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.

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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).

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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.

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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).

Table 1. Stations at the Cetina, Ruda, Grab and Rumin rivers with main information.

elevation Number Station Short-code Coordinates Dominant substrate (m a.s.l.) N 43° 58´ 36,1˝ gravel and sand, submersed 1. Cetina Spring CS 386 E 16° 25´ 48,6˝ vegetation N 43° 57´ 59˝ 2. Preočki most PM 371 gravel, sand E 16° 25´ 53˝ N 43° 57´ 35,7˝ 3. Crveni most CM 367 gravel, sand E 16° 25´ 46,6˝ N 43° 50´ 33,5˝ 4. Peruča reservoir PR 359 sand and silt E 16° 31´ 40,6˝ N 43° 40´ 01,4˝ cobbles, boulders covered 5. Obrovac Sinjski OS 301 E 16° 41´ 04,7˝ in green algae N 43° 36´ 21,1˝ 6. Trilj TR 296 sand and silt E 16° 43´ 46,2˝ N 43° 40´ 10˝ 7. Reservoir Prančević RP 285 sand and silt E 16° 44´ 28˝ N 43° 31´ 58,4˝ pebbles, pebbles covered in 8. Čikotina Lađa ČL 236 E 16° 44´ 42,3˝ moss N 43° 25´ 28˝ pebbles, pebbles covered in 9. Pavića most PAM 217 E 16° 45´ 23˝ moss N 43° 26´ 16,2˝ pebbles, pebbles covered in 10. Radmanove Mlinice RM 15 E 16° 45´ 11,3˝ moss N 43° 23´ 11,5˝ 11. Omiš OM 5 sand and silt E 16° 46´ 15,1˝ N 43° 40´ 07,1˝ 12. Ruda Spring RS 320 boulders, pebbles, moss E 16° 47´ 56˝ N 43° 40´ 17˝ 13. Ruda II RII 302 pebbles, boulders E 16° 46´ 20˝ N 43° 38´ 24,4˝ 14. Grab Spring GS 330 pebbles, boulders E 16° 46´ 33,9˝ N 43° 38´ 20,1˝ 15. Grab II GII 302 boulders E 16° 46´ 20,3˝ N 43° 46´ 38,2˝ 16. Rumin Spring RUS 350 boulders E 16° 39´ 00˝

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Table 2. Caddisfly distribution on the Cetina River (CS – Cetina Spring, PM - Preočki most, CM - Crveni most, PR - Peruča reservior, OS - Obrovac Sinjski, TR - Trilj, PR - Reservoir Prančević, ČL - Čikotina Lađa, PAM - Pavića most, RM - Radmanove Mlinice, OM – Omiš), and its tributaries, rivers Ruda (RS – Ruda Spring, RII - Ruda II), Grab (GS – Grab Spring, GII - Grab II) and Rumin (RUS – Rumin Spring)

Station CS PM CM PR OS TR RP ČL PAM RM OM RS RII GS GII RUS Species Cetina River upper part Cetina River middle part Cetina River lower part Ruda River spring area Grab River spring area Rumin River spring area

Family: RHYACOPHILIDAE 1. Rhyacophila balcanica Radovanović, 1953 29 80 3 79 2. Rhyacophila dorsalis plitvicensis Kučinić & Malicky, 2002 1 3. Rhyacophila fasciata delici Kučinić & Valladolid, 2020 40 108 146 120 14 2 88 12 27 200 112 13 4 34 4. Rhyacophila tristis Pictet, 1834 1 1 Family: GLOSSOSOMATIDAE 5. Glossosoma discophorum Klapálek, 1902 27 3 1 504 1 293 69 3 6. Synagapetus karawanyi Ulmer, 1938 23 1 Family: HYDROPTILIDAE 7. Hydroptila tineoides Dalman, 1819 2 8. Agraylea sexmaculata Curtis, 1884 1 17 8 1 35 Family: PHILOPOTAMIDAE 9. Wormaldia occipitalis Picet, 1834 29 2 10. Wormaldia subnigra McLachlan, 1865 2 2 96 62 2 Family: HYDROPSYCHIDAE 11. Hydropsyche dinarica Marinković-Gospodnetić, 1979 41 10 12. Hydropsyche incognita Pitsch, 1993 1 9 12 13 56 124 1 7 13. Hydropsyche instabilis (Curtis, 1834) 2 3 8 7 3 8 14. Hydropsyche pellucidula (Curtis, 1834) 11 Hydropsyche sp. (♀♀) 7 2 2 48 54 51 42 223 250 15 8 Family: POLYCENTROPODIDAE 15. Plectrocnemia conspersa (Curtis, 1834) 2 10 36 2 1 16. Polycentropus flavomaculatus (Pictet, 1834) 3 1 15 12 3 6 10 17. Polycentropus schmidi Novak & Botosaneanu, 1965 3 18. Cyrnus trimaculatus (Curtis 1834) 23 4 2 1 Family: PSYCHOMYIIDAE 19. Psychomyia klapaleki Malicky, 1995 23 4 155 103 27 61 193 2 7 20. Lype reducta (Hagen, 1868) 57 45 1 21. Tinodes braueri McLachlan, 1878 2 2 124 5 17 22. Tinodes dives (Pictet, 1834) 28 23. Tinodes waeneri (Linnaeus, 1758) 12 4 11 Family: PHRYGANEIDAE 24. Trichostegia minor (Curtis, 1834) 2 Family: LIMNEPHILIDAE ..continued on the next page Ecologica Montenegrina, 44, 2021, 69-95 73

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TABLE 2 25. Ecclisopteryx ivkeae (Previšić, Graf & Vitecek, 2014) 4 22 136 16 26. Limnephilus affinis Curtis, 1834 2 1 27. Limnephilus auricula Curtis, 1834 24 5 1 1 28. Limnephilus bipunctatus Curtis, 1834 1 29. Limnephilus flavicornis (Fabricius, 1787) 65 59 33 4 3 30. Limnephilus griseus (Linnaeus, 1758) 7 1 31. Limnephilus hirsutus (Pictet, 1864) 16 1 32. Limnephilus incisus Curtis, 1834 2 3 1 2 2 33. Limnephilus lunatus Curtis, 1834 679 401 292 4 17 6 1 3 3 1 34. Limnephilus marmoratus Curtis, 1834 23 41 8 3 35. Limnephilus rhombicus (Linnaeus, 1758) 1 1 36. Limnephilus sparsus Curtis 1834 3 37. Limnephilus vittatus (Fabricius, 1798) 53 13 4 1 38. Glyphotaelius pellucidus (Retzius, 1783) 1 39. Grammotaulius nigropunctatus (Retzius, 1873) 4 4 13 40. Potamophylax cingulatus-latipenis 6 41. Potamophylax nigricornis (Pictet, 1834) 2 42. Potamophylax pallidus (Klapálek, 1899) 2 43. Halesus digitatus (Schrank, 1781) 29 23 5 13 24 1 2 12 44. Halesus tesselatus (Rambur, 1842) 3 45. Mesophylax impunctatus McLachlan, 1884 3 2 1 1 46. Stenophylax meridiorientalis Malicky, 1982 1 47. Stenophylax permistus McLachlan, 1895 7 2 3 48. Micropterna fissa McLachan 1875 2 49. Micropterna nycterobia McLachlan, 1875 26 11 6 50. Micropterna sequax McLachlan, 1875 1 51. Micropterna testacea (Gemlin, 1879) 4 2 52. Allogamus uncatus (Brauer, 1857) 2 1 53. Hydatophylax infumatus (McLachlan, 1865) 7 54. Chaetopteryx fusca Brauer, 1857 1 55. Annitella apfelbecki (Klapálek, 1899) 79 Family: GOERIDAE 56. Silo piceus (Brauer, 1857) 2 165 33 Family: LEPIDOSTOMATIDAE 57. Lepidostoma basale (Kolenati, 1848) 399 291 9 58. Lepidostoma hirtum (Fabricius, 1775) 1 10 23 22 20 470 2 2 508 4 2 Family: LEPTOCEROIDAE 59. Athripsodes albifrons (Linnaeus, 1758) 3 11 5 6 47 1 1 60. Athripsodes dalmatinus Malicky, 1980 223 132 2 61. Ceraclea albimacula (Rambur, 1842) 3 1 2 6 2 1 ..continued on the next page 74

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TABLE 2 62. Ceraclea dissimilis (Stephens, 1836) 5 25 23 1 12 60 3 2 20 1 3 11 30 8 63. Mystacides azurea (Linnaeus, 1761) 1 2 1 2 5 10 64. Mystacides nigra Pictet, 1834 1 10 65. Triaenodes lefkas Malicky, 1974 3 66. Oecetis testacea (Curtis, 1834) 1 3 1 67. Adicella filicornis (Pictet, 1834) 2 68. Adicella syriaca Ulmer, 1908 1 1 Family: ODONTOCERIDAE 69. Odontocerum albicorne (Scopoli, 1769) 1 37 21 4 Family: SERICOSTOMATIDAE 70. Sericostoma flavicorne Schneider, 1845 124 110 193 12 2 49 145 29 12 6 23 Family: BERAEIDAE 71. Beraeamyia schmidi Botosaneanu, 1960 6 2 1

No. of specimens 1297 975 988 6 507 90 498 415 256 1007 13 1778 1567 418 140 93 No. of species 35 29 27 3 16 11 15 18 16 24 8 22 17 14 11 8 No. of genera 23 14 15 1 11 9 12 14 13 20 8 14 13 8 8 8 No of families 8 7 7 1 8 5 8 8 11 13 5 12 10 8 8 8

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Sampling work. During the years of 2004 and 2005, systematic collecting of Trichoptera was carried out along the Cetina River (upper, middle and lower parts) and its tributary the Ruda River; in 2007 and 2008 caddisflies were collected at two additional tributaries, the Grab and the Rumin rivers. Data obtained were supplemented and completed by additional sampling at some localities/stations (springs of the Cetina, the Ruda, the Grab and the Rumin rivers) after the above mentioned period, ending with the year 2018, the material of which was used for the DNA barcoding method (Tab. 4, Tab. 5). In order to define the seasonal dynamics of Trichoptera, fieldwork was performed monthly throughout the year. The specimens were collected in different types of habitats throughout the upper, middle and lower parts of the Cetina River, for 13 months. Four stations (the Cetina spring (CS), Preočki most (PM), Crveni most (CM) and the Peruča reservoir (PR)) were located in the upper reach, two stations (Obrovac Sinjski (OS) and Trilj (TR)) in the middle reach and another five stations (the Prančević reservoir (PR), Čikotina Lađa (ČL), Pavića most (PAM), Radmanove Mlinice (RM) and Omiš (OM)) in the lower reach of the Cetina River. In the tributary of the Cetina River, the Ruda River, the sampling was conducted at 2 stations in the upper reach (the Ruda spring (RS) and Ruda II (RII)); also at 2 stations on the Grab River in the upper reach (the Grab spring (GS) and Grab II (GII), and at 1 locality on the Rumin River (the Rumin spring (RUS)) (Fig. 1). During 2017 the genus Glossosoma was collected on the Neretva River near Žitomislić, downstream from the city of Mostar (Bosna and Herzegovina), in order for DNA barcoding to be performed and the correct faunistic composition in Croatia to be determined. The majority of caddisflies sampled for DNA barcoding and additional checking of identifications were collected in the period from 2014 to 2018 in the area of Cetina River. Caddisfly specimens were collected using light traps, with a 6W or 8W ultraviolet (UV) fluorescent tube powered by a 12 V battery with sampling effort of two hours. The tube was hung over an aluminium construction and covered with a white sheet. Adults were also collected with entomological nets during the day (leg. I. Vučković, M. Kučinić, A. Previšić, A. Popijač, I. Zrinski). The collected material was preserved and stored in 80% ethanol and absolute ethanol.

Laboratory work. Whole genomic DNA was extracted from legs using a GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, Germany) according to the manufacturer’s specifications and eluted in 60 µl of elution buffer. Full-length mtCOI DNA barcode regions were amplified using LCO1490/HCO2198 (Folmer et al. 1994) primer sets. The 50 μl polymerase chain reaction (PCR) mixture contained 1 x Go Taq®Reaction Buffer (containing 1.5 mM MgCl2, Promega), 0.2 mM of dNTP, 0.4 μM of each primer, 1.25 units of Go Taq®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). BOLD ID and accession number of the specimens used in analyses are given in Tab. 4. DNA barcoded specimens were deposited in the Croatian Natural History Museum in Zagreb. Four specimens of Rhyacophila fasciata collected in the Cetina catchment area, along with specimens from other areas, were used in describing of the subspecies R. f. delici. (Valladolid et al. 2020). All four specimens (paratypes) were morphologically analysed and DNA barcoded, and are deposited in the Museo Nacional de Ciencias Naturales, Madrid, Spain (MNCN) (Tab. 6). List of caddisflies with DNA barcoding data from the River Cetina are given in Tab. 6. Taxonomic identification of the collected adults was conducted using Malicky (2004) and Kumanski (1985, 1988). Collected specimens are deposited in the Trichoptera collection of the first author. Data analysis. The similarity of caddisfly assemblages among the stations was examined using non- metric multidimensional scaling (NMDS) ordination based on the Bray-Curtis similarity matrix. Species data were log transformed prior to analysis. NMDS analysis was performed using Primer 6 software package (Clarke & Gorley 2006). Prior to analysis, sequences were aligned using the program BioEdit (Hall 1999). Phylogeny-based identification was conducted with the use of three different methods of tree reconstruction: neighbor-joining (NJ) and maximum likelihood (ML) as implemented in MEGA 6.0. (Tamura et al. 2013) and Bayesian inference (BI). Prior to the ABGD analysis, pairwise genetic distances were calculated using the Kimura

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(K80) model with a transition/transversion rate of 2.0. For the range of prior intraspecific divergences (P), values were Pmin = 0.001 to Pmax = 0.08 and steps were set to 10. Relative gap width of X was set to 0.1. This analysis was performed on the ABGD webserver platform available at http://wwwabi.snv.jussieu.fr/public/abgd/abgdweb.html (Puillandre et al. 2012).

Results

Faunal and DNA barcoding features. During our research into the Cetina River catchment area, we recorded 71 caddisfly species at 11 stations on the Cetina River, 24 species at two stations on the Ruda River, 18 species at two stations on the Grab River and 8 species at one station on the Rumin River. On all Cetina tributaries, the Ruda, the Grab and the Rumin rivers, caddisflies were collected only in the spring areas, because they are short and generally are not divided into upper, middle and lower reaches. The species found at most of the stations in this research were Rhyacophila fasciata delici Kučinić & Valladolid, 2020 and Ceraclea dissimilis (Stephens, 1836), recorded at 14 stations, followed by the species Sericostoma flavicorne Schneider, 1845 and Limnephilus lunatus Curtis, 1834 registered at 11 stations (Tab 2). Sixteen (16) species were recorded only at one station, and these are: Rhyacophila dorsalis plitvicensis Kučinić & Malicky, 2002, Hydroptila tineoides Dalman, 1819, Wormaldia occipitalis Picet, 1834, Polycentropus schmidi Novak & Botosaneau, 1965, Tinodes dives (Pictet, 1834), Limnephilus sparsus Curtis, 1834, Glyphotaelius pellucidus (Retzius, 1783), Potamophylax cingulatus-latipenis, P. nigricornis (Pictet, 1834), P. pallidus (Klapálek, 1899), Halesus tesselatus (Rambur, 1842), Stenophylax meridiorientalis Malicky, 1982, Micropterna sequax McLachlan, 1875, Hydatophylax infumatus (McLachlan, 1865), Chaetopteryx fusca Brauer, 1857 and Annitella apfelbecki (Klapálek, 1899). The most abundant species were L. lunatus Curtis, 1834 (1407 specimens), Lepidostoma hirtum (Fabricius, 1775) (1058 specimens) and R. f. delici (920 specimens), while species recorded with only one specimen each were C. fusca, Glyphotaelius pellucidus (Retzius, 1783), L. bipunctatus Curtis, 1834, M. sequax and S. meridiorientalis.

Table 3. Number of species (S), number of specimens (N), the Margalef density index (D), the Pielou evenness index (J'), the Shannon-Weaver diversity index (H`) and Simpson's diversity index (1-λ´) at certain stations in the Cetina River basin (CS – Cetina Spring, PM - Preočki most, CM - Crveni most, PR - Peruča reservoir, OS - Obrovac Sinjski, TR - Trilj, RP - reservoirr Prančevići, ČL - Čikotina Lađa, PAM - Pavića most, RM - Radmanove Mlinice, OM – Omiš, RS - Ruda Spring, RII –Ruda, II, GS - Grab Spring, GII - Grab II and RUS – Rumin Spring).

Station / Index S N D J´ H´ 1-λ´ CS 35 1297 4,743 0,564 2,006 0,705 PM 29 975 4,068 0,636 2,144 0,791 CM 27 988 3,770 0,645 2,127 0,828 PR 3 6 1,116 0,790 0,8676 0,500 OS 16 507 2,408 0,579 1,607 0,742 TR 11 90 2,222 0,795 1,907 0,814 RP 15 498 2,254 0,615 1,664 0,729 ČL 18 415 2,820 0,695 2,066 0,821 PAM 16 256 2,705 0,745 2,029 0,799 RM 24 1007 3,326 0,638 1,992 0,750 OM 8 13 2,729 0,957 1,846 0,852 RS 22 1778 2,806 0,664 2,052 0,829 RII 17 1567 2,175 0,668 1,893 0,808 GS 14 418 2,154 0,473 1,250 0,496 GII 11 140 2,204 0,677 1,625 0,698 RUS 8 93 1,544 0,796 1,655 0,764

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Table 4. List of sequences of the genus Glossosoma used in DNA barcoding method. Each sample is accompanied by a sample ID, BOLD sequence ID, and location where specimens were collected.

BOLD ID Species name sample ID Location sequences Glossosoma bifidum McLachlan, 1879 TGBIF_1 NIPAG004-17 river Kupčina, spring, Mt Žumberak, Croatia Glossosoma conformis Neboiss, 1963 TGCON_1 CROTR210-19 creek Bliznec, Mt Medvednica, Croatia Glossosoma conformis TGCON_2 CROTR014-19 creek Bliznec, Mt Medvednica, Croatia Glossosoma discophorum Klapalek, 1902 TGDIS_1 CROAA004-18 river Tounjčica, spring, Croatia Glossosoma discophorum TGDIS_2 CROAA035-18 river Vitunjčica, spring, Croatia Glossosoma discophorum TGDIS_3 CROAA036-18 river Slunjčica, spring, Croatia Glossosoma discophorum TGDIS_4 CROAA036-18 river Una, spring, Croatia Glossosoma discophorum TGDIS_5 CROAA064-18 river Rumin, spring, Croatia Glossosoma discophorum TGDIS_6 CROTR057-19 creek Plitvice, spring, Croatia Glossosoma discophorum TGDIS_7 CROTR063-19 river Rumin, spring, Croatia Glossosoma discophorum TGDIS_8 CROTR090-19 river Grab, spring, Croatia Glossosoma neretvae Marinkovic, 1988 TGNER_1 CROTR052-19 river Neretva, Žitomislić, Bosnia and Herzegovina Glossosoma neretvae TGNER_2 CROTR059-19 river Neretva, Žitomislić, Bosnia and Herzegovina Glossosoma klotho Malicky, 2003 TGKLO_3a CROTR093-19 river Lumbardhi i Pejës, Pejë, Kosovo Agapetus ochripes TAOCH_2 CROTR010-19 creek Gospin potok, Šumarska kuća Duboka, Velika, Mt Papuk, Croatia

Species recorded in all four rivers were R. fasciata and C. dissimilis; only in the Cetina River we registered R. dorsalis plitvicensis Kučinić & Malicky, 2002, H. tineoides, W. occipitalis, P. schmidi, Cyrnus trimaculatus (Curtis 1834), Lype reducta (Hagen, 1868), Tinodes braueri McLachlan, 1878. T. dives, Trichostegia minor (Curtis, 1834), L. affinis Curtis, 1834, L. auricula Curtis, 1834, L. griseus (Linnaeus, 1758), L. hirsutus (Pictet, 1864), L. marmoratus Curtis, 1834, L. rhombicus (Linnaeus, 1758) and L. sparsus; only in the Grab River we recorded L. bipunctatus Curtis, 1834, P. nigricornis, P. pallidus, H. tesselatus, S. meridiorientalis, S. permistus McLachlan, 1895, Micropterna fissa McLachan, 1875, M. nycterobia McLachlan, 1875, M. sequax, M. testacea (Gemlin, 1879), Allogamus uncatus (Brauer, 1857), H. infumatus, C. fusca, A. apfelbecki, Athripsodes dalmatinus Malicky, 1980, Mystacides azurea (Linnaeus, 1761), M. nigra Pictet, 1834, Triaenodes lefkas Malicky, 1974, Oecetis testacea (Curtis, 1834), Adicella filicornis (Pictet, 1834) and Adicella syriaca Ulmer, 1908, while there were no species recorded exclusively in the Ruda and Rumin rivers. During this research 27 specimens of 17 species were DNA barcoded (Tab. 6). The most interesting barcoded species were R. fasciata delici, Agraylea sexamaculata Curtis, 1884, Hydroptila forcipata Dalman, 1819, Ecclisopteryx ivkae (Previšić, Graf & Waringer, 2014) and Athripsodes dalmatinus. Species E. ivkae and A. dalmatinus were barcoded for the frst time in the BOLD database. Because of a questionable finding of the species Glossosoma neretvae Marinkovic, 1988 (Malicky 2014) on the River Ruda, a detailed DNA barcode analysis was done for this finding, as well as for material from neighbouring countries.Phylogenetic analysis of specimen from Croatia based on the tree method confirmed identifications done according to their morphological features (Fig. 5). An unidentified larva (TGDIS_5) is clustered within the well supported clade with a male G. discophorum (TGDIS_7) collected at the same locality and Glossosoma discophorum Klapalek, 1902 from other localities. A male TGDIS_7 and TGDIS_5 larvae, together with G. discophorum specimens from the Mediterranean part of Croatia are separated into a different genetic line from the G. discophorum collected in the continental part of Croatia. At the spring of the river Tounjčica, a female TGDIS_1 was collected and was presumed to belong to the same species as a male of the species G. discophorum. The DNA barcode sequence of the female TGDIS_1 was successfully obtained. Results confirmed the female TGDIS_1 as G. discophorum due to the clustering within the well supported clade with other specimens of G. discophorum. A DNA sequence of specimen BHMKK157-12 from Montenegro was taken from the BOLD database. Specimen BHMKK157-12 was morphologically identified as G. neretvae, but grouped with the species G. discophorum. ABGD analysis classified specimens of G. discophorum

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(including TGDIS_5 larvae, female TGDIS_1 female and specimen BHMKK157-12 from Montenegro) into the same group (Group 3 on Fig. 5). Specimens of G. neretvae from Bosnia and Herzegovina are grouped by ABGD analysis in Group 4. The species G. neretvae proved to be a sister species of G. discophorum, and G. conformis Neboiss, 1963. In the ABGD analysis, G. klotho Malicky, 2003 and G. bifidum McLachlan, 1879 formed one group (Group 1). Results of inter and intraspecific genetic p-distances of the mitochondrial cytochrome oxidase I (DNA barcode region) gene fragments recorded for Glossosoma species from Croatia and BOLD database are shown in Tab. 5.

Table 5. Values of the genetic distances (uncorrected p‐distances) between groups of Glossosoma and outgroup species for DNA barcode region. Intraspecific values are marked in bold letters.

B&H

Italia

mis

Croatia

sp. larvae sp.

G. klotho

Montenegro

G. neretvae,

G. confor

G. discophorum

G. bifidum,

G. neretvae,

G. bifidum

Glossosoma

G. conformis 0-0.5 G. discophorum 11-11.4 0.2-0.8 Glossosoma sp., larvae 11-11.3 0-0.6 - G. neretvae, Montenegro 11.3 0.5-1.1 0.5 - G. neretvae, B&H 11.6 6.4-6.7 6.7 7.2 - G. bifidum, Italy 11 11.4-11.6 11.4 11.8 13.8 - G. bifidum, Croatia 11.6 12.1 11.9 12.2 14 1.4 - G. klotho 11.8 12 11.9 12.2 14 1.1 0.5 0 A. ochripes 23.4 24 24 24 25.4 22.3 21.9 21.9

Distribution features. In the upper reach of the Cetina River altogether 46 caddisfly species were recorded at four stations (Tab 2). Dominating families were Limnephilidae and Sericostomatidae. Faunistically, the most important caddisfly species in the upper reach were Glossosoma discophorum and Ecclisopteryx ivkae. In the middle reach of the Cetina River, 22 caddisfly species were registered (Tab. 2). Faunistically, the most interesting species in the middle reach were T. braueri and C. albimacula (Tab. 2). In the lower reach of the Cetina River 36 caddisfly species were recorded (Tab. 2) Faunistically, the most interesting species in the lower reach were R. dorsalis plitvicensis, H. tineoides, A. dalmatinus and T. lefkas (Tab. 2). In the caddisfly fauna of the Ruda River, 22 species belonging to 14 families were recorded (Tab. 2). Faunistically the most interesting species of the Ruda River was Hydropyche dinarica (Tab. 2). At the spring of the Grab River 13 caddisfly species were recorded with 418 specimens (Tab. 2). Faunistically the most interesting species of the Grab River was also H. dinarica (Tab. 2). Since the Rumin River is only a bit longer than 1 kilometer, the research was conducted only at the spring, where 93 specimens belonging to 8 families were collected (Tab. 3), and faunistically the most interesting species was G. discophorum (Tab. 2).

Ecological features. Stations on the Cetina River were compared using the Shannon-Weaver (H`) and Simpson diversity indices for small samples (1-λ´). The Pielou evenness index (J') and the Margalef index (D) of population density were calculated. The greatest value of the Margalef index (D) of density was at the sation IC (5,027). Values of the Pielou evenness index ranged from 0.564 at station CS to 0.957 at station OM. The greatest value of the Shannon-Weaver diversity index was at station PM (2.144). Values of the Simpson diversity index ranged from 0.500 at station PR to 0.852 at station OM (Tab. 3). At the stations of the left tributaries of the Cetina River, the Shannon-Weaver index (H`), Simpson diversity index (1-λ´), Pielou evenness index (J') and the Margalef index (D) of population density were also calculated. Values of the Margalef index of density ranged from 1.544 at station GS to 2.806 at the RS. Values of the Pielou evenness index ranged from 0.473 at station IG to 0.796 at station RUS. Values of the Shannon-Weaver

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CADDISFLIES OF RIVER CETINA diversity index ranged from 1.250 at station GS to 2.052 at station RS. Values of the Simpson's diversity index ranged from 0.496 at station GS to 0.829 at station RS (Tab. 3). Results of the Sørensen similarity index showed that the similarity between the upper and middle parts of the Cetina River, in terms of the caddisfly community, amounts to 50% between middle and lower parts 45%, and between upper and lower parts 32%. The similarity between the spring of the Cetina River and the other study springs are as follows: spring of the Rumin River 31%, the spring of the Grab River 24%, and the spring of the Rumin River 27%. Results of the Sørensen similarity index based on the caddisfly community showed that the similarity between the springs of the Ruda and Grab rivers is 55%, between the springs of the Ruda and Rumin rivers is 33%, and between springs of the Grab and the Rumin rivers is 36%. In the Cetina River and its tributaries, 71 caddisfly species were recorded, and 28 of them are common to the Cetina and its tributaries, which gives them an index of faunistic similarity of 38.9% Cluster analysis (Fig. 2) and the method of nonmetrical multidimensional scaling (NMDS) (Fig. 3) were used in order to determine similarities in caddisfly communities based on the species for each river. Stress level of 0.11 in the MDS analysis corresponds to a good data ordination and enables good interpretation of data obtained. The cluster analysis showed separation (grouping) of stations in the upper part of the Cetina River - CS, PM and CM. Higher similarity was shown between stations CM and PM, while station CS was less similar to these stations. The PR station, also on the upper part of the Cetina River, showed a significant divergence from the other stations of that part. In the middle part, station TR was distinct, while the other station in the middle part, OS, was grouped with station RUS in its immediate vicinity. Stations in the lower part were also grouped together (RP, ČL, PAM and RM). Station OM in the brackish part of the lower course of the Cetina River was disctinct on its own. Stations on the Ruda River grouped together (RS and RII), with those on the Grab River (GS and GII) and the Rumin River spring RUS (Fig. 2).

Figure 2. Cluster analysis of caddisfly fauna similarity at stations on the rivers Cetina, Ruda, Grab and Rumin.

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Figure 3. MDS analysis of caddisfly fauna similarity at stations on the Cetina, the Ruda, the Grab and the Rumin rivers.

Out of all collected caddisfly species in the Cetina River catchment area, 62 species belong to the group of summer species. Ten species are present exclusively from September to November (Tab. 7), while in spring only one caddisfly species was recorded (Hydatophylax infumatus) (Tab. 7). Autumn species in this area were: P. cingulatus-latipennis, P. pallidus, S. meridiorientalis, M. fissa, M. sequax, M. testacea, Mesophylax impunctatus, Allogamus uncatus, and autumn-winter species were C. fusca and A. apfelbecki. The longest acitivity period was registered for the species R. f. delici, Hydropsyche cf. incognita and females from the genus Hydropsyche, T. braueri, L. lunatus, L. marmoratus, L. hirtum, Odontocerum albicorne and S. flavicorne, while only in one month we recorded the following species R. tristis, Hydroptila tineoides, L. bipunctatus, Glyphotaelius pellucidus, P. cingulatus-latipennis, P. nigricornis, P. pallidus, S. meridiorientalis, M. fissa, M. sequax, Allogamus uncatus, H. infumatus, C. fusca, O. testacea and A. filicornis (Tab. 7). The seasonal dynamics analysis showed that the greatest number of species was recorded in June (49 species), May (36 species) and July (33 species) (Tab. 7), and the lowest in the winter months (December and January) when only one species was recorded in each month. The majority of the species have a 4 – 6 months dynamics (Tab. 7). The most common species with respect to seasonal dynamics was R. f. delici registered during 11, followed by L. lunatus, registered during 8 months.

Discussion

Faunistic features. During this research 71 caddisfly species were recorded in the Cetina River catchment area (Tab. 2). Data obtained in our research were completed by data from the faunistic literature of H. Malicky (2014a), for the sampling station of Radmanove Mlinice, with the following species: Hydroptila sparsa Curtis, 1834, Hydroptila vichtaspa Schmid, 1959, Polycentropus irroratus Curtis, 1835, Tinodes pallidulus McLachlan, 1878, Leptocerus interruptus (Fabricius, 1775), Oecetis lacustris (Pictet, 1834), which makes the total number of caddisfly species for the Cetina River 77. Such a high number represents the highest number of species in the individual catchment area in the Mediterranean part of Croatia (Kučinić et al. 2011; Ridl et al. 2015; Šalinović 2019).

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Table 6. List of caddisflies with DNA barcoding data from the River Cetina: specimens ID marks in the Trichoptera DNA barcoding collection; Locality; BOLD Sequence ID.

Species Specimen ID Locality BOLD Sequence ID

Rhyacophila fasciata delici MNCN:ADN 96485 River Cetina, Vukovića spring MT559347 Rhyacophila fasciata delici MNCN:ADN 96486 River Cetina, Vukovića spring MT559348 Rhyacophila fasciata delici MNCN:ADN 103411 River Cetina, Brkljača channel, MT559358 Udovičić Rhyacophila fasciata delici MNCN:ADN 103412 River Cetina, tributary, Civljani MT559359 Glossosoma discophorum TGDIS_5 spring of the River Rumin CROAA064-18 Glossosoma discophorum TGDIS_7 spring of the River Rumin CROTR063-19 Glossosoma discophorum TGDIS_8 spring of the River Grab CROTR090-19 Agraylea sexamaculata TRIC_1 River Cetina, Čikotina Lađa CROTR093-19 Allotrichia pallicornis TAPAL_2 River Cetina, Čikotina Lađa CROTR092-19 Hydroptila forcipata THFOR_2 River Cetina, Čikotina Lađa CROTR095-19 Hydroptila forcipata THFOR_4 River Cetina, Radmanove Mlinice CROTR127-19 Hydropsyche instabilis THINS_3 River Cetina, Čikotina Lađa CROTR162-19 Hydropsyche instabilis THINS_6 spring of the River Grab CROTR091-19 Hydropsyche instabilis THINS_7 River Cetina, Čikotina Lađa CROTR151-19 Agrypnia varia TAVAR_2 Spring of the River Grab CROTR078-19

Silo piceus TSPIC_2 River Cetina, village Totići CROTR100-19 Silo piceus TSPIC_3 River Cetina, village Totići CROTR101-19 Lepidostoma basale TLBAS_3 spring of the River Grab CROTR122-19 Lepidostoma hirtum TLHIT_5 River Cetina, Radmanove Mlinice CROTR218-19 Lepidostoma hirtum TCALB_3 River Cetina, Radmanove Mlinice CROTR244-19

Ecclisopteryx ivkae TEIVK_1 River Cetina, spring Glavaš CROAA106-18 Glyphotaulius pellucidus TRBAL_3 River Cetina, spring Nela CROTR064-19 Limnephilus lunatus TLLUN_3 spring of the River Grab CROTR233-19 Athripsodes dalmatinus TADAL_1 River Cetina, kanal Brkljača CROAA066-18 Mystacides azurea TMAZU_3 River Cetina, Radmanove Mlinice CROTR126-19 Mystacides niger TMYS_3 River Cetina, Čikotina Lađa CROTR079-19 Oecetis testacea TONOT_6 River Cetina, Radmanove Mlinice CROTR135-19

With respect to other parts of Croatia where longlasting research has been conducted, a higher number of species was recorded only in the Dobra River catchment area, where 81 species were recorded (Cerjanec et al. 2020) and in the area of Plitvice Lakes NP, with 89 caddisfly species (Kučinić et al. 2017). According to the faunistic results from the research done in the Cetina River catchment area in the last few years, especially for the purpose of DNA barcoding, no new species were registered, which shows that this study, combined with results of Malicky (Malicky, 2014a), determined over 90% of potential caddisfly fauna of this area. Beside faunistic research, the Trichoptera of the Cetina River have also been the subject of taxonomic studies: description of larvae of T. braueri (Graf et al. 2008a), A. apfelbecki (Waringer et al. 2009; Previšić et al. 2014), description of a female of A. apfelbecki (Vučković et al. 2011), and the finding and species description of E. ivkae (Previšić et al. 2014). Faunistically, the most interesting species in the study area are: R. d. plitvicensis, G. discophorum, H. vichtaspa, A. sexmaculata, T. minor, E. ivkae, L. sparsus, P.nigricornis, H. infumatu, Leptocerus interruptus, T. lefkas, O. lacustris (Graf et al. 2008a; Kučinić et al. 2020a, 2020b; Malicky, 2014a; Previšić et al. 2014; Vučković, 2011; Waringer et al. 2009).

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The species A. sexmaculata, T. minor, L. sparsus, P. nigricornis, H. infumatus, L. interruptus and O. lacustris were recorded for the first time in the Mediterranean part of Croatia, and the Cetina River has been identified as the westernmost point of the distribution area of the species T. lefkas (Kučinić et al. 2020a). The finding of only one specimen of the subspecies R. d. plitvicensis during the 15 years of research into the Cetina River and its tributaries indicates that this subspecies, distributed in the central-mountainous part of Croatia (Kučinić & Malicky 2002; Kučinić et al. 2017), is perhaps not a regular member of Trichoptera fauna of the Cetina catchment, but was more likely transported here by car from the area of Plitvice Lakes and their surroundings, where it occurs naturally. According to our faunistic data, supported by data obtained by DNA barcoding, G. neretvae is distributed only in a small area on the Neretva River near Mostar (Marinković Gospodnetić 1988), while G. discophorum inhabits a much larger area (Cerjanec 2002; Marinković-Gospodnetić 1988). H. vichtaspa was recorded only at the locality RM (Malicky 2014a) and this is the only finding of this species in Croatia. According to Fauna Europaea (accessed on 12th May 2021) it is distributed in the southern parts of Europe (Fig. 4) (Malicky 2005; Fauna Europaea, 2021).

Figure 4. Distribution of species Hydroptila vichtaspa according to Fauna Europaea (accessed on 12th May 2021) with location of the first finding in Croatia.

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The faunistically and taxonomically important endemic species E. ivkae, described in 2014, occurs in study area (Previšić et al. 2014). Our research showed the presence of this species, not only in the spring area of the Cetina River (Previšić et al. 2014), but also a few kilometers downstream in the upper part of the Cetina (Vučković et al. 2016). In this research, the above mentioned species was recorded only in this area, and according to data from several other faunistic researches in Croatia (e.g. Cerjanec et al. 2020; Kučinić et al. 2014, 2017; Previšić et al. 2012, 2013; Previšić & Popijač 2010), where E. ivkae was not found, we can assume that the mentioned species is a microendemic, with a distribution only in the upper part of the Cetina River.

Molecular data. Regarding the DNA barcoded specimens from the Cetina River area, two species new for the BOLD database are especially interesting E. ivkae and R. fasciata delici. There are also interesting data considering the genus Glossosoma. A very interesting finding was the species G. discophorum, confirmed by DNA barcoding, recorded at the springs of the Cetina, the Rumin, the Ruda and the Grab rivers. There were certain indications (morphological characteristics of adults) that the spring of the Ruda River is inhabited by the species G. neretvae, but the analysis of collected material for DNA barcoding in the Cetina River catchment area, and of specimens of G. neretvae collected at the type locality in Bosnia & Herzegovina at the Neretva River, showed that this was undoubtedly the species G. discophorum (Kučinić et al. 2020b). This is visible from the maximum likelihood phylogram (Fig 5), which shows that G. discophorum and G. neretvae are forming different cluster as well as two distinct groups identified in ABGD analysis (Group 3 G. discophorum and Group 4 G. neretvae) with interspecific difference ranging from 6.4% to 6.7%. This interspecific difference is in the range of interspecific difference among caddisfly species noted in the literature (eg. 7.8% Barcelos-Silva et al. 2018) (Tab. 5, Fig. 5).

Figure 5. Maximum likelihood phylogram based on a fragment of COI (DNA barcode region) showing the related relationships of the genus Glossosoma. The bootstrap values (BS) are marked on the branches in the order NJ/ML. BS values less than 80 are not shown. The groups delineated by ABGD approach are shown on the right side of the tree. Specimens which genomic DNA was extracted in this study are written in bold letter.

There are also very interesting results regarding species G. klotho (Fig. 5) showing that in the ABGD analysis, specimens of this species from Kosovo and Bulgaria group with specimens of Glossosoma bifidum from Croatia and Italy into one species (Fig. 5). Genetic distance between species G. klotho and G.

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VUČKOVIĆ ET AL. bifidum is in range of intraspecific values recorded for the genus Glossosoma (0,51-1,54%, Ćukušić 2019; Zhou et al. 2010). Additional morphological and molecular analyses are needed to determine the exact taxonomical status of populations of G. klotho, as well as a comparison with specimens from Greece, as one of the type localities from which the species was described (simultaneously based on the material from Greece, Montenegro and Macedonia) (Malicky, 2003). We also DNA barcoded 27 specimens of 17 species of caddisflies from the area of the Cetina River and its tributaries (Tab. 6), and four specimens were used as paratypes in describing the subspecies R. f. delici (Valladolid et al. 2020, Tab. 6). The maximum value of uncorrected genetic distances between the larvae of the genus Glossosoma sp. TGDIS_5 and G. discophorum species is 0.6% (Tab 6), which is in the range of recorded intraspecific values in the genus Glossosoma (0.51-1.54% Zhou et al. 2010). Larvae of G. discophorum have not yet been morphologically described, and therefore are not included in the identification keys. So far, taxonomy has been able to identify the genus level of the larval stage. After phylogenetic analysis has confirmed beyond a doubt that specimen TGDIS_5 represents the species G. discophorum, the next step will be finding morphological characteristics specific for this species and a taxonomic description of the larva. Specimen BHMKK157-12 from Montenegro identified as G. neretvae according to p-distance differs from G. discophorum by 0.5% to 1.1%. That value is similar to the value of intraspecific difference for DNA barcode region recorded in this study (0-0.8%) and in other Glossomatidae (0.51-1.54%, Zhou et al. 2010) and another caddisfly (eg. 0.2% Graf et al. 2015). This indicates a possibility that BHMKK157-12 is taxonomically misidentified or there was an error in labeling the specimen. Molecular research of the species Potamophylax cingulatus-latipennis indicates a complex situation. Results based on the mtCOI gene DNA barcoding region point out the possibility that this is one species with several subspecies (Ćukušić 2019). According to the research in Croatia until now two taxa from the complex Potamophylax cingulatus-latipennis were recorded - Potamophylax cingulatus alpestris in the northern part of Croatia (Ćukušić 2019) and Potamophylax depilis from the area of Gacka River (Oláh et al. 2018). The exact (taxonomic) status of this species will be clarified by further research into integrative taxonomy which includes morphological and molecular analyses (Kučinić et al. 2013; Previšić et al. 2014; Santos et al. 2016).

Ecological features. The biodiversity of the caddisflies fauna at the study stations is conditioned by their biological characteristics; however, the interplay of suitable physico-chemical water parameters, general karst hydromorphology and diversity of microhabitats, most likely resulted in the rich and diverse caddisfly assemblages (e.g. Bonacci et al. 2020; Bonada et al. 2007; Cerjanec et al. 2020; Doledec & Statzner 2008; Habdija et al. 2002; Graf et al. 2008b; Moog 2002; Statzner 2008; Statzner et al. 2007, 2008; Šemnički et al. 2012; Urbanič et al. 2005; Waringer 1991). The greatest numbers of specimens collected on the Cetina River were at stations CS and RM, while in the Cetina tributaries the greatest numbers were at stations RS and RII (Tab. 2). At stations CS, RM and RS there are specific hydrological characteristics, because the substrate is very diverse, mostly covered with moss and pebbles, which make for a wide spectrum of microhabitats and hypersaturation (e.g. Graf et al. 2008b; Habdija 1988; Kučinić et al. 2007; Moog 2002; Urbanič et al. 2005). The values of diversity indices (Shannon-Weaver and Simpson) are the highest at stations in the upper part of the Cetina, PM and CM, and at the stations in the lower reach of the Cetina River, RM and PM, while the lowest values of diversity indices were measured for the station PR (Tab. 3). On the tributaries the highest values were at stations on the Ruda River RS and RII (Tab. 3). These results are specific for the researched area, as it is common that upper parts of rivers have lower diversity than the lower and middle parts (Kučinić 2002; Kučinić et al. 2017), while here the greatest diversity was recorded in the upper reach; the lowest in the middle reach and at PR (reservoir) station in the upper reach and the RP in the lower reach. These results are most probably a consequence of the significant anthropogenic influence, most prominent in the middle reach of the Cetina River (Vučković et al. 2009). Anthropogenic influence as an abiotic factor has a negative influence on aquatic and riparian ecosystems as large human population, unresolved waste waters, exploitation of water for drinking and generation of electricity, building roads and human settlements, and other operations in the environment irreversibly change the watercourse and the balance within the ecosystem (Boyle & Fraleigh 2003; Nedeau et al. 2003; Neumann et al. 2003a, 2003b), while changes in biological conditions lead to reduced diversity and abundance within the aquatic ecosystem (Běche et al. 2006). Research conducted on the caddisfly larvae from the Cetina River also showed the greatest caddisfly abundance and diversity at RS and RM (Vučković, 2008), while at station CS there was lower number of

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CADDISFLIES OF RIVER CETINA caddisfly larvae than of the imago stage. High numbers of caddisflies in the spring parts of the Cetina and the Ruda rivers were expected, but the high numbers of species recorded in the lower part of Cetina at the station of RM were surprising; it was influenced by specific geological and hydrological characteristics at this locality (Graf et al. 2008b; Kučinić et al. 2017; Stanić-Koštroman et al. 2012, 2015), where the habitat of the Cetina River becomes more similar to the upper parts of watercourses due to the gravelly bottom, hypersaturation and large amounts of moss. Research into other groups of aquatic insects (e.g. Plecoptera: Popijač & Sivec 2009, 2010 and Ephemeroptera: Vilenica et al. 2016a, 2016b) in the Cetina River catchment area confirm their relatively high number at these stations. Water temperature is another major factor structuring the composition of caddisfly assemblages (e.g., Graf et al. 2008b; Moog 2002; Ridl et al. 2015; Šemnički et al. 2012). The numerous lateral springs (UNEP/MAP/PAP 2000) along the drainage probably have a strong influence on water temperature. In the MDS analysis in the Cetina River catchment area, certain stations were grouped together according to the faunistic characteristics of caddisflies. The analysis grouped the upper stations IC, PM and CM, as well as those in the lower course: reservoir AP, ČL, PAM and RM, while stations PR and OM were distinct from them, which was expected (Fig. 3). The PR station is distinct because of the small number of recorded caddisflies, caused by anthropogenic influence, i.e. the building of the dam and the formation of the reservoir, which led to significant hydromorphological changes where part of the watercourse became stagnant, while station OM is distinct because of the small number of caddisflies collected and the strong influence of the sea. Station TR, in the middle part of the watercourse, also stood out because of the anthropogenic influence (it is located 500 m below the city of Trilj’s wastewater discharge). Sampling station OS in the middle part of the Cetina River has the characteristics of stations from the upper part of the watercourse, and it was grouped with station the Ruda. River flows into the Cetina River a few kilometers upstream. Stations on the Grab River, GS and GII, grouped together because they are very close to each other, as are stations on the Ruda River, RS and RII. The distance between those two springs is 5 km. They differ in hydrological features, since the Ruda River receives significantly more water from Buško blato, which makes its spring significantly richer than the spring of the Grab River which also receives majority of its water from Buško Blato; they grouped close together with a similar caddisfly fauna (Fig. 2, 3).

Sesonal distribution features. The activity of adult caddisfly stages, i.e. their biological cycle, differs very much from species to species. Beside biological features and peculiarities of each species, genus or family, life cycles are influenced by various environmental factors, from physical-chemical characteristics of certain parts of a watercourse, climate, to surrounding vegetation, and to various relations between species in the watercourse and species around it (Malicky 2004; Kučinić et al. 2017; Previšić et al. 2007a). Croatia lies in the temperate zone in which the majority of collected caddisflies emerge during the warm months of the year (end of spring, beginning of summer) (Kučinić 2002; Kučinić et al. 2011, 2017; Previšić et al. 2007a; Stanić-Koštroman et al. 2015), as confirmed by this research into the Cetina River catchment area. The majority of caddisfly species were recorded in May and June, when also the greatest abundance was noted. Some species were recorded only once, while others were recorded in many months (Tab. 7). In most months (in 11 months altogether) the species R. fasciata was identified, which is in line with the results of previous research (Kučinić et al. 2011, 2017; Previšić et al. 2007a) as well as the hypothesis presented by Otto (1981) according to which Trichoptera species with predatory larval types have a very long emergence period (Otto 1981) and occurrence in a larger number of months throughout the year. Beside spring-summer and summer species in the Cetina River catchment area we registered a species whose imago stage shows autumn and autumn-winter activity. This is a caddisfly species characteristic for springs, Annitella apfelbecki, recorded only in October, November and December and only at CS (Vučković et al. 2011; Waringer et al. 2009). The appearance of certain groups of hexapoda (Collembola, Diptera, Coleoptera, Lepidoptera, Plecoptera and Trichoptera) in winter is very interesting and requires a lot of species adaptation to very low air temperatures in which they have to move, reproduce, lay eggs and perform other metabolic functions (Hågvar 2010; Szivák et al. 2017). Regarding the ethology of each group, it is important to emphasize in which area the research is conducted, because there are very big differences in e.g. temperate, subtropical and tropical areas. Winter in the temperate zone requires special adaptations, also visible in Trichoptera (Hågvar 2010). Many of their

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VUČKOVIĆ ET AL. genera and species evolved in a cold period, and they have maintained the feature of being active in low temperatures. It should also be emphasized that during that period there are far fewer predators around (Otto 1981; Previšić et al. 2007a), so causes for that type of behaviour can be found in ethology which has most likely conditioned the evolution of these taxa.

Table 7. Seasonal dynamics of caddisflies by months in the researched area of the Cetina River and its left tributaries the Ruda, the Grab and the Rumin rivers.

Season Autum Winter Spring Summer Species/Month IX X XI XII I II III IV V VI VII VIII Rhyacophila balcanica ● ● ● ● ● Rhyacophila dorsalis plitvicensis ● Rhyacophila f. delici ● ● ● ● ● ● ● ● ● ● ● Rhyacophila tristis ● Glossosoma discophorum ● ● ● ● ● Synagapetus karawany ● ● Hydroptila tineoides ● Agraylea sexmaculata ● ● ● Wormaldia subnigra ● ● ● ● ● Wormaldia occipitalis ● ● Hydropsyche dinarica Hydropsyche incognita ● ● ● ● ● ● ● Hydropsyche instabilis ● ● ● ● Hydropsyche pellucidula ● ● Hydropsyche sp. (♀♀) ● ● ● ● ● ● ● Plectrocnemia conspersa ● ● ● ● ● Polycentropus flavomaculatus ● ● ● ● ● Polycentropus schmidi ● ● Cyrnus trimaculatus ● ● ● ● ● Psychomyia klapaleki ● ● ● ● Lype reducta ● ● ● Tinodes braueri ● ● ● ● ● ● Tinodes dives ● ● ● ● ● Tinodes waeneri ● ● ● Trichostegia minor ● ● Ecclisopteryx ivkae ● ● ● ● Limnephilus affinis ● ● Limnephilus auricula ● ● Limnephilus bipunctatus Limnephilus flavicornis ● ● ● ● ● ● Limnephilus griseus ● ● Limnephilus hirsutus ● ● Limnephilus incisus ● ● Limnephilus lunatus ● ● ● ● ● ● ● ● ..continued on the next page

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TABLE 7. Limnephilus marmoratus ● ● ● ● ● ● Limnephilus rhombicus ● ● ● Limnephilus sparsus ● ● Limnephilus vittatus ● ● ● ● ● Glyphotaelius pellucidus ● Grammotaulius niropunctatus ● ● ● Potamophylax cingulatus-latipennis ● Potamophylax nigricornis ● Potamophylax pallidus ● Halesus digitatus ● ● ● ● ● Halesus tesselatus ● ● Mesophylax impunctatus ● ● Stenophylax meridiorientalis ● Stenophylax permistus ● ● ● ● Micropterna fissa ● Micropterna nycterobia ● ● ● ● Micropterna sequax ● Micropterna testacea ● ● Allogamus uncatus ● Hydatophylax infumatus ● Chaetopteryx fusca ● Annitella apfelbecki ● ● ● Silo piceus ● ● ● ● ● Lepidostoma basale ● ● Lepidostoma hirtum ● ● ● ● ● ● ● Athripsodes albifrons ● ● ● ● ● Athripsodes dalmatinus ● ● ● ● Ceraclea albimacula ● ● ● Ceraclea dissimilis ● ● ● ● ● Mystacides azurea ● ● ● ● ● Mystacides nigra ● ● ● Triaenodes lefkas ● ● Oecetis testacea ● ● Adicella filicornis ● Adicella syriaca ● ● Odontocerum albicorne ● ● ● ● ● Sericostoma flavicorne ● ● ● ● ● ● Beraemyia schmidi ● ● Total number of species 30 22 12 2 4 1 8 11 35 49 32 24

Conservation issues. In the Balkan Peninsula, including some areas in Croatia, considerable numbers of hydropower plants have been planned, many of which are on mountain springs or in spring areas due to their natural hydrological potential. The construction of hydroelectric facilities can cause changes in

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VUČKOVIĆ ET AL. morphological and hydrological characteristics at the construction station; consequently, the changed environmental conditions may lead to the endangerment or even loss of certain species. The protection of these habitats is possible, among other things, through protection of endemic and rare species in this area (Duplić et al. 2015; Mrakovčić et al. 2004; Previšić et al. 2014; Šašić et al. 2015; Vučković et al. 2016).

Conclusion

Faunistic features of the Cetina River catchment area are conditioned by its geographical position and relative isolation due to the mountain massifs of Mt Svilaja in the west and Mt Dinara and Mt Kamešnica in the east. In this study we have established the characteristics of the Trichoptera fauna of the Cetina River and its tributaries, as an initial state for all aspects of future research. The results obtained are also very valuable in the context of the newly founded Dinara Nature Park, which encompasses parts of the upper course of the Cetina River. Because of the significant anthropogenic influence, in the future adequate protection of the entire course of the Cetina River should be ensured, at least at the level of the Nature Park. The DNA barcoding method has proved to be indispensable in a modern approach to faunistic and taxonomic research (e.g. Kučinić et al. 2020b; Ruiter et al. 2013; Santos et al. 2016; Valladolid et al. 2020).

Acknowledgements This research was supported by funding from the Croatian Waters, the University of Zagreb Natura 2000 integration project and the scientific project “DNA barcoding of Croatian faunal biodiversity” (IP-2016-06- 9988) funded by the Croatian Science Foundation.

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