A FIRST RECORD OF EPHORON VIRGO (OLIVIER, 1791) (EPHEMEROPTERA: POLYMITARCYIDAE) FROM THE SAVA RIVER, WITH NOTES ON ITS ECOLOGICAL PREFERENCES AND RARITY OF FINDINGS IN THE REGION

Ephoron virgo (Olivier, 1791) is an European burrowing filter-feeding mayfly species. Once common in large rivers, in XX century it became rare due to increased negative anthropogenic influences. In Serbia there was only one recent report of its presence, from the Danube River (Iron Gate area). The aim of this paper is to report on the first finding of E. virgo from the Sava River, and to discuss its scarce findings in the region. Three larval specimens were found in the Belgrade region in grab sample taken in 2014. In order to assess the development of the population in the region, a comprehensive investigation should be conducted.


Introduction
Ephoron virgo (Olivier, 1791) is one of only two European representatives of world wide distributed group Polymitarcyidae (pale burrowers). The species is considered as a Siberian (or Amur-Siberian) faunistic element, probably of polycentric origin, distributed throughout Palaearctic, with records in Northern Africa, Asia Minor, Europe (except British islands, while in Fennoscandia and the Baltics it has been replaced by the other species Ephoron nigridorsum (Tshernova, 1934)) and the Eastern Palaearctic (Bauernfeind & Soldan 2012). This large potamal and hiporhitral mayfly has univoltine life cycle, either of winter (overwinters in the nymphal stage) or summer (overwinters in the egg stage) type (Clifford, 1982). In western Palaearctic (Europe) the univoltine summer type seems to be more common, where the most of the lifetime (up to 9 months including winter) is spent in the egg with a dormant embryo. Larvae grow within 3-4 months, while as an adult it lives only a few hours during the night. Subimago emerges in late summer (from the end of the July to the middle of September) and the flight activity is positively correlated with the air temperature (Ibanez et al, 1991;Kureck 1996;Haybach et al, 2004;Kureck & Seredszus 2007). The adults are strongly Ecologica Montenegrina 13: 80-85 (2017) This journal is available online at: www.biotaxa.org/em attracted by light traps (Bauernfeind & Soldan 2012). The larvae (body length 12 -20 mm) are of the "burrowing" type with pronounced mandibular tusks projecting far beyond anterior head margin, with numerous tubercles and prominent spines on the upper surface. They have frons with a pronounced, conical, protuberance mediodistally (longer and thiner than in E. nigridorsum) and frontoclypeus with a transversal row of long, bipectinate bristles. More morphological details are provided in literature (Nilsson, 1996;Oscoz et al, 2011;Bauernfeind & Soldan 2012). During their investigation of this species in Germany (the Rhine and Main rivers) Schleuter et al (1989) found that the larvae live in the littoral zone (down to a depth of 6 m) and prefer mixed substrate of coarse and fine material. The larvae construct U-shaped burrows and feed on small organic particles (seston) including bacteria and algae. and pumping water through their burrows and filtering small particles, they enhance aerobic microbial activity by oxygenating river sediments (Cid et al, 2008;Kureck et al, 2014).
E. virgo disappeared from many European rivers in the XX century, but returned in the late XX and the beginning of XXI century, with the improvement of water quality , becaming a symbol for the recovery of polluted rivers. In the Danube basin numerous are reported recent findings of this mayfly e.g. from the Danube (Slovakia: Elexová, 1998;Adamek et al, 2007;Hungary: Szaz et al, 2015;Bulgaria: Vidinova & Russev, 1997), and the Tisza (Hungary: Galdean, 1997;Kovacs et al, 2001). In Serbia, however, the findings of this unmistakable mayfly species are scarce. According to our knowledge the taxon was recorded only once, in the Danube (Iron Gate area) (Paunović et al, 2005). In the recent update of check-list of Ephemeroptera from Serbia (Petrović et al, 2015), the presence of this taxon was reported from "Danube small direct tributaries" without any additional specifications.
Herein we report a new finding of E. virgo from Serbia which is the first record of this species from the one of the largest Danube tributaries, the Sava River.

Material and Methods
As a part of yearly investigation of large rivers in the Belgrade region, aquatic macroinvertebrates samples were taken on June 29 th , 2014. at the sampling locality "Duboko" situated on the right riverbank of the Sava River (25th rkm) (Fig. 1). Sampling was conducted by Van Veen grab sampler in littoral zone (5-8 m from the riverbank, an average water depth of 3-5 m). Samples were preserved in 95% ethanol and taken to the laboratory of the Department of Hydroecology and Water Protection of Institute for Biological Research "Siniša Stanković" Belgrade for further processing. The main physico-chemical parameters of water were measured in situ (pH, T, conductivity and dissolved oxygen), while selected nutrient concentrations were measured in the laboratory (Table 1). The investigated river stretch could be described as a free flowing, less impacted by hydromorphological degradation than other sectors of the Sava River and relatively natural in respect to riparian vegetation characteristics, with greenbelt of Populus and Salix trees on the bank. In the close vicinity settlements of Umka (downstream) and Bariĉ (upstream) are situated, while upstream there are small townObrenovac (with TP plant Obrenovac) and mouth of the Kolubara River (the right Sava tributary). As the main treats to the locality communal and industrial wastewaters and influence of TP Obrenovac could be singled out. The Kolubara River, which flows throughout one of the larger Serbian coal-mining areas, could deliver additional coal-based minning pollution. River bottom/sediment near the bank (3 to 5 m depth) consists mainly of mud and clay substrate.

Results and discussion
During processing of samples taken from the "Duboko" locality at the Sava River near Belgrade, three larval specimens of E. virgo (Fig. 2) were found. Besides findings from the Danube (upstream the Iron Gate; Paunović et al, 2005), this finding represents the second confirmed report of this species from Serbia, and the first finding of this mayfly species from the Sava River Basin. Collected specimens are young larvae, about 7 mm in length and with very short wing pads (Fig. 2).This burrowing mayfly is considered as typical for a clay bottom of potamal rivers (Vidinova et al, 1997), but could be found also on the sand, gravel, mud and corophium mud (Ibanez et al, 1991;Vidinova et al, 1997). Our finding is in accordance with these literature data. The finding at some distance from shore (water depth 3 to 5 m), corresponds with similar reports from Spain (Ebro River), where it was found that the species prefer more stable, deeper river zones (Ibanez et al, 1991). Such more stable habitat is important in cases of often and/or extreme water-level fluctuations, such as was case with the Sava River in the 2014 with spring floods, and generally higher and more fluctuating water-level (Hydrological Yearbook, 2014). As a majority of mayflies E. virgo has rather narrow abiotic valences/requirements, as being sensitive to various pollutants, particularly organic/eutrophic conditions (Oscoz et al, 2011). Due the pollution in European rivers from the middle of the XX century, a significant decline of its populations was recorded. This species is probably even more sensitive because of its burrowing life style in sediment, which, although being more stable environment than water-column, acts as a collector of various pollutants, especially heavy metals (Milenković et al, 2005). Various studies (e.g. Greve et al, 1999;van der Geest et al, 2000;Nguyen et al, 2012) pointed to sensitivity of this mayfly to some heavy metals (e.g. lead (Pb)) and thus applicability of the species in sediment toxicity assays. However, the sediment pollution rarely is only limiting factor affecting more sensitive insects/biota (de Haas & Kraak, 2008). Regarding other limiting factors, a more aerated environment preference (with 5.55 mg/l O 2 as lower threshold; Vidinova et al, 1997) of this potamal burrowing species, should be mentioned. Additionally, in case of short-living adults, problem could arise from light-pollution in urban areas. Szaz et al (2015) found in their investigation in Hungary particularly negative impact of lights on bridges. As the Duboko locality characterizes well aerated environment (Table 1), and with low organic pollution (except ammonia (NH 4 )), it is clear that this locality meets the main abiotic (physic-chemical) requirements as the habitat for E. virgo. Moreover, as this site has relatively low light pollution it represents a favourable environment for adults of this mayfly as well.
Besides sensitivity to pollution, interactions with invasive species could influence this species. A large predatory amphipod Dikerogammarus villosus (Sowinsky, 1894) nowadays is one of dominant gammarids in the largest Serbian waterway -the Danube River (Paunović et al, 2007;Borza et al, 2015), and present in other larger rivers such as the Tisa (Zorić et al, 2015) and the lower parts of the Sava (Paunović et al, 2012) and the Velika Morava (Marković et al, 2015). Kureck et al. (2001) found a significant decrease of the E. virgo in presence of D. villosus because of predation (from Haas et al, 2002). Although spreading upstream the Sava River, D. villosus still has not reached such abundance and dominance as in the Danube, the Sava River could be considered as a more favourable environment for these mayflies. A competition for resources, particularly food, with aggressive and often in larger rivers very abundant invasive filtrators, such as bivalvs (e.g. Corbicula fluminea (Müller, 1774), Sinanodonta woodiana (Lea, 1834)) and crustaceans (e.g. Chelicorophium curvispinum (Sars, 1895)), could be another reason for declining of E. virgo (Kureck et al. 2014). The food competition often is considered as one of the most important factors limiting distribution of this species, although Kureck et al (2014) clearly stated importance of various environmental factors synergy.
Serbian large rivers (Danube, Sava, Tisa and Velika Morava), as potential habitats for E. virgo could be considered as a rather hostile and unfavourable environments in terms of previously discussed limitations. All these rivers, and particularly the Danube, are under heavy anthropogenic pressures (pollution, hydrological alterations, invasive species etc.). As more favourable habitats for this species remain a few "ecological refugia", such as the Duboko locality on the Sava River. In order to assess the development of the populations in the region, a comprehensive investigation should be conducted.