Nodosilinea svalbardensis Davydov et Shalygin, 2020

Davydov, Denis, Shalygin, Sergei & Vilnet, Anna, 2020, New cyanobacterium Nodosilinea svalbardensis sp. nov. (Prochlorotrichaceae, Synechococcales) isolated from alluvium in Mimer river valley of the Svalbard archipelago, Phytotaxa 442 (2), pp. 61-79 : 64-68

Nodosilinea svalbardensis Davydov et Shalygin sp. nov. ( Fig. 3 and 4 )

Diagnosis:— Akin to Nodosilinea nodulosa , distinguished from it by pseudobranched filaments and its occurrence on wetted sand. Different from any other representatives of the genus Nodosilinea by its phylogenetic position based on 16S rRNA and 16S-23S ITS rRNA gene phylogenies, p-distance analysis, and differences in secondary structures of Box-B helices.

Description:— In liquid medium form macroscopic , blue-green loose aggregates floating on the surface or settle to the bottom of the glass. In agar plate strain forms a tight blue-green mat. In natural populations filaments are quite long, occasionally slightly waved, mostly unbranched, rarely with single pseudobranchings. Nodules present—up to 4 µm wide in low light in the culture only. Sheaths soft, thin, colorless, adherent to the edges of the cells, sometimes widened, hyaline. Trichomes are distinct constricted at the cross walls, with cells dividing throughout the trichome length. Cells in natural populations are elongate, in cultures shorter to longer than wide 1.2–2.1 µm long (on the ends of trichomes longer than in the middle parts), 1.2–1.7 µm wide, blue-green, pale blue-green when old, often with one prominent granule per cell. At the end of trichomes cells are rounded, similar to intercalary cells. Hormogonia and necridic cells are (rare) presented.

Etymology:— N. svalbardensis N.L. fem. Adj. = the name is given from species type locality—the Svalbard archipelago.

Type Locality: — the Svalbard archipelago, West Spitsbergen Island, west coast of Billefjorden bay, west coast of Mimerbukta bay, the valley of Mimer river, fluvioglacial valley, sand band. 78.6514 N, 16.3371 E, elevation: 2 m., collected by D. Davydov on August 2, 2013.

Habitat:— Sand wetlands, an upper surface of the sand.

Holotype:— KPABG 3220 ! in the herbarium of Polar-Alpine Botanical Garden-Institute of Kola Science Centre of RAS, Kirovsk, Murmansk region, Russia.

Reference strain:— LID- 610001 in the Collection of Cyanoprokaryotes of Polar-Alpine Botanical Garden-Institute of Kola Science Centre of RAS, Kirovsk, Murmansk region, Russia.

Phylogenetic analysis: — The Bayesian analysis of 181 Synechococcalean OTUs and Gloeobacter kilaueensis J.W. Saw et al. (2014: 8) strain JS1( T) as an outgroup taxon yielded a tree in which Nodosilinea species are obviously separated from any other genera in the order. Halomicronema was a sister taxon to Nodosilinea among other strains included in the study ( Figs. 5 , S 1 ). Comprehensive, highly supported 16S rRNA phylogeny shows Nodosilinea svalbardensis in the sister position to N. epilithica , N. bijugata , N. ramsarensis , N. chupicuarensis , and the type species of the genus - N. nodulosa . Other Nodosilinea species such as N. conica and N. radiophila appeared to be more distantly related.

Nodosilinea svalbardensis formed an unsupported clade together with two undescribed, uncertain, unpublished taxa: “ Leptolyngbya cf. foveolarum ” SAG 24.13 ( Brinkmann et al. 2015) and “ Oscillatoria ” sp. BS594 ( KM 019975) with 98.74% identity (Table 1). Both of them are lacking unambiguous information concerning ecological preferences and importantly morphology. In addition, ITS is not available for these taxa. With p-distance difference of ~ 98.5 and other lines of evidence, it is impossible to affiliate “ Leptolyngbya cf. foveolarum ” SAG 24.13 and “ Oscillatoria ” sp. BS594 to any existed species including Nodosilinea svalbardensis .

TABLE. 1. Similarity matrix (p-distance) generated using 16S rRNA region among N. svalbardensis and most closely related taxa

NCBI accession numbers are given within the parenthesis.

According to the 16S p-distance analysis, Nodosilinea svalbardensis have a minimum ~ 97.4% (range 96–97.4) similarity compared with other Nodosilinea species (Table 1), which is well enough for the species delineation. Overall Nodosilinea spp. are in the range of 4% dissimilarity on 16S p-distance, which is quite high for the genera within Synechococcales ( Mai et al. 2018) .

16S-23S ITS rRNA Bayesian phylogeny has the same signal as 16S rRNA one ( Fig. 6 ). On that, Nodosilinea svalbardensis formed separate unsupported lineage in the sister position to the clade includes N. bijugata . The p-distance similarity in the 16S-23S ITS rRNA region among strains Nodosilinea was quite low, maximally up to 90.89% ( Table 2). The long branch on the phylogenetic tree and high 16S-23S ITS rRNA p-distance dissimilarity are confirming that N. svalbardensis belongs to a separate species.

Analysis of the 16S-23S ITS rRNA has shown that D1-D1ʹ helices were quite similar across selected taxa ( Fig. 7 ), which is typical for many cyanobacterial taxonomic groups. Conversely, Box-B structures appeared to be drastically different within the chosen set of sequences ( Fig. 8 ). In terms of the sequential differences, D1-D1ʹ helix of Nodosilinea svalbardensis possessed 4 point mutations in the different parts compared to selected species individually ( Fig. 7 ). Most of the sequential differences between species occurred within the terminal loop of the D1-D1ʹ helices. The secondary structure of the D1-D1ʹ helix of N. svalbardensis was identical to the structural motifs found in strains N. epilithica Kovácik 1990 /52, N. chupicuarensis PC 471 and N. nodulosa UTEX 2910 — type species of the genus. This fact is not essential to conclude that N. svalbardensis belongs to any of listed species. 16S rRNA, 16S-23S ITS rRNA phylogenies, p-distance analysis and differences in Box-B helices insured that N. svalbardensis is a new lineage within the genus Nodosilinea . The secondary structures of the N. svalbardensis , N. epilithica Kovácik 1990 /52, N. chupicuarensis PC 471 and N. nodulosa UTEX 2910 differed from D1-D1ʹ helix of N. bijugata since it had a longer terminal loop ( Fig. 7 ) due to point mutation (U to A) that prevented AA: UU paring. The length of the sequence of BoxB helix of N. svalbardensis was 47 bp long which is ~ 7 bp longer than typical Box-B helices within genus Nodosilinea ( Fig. 8 ). The long length is due to a 7 bp insertion mutation occurred on the 5ʹ stand adjacent to the following sequence 5ʹ–AGUGUG –3ʹ. A sequential peculiarity of the Box-B of the N. svalbardensis determined its structural difference compared to other species of the genus. In that fashion, Box-B helix of the N. svalbardensis possessed 3 internal bulges which were not usual for other sequences in the chosen set of taxa. Terminal loop of the Box-B helix of the N. svalbardensis akin to one in N. chupicuarensis PC 471 with the difference in one insertion point mutation of the nucleotide “G” in the latter. Overall terminal loops were quite diverse in the chosen taxa which were in agreement with the general trend. Analysis of the ITS region confirmed that N. svalbardensis is a new species of the genus.

Figures

FIGURE 3. DIC micro-photographs of Nodosilinea svalbardensis. A. Demonstration of the nodules. B. Illustration of the single false branching. C. Close up look on the peripheral thylakoids arrangement. Numbers indicate morphological features: 1—nodules, 2—granules within the cells, 3—false branching, 4—necridia.

FIGURE 4. Line drawings of Nodosilinea svalbardensis.A. Culture material. B. Natural populations. Note clearly visible sheaths in the natural populations. Numbers indicate morphological features: 1—false branching, 2—nodule, 3—attached hormogonia, 4—necridia, 5—granules.

FIGURE 5. 16S rRNA Bayesian phylogeny with a total of 181 sequences from order Synechococcales/Gloeobacterales, including 75 sequences from genus Nodosilinea, showing evolutionary lineage corresponds to new species Nodosilinea svalbardensis, Symbol “-” show support less than 50% on representative nodes. The type sequences of established Nodosilinea spp. are given in bold font. Taxa in the quotation mark needs to be revised.

FIGURE 6. Bayesian 16S-23S ITS rRNA phylogeny showing the position of the Nodosilinea svalbardensis. Symbol “-” show support less than 50% on representative nodes. Taxa in the quotation mark needs to be revised. Note type species of Nodosilinea, N. nodulosa UTEX 2910.

FIGURE 7. Color-coded secondary structure of D1-D1’ helices of the 16S-23S ITS region and its alignment for 5 representative species of Nodosilinea. Circles near N. svalbardensis indicate differences with closely related N. bijuigata.

FIGURE 8. Color-coded secondary structure of Box-B helices and alignment of the selected set of sequences of Nodosilinea. The black color with white letters indicates conservative regions, gray areas denotes variable parts. Notice that sequences of the terminal loop are placed in the boxes within the alignment

FIGURE. 1. Map showing sampling site (red circles) in the Svalbard archipelago. Free products of ©Norwegian Polar Institute (http:// www.npolar.no) were used to reproduce the map.