Spelaeonaias floccida Lamprinou, Christodoulou, Hernández -Mariné et Economou-Amilli, sp. nov. (Figs 1–4)

Thallus woolly, partly growing subaerophytically with densely coiled and/or parallel-arranged filaments. Filaments long, isopolar, very slightly attenuated towards the ends. Sometimes heteropolar young filaments are obvious germinating from hormogonia with a basal heterocyte (Fig. 1I). Filaments true-branched with a Y-like type of branching (Figs 1 A-B,1F, 4A), rarely with false scytonematoid branching (Figs 1C, 4B). Y-type of branching originating from the oblique division of an intercalary cell followed by a second oblique division perpendicular to the previous one but without lateral displacement of the branch point (Figs 1B, 4C). Heterocytes intercalary (Figs 1D, 1F), 5.2–10.7 μm (8.15 μm ± 1.63, n=30) wide and 6.7–15 μm (9.62 μm ± 2.85, n=30) long with a pale yellow content. Mature filaments 7.8–12 μm (9.55 μm ± 0.99, n=30) wide. Branches narrower than the main filament, 4.84–8.1 μm (6.58 μm ± 0.91, n=30) wide. Sheath thin and firm, colorless. Trichomes constricted at the cross walls and extending slightly attenuated at the ends. Cells cylindrical or dolichoform to barrel-shaped, 5.16–13 μm (8.2 μm ± 1.9, n=30) long at the mature filaments, and 8.17–15.6 μm (10.98 μm ± 2.34, n=30) long at the young filaments, with smooth finely granulated content, blue-green to violet-brownish due to the phycoerythrin predominance (Fig. 5). End cells more or less rounded. Ensheathed hormogonia 32.74–73.69 μm (53.5 μm ± 11.49, n=30) long and 6.73–9.07 μm (7.88 μm ± 0.76, n=30) wide, maintaining 4–17 cells and growing subaerophytically at the ends or at intercalary sites of the trichomes (Figs 1H, 1I). Necridic cells sometimes present.

Habitat:— Dim-light calcareous substrata, in caves.

Etymology:— floc’.ci.da (adj. femin; Lat. floccidus –a, –um); from floccus (pl. flocci) = tuft of hair or wool.

Strains:—KY018918

Type:— GREECE. Cave ‘ Vlychada’, part of ‘ Diros’ cave complex (36 ο 38.316’ N, 022 ο 22.709’ E), (holotype, ATHU-CY 3375 (Herbarium of Botanical Museum of the Athens University, Greece), Reference strain: Spelaeonaias floccida PH 00323987 (culture collection in PH; Academy of Natural Sciences, USA).

SEM observations: —SEM examination confirms the morphology of the trichomes as being cylindrical or dolichoform, the presence of Y-like branching (Figs 1E, 1G) as well as the presence of hormogonia (Fig. 1J).

TEM observations:— ΤΕΜ ultrastructure of Spelaeonais is seen in Figs 2A–F, 3A–D. The cells are bound by a sheath formed by layers of different electron densities; the thick inner layer is parallel to the cell wall while the external ones are diffluent. Outside the plasma membrane, the cell wall comprises a thin layer of peptidoglycan (c. 75 nm) and an outer membrane (OM) (Figs 2A–B), which is not part of the septum. The peptidoglycan layer is crossed by pores, but plasmodesmata were not observed (Fig. 2D). The OM, together with the sheath, maintains the structure of the branched filament, even if the cells are no longer in contact. Nucleoid region is scattered throughout the cyatoplasm (Figs 2E–F). In this region there are ribosomes, carboxysomes (surrounded by an outer shell) (Fig. 2C) and storage inclusions such as polyphosphate bodies, appearing black or as electron transparent reserve spaces. It is conspicuous that the thylakoidal system surrounds the nucleoid regions with the thylakoids arranged in small curved or whorled parallel groups (Figs 2A, 2F). Heterocytes are developed either in the main filament or in the lateral branches; their shape and size were similar to the originating cells (Fig. 3B). Cell division proceeds by the formation of a septum, which is continuous with the peptidoglycan layer (Fig. 3C). At a certain point of the filament, a cell is dividing diagonally and the resulting two cells change division polarity thus forming one or two secondary branches perpendicular to the main axis (Figs 3A, 3E–D).

Sequence data and phylogenetic analyses:— All 11 clones of the single PCR product sequenced have generated identical sequences. The length of the generated sequence was 631 bp (GenBank accession number: KY 018918 after the acceptance of the manuscript). The aligned dataset was composed of 72 sequences and was 2164 bp long. The length difference between the generated sequence and the aligned dataset is due to the fact that for the taxa retrieved from GenBank the longest possible portion of their 16 S rRNA sequence was retained. In the aligned dataset 1195 sites were variable, 609 of which were parsimony informative. The overall mean distance was 0.08 (8%).

The phylogenetic tree inferred from MrBayes, representing the evolutionary relationships of our sample to the remaining 71 sequences, is provided in Fig. 6. The posterior probabilities of the BI analysis are shown on the nodes. The tree is relatively well resolved in its greater part. The respective tree generated from BEAST v.1.8.2. is provided in Fig. S1 (see supplementary file). According to the trees inferred both from MrBayes and BEAST, Spelaeonaias forms a well-supported monophyletic clade with the strains “ Stigonematales cyanobacterium SA1301” and “ Stigonematales cyanobacterium SP 302” (GenBank codes HQ917695 and HQ917696 respectively) described from Maltese catacombs (Zammit et al. 2010). However, the relationship of this group with other taxa belonging to the family Symphyonemataceae (incl. Scytonemataceae) is moderately resolved since some of true-branched strains of the genera Iphinoe, Symphyonema, Symphyonemopsis and Mastigocladopsis seem to be related to the not truly branched ones, i.e. Brasilonema spp. and Scytonema hoffmannii (Hauer et al. 2014, Komárek et al. 2014).