinocras quality status (Table 3). cephalotes (Curtis, 1827) (Elbrecht et al. 2014) and In Table 4 we showed sympatric species of cad- other caddisflies, e.g., Lectrides varians Mosely, 1953 disflies collected at the locations on which we found (Wickson et al. 2014), Plectrocnemia conspersa (Curtis, A. balcanica or A. cremisa. 1834) (Wilcock et al. 2005). Further evidence for such pattern is provided when genetic distance of A. cremisa Discussion specimens from two populations in Croatia are com- pared, i.e., the p−distance between specimens from Zr- So far three species of the genus Adicella have been manja and Krapina rivers is only 0.3 % (Table 2). Sim- recorded in Croatia: A. filicornis Pictet, 1834, A. re- ilarly as in A. cremisa, low intraspecific variability in ducta (McLachlan, 1865) and A. syriaca Ulmer, 1907 the DNA barcode region was observed in the mayfly (Kučini´c et al. 2012). Acidella cremisa and A. balcanica Rhithrogena braaschi Jacob, 1974 within the Western represent new records for the Croatian caddisfly fauna Dinaric Balkan ecoregion (Vilenica et al. 2016). On the (e.g., Cuk´ & Vučkovi´c 2010, 2014; Cuk´ et al. 2015; Pre- contrary, high intraspecific variability (p-distances up viši´c et al. 2009, 2013a, b, 2014; Previši´c&Popijač to 6.3%) was recorded in Drusus endemics (Drusinae, 2010; Kučini´c et al. 2011, 2012, 2014, 2015, 2016). Limnephilidae) in the Western Balkans (e.g., Previši´c et al. 2009; Pauls et al. 2006). Although some of these Genetic identification and interspecific distance of Adi- studies use a different fragment of the mtCOI gene cella species (mtCOI-3P and mtCOI-5P), Graf et al. (2015) showed No study examined the interspecific p-distances of COI that differentiation level of these two mtCOI regions is sequences within the genus Adicella, but maximum very similar in Limnephilidae. The ecology of partic- value of the p-distances similar to ours were recorded ular species (e.g., dispersal behaviour, habitat prefer- in the genus Smicridea, Hydropsychidae, also for a bar- ences etc.) is often reflected in the existence/absence of code region (20%, Pauls et al. 2010). The p-distance be- the contemporary gene flow. For instance, in contrast tween A. cremisa and A. balcanica are higher than min- to lowland Adicella species (Graf et al. 2008, 2016), imum values of the interspecific variability usually ob- Drusus species inhabit montane springs and streams served in caddisflies for the barcode region (8% Pauls andthusshowhighdegreeofendemismandgenetic et al. 2010; 5.3% Zhou 2009; 8.2% Graf et al. 2015). isolation (e.g., Previši´c et al. 2009; Pauls et al. 2006, Thus, results of the DNA barcode method presented 2009; Ibrahimi et al. 2015, 2016; Vitecek et al. 2015a, here support the morphological identification of a cad- b; Waringer et al. 2015). disfly species new for the Croatian fauna. The ability of the DNA barcode to delimit closely related caddisfly Distribution of A. balcanica and A. cremisa species is shown in many studies where a single locus The distribution of A. balcanica and A. cremisa,is was used (mtDNA COI-5P, e.g., Pauls et al. 2010; Jack- insufficiently investigated, and data are still scarce. son et al. 2014). Adicella balcanica was described from specimens col- lected in Bosnia and Herzegovina (Botosaneanu & No- Intraspecific distance of A. cremisa specimens indicates vak 1965) and A. cremisa is described from its type lo- gene flow between South European populations cality in Austria (Malicky 1972). According to Graf et High similarity of A. cremisa specimens from Croatia al. (2008, 2016) A. cremisa has been recorded in ecore-

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

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Graf et al. (2008, 2016) indicate that A. cremisa 2016). However, further studies need to be conducted occurs mainly in plains (< 300 m), rarely at higher due to the small number of collected specimens within altitudes (up to 1000 m). In Croatia, we found it in ac- this study. cordance with its main distribution preference, i.e. at low altitude (around 200 m a.s.l.). Adicella balcanica Conclusions occurs at higher altitudes (300–1900 m a.s.l., Graf et al. 2008, 2016) and it is mostly a eucrenal species, also In the current study we present the first record of occurring in hypocrenal and epirhithral region (Graf et two Adicella species in Croatia and contributed to the al. 2008, 2016). We found A. balcanica at 105 m a.s.l. in knowledge of the Croatian caddisfly fauna. We also ac- the Krupa canyon in the middle to lower river sections. centuated the importance of the Western Balkan region It is typical for the Mediterranean kasrt rivers in the as a hot-spot of freshwater diversity, as is also pointed Dinaric region which do not follow the River Contin- out by numerous previous studies (Kučini´c et al. 2014; uum Concept presented by Vannote et al. (1980) (e.g., Previši´c et al. 2014; Ibrahimi et al. 2015; Vitecek et al. Pinna et al. 2003; Vučkovi´c 2011). On the other hand, 2015a, 2017). We used DNA barcode method to confirm there are several underground springs along the Krupa morphological identification. This enabled a further in- River bed (Šafarek & Šoli´c 2011) changing the condi- sight into the genetic connectivity of A. cremisa popu- tions more favourable for crenal species. Furthermore, lations in Croatia and the region. Furthermore, we pre- records of adults only do not provide certain evidence sented valuable information on distribution and ecology of stream zonation preference since larvae were not col- of insufficiently studied Adicella species. lected within this study. For A. cremisa stream zonation preference is un- Acknowledgements known. However, current findings suggest that it pos- sibly occurs in the epirhithral and metarhithral zone, We are very grateful to employees of the Central Water similar to other Adicella species presented in Graf et Management Laboratory of Hrvatske vode for providing the al. (2008). This conclusion is based on results of the results of physico-chemical parameters and Marwan Zeitoun current study. Within the study we sampled at the Zr- (Zagreb) who helped us in the field collections. This study manja River at eucrenal, hypocrenal and epipotamal was supported by the “EU Natura 2000 Integration Project (NIP)” funded by the Croatian Ministry of Environmen- part of the river, but there were no Adicella species. tal and Nature Protection, by Croatian Science Foundation According our physico-chemical water parameters, under the project (IP-2016-06-9988, DNA Barcoding of Di- we can assume that A. balcanica and A. cremisa pre- versity of Croatian Fauna, by M. Kučini´c) and University fer alkaline environment. Regarding preference to pH of Zagreb funding. For technical help thank you to prof. value of two widely distributed Adicella species, A. fi- Halil Ibrahimi from University of Pristina. We are also very licornis prefers neutral to alkaline environment, while grateful to two anonymous reviewers. A. reducta is indifferent (Graf et al. 2008, 2016). The larva of A. cremisa was roughly described References by Cianficconi & Moretti (1987). 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