Records of new algal taxa within various aquatic and aerophytic habitats in Slovenia

. In Slovenia, the first algal research dates back to the year 1845, when Kützing referred to several localities in Slovenian territory in his work »Phycologia germanica«. The present knowledge of diversity, variety and abundance of algae, however, is still incomplete, which applies both to aquatic and terrestrial species. The presented study carried out in 2005 and 2006 included 30 different aquatic and 5 terrestrial environments. It embraced data on species composition and distribution of benthic species in aquatic ecosystems and terrestrial environments. 634 taxa from ten classes were identified, 170 of which were discovered in Slovenia for the very first time. Most new identified taxa in Slovenia belong to Bacillariophyceae, specifically Navicula and Nitzschia. The genera of Amphidinium, Bumilleria, Heterothrix, Stylopyxis, Entomoneis, Stenopterobia, Chlorophysema, Podohedra and Poloidion had not been recorded in Slovenia prior to this study.


Introduction
At present, algology is a deficient sub-discipline of biology and ecology in Slovenia.There are many reasons for this state of affairs, including the fact that today the research is largely dedicated to polluted aquatic ecosystems (lakes, dams, rivers), mainly as monitoring of potential contamination or changes in trophicity.The algae of distinct (including extreme) environments, such as peat bogs, fens, marshy meadows, thermal springs, pools, brackish waters, soil, humid rocks, rock walls, stumps, tree trunks, etc., are particularly poorly investigated (Krivograd Klemenčič 2007).
A better knowledge of algal diversity is important in the processes of natural heritage conservation.Algae are key indicators of anthropogenic pressures on aquatic ecosystems and mineralization level.The presence, absence and abundance of algae are the parameters for the assessment of conditions of aquatic ecosystems (Wetzel & Likens 1995).The importance of algae is even greater in extreme conditions, such as blooming lakes (Sedmak & Kosi 1997).Stevenson (1996) estimates that about 26,000 algal species inhabit inland waters.In Slovenia, 2,067 species of algae and cyanobacteria have been identified so far (Vrhovšek et al. 2006).The data indicate great diversity of aquatic ecosystems; however, it is difficult to verify this number, considering that many of the localities of aquatic ecosystems studied in the past do not exist anymore or have been severely altered or polluted (Vrhovšek et al. 2006).At the same time, the new recorded species increase this number.

Selection of sampling points
The Excel computer database of freshwater and terrestrial algae of the Limnos Company was the basis for the selection of 30 sampling sites in aquatic and 5 in terrestrial environments (Fig. 1, Tab. 1).The examined habitats presented in this study, previously algologically poorly examined, were springs, waterfalls, oxbows, bogs, mires, brackish waters and terrestrial aerophytic habitats.Additional criterion for the selection of sampling sites applied in this study was type of substratum on which algae were attached in aquatic environments, limestone, silicate, flysch and other substrates, such as decayed wood stump, limestone rocks, concrete and soil in terrestrial habitats.1 for sampling sites codes.Slika 1. Zemljevid Slovenije z označenimi vzorčnimi mesti.Oznake vzorčnih mest so prikazane v Tabeli 1. Brdo, a waterfall on the Bistrica tributary).Samples of silt were taken from the bottom of both puddles.Water at the springs at Koroški Selovec and Kopanj is directed through a pipe into a wooden trough, which was scraped for floristic analysis (Tab.1).
In aerophytic habitats, surfaces with algal mats were scraped using a sharp knife.
Specimens were afterwards resuspended in a small quantity of distilled water.
Samples were preserved in the field with 35 % formaldehyde (Merck) with the final concentration of 4 %.Samples from bogs, mires and the constructed wetland were examined twice, prior to and after fixation with formaldehyde.Such samples often contain flagellates, which change their morphology to indistinguishable level when formaldehyde is applied (Wołowski & Hindák 2005).
Samples were examined in the laboratory using light microscopes Nikon Eclipse E400 and Nikon Eclipse TE300, equipped with digital camera Nikon Digital Camera DXM 1200, Japan.
Images were processed using software Lucia 4.6 (Laboratory Imaging s.r.o., Czech Republic).
In order to determine the diatoms to the level of species, the samples first had to be treated suitably with concentrated HNO 3 (Schaumburg et al. 2004).Permanent slides with diatoms were made from the treated samples by Naphrax (Schaumburg et al. 2004).Slides were examined under a magnification of 1000 and 1200×, and phase contrast microscopy was applied where necessary.Individual algal cells were photographed to observe cell structures in detail.Abundance of individual taxa was estimated using the method described by Grbović (1994) (Tab.2).

Results and discussion
In the present study, 464 algal taxa were identified of a total of 2,067 taxa from the list of freshwater and terrestrial algae in Slovenia (Vrhovšek et al. 2006)

Taxa
A 1

Sampling site
A 1

Springs
A total of 20 taxa were identified in four springs, which had not been registered before in Slovenia.18 belonged to Bacillariophyceae and 2 to Xanthophyceae.Most of them were from the genera of Achnanthes (4), Navicula (4) and Nitzschia (4).Especially rich site regarding the »new« species was the spring of Šica (13), where both new species of the Xanthophyceae class (Bumilleria spirotaenia and Heterothrix quadrata) were recorded.B. spirotaenia occurs as green mat on silted bottoms of water bodies (Ettl 1978); something similar was observed at the sampling point in the Šica spring.

Waterfalls
18 new taxa for Slovenia were identified in five waterfalls.Of the newly found taxa in Slovenia, 16 were from Bacillariophyceae, Homoeothrix janthina from Cyanobacteria and Heterodendron squarrosum from Xanthophyceae.Most new species belonged to the genera of Navicula (4) and Achnanthes (3).

Oxbows
In the oxbows of the Mura River, a total of 25 taxa were identified, which had not been known before in Slovenia.Of the new taxa, 19 belonged to diatoms, Borzia curta and Woronichinia robusta to Cyanobacteria, Bumilleria klebsiana and B. spirotaenia to Xanthophyceae, while Characium ensiforme and Microspora abbreviata belonged to Chlorophyceae.

Bogs and mires
Samples from Radensko polje contained 12 taxa, previously not identified in Slovenia.
Eight new taxa belonged to diatoms, three to Chlorophyceae, and Peroniella minuta to Xanthophyceae.
In mires and in the lake of Črno jezero, 85 new taxa were identified, 44 of which belonged to Bacillariophyceae, 14 to Zygnematophyceae, 12 to Cyanophyceae, eight to Chlorophyceae, three to Xanthophyceae, two to Euglenophyceae, one to Dinophyceae, and one to Chrysophyceae.The most recently identified taxa belonged to the genera of Navicula (12), Pinnularia (9) and Eunotia (6).There were 15 new taxa for Slovenia identified in the bog at Koroški Selovec, 15 in the peat bog of Žejna dolina (Krivograd Klemenčič & Paradiž 2006), 11 in the peat bog at Holmec, six in the peat bog of Mali plac at the Ljubljana bog, 12 in the peat bog of Ledina, 27 in the lake of Črno jezero, 10 in the bog above the Tinče cottage, eight in the bog I on the way to Osankarica, and nine in the bog II on the way to Osankarica.
In puddles at Jelovica and Navrški vrh, nine taxa were identified that had not been recorded before in Slovenia.Four new taxa belonged to Chlorophyceae, three to Bacillariophyceae, and two to Zygnematophyceae.

Brackish water
36 taxa were identified as new records in Slovenia.33 new taxa belonged to diatoms and three to Cyanobacteria.

Constructed wetland
14 taxa were identified (Krivograd Klemenčič 2008), but there were no new records for Slovenia.

Aerophytic habitats
In total, 18 new taxa were recorded in aerophytic habitats, which have not been known until now in Slovenia.12 taxa belonged to Bacillariophyceae, four to Chlorophyceae.
Gloeocapsa lignicola and Phormidium papyraceum belonged to Cyanobacteria.The majority of identified species were typical aerophytes.Especially rich in »new« species were humid rocks at the entrance to the cave of Huda luknja.At this sampling site, a green alga Poloidion didymos was identified, which is the only representative of the Poloidion genus often inhabiting humid soils and mosses from the liverworts group (Ettl & Gärtner 1995).The other three species of the Chlorophyceae class (Podohedra bicaudata, P. falcata and Trentepohlia annulata) were identified on a pine stump on the Jelovica plateau.All three species are rather common in Europe, but they were identified for the first time in Slovenia, because this habitat (tree stumps) had simply been overlooked in previous studies.
3. Inadvertence of particular algal species.The authors may have incorrectly identified many species of algae, in particular the small ones (under 10 µm).They might have overlooked them or even lost them due to an unsuitable sampling procedure.In this study, the scanning electronic microscopy (SEM) was not used for identification of diatoms owing to the problems connected with SEM sample preparation.Specifically, most samples were taken from stagnant water from a siliceous substratum, which contained much organic material.This material cannot be removed in the preparation of diatom samples by concentrated HNO 3 to the extent that enabled the visibility of the cell surface structure by SEM.It is reasonable to assume that by the application of a more suitable procedure for the preparation of diatoms for SEM and subsequent identification of algae by the means of SEM, a much larger number of new records of diatoms would have been identified.

Table 1 .
Sampling sites with codes Tabela 1. Seznam vzorčnih mest z oznakami In bogs, mires, oxbow lakes, springs, waterfalls and estuaries, scrapes of algal mat were sampled from the surface of pebbles, stones, rocks, macrophytes and submersed wood.Algae associated with mosses were sampled by squeezing the submerged talus of a moss (mires, e.g. a spring at Koroški Selovec, a spring at Medvedje