Biologia 72/7: 796—806, 2017Section ZoologyDOI: 10.1515/biolog-2017-0087

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

Anđela Cukušic1, Renata Cuk2, Ana Previšic3, Martina Podnar4, Antun Delic5

& Mladen Kučinic3*1Geonatura Ltd. Consultancy in Nature Protection, Fallerovo šetalište 22, 10000 Zagreb, Croatia2Hrvatske vode, Central Water Management Laboratory, Ulica grada Vukovara 220, 10000 Zagreb, Croatia3Department of Biology, Laboratory for Entomology, Faculty of Science, University of Zagreb, Rooseveltov trg 6, 10000Zagreb, Croatia; e-mail: mladen.kucinic@biol.pmf.hr4Croatian Natural History Museum, Demetrova 1, 10 000 Zagreb, Croatia5Faculty 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, wererecorded in the summer period of 2014 which represent the first records of these species in Croatia. The former was collectedat two relatively distant sites, the Krapina and the Zrmanja rivers, while the later was collected at the Krupa River. Bothspecies were identified by morphological characteristics as well as DNA barcoding method. We calculated the uncorrectedpairwise distances within Adicella and used molecular phylogenetic approach to delimit species. A. cremisa from Croatiashowed no significant difference in the mtCOI region and they are highly similar to A. cremisa from Italy. Additionally, theecological preferences and distribution of Adicella species are presented. Our findings represent a significant contribution tothe aquatic biodiversity of the Western Balkans.

Key words: molecular identification; aquatic insects; caddisfly

Introduction

DNA barcoding method is used to identify species ofdifferent groups of organisms (animals, plants, fungi)and is based on sequencing of the standardized segmentof the mitochondrial (mt) cytochrome c oxidase subunit1 (COI) gene (Hebert et al. 2003). Advantages of us-ing DNA barcodes in species identification comparedto 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 widelyused robust primers able to successfully amplificate thebarcode region in variety of taxa (Hebert et al. 2003).DNA barcoding of caddisflies has overall wide ap-

plication in association of previously unknown larvalstages 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 regionwas used in studies mainly to support description ofnew species and to associate previously unknown lar-val stages (e.g., Gíslason et al. 2015; Graf et al. 2015;Waringer et al. 2015; Vitecek et al. 2015b) and to per-form phylogenetic analyses (Kučinic et al 2013; Viteceket al. 2015a, 2017).

The Croatian caddisfly fauna was encompassed inseveral papers using the mtCOI gene in similar con-text (e.g., Previšic et al. 2009, 2014; Kučinic et al.2010), however, not the “barcode region” (mtDNACOI-5P). The barcode region was used only to sup-port the description of the new species Chaetopteryxbucari Kučinic, Szivak & Delic, 2013 (Kučinic et al.2013) and the new record of Tinodes antonioi Boto-saneanu & Taticchi-Vigano, 1974 (Kučinic et al. 2016).This paper presents the first finding of the two

caddisfly species from the genus Adicella: A. cremisaMalicky, 1972 and A. balcanica Botosaneanu & No-vak, 1965 in Croatia. The genus Adicella MacLachlan,1877 belongs to the tribe Triaenodini Morse, 1981 ofthe long-horned caddisfly family Leptoceridae. FamilyLeptoceridae has a cosmopolitan distribution and it isthe second largest family of caddisflies in the world witha total of 1567 described species (Morse 2003; Holzen-thal & Pes 2004). This family has two subfamilies, thesubfamily Triplectidinae distributed in southern hemi-sphere and the more cosmopolitan subfamily Leptoceri-nae, 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

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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 & Andersen1997). According to Graf et al. (2008, 2016) there are 14species of the genus Adicella in Europe, two of whichA. filicornis (Pictet, 1834) and A. reducta (McLach-lan, 1865), are widely distributed, including Croatia(Kučinic et al. 2012; Robert 2015). Some ecologicalpreferences are known for A. cremisa, unlike A. bal-canica whose both larva and ecological preferences areunknown so far. To confirm the morphological identifi-cation of collected specimens, we utilised sequence dataof the mtCOI gene (i.e., the barcode region, mtDNACOI-5P). As one of the first examples of the use of thebarcode methodology in Croatian caddisflies, we furtherhighlight the importance of a simple and fast molecu-lar method in identification of freshwater invertebratetaxa. Additionally, we give environmental data on theirhabitats, and discuss the ecology and the distributionof these invastegated species, both insufficiently inves-tigated not only taxonomically (unknown morphologyof larvae; Waringer & Graf 2011) but also ecologically(Graf et al. 2008). Finally, we present data of caddis-flies species syntopically collected with the two Adicellaspecies.

Material and methods

Research areaThe Krapina River is situated in the Pannonian ecoregion(ER11) (Illies 1978) (Pannonian-peripanonian part of Croa-tia according to Bertic et al. 2001) and is about 70 kmlong. It springs at Ivanščica Mt., flows through the Hrvatskozagorje region and then into the Sava River near Zaprešic(Šafarek & Šolic 2011). The study site at the Krapina Riverwas in the village Krapina Selo (46◦4.34′ N, 16◦12.00′ E,

168 m a.s.l.) (Fig. 1A). According to the Croatian typol-ogy, this part of the Krapina River is classified as “smalllowland rivers with gravel and pebble substrate” (Narodnenovine 2013, 2014).

The Zrmanja River is 69 km long and situated in theDinaric ecoregion (ER5) (Illies 1978) (Mediterranean partof Croatia according to Bertic et al. 2001), it originates un-derneath Poštak peek and flows into the Adriatic Sea closeto the town Obrovac. Its catchment area is mostly built ofkarstic 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 temporaryflow (Šafarek & Šolic 2011). The study site at the ZrmanjaRiver 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 theZrmanja River and it is situated in the Dinaric ecoregion(ER5). The Krupa River is 11.5 km long with all characteris-tics of karst river (karst spring, river channel made of karsticcarbonate rocks with subterranean channels through whichgroundwater flows in conduits) (Šafarek & Šolic 2011).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 Riveris classified as “short-flowing lowland rivers with a channeldrop > 5‰” (Narodne novine 2013, 2014).

Sampling and laboratory methodsSamples of adult caddisflies were collected using entomo-logical net and UV light traps. The samples were storedin 96% ethyl alcohol. Seven specimens of A. cremisa andone specimen of A. balcanica have been deposited in theNIP Trichoptera collection (collection formed as a part of

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798 A. Cukušic et al.

Table 1. Details of the specimens used in analysis with assigned species name, geographic origin, sample ID, BOLD Sequence IDnumber 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 KX5554702 A. cremisa Croatia TACRE 1 NIP009-16 BankIt1936914 TACRE 1 KX5554713 A. cremisa Croatia TASYR 1 NIP008-16 BankIt1936914 TASYR 1 KX5554724 A. cremisa KKCAD-0425 KKCAD417-075 A. cremisa Italy 08HMCAD-149 HMTRI149-086 A. cremisa Italy 08HMCAD-176 HMTRI176-087 A. cremisa Italy 08HMCAD-153 HMTRI153-088 A. cremisa Italy 08HMCAD-152 HMTRI152-089 A. cremisa Italy 08HMCAD-151 HMTRI151-0810 A. cremisa Italy 08HMCAD-150 HMTRI150-0811 A. filicornis Austria 12HMCAD-059 BHMKK222-1212 A. filicornis France 10OFSI-0187 OFTRI186-1013 A. filicornis Germany BCZSMAQU00856 FBAQU1141-1214 A. reducta Germany BCZSMAQU00857 FBAQU1142-1215 A. reducta Portugal HMCAD0810-3 HMKKT630-1016 A. reducta Spain 09MNKK0411 KKUMN419-1017 A. reducta Belgium UA-SG-TRICH-C18 TFLAN061-1118 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 (Malicky2004a). Systematic presentation follows Morse (2017).

All physico-chemical parameters were analysed accord-ing to standard analytical methods for assessment of surfacewater 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, one A. balcanica and one Rhya-cophila vulgaris (Pictet, 1834), which are kept as a voucherin the Trichoptera DNA Barcode collection in Natural His-tory Museum in Zagreb. Whole genomic DNA was extractedusing GenElute Mammalian Genomic DNA Miniprep kit(Sigma-Aldrich, Germany) according to the manufacturer’sspecifications and eluted in 100 µl of elution buffer.Full-length COI-5P DNA barcodes were amplified usingLCO1490/HCO2198 (Folmer et al. 1994) primer sets. The50 µl polymerase chain reactions (PCR) mixture contained1x Go Taq� 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� DNA Polymerase (Promega) and 5 µlof DNA eluate. PCR cycling conditions comprised an initialdenaturation step (94◦C for 2 min) followed by 35 cycles ofdenaturation at 94◦C for 30 s, annealing at 50◦C for 30 sand elongation at 72◦C for 90 s and a final extension step of72◦C for 7 min. Product purification and bidirectional se-quencing was performed by Macrogen Inc. sequencing ser-vice (Seoul, South Korea) using the amplification primers.Sequences were edited manually and aligned using the pro-gram BioEdit (Hall 1999). DNA sequences were submittedto Barcode of Life Data Systems (BOLD, Ratnasingham &Hebert 2007) and GenBank. BOLD ID and accession num-ber to GenBank are given in Table 1.

Data set for phylogenetic analysis comprised the DNAbarcodes amplified from A. cremisa adults from both loca-tions 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 (Ta-ble 1). The sequence of R. vulgaris was included as outgroup.First we compared our DNA sequences with the ones avail-able in the BOLD database using BOLD Identification En-gine (accessed March 2016), second, the Neighbor-Joining(NJ) tree based on the Kimura-2-Parameter (K2P) distancemodel are built using MEGA 6.0. (Tamura et al. 2013) phy-logenetic software. The robustness of branching was assessedby bootstrapping analysis (2000 replicates). Finally, we cal-culated the uncorrected pairwise distances between speci-mens (p-distances) based on the mtCOI barcode sequencesusing 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 20th 2014(7 specimens: 5 males, 2 females) and the ZrmanjaRiver at Palanka on August 11th 2014 (2 specimens:2 males). Figure 3 presents the male genitalia (Figs 3A,

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DNA barcoding and first records of two Adicella species in Croatia 799

Fig. 3. A. cremisa: A, B – male genitalia; C, D – female genitalia.

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 Rivernear 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 ofA. balcanica and A. cremisa.According to BOLD Identification Tree,A. cremisa

specimens from Croatia are highly similar to A. cremisafrom Italy, from the Friuli-Venezia Giulia region inItaly (top similarity matches with A. cremisa in BOLDdatabase is 100%). The NJ tree additionally sup-ports all morphological identifications, i.e. A. cremisaspecimens from both sites cluster within highly sup-ported monophyletic A. cremisa clade. The sister taxarelationship of A. balcanica and A. cremisa is alsohighly supported (Fig. 5, electronic supplementary file– Fig. 1S). The maximum value of the uncorrectedpairwise distance (p-distance) between Adicella speciesis around 21% (Table 2). The p-distance betweenA. cremisa and A. balcanica is around 14% (Table 2).

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800 A. Cukušic 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 forAdicella species from Croatia and BOLD database.

A.cremisa A.cremisa A.balcanica A.reducta A. filicornis A. syriacaZrmanja 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 inCroatia.

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 13.5pH 8 8.1 8.2 8.1 8.2 8.3 8 8.1 8.2Conductivity (µS cm−1) 579 626 711 335 354 370 350 382 420Alkalinity (mgCaCO3 L−1) 304 313.8 334 180 185.2 191 181 202 223Dissolved oxygen (mgO2 L−1) 7.5 9.5 11.3 9.1 10.9 11.4 10.7 11.2 11.4Oxygen saturation (%) 77.2 86.2 103.1 80.2 97.8 106.2 97.1 101.7 108.7BOD5 (mgO2 L−1) 0.7 1.7 4 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5 < 1.5COD-Mn (mgO2 L−1) 2.1 3.6 7 < 0.6 < 0.6 0.6 < 0.6 < 0.6 0.7Ammonia (mgN L−1) < 0.01 0.0854 0.19 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 0.011Nitrites (mgN L−1) 0.004 0.0202 0.045 < 0.0015 0.0018 0.0025 < 0.0015 < 0.0015 < 0.0015Nitrates (mgN L−1) 0.56 0.8583 1.23 0.294 0.3392 0.378 0.172 0.2097 0.239Total nitrogen (mgN L−1) 0.9 1.1317 1.52 0.2956 0.3507 0.415 0.172 0.2378 0.341Ortophosphates (mgP L−1) 0.008 0.032 0.065 < 0.006 < 0.006 0.007 < 0.006 0.009 0.015Total 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 tothe upper courses of the investigated rivers, the Krapina

and the Zrmanja rivers, while the record of A. balcanicabelong to the middle to lower course of the Krupa River.

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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 withA. cremisa at the KrapinaRiver by Krapina Selo

Ithytrichia lamellaris Eaton, 1873 22 adult male 14 adult female UV light trapMystacides nigra (L., 1758) 1 adult male entomological netPotamophylax rotundipennis (Brauer, 1857) 2 adult female UV light trapCyrnus trimaculatus (Curtis, 1834) 1 adult male entomological netLype reducta (Hagen, 1868) 14 adult male entomological net

Sympatric species withA. cremisa at Palanka siteof the Zrmanja River

Hydropsyche instabilis (Curtis, 1834) 1 adult male UV light trapPsychomia klapaleki Malicky, 1995 5 adult male 1 adult female entomological netRhyacophila fasciata Hagen, 1859 1 adult male UV light trap

Sympatric species withA. balcanica at KrupaMonastery at the KrupaRiver

Athripsodes bilineatus (L., 1758) 1 adult male entomological netWormaldia subnigra McLachlan, 1865 1 adult male entomological netPolycentropus irroratus Curtis, 1835 1 adult male UV light trapLype reducta (Hagen, 1868) 5 adult male 2 adult female UV light trap

Relatively high pH values (> 8) were recorded atall sampling sites and other physico-chemical param-eters show relatively high variability (Table 3). Basedon the measured physico-chemical parameters all treesampling sites were classified into high or good waterquality status (Table 3).In Table 4 we showed sympatric species of cad-

disflies collected at the locations on which we foundA. balcanica or A. cremisa.

Discussion

So far three species of the genus Adicella have beenrecorded in Croatia: A. filicornis Pictet, 1834, A. re-ducta (McLachlan, 1865) and A. syriaca Ulmer, 1907(Kučinic et al. 2012). Acidella cremisa and A. balcanicarepresent new records for the Croatian caddisfly fauna(e.g., Cuk & Vučkovic 2010, 2014; Cuk et al. 2015; Pre-višic et al. 2009, 2013a, b, 2014; Previšic & Popijač2010; Kučinic et al. 2011, 2012, 2014, 2015, 2016).

Genetic identification and interspecific distance of Adi-cella speciesNo study examined the interspecific p-distances of COIsequences within the genus Adicella, but maximumvalue of the p-distances similar to ours were recordedin the genus Smicridea, Hydropsychidae, also for a bar-code region (20%, Pauls et al. 2010). The p-distance be-tween A. cremisa and A. balcanica are higher than min-imum values of the interspecific variability usually ob-served in caddisflies for the barcode region (8% Paulset al. 2010; 5.3% Zhou 2009; 8.2% Graf et al. 2015).Thus, results of the DNA barcode method presentedhere support the morphological identification of a cad-disfly species new for the Croatian fauna. The abilityof the DNA barcode to delimit closely related caddisflyspecies is shown in many studies where a single locuswas used (mtDNA COI-5P, e.g., Pauls et al. 2010; Jack-son et al. 2014).

Intraspecific distance of A. cremisa specimens indicatesgene flow between South European populationsHigh similarity of A. cremisa specimens from Croatia

with A. cremisa population in Italy (as it shown inBOLD illustrated Identification Tree) implies the exis-tence of the contemporary or recent gene flow betweendisjunctly distributed populations in the wider region,as recorded in other taxa, e.g., the stonefly Dinocrascephalotes (Curtis, 1827) (Elbrecht et al. 2014) andother caddisflies, e.g., Lectrides varians Mosely, 1953(Wickson et al. 2014), Plectrocnemia conspersa (Curtis,1834) (Wilcock et al. 2005). Further evidence for suchpattern is provided when genetic distance of A. cremisaspecimens from two populations in Croatia are com-pared, i.e., the p−distance between specimens from Zr-manja and Krapina rivers is only 0.3 % (Table 2). Sim-ilarly as in A. cremisa, low intraspecific variability inthe DNA barcode region was observed in the mayflyRhithrogena braaschi Jacob, 1974 within the WesternDinaric Balkan ecoregion (Vilenica et al. 2016). On thecontrary, high intraspecific variability (p-distances upto 6.3%) was recorded in Drusus endemics (Drusinae,Limnephilidae) in the Western Balkans (e.g., Previšicet al. 2009; Pauls et al. 2006). Although some of thesestudies use a different fragment of the mtCOI gene(mtCOI-3P and mtCOI-5P), Graf et al. (2015) showedthat differentiation level of these two mtCOI regions isvery similar in Limnephilidae. The ecology of partic-ular species (e.g., dispersal behaviour, habitat prefer-ences etc.) is often reflected in the existence/absence ofthe contemporary gene flow. For instance, in contrastto lowland Adicella species (Graf et al. 2008, 2016),Drusus species inhabit montane springs and streamsand thus show high degree of endemism and geneticisolation (e.g., Previšic 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. cremisaThe distribution of A. balcanica and A. cremisa, isinsufficiently investigated, and data are still scarce.Adicella balcanica was described from specimens col-lected in Bosnia and Herzegovina (Botosaneanu & No-vak 1965) and A. cremisa is described from its type lo-cality in Austria (Malicky 1972). According to Graf etal. (2008, 2016) A. cremisa has been recorded in ecore-

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802 A. Cukušic 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 newfindings in Croatia (red points presents A. cremisa and blue point A. balcanica).

gions ER3 (Italy, Corsica and Malta), ER5 (DinaricWestern Balkan) and ER9 (Central Highlands, sensuIllies 1978). Adicela balcanica has a wider distributionin the Balkans, in the ER5, ER4 (Alps), ER6 (HellenicWestern Balkan) and ER11 (Hungarian lowlands) (Grafet al. 2008, 2016). However, according to Robert (2015),both species have more localised distribution only insome parts of the mentioned ecoregions. The sites atthe Zrmanja River and Krupa River are situated in theER5 while the site at the Krapina River is situated onthe border between two ecoregions, ER5 and ER11. InFig. 6 we present distribution of A. cremisa and A. bal-canica in Europe according to Fauna Europaea (Mal-icky 2013) BOLD data, freshwaterecology.info (Graf etal. 2016) with records ofA. cremisa in Italy (Cianficconi2002; Cianficconi & Moretti 1987), Austria (Graf 2002)and Slovenia (Krušnik & Urbanič 2002) and records ofA. balcanica in Bosnia and Herzegovina (Botosaneanuand Novak 1965), Serbia (Zivic 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 rep-resent an important contribution to the knowledge ontheir distribution ranges. A systematic inventory of cad-disflies in Croatia was conducted on more than 220sites in different geographical parts of Croatia in thelast 20 years in different periods (e.g., Cerjanec 2012;Kučinic 2002; Kučinic et al. 2011; Vučkovic 2011). In

these investigations including this study, A. cremisawas recorded at only two sites and A. balcanica at onlyone. All these data indicate that these two species arevery rare in the fauna of Croatia and show a disjunctdistribution (Fig. 6). Relatively high pH value (> 8)recorded at all sampling sites could be one of the prefer-ences determining the distribution of these two species.

Ecology of A. balcanica and A. cremisaAccording to Graf et al. (2008, 2016) Adicella specieshave a wide ecological valence what contributes to widedistribution of A. reducta and A. filicornis in Europe.The physico-chemical parameters at sites included inthe current study also show seasonal variability (e.g.,water temperature at the Krapina River varied morethan in the Zrmanja and Krupa rivers; lowest wa-ter temperature was recorded at the Krapina River,but also highest values of conductivity and alkalinity(Table 3). These data suggest that A. balcanica andA. cremisa also have wide ecological valence. No recordsof larvae at any investigated site could be related topotentially small populations and specific microhabi-tat preferences (both are organic habitat specialists,Graf et al. 2008, 2016), therefore, it can be hard torecord them even at sites where they normally occur.Consequently, we can assume that both species havewider distribution than currently known according tothe available records (Fig. 6).

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DNA barcoding and first records of two Adicella species in Croatia 803

Graf et al. (2008, 2016) indicate that A. cremisaoccurs mainly in plains (< 300 m), rarely at higheraltitudes (up to 1000 m). In Croatia, we found it in ac-cordance with its main distribution preference, i.e. atlow altitude (around 200 m a.s.l.). Adicella balcanicaoccurs at higher altitudes (300–1900 m a.s.l., Graf etal. 2008, 2016) and it is mostly a eucrenal species, alsooccurring in hypocrenal and epirhithral region (Graf etal. 2008, 2016). We found A. balcanica at 105 m a.s.l. inthe Krupa canyon in the middle to lower river sections.It is typical for the Mediterranean kasrt rivers in theDinaric region which do not follow the River Contin-uum Concept presented by Vannote et al. (1980) (e.g.,Pinna et al. 2003; Vučkovic 2011). On the other hand,there are several underground springs along the KrupaRiver bed (Šafarek & Šolic 2011) changing the condi-tions more favourable for crenal species. Furthermore,records of adults only do not provide certain evidenceof stream zonation preference since larvae were not col-lected within this study.For A. cremisa stream zonation preference is un-

known. However, current findings suggest that it pos-sibly occurs in the epirhithral and metarhithral zone,similar to other Adicella species presented in Graf etal. (2008). This conclusion is based on results of thecurrent study. Within the study we sampled at the Zr-manja River at eucrenal, hypocrenal and epipotamalpart of the river, but there were no Adicella species.According our physico-chemical water parameters,

we can assume that A. balcanica and A. cremisa pre-fer alkaline environment. Regarding preference to pHvalue of two widely distributed Adicella species, A. fi-licornis prefers neutral to alkaline environment, whileA. reducta is indifferent (Graf et al. 2008, 2016).The larva of A. cremisa was roughly described

by Cianficconi & Moretti (1987). However, the headcolouration pattern and case design are very close toA. reducta (Waringer & Graf 2011). Therefore, re-description of the larva should probably be taken intoconsideration. Larvae of A. cremisa are usually foundin slow flowing streams and lentic zones, preferably oc-curring in macrophytes, mosses and woody debris (Grafet al. 2008). The larva of A. balcanica is unknown.Moreover, larvae of all Adicella species, including

A. cremisa and A. balcanica, are organic habitat spe-cialists (Graf et al. 2008, 2016). Even though larvaewere not collected within the current study, habitatspreferred by these species are present at sampling siteswhere adults were collected, e.g., woody debris, macro-phytes 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). Emer-gence/flight periods recorded for both species in thecurrent study were therefore mainly in line with typicalperiods of Adicella species. Both species were recordedonly in the summer period even though the sites werevisited three times throughout 2014. Emergence/flightperiod for A. balcanica is, therefore, shorter for oneperiod (spring) than presented by Graf et al. (2008,

2016). However, further studies need to be conducteddue to the small number of collected specimens withinthis study.

Conclusions

In the current study we present the first record oftwo Adicella species in Croatia and contributed to theknowledge of the Croatian caddisfly fauna. We also ac-centuated the importance of the Western Balkan regionas a hot-spot of freshwater diversity, as is also pointedout by numerous previous studies (Kučinic et al. 2014;Previšic et al. 2014; Ibrahimi et al. 2015; Vitecek et al.2015a, 2017). We used DNA barcode method to confirmmorphological identification. This enabled a further in-sight into the genetic connectivity of A. cremisa popu-lations in Croatia and the region. Furthermore, we pre-sented valuable information on distribution and ecologyof insufficiently studied Adicella species.

Acknowledgements

We are very grateful to employees of the Central WaterManagement Laboratory of Hrvatske vode for providing theresults of physico-chemical parameters and Marwan Zeitoun(Zagreb) who helped us in the field collections. This studywas supported by the “EU Natura 2000 Integration Project(NIP)” funded by the Croatian Ministry of Environmen-tal and Nature Protection, by Croatian Science Foundationunder the project (IP-2016-06-9988, DNA Barcoding of Di-versity of Croatian Fauna, by M. Kučinic) and Universityof Zagreb funding. For technical help thank you to prof.Halil Ibrahimi from University of Pristina. We are also verygrateful to two anonymous reviewers.

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