The genus Dixa (Diptera, Dixidae) in Croatian lotic habitats, with a checklist of species and relationships with the fauna of neighbouring countries

Abstract Invertebrate surveys in Croatia conducted between 2005 and 2018 included 39 sampling sites yielding bycatch samples of Dixidae (Diptera). All records of this family from the territory of Croatia are summarized, including previously unpublished data. Collections contained six species of Dixa Meigen – D. dilatata Strobl, D. maculata Meigen, D. nebulosa Meigen, D. nubilipennis Curtis, D. puberula Loew, and D. submaculata Edwards, with Dixa dilatata reported from Croatia for the first time. Information relating to the ecoregions in which species were found and specific species traits are provided. Compared to neighbouring countries, the Croatian species assemblage is most similar to the fauna of Italy and least similar to that of Serbia and Montenegro.


Introduction
The Dixidae, or meniscus midges, are one of the smallest families of Diptera in Europe, with only two genera, Dixa Meigen and Dixella Dyar and Shannon, and 32 species recorded (Oosterbroek 2007;Pape and Beuk 2012). Approximately 190 species are recognized worldwide (Wagner et al. 2008;Pape et al. 2011; Moulton 2016Moulton , 2017. They are nematocerous flies belonging to the superfamily Culicoidea, which also includes the Culicidae, which they most closely resemble (Wiegmann et al. 2011;Borkent 2012). Adults are small, frail, and do not feed. They remain near their biotopes (streams, ponds), and rest in the vegetation. Males of some species form swarms. Eggs are deposited in masses at the water's edge, and the life cycle includes four larval instars and the pupa. The larvae are filter feeders that rest on the water surface where they take on a characteristic, reversed U-shaped posture. Pupation takes place on emergent substrates. Larvae are feeding on microorganisms and decaying plant or animal material trapped in the water column or on the surface film (Wagner et al. 2008, Wagner 2011. Larvae of Dixa prefer running water, while those of Dixella occur in both stagnant or slow-moving water (Oosterbroek 2007). Some species are restricted to bog or mesotrophic lakes and are appropriate bioindicators. Species diversity is highest in springs and in headwater streams (Wagner et al. 2008). Dixid larvae are sometimes a significant component of invertebrate drift in streams (Elliott and Tullett 1977;Sertić Perić et al. 2014). They are extremely sensitive indicators of the presence of surfactant or oil-borne pollutants in streams (Thomas 1979). Larval mortality increases with decreasing surface tension of water (Fowler et al. 1997). Disney (1999) published an exceptionally fine compilation of West Palaearctic Dixidae that can be used worldwide as a basic information resource.
So far there have only been two studies dealing with Dixidae in Croatia, and the only records are those in Ivković et al. (2017) from the Krka River and  from Plitvice Lakes National Park resulting from a study of the emergence patterns and ecological preferences of Dixidae.

Materials and methods
Study site. Croatia is a relatively small country with a surface area of 56,594 km 2 situated at the crossroads of Central and Mediterranean Europe and the Balkan Region. It is divided into two ecoregions, the Dinaric western Balkan (ER5) and the Pannonian lowland (ER11) (Illies 1978), and forms part of two drainage basins, the Black Sea Basin and the Adriatic Sea Basin.
Specimen records. This paper is based on unpublished data from our own research and published data gleaned from the literature. Each record was georeferenced using ArcGIS software. The literature used for identifications included Shtakel'berg (1989) and Disney (1999). We followed the current classification of Pape and Beuk (2012). Locality records are listed for each species. A list of locality names including latitude, longitude, altitude, and number code for each locality is given in Table 1, and a map with all sites plotted is provided as Figure 1. Specimens were collected from lotic freshwater habitats throughout Croatia. Adult specimens were collected using emergence traps (details in Ivković et al. 2013), sweep nets, yellow pan traps and aspirators, whereas larvae were collected by Surber sampler (25 × 25 cm) and kick-net sampler (25 × 25 cm). Larval samples were collected as a result of several macroinvertebrate surveys conducted between 2005 and 2018. Specimens were preserved in 80% or 96% ethanol (EtOH). For identification of adults, male and female terminalia were dissected, if needed. In some cases, terminalia (and preceding abdominal segments) were cleared in 10% KOH, neutralized with acetic acid, and rinsed in water to improve visualization and facilitate identification. For larvae, all available structural characters were used for identification. Taxonomic diversity was considered at the level of species according to Pape and Beuk (2012). European ecoregions were defined according to the Limnofauna Europaea (Illies 1978). Data analysis. A list of species was compiled from all specimen data ( Table 2). Comparison of species richness and assemblage composition with surrounding countries (Italy, Hungary, Serbia and Montenegro) was conducted by compiling species lists for those countries taken from the Fauna Europaea (Pape and Beuk 2012). Slovenia and Bosnia and Herzegovina were not included in the comparison matrix, as there are no Dixa species records (Pape and Beuk 2012). A species-by-country matrix was constructed and a Sørensen Index of Similarity of each pairwise comparison calculated using Primer v6 software (Clarke and Gorley 2006).  Table 1 for codes). Table 2. Croatian Dixa. Species niche traits. Key: habitat type 1 = spring or eucrenal zone, 2 = stream, 3 = river, 4 = tufa barrier (barrage lake outlet); voltinism U = univoltine, B = bivoltine, M = multivoltine; occurrence Sp = Spring, Su = Summer, A = Autumn, AYR = all year round;. distribution in Europe wd = widely distributed. NA -not applicable. European Ecoregions are taken from Illies (1978); Dinaric western Balkan (5), and Pannonian lowland (11).

List of Dixa species of Croatia
The following format is used for the distribution data: literature references (name of the site and in parentheses the citation of the reference and site ID); new records (life stage in which the identifications were made, i.e., adult ♂, ♀ and larvae, name of the site and in parentheses the site ID, date of collection and the collector). All the sites and their numbers are listed in Table 1.

Species richness and assemblage composition
In total six species of Dixa (Table 2) are recorded from Croatia, collected from 39 sites (Fig. 1, Table 1). Dixa nebulosa is found at the greatest number of sites (25) while Dixa dilatata was the rarest, found only at two sites. All six species occur in the Dinaric western Balkan (Ecoregion 5), while four species occur in the Pannonian lowland (Ecoregion 11). All recorded Dixa species are widely distributed in Europe ( Table 2). Six of the 12 recognized Dixa species in Europe (Pape and Beuk 2012) are now reported from Croatia. Some of the species, e.g., Dixa puberula and D. nebulosa, may eventually prove to be members of a species complex, rather than a single species (J.K. Moulton and R. Wagner, pers. comm.). Available seasonal phenological data (Table 2) revealed two species (Dixa puberula and Dixa submaculata Edwards) are multivoltine and present all year round. Dixa maculata and D. nebulosa occurred from spring through autumn. Dixa nubilipennis was only collected during summer.
Italy and Hungry have seven and five, respectively, recorded species of Dixa, while Serbia and Montenegro each have only a single recorded species, Dixa nebulosa (Fig.  2). The Sørensen Index of Similarity showed that the Dixa fauna of Croatia is most similar to that of Italy followed by Hungary, whereas it is least similar to that of Serbia and Montenegro (Table 3). These results were to be expected due to the low number of species recorded for Serbia and Montenegro.
Comparing our list of Croatian species with published records in the Fauna Europaea (Pape and Beuk 2012) revealed that none of the six species treated here were previously recorded from Croatia until Ivanković et al. (2019) reported D. maculata, D. nebulosa, D. nubulipennis, D. puberula, and D. submaculata. Herein, we report Dixa dilatata as new to the dixid fauna of Croatia.

Concluding remarks
All the recorded species have a wide European distribution and none is restricted to Croatia or to the Balkan Region. There may be a few more species of Dixa yet to be recorded, and, because of the high endemicity of the Dinaric area (Ivković and Plant 2015) and especially of the aquatic Diptera (Ivković et al. 2012;Pont and Ivković 2013;Kvifte et al. 2013;Kvifte and Ivković 2018), it is possible that undescribed species of Dixa may yet be found. In the future, collecting should be focused not only on lotic habitats but also on lentic habitats so that Dixella species can also be studied.
samples from which Dixidae larvae were isolated. We are especially grateful to reviewer J.K. Moulton and subject editor Art Borkent for improving the manuscript with their helpful comments and corrections.