X-ray Fluorescence Techniques in Determining the Habitat Preferences of Species-Ulva pilifera (Ulvales, Chlorophyta) from Montenegro Case Study.

The paper presents four new sites where bright green Ulva thalli were found inhabiting freshwater (a river, a ditch, the Milet Canal) and marine (on the rocky shore of the Adriatic Sea) habitats in Montenegro. The aims of this study were to determine, for the first time, whether specimens of Ulva pilifera collected in Montenegro are phylogenetically and morphologically the same species as the one occurring in Europe. Using total reflection X-ray fluorescence (TXRF) and wavelength dispersive X-ray fluorescence (WDXRF) techniques it assessed the elemental composition of their thalli and its influence to colonise new habitats. Elements: Al, As, Ba, Br, Ca, Cl, Cr, Cu, Fe, Hf, I, K, Mg, Mn, Na, Ni, P, Pb, Rb, S, Si, Sr, Ti, V, and Zn were determined. The highest elemental concentrations were found for Ca = 16.3% (using WDXRF) and for Sr = 292 ppm (using TXRF) in the Ulva thalli. Ulva pilifera analysed from Montenegro, based on classical morphological methods and molecular techniques, are closely related to the same species from inland and coastal waters throughout Europe. The analysis of trace elements showed that the metal content in Ulva thalli is correlated with the trace elements in water and sediments. Ulva pilifera fits numerous features that make it one of the bioindicators of marine pollution, thanks to its worldwide distribution and capacity to accumulate trace elements.

Phylogenetic analysis of cultivation-resistant terrestrial cyanobacteria with massive sheaths (Stigonema spp. and Petalonema alatum, Nostocales, Cyanobacteria) using single-cell and filament sequencing of environmental samples.

Molecular assessment of a large portion of traditional cyanobacterial taxa has been hindered by the failure to isolate and grow them in culture. In this study, we developed an optimized protocol for single cell/filament isolation and 16S rRNA gene sequencing of terrestrial cyanobacteria with large mucilaginous sheaths, and applied it to determine the phylogenetic position of typical members of the genera Petalonema and Stigonema. A methodology based on a glass-capillary isolation technique and a semi-nested PCR protocol enabled reliable sequencing of the 16S rRNA gene from all samples analyzed. Ten samples covering seven species of Stigonema from Europe, North and Central America, and Hawaii, and the type species of Petalonema from Slovakia were sequenced. Contrary to some previous studies, which proposed a relationship with heteropolar nostocalean cyanobacteria, Petalonema appeared to belong to the family Scytonemataceae. Analysis of Stigonema specimens recovered a unique coherent phylogenetic cluster, substantially broadening our knowledge of the molecular diversity within this genus. Neither the uni- to biseriate species nor the multiseriate species formed monophyletic subclusters within the genus. Typical multiseriate species of Stigonema clustered in a phylogenetic branch derived from uni- to biseriate S. ocellatum Thuret ex Bornet & Flahault in our analysis, suggesting that species with more complex thalli may have evolved from the more simple ones. We propose the technique tested in this study as a promising tool for a future revision of the molecular taxonomy in cyanobacteria.