LOCAL ENDEMIC AND THREATENED FRESHWATER HYDROBIIDS OF WESTERN GREECE: ELUCIDATION OF ANATOMY AND NEW RECORDS

Islamia trichoniana Radoman, Pseudoislamia balcanica Radoman and Trichonia trichonica Radoman are local endemics and threatened hydrobiids living in Lake Trichonis, Western Greece. The rough initial description of their reproductive organs (i.e. penis, bursa copulatrix and receptacula seminis) is supplemented with new data. The radulae of I. trichoniana and P. balcanica are described in detail for the first time. New records of the three species are given and preliminary data about their feeding regime are provided. KeywoRds: Islamia trichoniana, Pseudoislamia balcanica, Trichonia trichonica, freshwater periphytic diatoms, Lake Trichonis


INTRODUCTION
The Mediterranean Basin Biodiversity Hotspot (MBBH) is well known for its globally important freshwater biodiversity; the freshwater bodies of Greece are part of its Balkan sub-region supporting some of the most diverse and heavily threatened ecosystems (daRwall et al. 2014).However, the freshwater fauna of Greece, especially Truncatelloidea, remains poorly inventoried; the distributional information on endemic and threatened species is often out of date or unavailable (Radea et al. 2013, daRwall et al. 2014).
It is known that in brackish and marine systems the Hydrobiidae are regarded as deposit feeders and/or herbivores feeding on periphytic algae (e.There are no detailed data on the diet of the freshwater Hydrobiidae based on diatoms, though diatoms are among the dominant members of the littoral microalgae communities constituting the phototrophic biofilms in both lotic and lentic freshwater habitats (molIno & wetheRBee 2008, sheath & wehR 2015).
The aim of this study was to expand the knowledge about the soft body anatomy and distribution of the threatened local endemics I. trichoniana, P. balca nica, and T. trichonica and to provide preliminary data on their food items.

MATERIAL AND METHODS
Etoloakarnania has an extensive aquatic system in Western Greece including natural and artificial lakes, rivers, lagoons, and springs.Seven localities in Lake Trichonis and adjacent springs and streams were selected for sampling (Table 1) in the spring and autumn of 2014, 2015 and 2016.The snails were hand-collected from stones, gravel, mosses and dead leaves, and were fixed 'unrelaxed' in 70% ethanol for safer preservation of radulae and reproductive organs.Before dissection, the shells were removed by soaking in Pereny's solution.Radulae were cleaned with KOH solution (5 g l -1 ) at room temperature, rinsed in distilled water and air-dried before being mounted on stubs and spray-coated in gold-palladium for Scanning Electron Microscopy (SEM Jeol JSM-35 operating at 25 kV).Digital pictures of reproductive organs were taken using a Canon EOS 1000D camera attached to a stereomicroscope Stemi 2000-C, Zeiss.During this procedure, the specimens were submerged in water in order to avoid distortion of important taxonomic features that could be caused by the long-term tissue preservation buffers.
The digestive system contents were analysed by isolating both the intestinal faecal pellets and the stomach of the hydrobiids examined.For detailed examination, faecal pellets and stomach of four specimens fixed in ethanol (one I. trichoniana, one T. trichon ica, and two P. balcanica, one from Lake Trichonis and one from Aghia Sophia springs) were extracted and treated separately on microscope slides.A few drops of 50% hydrochloric acid (HCl) were added to eliminate any ingested carbonates (limestone, shell fragments).Then, a few drops of hydrogen peroxide H 2 0 2 (30%) were added to oxidise the organic matter (stomach walls, algal cell contents and any organic debris).The slides were left in Petri dishes for two days in direct sunlight (for faster organic oxidation); from time to time hydrogen peroxide was added to avoid drying.Digital pictures of diatoms were taken using an AmScope MU503 Real-Time Live Video Microscope Digital Camera attached to a Carl Zeiss Axiolab E re Microscope.

DIATOM FOOD ITEMS IN STOMACH CONTENTS AND FAECAL PELLETS
The diatom Cocconeis placentula Ehrenberg sensu Jahn et al. (2009) (Figs 26,28,29) was abundantly ingested by P. balcanica (Aghia Sophia) and in smaller quantities by T. trichonica (Lake Trichonis).A few valves of Cyclotella trichonidea var.parva Economou-Amilli (Fig. 27) and Mastogloia elliptica (Agardh) Cleve (Figs 30,31) were found in the digestive system of T. trichonica (Lake Trichonis).No diatom remains were observed in the digestive system of I. trichoniana and P. balcanica from Lake Trichonis.Our knowledge about the internal morphology of the three endemic species I. trichoniana, P. balca nica and T. trichonica is mainly based on the short descriptions and drawings of Radoman (1973Radoman ( , 1978Radoman ( , 1983)).The detailed description of both the unknown radu lae and the poorly known reproductive organs of I. trichoniana and P. balcanica presented here supplement the species descriptions.

Islamia trichoniana
The genus Islamia is characterised by the presence of either one or two pairs of basal cusps on the central radular tooth (Radoman 1983, Bodon et al. 2001).I. trichoniana is included among those having a single pair of basal cusps on the central tooth; this character state is also observed in the European I. at teni, I. gaiteri, I. globulus, I. valvataeformis and Islamia sp.(Bodon et al. 1995, 1996, 2001, aRConada & Ramos 2006).In the former Rissooidea, the single pair of basal cusps was assumed to be plesiomorphic (PondeR 1985).
The reproductive organs of I. trichoniana were not described by Radoman (1978Radoman ( : 25, 1983: 127): 127).He only mentioned that the penis of this species was similar to that of Islamia graeca Radoman, 1973, a species described from a lake (L.Amvrakia) near Lake Trichonis.However, this is not confirmed by our findings.The penis of I. graeca bears "small, short branches and a large fold on the ventral side" according to Radoman (1983: 125, fig. 70C).On the contrary, the penis of I. trichoniana has no short branches and it bears a large penial lobe overgrowing the penis apex proper; the penial duct is clearly visible and the muscular pleat ("fold" according to Radoman) is of medium size.The muscular pleat on the ventral side of penis and the well-developed penial lobe show that I. trichoniana belongs to the "oriental" group of Islamia species which live in the Balkan Peninsula, Turkey and part of Italy (Bodon et al. 1995).

Pseudoislamia balcanica
The central radular tooth of P. balcanica bears a single pair of basal cusps (similarly as in I. trichoniana).
The cusps of the central and lateral teeth are narrower and more pointed compared to those of I. tricho niana (present study) and T. trichonica (szaRowsKa 2006: 112, fig. 130).
The male and female reproductive organs differ from those described and illustrated by Radoman (1978Radoman ( : 23-24, fig. 1, 1983: 83-84, fig. 44): 83-84, fig. 44); namely, the pointed penial apex, the lower position of the outgrowth, the presence of a grey-black pigmented area on the dorsal side of penis, as well as the larger size and the semi-globular shape of bursa copulatrix are notable differences.
During the present study, P. balcanica was found in two new localities, a stream close to Pantanassa on the N. shore of Lake Trichonis and a spring 20 km farther (in Varasova).Additionally, it was re-found in the springs of Aghia Sophia, where it had been recorded for the first time by Radea et al. (2013).The number of the species' localities is now four.These records corroborate the earlier suggestion (Radea et al. 2013) that P. balcanica is more widespread than previously thought and that the species inhabits both lentic and lotic waters.
It is remarkable that in the majority of specimens from Lake Trichonis the reproductive organs were misshapen: the males had dwarf penes and the female reproductive organs were damaged by trematodes.There is a distinct interaction between pollutants and trematode parasitism in freshwater gastropods; pollution can modulate levels of parasitic infections by suppressing host immunity (hoCK & PoulIn 2012).Lake Trichonis receives pollutants from various sources, especially from intensive agri-cultural practices, urban sewage, stock grazing and small industries (BeRtahas et al. 2006).
In the last three decades, T. trichonica was found alive in Lake Trichonis three times, in 1985(szaRowsKa 2006), 2009and 2012(Radea et al. 2013).During 24 years the species was considered to be extinct from the lake (RegnIeR et al. 2009).In the present study, T. trichonica was collected in a new locality (Neromanna), a spring 3 km NE of Trichonis as well as from a new site in Lake Trichonis, west of that reported in Radea et al. (2013).As in the case of P. balcanica, the distribution of this species is wider than previously thought.

INGESTED DIATOMS AND MORPHOLOGY OF RADULAR CUSPS
The main ecological characters of the diatom taxa found in the stomach and the faecal pellets of the hydrobiids examined are presented below.

Mastogloia elliptica (Agardh) Cleve
Mastogloia elliptica is regarded as new for Lake Trichonis, and new for Greece.In Lake Trichonis, it was found living on the substratum (Fig. 32) with no obvious extracellular polysaccharide structures and restricted motility, thus belonging to the low profile ecological guild.

Cocconeis placentula Ehrenberg sensu Jahn et al. (2009)
C. placentula is a common periphytic diatom of Lake Trichonis that abundantly colonises all kinds of substrata; it has been found as epizoic on the gastropod Potamopyrgus antipodarum (J.E. Gray, 1843), epiphytic on aquatic plants (Myriophyllum spicatum L. and Nasturtium officinale R. Br. (Radea et al. 2008) and on abiotic substrata (louvRou & eConomou-amIllI, unpublished).The species is included in the low profile ecological guild since it is prostrate, i.e. adheres firmly to the substratum through the entire raphe valve surface (Passy 2007).Aquatic gastropods with taenioglossate radula which are adapted to 'rasping' and 'scraping' the biofilms are able to feed on specific growth forms of microalgae (crustose, prostrate, gelatinous, and some of the stalked and short filamentous forms) (steInman 1996, VenKatesan et al. 2016).
P. balcanica, with its narrow and pointed cusps on the central and lateral teeth (Figs 21-23) might be able to feed on organic debris and protists' cells by rasping and by piercing and tearing fleshy algae (hawKIns et al. 1989, PadIlla 2004).
T. trichonica has wide and blunt radular cusps on the central and lateral teeth (szaRowsKa 2006: p. 112, fig.130), a structure offering more surfaces in contact with the substratum when feeding.This radula type is eminently effective for rasping and removing loose material from the substratum (PadIlla 1985(PadIlla , 2004)).
Our findings on the diatom food items (Figs 27-31) and their mode of attachment to the substratum are in accordance with the above views on the function mode of the radula of P. balcanica and T. trichonica.

BIODIVERSITY OF LAKE TRICHONIS
In the "Project Aqua" Trichonis was characterised as an area of high potential research value because of its high number of endemic algae, molluscs and fishes (lutheR & RzosKa 1971).It was designated as Special Area for Conservation (SAC) and, finally, included in the European Ecological Network Natura 2000 due to the priority habitat of calcareous fens.
This lake is known as a hotspot of freshwater biodiversity in Greece, particularly of molluscs.It is remarkable that the index of gastropod endemism es-timated for the Lake Trichonis Basin is close to such values for Lake Baikal, Russia, and Lake Biwa, Japan, and is only exceeded by Lake Ohrid, Macedonia/ Albania, and the ancient lakes of Sulawesi, Indonesia (alBReCht et al. 2009).
The continuous presence of the local endemic hydrobiids I. trichoniana, P. balcanica and T. trichonica, together with the presence of diatoms regarded as endemic (C.trichonidea and C. trichonidea var.parva) is a valuable indication of the biodiversity preservation in Lake Trichonis.
During the last thirty years, Lake Trichonis has shown a general stability of its physicochemical parameters and has retained its oligo-to mesotrophic character.However, the presence of certain zooplanktonic species which are typical of eutrophic lakes could reflect a possible alteration of its trophic status (doulKa & KehayIas 2008, KehayIas & doulKa 2014).Being mainly hydrology-dependent, the trophic status of Trichonis is unpredictable.Consequently, effective management targeting both elimination of nutrient pollution loads and controlled water extraction is necessary (alBReCht et al. 2006, BeRtahas et al. 2006) in order to protect the lake's biodiversity.

ACKOWLEDGEMENTS
The field work was partly funded by the project "Monitoring and assessment of the conservation status of invertebrate species of EU interest in Greece", contract entity: "Ministry of Environment and Climate Change" of Greece.The manuscript was improved by the helpful comments and suggestions of the Editor a. lesICKI and two anonymous reviewers.