Stenomitos hiloensis Johansen, Gargass et Shalygin, 2020

Shalygin, Sergei, Shalygina, Regina R., Redkina, Vera V., Gargas, Cory B. & Johansen, Jeffrey R., 2020, Description of Stenomitos kolaenensis and S. hiloensis sp. nov. (Leptolyngbyaceae, Cyanobacteria) with an emendation of the genus, Phytotaxa 440 (2), pp. 108-128 : 114-120

publication ID	https://doi.org/ 10.11646/phytotaxa.440.2.3
persistent identifier	https://treatment.plazi.org/id/FF1C87AB-ED03-FFAC-15B5-3E9EFA13FA90
treatment provided by	Felipe
scientific name	Stenomitos hiloensis Johansen, Gargass et Shalygin
status	sp. nov.

Treatment

Stenomitos hiloensis Johansen, Gargass et Shalygin sp. nov. ( Figs. 2 View FIGURE 2 , 3 View FIGURE 3 D-G)

Filaments long to short, arranged in parallel or spiral, with or without sheaths, 2 µm wide. Sheath firm, colorless, thin, usually visible only during or following hormogonia formation. Trichomes isopolar, unbranched, untapered, clearly constricted near the transverse cell walls, 0.7–1.8 µm wide. Cells isodiametric, blue-green, not granulated, 0.5–1.2 µm long. Apical cells rounded, same size as regular cells. Hormogonia short, 2-8 celled usually with appressed cells. Necridia frequent. Thylakoids parietal.

Holotype here designated: dried specimen deposited into herbarium of Bernice Pauahi Bishop Museum , Honolulu, Hawaii, USA under following accession number: BISH 776187 About BISH .

Type locality: Seep wall on Akeola Road (19.7035°N, 155.136°W) in the tropical rain forest, Hilo, Hawaii, sampled 22 May 2010 by Rex Lowe, Pat Kociolek and Melissa Vaccarino.

Reference strain: HA6792-KK3 (isolated into culture by Katie Kavulic), deposited in the Cyanobacterial Culture Collection at John Carroll University.

Etymology: Pertaining to the city of Hilo.

NCBI GenBank Accession number: MN152980.

Comparison with other taxa:— Up until this manuscript, Stenomitos contained only three species: S. rutilans Miscoe et J.R. Johansen in Miscoe et al. (2016: 85), S. tremulus (J.R. Johansen et Casamatta in Casamatta et al. 2005: 420) Miscoe et J.R. Johansen in Miscoe et al. (2016: 86), and S. frigidus ( Fritsch 1912: 31) Miscoe et J.R. Johansen in Miscoe et al. (2016: 86). Stenomitos kolaensis and S. rutilans are easily distinguished under the microscope via differences in coloration: blue-green versus red-brown, respectively, as well as differences in diameter and length of the cells, and the occurrence of necridia in S. kolaensis ( Table 1). It is unlikely that geographical distribution for the S. kolaensis from the near Arctic will overlap with Hawaiian taxa in tropical rainforest climate. S. tremulus was isolated from a pond in Bylot Island, Nunavut, Canada at a latitude very similar to the site in the Kola Peninsula from which S. kolaensis originates. S. kolaensis was isolated from soils, has wider trichomes and shorter cells than S. tremulus , and shares only 97.75% genetic identity based on the 16S rRNA gene; it is consequently easily separated from that species.

S. hiloensis is ecologically similar to S. rutilans , but shows clear phylogenetic and morphological separation from S. rutilans . S. hiloensis has blue-green cells in contrast to red-brown in S. rutilans , additionally filament were little bit wider. S. hiloensis is thinner and has shorter cells than S. kolaensis ( Table 1.).

The numerous Antarctic strains ascribed to Stenomitos frigidus (= Leptolyngbya frigida ( Fritsch 1912: 31) Anagnostidis & Komárek (1988: 391)) represent three different species, which are clearly phylogenetically and ecologically separated from the other species in the genus and from each other. One of these is likely equivalent to Fritsch’s original taxon. The CANT/CAU and WJT strains are all isolated from arid soils, and undoubtedly represent an undescribed species. A detailed study of these “ L. frigida ” strains, with morphological observations and further characterization of their ITS regions, would certainly uncover further species-level diversity in the genus. Revisiting S. tremulus and characterizing its ITS region would also be helpful in the revisionary work that is needed.

Phylogeny:— All branches on the 16S rRNA phylogenetic tree clustered into five major families within Synechococcales ( Fig. 4 View FIGURE 4 ). Asterisks (*) indicate maximum support values, hyphens (-) indicate support values ≤ 50, support values are given in parentheses after each clade in the following order: BI/ML. Clades referring to the following families Pseudanabaenaceae (*/99), Prochlorothricaceae (0.95/-), and Trichocoleusaceae (*/98), had high support values, at least in the BI analysis. The clade containing the Leptolyngbyaceae was unsupported at the family level (0.73/-), possibly because of the unstable position of the Pantanalinema, Pinnochia , and Limnolyngbya / Scytolyngbya nodes. The Oculatellaceae was only weakly supported in our analyses (0.82/-). The unstable groups in the Leptolyngbyaceae did not affect the position of the genus Stenomitos within the family in any of our analyses. Additionally, the “AGC” triad in Helix 23 and the “A:U” pairing in the apical portion of Helix 27, both in the 16S rRNA molecule, were found in Stenomitos , supporting the affiliation of that genus within the Leptolyngbyaceae (see Mai et al. 2018). The Stenomitos clade was highly supported (*/93; Fig. 4 View FIGURE 4 ). The Stenomitos clade consisted of three Antarctic clusters ( Fig. 4 View FIGURE 4 : ANT and CANT labeled taxa), Mojave Desert species ( Fig. 4 View FIGURE 4 : WJT24-NPBG20) and additional taxa from a wide range of geographic locations (Hawaii, Russia, subarctic, arid Europe). All species and strains within the Stenomitos clade possessed>97% genetic identity based on 16S rRNA gene sequence ( Table 2), supporting recognition of a single genus. Many strains, including those assigned specific species epithets had genetic identities ≥98.7%, the proposed threshold for species recognition within prokaryotes ( Yarza et al. 2014). The sister taxon to Stenomitos , Neosynechococcus sphagnicola Dvořák et al. (2014: 26) , was also above the recognized generic threshold of 94.5% identity for all Stenomitos strains. Consequently, 16S genetic identity alone does not provide clear evidence that these the Stenomitos species are separate taxa, or that the genus Stenomitos is separate from Neosynechococcus . The phylogeny based on the 16S rRNA gene region showed S. kolaensis and S. hiloensis to be members of the subclade containing S. rutilans and S. tremulus . Leptolyngbya frigida ( Fig. 4 View FIGURE 4 ), which we designate S. “frigidus ” due to its uncertain placement in that species is polyphyletic and appears to represent at least three different species. The ITS phylogeny indicated that the Stenomitos taxa for which we have ITS sequence separate into two clades. S. kolaensis is more closely related to S. rutilans , while S. hilosensis is in the clade containing several S. frigidus ( Fig. 5 View FIGURE 5 ).

Secondary structures of 16S–23S ITS:— ITS analysis included a comparison of the conserved domains (D1-D1ʹ, Box-B, V2 and V3 regions) of eight strains of Stenomitos ( Figs 6 View FIGURE 6 , 7). The structure of the D1-D1ʹ helices was found to be highly conserved across all eight strains, with the exception of a desert soil crust strain, Stenomitos sp. WJT24- NPBG20, which had an altered structure in the terminus of the helix due to several nucleotide substitutions ( Fig. 6 H View FIGURE 6 ). Although the D1-D1ʹ helix structure was conserved, there were significant differences in the actual sequences in this helix, particularly in the 28 nucleotides forming the terminus of the helix. No two D1-D1ʹ helices were identical in sequence, but some pairs differed only in a single nucleotide. The V2 helix situated between the two tRNA genes was highly divergent among strains, and consisted of either a long helix ( Fig. 6 J View FIGURE 6 , L-N, P) or a very short helix that may not even form ( Fig. 6 I, K, O View FIGURE 6 ). The long helices were quite different in sequence, length, and structure. The short helices differed in sequence but not structure. No two V2 helices were identical in sequence.

. S. S S. S. S.. S. S S. S.. S S. S.. S. cf Unc kolaensis tremulus hiloensis SAG species tenuis PMC rutilans HA WJT species frigidus frigidus frigidus frigidus frigidus frigidus Stenomitos . Pasv UTCC 6792 HA 304 7619 - 24 ANT. L CANT CAU10. L ANT ANT. CIP 3 RJ RS 28 471 KK - 37.90 -7 - 2 LM NPBG 3. B 52 10 8.1 1 REIDJ. 088 3 20

Stenomitos kolaensis

Pasv RS28

Stenomitos tremulus 99.0

UTCC471

Stenomitos hiloensis 99.2 99.3

HA6792-KK3

Stenomitos species 99.1 99.0 99.2

SAG 37.90

The Box-B helices of all Stenomitos strains differed in sequence. Most also differed in structure, although three were almost identical ( Fig. 7 A, D, E). The basal clamp was 3 bp longer than illustrated for most other Box-B helices. As these extra bases always were able to pair in Stenomitos we assumed that in vivo they also pair, and have shown them ( Fig. 7 A-H). The V3 helices displayed a high degree of divergence in terms of structure and sequence ( Fig. 7 J-O). Of the examined sequences, only the basal clamp had a clear consensus sequence (5ʹ-UGUCAGGUAGA— UCAYAGACA-3ʹ).

Both S. kolaensis and S. hiloensis have ITS sequences and structures that are distinct from each other and the other known strains of Stenomitos . These differences support the description of our new species. Stronger support for recognition of our species and putative new species not described in this work exists in the percent dissimilarity of the aligned 16S-23S ITS sequences for Stenomitos strains ( Table 3). According to several studies ( Erwin & Thacker 2008, Osorio-Santos et al. 2014, Pietrasiak et al. 2014, Becerra-Absalon et al. 2018, González-Resendiz et al. 2018, Mai et al. 2018, Pietrasiak et al. 2019), greater than 7% dissimilarity in orthologous ITS regions can be considered strong evidence that strain pairs belong to separate species. Most of the Stenomitos strains appear to be distinct species as pairwise comparisons are>10%, with the exception of four Antarctic strains identified as S. frigidus that show 0.0% dissimilarity ( Table 3).

1 1

3..

28 KK- LM

2 B 1.. B 2 52 LB LB 64

Pasv

RS 6792

7619 -

HA SAG 37.90. 8.1 L ANT ANT 53. L ANT 53 L. 52.3.L ANT ” ANT. ANT. ”

kolaensis hiloensis rutilans species frigidus frigidus frigidus frigidus “ frigidus frigidus “

..........

S S S S S S S S S S Stenomitos kolaensis Pasv RS 28

Stenomitos hiloensis HA 6792-KK3 10.6