Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from mats collected on Signy Island, South Orkney Islands, Antarctica.

Terrestrial cyanobacteria are very diverse and widely distributed in Antarctica, where they can form macroscopically visible biofilms on the surfaces of soils and rocks, and on benthic surfaces in fresh waters. We recently isolated several terrestrial cyanobacteria from soils collected on Signy Island, South Orkney Islands, Antarctica. Among them, we found a novel species of Nodosilinea, named here as Nodosilinea signiensis sp. nov. This new species is morphologically and genetically distinct from other described species. Morphological examination indicated that the new species is differentiated from others in the genus by cell size, cell shape, filament attenuation, sheath morphology and granulation. 16S rDNA phylogenetic analyses clearly confirmed that N. signiensis belongs to the genus Nodosilinea, but that it is genetically distinct from other known species of Nodosilinea. The D1-D1´ helix of the 16S-23S ITS region of the new species was also different from previously described Nodosilinea species. This is the first detailed characterization of a member of the genus Nodosilinea from Antarctica as well as being a newly described species.

Introduction

Cyanobacteria are a widely distributed group of oxygenic photosynthetic prokaryotes that possess chlorophyll a and phycobiliproteins [1]. Despite their widespread occurrence and ecological importance, the taxonomy of cyanobacteria remains problematic. Cyanobacterial classification has recently undergone rapid revision based on the use of polyphasic approaches to define new taxa [2]. These approaches combine molecular characterization with cytomorphological and ecological characteristics and have been used to erect and describe new taxa and for the validation of classically described taxa [3, 4].

Amongst the different cyanobacteria groups, Leptolyngbyaceae (Synechococcales) is one of the most taxonomically challenging. Representatives of this family are morphologically simple, often with ambiguous and indistinct morphological features that lead to uncertainty in identification [5]. However, species within the group can be distinguished based on molecular evaluation. Ongoing taxonomic revisionary work to date has resulted in over 21 new genera being erected from the original genus Leptolyngbya [6-23]. The new erected genus were listed in various literature [6-11] and others are described [12-23].

The genus Nodosilinea [8] shows a clear phylogenetic separation from Leptolyngbya based on molecular assessment. The genus has been reported to possess a unique capability to form short convoluted lengths of trichomes within the bounding sheath, termed nodules. This was first documented in L. nodulosa [24]. The generic assignment of L. nodolusa was subsequently revised with the support of molecular phylogenetics and this species became the first member of the newly erected genus Nodosilinea, as N. nodulosa [24]. Subsequent studies have reported the ability to form nodules in all members of the genus when exposed to low-light conditions of < 4-μmol m-2 s-1 for 4 weeks. Some species have also been reported to perform nitrogen fixation [8, 24, 25]. Apart from nodule formation, the characteristics of the genus resemble those of Leptolyngbya, with uniseriate trichomes ranging from 0.5 to 3.5 μm wide within a thin sheath, similar vegetative and apical cell shapes, constriction at each cross wall and containing granules [26].

Representatives of the genus have been recorded from various habitats. Nodosilinea sp. LEGE 13457 and Nodosilinea sp. TM-3.1 were both isolated from the McMurdo Dry Valleys, Antarctica [27]. N. ramsarensis and N. radiophila were recorded from a thermal spring in Iran [28] and N. nodulosa from marine phytoplankton in the China Sea [24]. Other species are subaerial on walls (N. epilithica [8], N. chupicuarensis [25]) and occur in soils (N. conica [8]) and freshwater (N. bijugata [8, 29]).

In Antarctica, cyanobacteria are the most important primary colonizers of soils [30]. They play vital roles in soil stabilisation, photosynthetic carbon fixation, and the release of fixed nitrogen, whilst forming the base of the terrestrial food web [31]. Cyanobacteria are found across all geographical regions of Antarctica where they can form macroscopically visible mats, crusts or thin biofilms on the surfaces of soils and rocks and in streams, ponds and lakes, as well as occupying endolithic niches [32, 33].

Leptolyngbya sensu stricto is one of the most widely recorded genera in Antarctica [34-41]. However, detailed characterization combining morphological, ultrastructural and molecular approaches has been performed to date on only three named species (L. bijugata [42], L. borchgrevinkii and L. frigida [43]) and two unidentified species [44].

Three species of Leptolyngbya (i.e. L. foveolarum, L. notata and L. perelegans) were identified in a broad-scale floristic survey conducted on Signy Island, South Orkney Islands, in the 1970s [34]. Their taxonomic assignments, however, were based solely on morphological traits and have not subsequently been subjected to molecular assessment.

In this study, we report the polyphasic characterization of a cyanobacterial strain superficially resembling Leptolyngbya, recently isolated from soils collected on Signy Island.

Materials and methods

Ethics statement

New algal material was collected from Signy Island in the 2015/16 austral summer season under permit number 46/2015 issued under the United Kingdom Antarctic Act. The Department of Quarantine and Inspection Services Malaysia (MAQIS) provided import permit JPK1412016065849 to permit the samples to be imported to Malaysia (https://www.maqis.gov.my/). No other specific permission was required for any other locations or activity included in this study. The locations are not privately owned or protected in any way. This study did not involve any endangered species or protected species.

Sample origin and culture conditions

Mat samples were collected under permit as listed above from cracks and crevices in rocks and beneath loose fragments of stone on a west-facing slope below Robin Peak (60.6833° S, 45.6333° W) on Signy Island during the expedition of British Antarctic Survey in austral summer of 2015/16. All apparatus used for obtaining the samples was sterile and samples were stored and subsequently transported frozen (-20°C) in sterile containers to the Universiti Sains Malaysia. Cyanobacterial strains were then brought into culture by inoculation on 1% agarised full strength BG-11 medium in Petri dishes [45]. Culture media were supplemented with 100 μg mL-1 cycloheximide to prevent growth of eukaryotes [46]. Cultures were incubated at 15 ± 2°C with 24 h light supplied by cool white fluorescent lamps at < 4 μmol m-2 s-1. After 4 weeks of incubation, algal growth developing on the plates was examined microscopically.

Morphological characterization

Morphological examination was conducted using an Olympus BX-53 light microscope (Olympus America Inc., Center Valley, PA, USA) at 100 – 2000X magnification. Photomicrographs were taken. Illustrations were made with the aid of a camera lucida. Specimens were analysed based on morphological characteristics, particularly filament and trichome width, sheath morphology, cell colour, shape of intercalary and apical cells and presence of granules. Size measurements were made on 30 randomly chosen replicate specimens for each morphospecies. The characteristics of the strain studied were compared with descriptions in the literature [8, 25, 26].

Transmission electron microscopy

Transmission electron microscopy (TEM) samples were fixed in McDowell-Trump fixative solution [47] prepared in 0.1 M phosphate buffer and later post-fixed with 1% osmium tetroxide. The fixed material was dehydrated in an ethanol series (50%, 75%, 95%, and 100%) and embedded in Spurr’s resin [48] mixed in a rotator overnight. The ultrathin sections were treated with uranyl acetate and lead citrate to improve contrast [49]. Thin sections were collected on copper grids and were observed using a JEM 2000FX (JEOL, Tokyo, Japan) operating at 100 kV.

Molecular analyses

DNA was extracted using the G-spin for bacteria genomic DNA extraction kit (iNtRON Biotechnology, Korea) following the manufacturer’s protocol. DNA sample concentration was measured using a Thermo Scientific NanoDrop instrument. The 16S rDNA gene and the 16S–23S internal transcribed spacer (ITS) region were amplified using the polymerase chain reaction (PCR) and the combination of primers 2 (5’–GGG GGA TTT TCC GCA ATG GG– 3’) and 3 (5’–CGC TCT ACC AAC TGA GCT A– 3’) for the 16S rRNA gene and primers 1 (5’- CTC TGT GTG CCT AGG TAT CC– 3’) and 5 (5’–TGT AGC TCA GGT GGT TAG– 3’) for the ITS region [50]. This resulted in products of approximately 1,600 bp for the 16S rRNA gene and 600 bp for the ITS region. The reaction mix comprised 2 μL of extracted DNA used in 50 μL reactions containing 1 μL of each forward and reverse primer, 21 μL of ultrapure water and 25 μL of MyTaq™ Red Mix, which is a pre-prepared mixture of buffer, dNTPs and Taq polymerase (Bioline, United Kingdom). PCR was carried out using a Bio-Rad Thermal Cycler with standard parameters set as follows: 95°C for 2 min, 95°C for 15 sec, 55°C for 15 sec (30 cycles), and 7 min 20 sec extension at 72°C. Once the reaction was completed, the integrity of the PCR product was verified using a 2% agarose gel.

Phylogenetic analyses and ITS folding

All sequences were edited and assembled using the Geneious 11.0 software package (Biomatters, http://www.geneious.com). Sequence alignments were prepared using the MUSCLE algorithm in Geneious 11.0 and then manually checked by eye. The dataset included 47 OTUs, consisting of sequences newly obtained in this study together with additional sequences retrieved from GenBank of closely related species of Nodosilinea, more distantly related species of Leptolyngbya sensu stricto and one outgroup taxon (Gloeobacter violaceus FR798924). Some Leptolyngbya sequence that were retrieved from GeneBank currently continue to be known as Nodosilinea and we identify these sequences with “Leptolyngbya” in the phylogenetic tree. All new sequences generated in this study have been deposited in GenBank under accession numbers USMFM MN585774 and USMFM MN585775.

Phylogenetic analyses were performed using two different methods: maximum likelihood (ML) and Bayesian inference (BI). Before ML and BI analyses, the best-fit model of DNA substitution was determined using the program Kakusan4 [51]. ML analyses were performed using with RaxML v7 [52] in Geneious 11.0 using the general time-reversible invariant-sites (GTRI) nucleotide substitution model with the default parameters. The bootstrap probability of each branch was calculated using 1000 replications. BI analyses were performed with the program MrBayes v3.1.2 [53]. Two independent analyses, each consisting of four Markov chains, were run simultaneously for 3,000,000 generations, sampling every 100 generations. Log likelihood and parameter values were assessed with Tracers ver. 1.5 [54]. A burn-in of 25% of saved trees was removed, and the remaining trees were used to calculate the Bayesian posterior probability values. ML and BI trees were edited with the program FigTree v1.3.1 [53].

The 16S–23S ITS region was used for modelling of secondary structure folding. The tRNA genes were identified using tRNAscan-SE 2 [55]. The secondary structure of the D1–D1´ helix was modelled using the Mfold WebServer with default conditions.

Results

A single strain was successfully isolated from one sampling site and showed the diagnostic traits of the genus Nodosilinea. However, it has morphological characteristics that did not correspond to any previously described species. In addition, our phylogenetic study confirmed that this strain forms a distinct lineage within the genus with respect to the nucleotide sequences of both the 16S rDNA gene and the 16S-23S ITS region. We hereafter refer to this strain under the name Nodosilinea signiensis sp. nov.

Class Cyanophyceae

Order Synechococcales

Family Leptolyngbyaceae

Genus Nodosilinea

Nodosilinea signiensis sp. nov.

Description. (Culture conditions) Mat creeping on agar, pale blue-green to olive-green in colour. Filaments long, immotile, solitary, occasionally forming spirals under normal light conditions of 27 μmol m-2 s-1 (Fig 1A and 1C). Under low light intensity of <4 μmol m-2 s-1, uniseriate trichomes can lie parallel or twisted around one another within a common sheath (Fig 1D and 1E). This resembles nodule formation whereby filament width in these areas becomes wider, up to 5.0 μm wide (Fig 2C–2E). Cells 1.0 (1.5)– 2.0 μm wide, 1.0–2.0 (2.3) μm long, shorter to longer than wide, discoid to barrel-shaped (Fig 2A–2G). Apical cells rounded, non–capitate, without calyptra and lacking granules (Fig 2G). Sheath colourless, thin.

Fig 1

Nodosilinea signiensis sp. nov. grown in culture.

(A), filaments. (B), rounded apical cell (arrow). c, spiral formation (arrow). (D) and (E), trichomes twisted within the enclosing sheath, young trichome forming nodules (arrow). Scale bars: 20 μm.

Fig 2

Morphological characteristics of Nodosilinea signiensis sp. nov.

(A)–(E), trichome variously curved, sometimes straight, tangled together or twisted, characteristic nodules (arrow). (F) and (G), trichomes lying together within a common sheath, mature rounded apical cells (arrow). Scale bar: 10 μm.

Etymology. Nodosilinea signiensis, Nodosilinea = “Knotted line” Perkeson et al. (2011); signiensis (sig.nie’n.sis) adj. signiensis = originated from Signy Island.

Habitat. Mats in cracks and crevices in rocks and beneath loose fragments of stone collected from west-facing slope below Robin Peak (60.6833° S, 45.6333° W).

Occurrence. South Orkney Islands, Signy Island.

Observations. In cultures incubated for 4 weeks under low light, filaments are entangled, curved or very occasionally form a spiral. Cell division without lengthening of the enclosing sheath causes the trichomes to sometimes bend and fragment to form two or three twisted structures that resemble nodules, although this was rarely observed.

Morphological assessment. The strain displays similar nodule formation to that of the seven currently described species within the genus. Nodosilinea sp. LEGE 13457 and Nodosilinea sp. TM-3.1, both originating from Antarctica [27], are not included here in the comparison as both strains lack detailed taxonomic characterization. Cell width of Nodosilinea signiensis sp. nov. falls within the range of all previously recorded species of Nodosilinea (Table 1). Cell length closely resembles five of the seven described species (N. conica, N. chupicuarensis, N. nodulosa, N. radiophila and N. ramsarensis), while N. epilithica and N. bijugata have longer cells at 8 μm and 6.2 μm, respectively (Table 1). The vegetative cell shape of N. signiensis was distinct, being the only species showing a variety of cell shapes ranging from isodiametric to longer than wide or shorter than wide. Cell constriction changed from slightly constricted to distinctly constricted as trichomes matured. In all other species of Nodosilinea, the cell shape was reported to be stable throughout the development of the trichome (Table 1). Trichomes of the present strain showed no attenuation towards the apex, in contrast to the abrupt tapering in N. conica (Table 1). Nodosilinea signiensis sp. nov. is the only species lacking cell granulation. Sheath development was very similar to N. conica and N. chupicuarensis, with the thin, colourless sheath occasionally becoming wide in mature filaments (Table 1). Three species, N. conica, N. radiophila, and N. ramsarensis, have been recorded from extreme environments (Table 1).

Table 1

Comparison of characteristics of the previously described eight species of Nodosilinea [8, 24, 25, 28] and N. signiensis sp. nov. (‘?’ indicates feature unknown).

Characters	Nodosilinea signiensis sp. nov.	Nodosilinea epilithica	Nodosilinea bijugata	Nodosilinea conica	Nodosilineachupicuarensis	Nodosilineanodulosa	Nodosilinearadiophila	Nodosilinea ramsarensis
Cell length (μm)	1.0–2.0 (2.3)	1.0–8.0	1.5–6.2	0.9–2.4	1.1–1.3	1.1–1.5	1.0–2.0	(0.8) 1.0–1.5
Cell width (μm)	1.0 (1.5)	1.5–2.5	1.5–1.7	2.5–2.7	1.2	1.2–2.4	2.0–5.0	1.0–2.0
Cell shape	Isodiametric, longer than wide/ barrel shape	Barrel shaped, shorter to longer than wide	Isodiametric, longer than wide	Isodiametric, shorter than wide	Isodiametric	Isodiametric, longer than wide	Isodiametric, longer than wide	Isodiametric, longer than wide
Cross-wall	Slightly constricted to strongly constricted	Distinctly constricted	Slightly constricted	Slightly constricted	Constricted	Slightly constricted to strongly constricted	Distinctly constricted	Distinctly constricted
Filaments	Solitary, immotile, forming spiral	Forming nodules in low light	Rarely forming nodules	Rarely forming nodules	Multiseriate, motile, forming nodules	Forming nodules	No formation of nodules	Rarely forming nodules
Attenuated	Absent	?	?	Tapering abruptly	?	?	?	?
Apical cells	Rounded	Rounded	Rounded	Rounded	Dome-shaped	Rounded	?	?
Granule	Ungranulated	Granulated	Granulated	?	Granulated at cross walls	Granulated at cross walls	Granulated at cross walls	Granulated
Sheaths	Very thin, colourless	Thin, colourless, occasionally becoming wide and diffluent	Often absent, thin, colourless	Soft, thin, colourless	Thin, clear	Thin, colourless, occasionally becoming wide and diffluent	Thin, colourless	Thin, colorless
Special features	Uniseriate trichome lie parallel or twisted around one another within a common sheath that resembles nodules	Cells typically barrel shaped after cell division, cylindrical in nondividing trichomes	Cells typically barrel shaped to spherical; inflated sheaths	Abundant nodule formation	Filament forming a tight spiral	?	?	?
Occurrence	Soil—Signy Island, Antarctica	House wall -Peninsula Gargano, town of Vieste (Foggia), Italy.	Littoral zone–Eutrophic Lake Piaseczno, Poland	Sevilleta Long Term Ecological Research, New MexicoSoil -Chihuahuan Desert, USA	Stone monument surface—Central Mexico	Marine—South China Sea	Benthic mat in thermal spring (27 <C)—Talesh Mahalleh, Ramsar Iran	Soil around thermal spring (32 <C)—Khaksefid, Ramsar, Iran.

Transmission electron microscopy. Thylakoids were parietal (4–5 per cell), visible in longitudinal section but poorly seen under cross section (Fig 3). Nodule formation was not observed under TEM. Cyanophycin granules were visible among the thylakoids but carboxysomes were absent. The cell wall was simple, similar to Leptolyngbya species, and the sheath was distinct.

Fig 3

TEM of Nodosilinea signiensis.

(A), cross-section of a cell within its surrounding sheath. (B) longitudinal section of a trichome. Scale bar: 1 μm. s = sheath; t = thylakoids; n = nucleoplasm. Thylakoids are arranged more or less parallel in a parietal position.

Phylogenetic analysis. The sequence dataset consisted of 1,482 bp, including gaps. Our tree suggests that “Leptolyngbya antarctica” should be reclassified into the genus Nodosilinea, as with several other sequence of “Leptolyngbya” deposited in GenBank. Partial sequences of the 16S rDNA of N. signiensis were distinct from other Nodosilinea sequences including “Leptolyngbya antarctica” by at least 2% (≥ 29 differences). Both ML and BI analyses yielded an identical topology, and therefore only the ML tree with a–ln L score of 11654.762 is presented (Fig 4). The 16S rDNA phylogeny clearly indicated that our strain was nested within the genus Nodosilinea (98% ML bootstrap percentage (BP) and 1.00 posterior probability (PP). The relationships within the Nodosilinea clade are complex and showed several low- to well-supported monophyletic groups. Nodosilinea signiensis forms a monophyletic group with two sequences of two specimens previously identified as “Leptolyngbya” sp. and one sequence of Nodosilinea sp. with high support from both ML (BP = 79%) and BI (PP = 93%).

Fig 4

16S rRNA gene-based maximum likelihood phylogenetic tree showing the genetic distinctiveness and phylogenetic position of Nodosilinea signiensis sp. nov.

ML bootstrap values (left) followed by Bayesian posterior probabilities (right) on branches. Dashes indicate support values less than 50%. The sequence generated in this study is in boldface. Gleobacter violaceous FR798924 was the outgroup.

ITS Secondary structure. The D1–D1´ helix, a semi-conserved subregion of the 16S–23S ITS region, was examined in the genus Nodosilinea. The putative secondary structures of the D1–D1´ helix of N. signiensis sp. nov., together with those of other species of Nodosilinea, are presented in Fig 5. The D1–D1´ helix of eight Nodosilinea species contained 62–64 nucleotides, and seven of them (N. signiensis sp. nov, N. radiophila TMS2B, N. ramsarensis KH-S S2.6, N. chupicuarensis PCA471, N. epilithica Kovacik 1998/7, N. nodulosa UTEX 2910, N. bijugata Kovacik 1986/5a) possessed a 6 nucleotide unilateral bulge with highly conserved sequence (CACUCU), and shared the same basal stem structure (GACC–GGUC) (Fig 5). Despite their similar D1–D1´ helix structures, the sequences of N. signiensis sp. nov. differed from those of the seven previously described species.

Fig 5

Secondary structure based on 16S – 23S internal transcribed spacer (ITS) for eight species of Nodosilinea.

Nodosilinea signiensis sp. nov. shared an identical unilateral bulge and basal stem structure with other species, except for N. conica SEV4-5-c1, but differed from other species in sequence detail.

Discussion

Our study on a cyanobacterial strain superficially resembling Leptolyngbya, isolated from soils obtained on Signy Island, has resulted in the identification of a new species of Nodosilinea. Nodosilinea signiensis sp. nov. is differentiated from other previously described species based on cell size, cell shape, filament attenuation, sheath morphology, granulation and geographical distribution. Due to the simple morphology of this genus, some characteristics overlap between the species. However, the distinct cell shapes and the lack of filament attenuation separate N. signiensis sp. nov. from all previously described species. Cultivation clearly showed that nodule formation is only facilitated in low light conditions. Therefore, strains assigned to similar genera that have not been exposed to low light could have been misidentified. The occurrence of this species in an Antarctic terrestrial habitat further supports its separation from previously described species, based on the strikingly different biotope.

Our genetic data further indicate that N. signiensis sp. nov. is distinct from other species of Nodosilinea. The 16S rDNA phylogenetic results are congruent with morphological examination in confirming that N. signiensis sp. nov. is a member of the genus Nodosilinea. Within Nodosilinea, N. signiensis sp. nov. showed similarity values of < 98% compared with species from the basal subclade. Stackbrandt and Goebel [56] recommended that the similarity cut-off for bacterial species recognition should be 97%. Recently, Stackbrandt and Ebers [57] raised the cut-off to 98.7%, a value later supported by Yarza et al. [58]. On this basis, N. signiensis sp. nov. represents a species distinct from the seven previously described Nodosilinea species. Leptolyngbya antarctica is recognised as the sister clade within the genus Nodosilinea. Morphological study of L. antarctica [59] and the genetic evidence confirm that N. signiensis sp. nov. and L. antarctica are clearly distinct from each other. The evolutionary relationships of N. signiensis sp. nov. within the genus remain largely unclear, possibly due to the limited number of variable sites in the 16S rDNA gene. Future use of multiple genetic makers is required to fully clarify phylogenetic relationships within Nodosilinea.

Modelling of the secondary structure of the ITS region indicated that the structure of the D1-D1’region of N. signiensis sp. nov. was closely similar to that of other species of Nodosilinea, even though the genetic sequences were distinctly different. Previous studies have shown that analysis of 16S rDNA gene phylogeny coupled with the secondary structure of the ITS region provides a suitable tool for separation of cyanobacteria species [8, 60, 61, 62, 63]. We note that the secondary structure of the ITS region in Nodosilinea is conserved among species. This study demonstrates that the integration of other lines of evidence, from morphological and genetic sequence data, provides an effective means to improve the taxonomy of species of Nodosilinea.

25 Jul 2019

PONE-D-19-15186

Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from soil collected on Signy Island, South Orkney Islands, Antarctica

PLOS ONE

Dear Dr Merican,

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Additional Editor Comments (if provided):

Thank you for you well written and conducted study. The manuscript has been evaluated by one reviewer and I have also carefully read the manuscript.

I commend you on the polyphasic approach and the careful photography and drawings. The study is quite limited in scope and it is probably better suited to an algal or taxonomic style journal. I have marked a few small corrections, suggestions and questions on the PDF and ask that you respond to each of these.

The reviewer has raised a number of major concerns related to single strain and limited corresponding environmental data. Please carefully consider these concerns and provide responses to each.

Thank you for submitting your work to PLoS One. I look forward to receiving you responses.

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: N/A

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Reviewer #1: No

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Reviewer #1: Dear Authors,

The manuscript of Radzi et al.: “Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from soil collected on Signy Island, South Orkney Islands, Antarctica” introduce a new cyanobacterial species description: Nodosilinea signiensis. The manuscript is well written and well structured. However, the sampling size is not appropriate and does not support the conclusions of the study. I will not recommend this manuscript for publication as it is.

So far, the definition of bacterial ‘species’ is still debated. So, the combination of molecular and morphological characterization method is a good approach and a good choice. Nevertheless, my main concern about this manuscript is more about the material. Indeed, all the description of this ‘new’ taxa is based on the characterization of only one isolate in culture. This is not enough to define a new species under botanical or bacteriological codes. Also, the characterization of a new lineage requires the characterization of the organism in multiple environmental samples from diverse location, as well as the characterization of different strains isolated from diverse samples.

My second concern is about the fact that lineages belonging to Nodosilinea genus are very common in Antarctica. Thus, small non heterocytous filamentous cyanobacteria forms conspicuous mats in Antarctica and are well described in the literature (including strains) (eg. Taton et al 2006, Komarek et al 2007). So far, Letpolyngbya antarctica was reported as the most dominant taxa observed in mats. Recently, it was found that one of the clusters composing this polyphyletic taxon (firstly described by Taton et al. 2006) was the most abundant in saline lakes and was related to Nodosilinea (see Pessi et al. 2018). This taxon was also observed and strains were isolated from sample of sub Antarctic islands. They may be related to your isolate, and the latitude may partly explain their relatedness. For examples I know some strains from James Ross islands that are placed more or less in the same phylogenetic position. To my knowledge there are at least 5 strains available in collection culture which belong to this cluster from both maritime and continental Antarctic. I suggest you include the analysis of theses strains in your study.

I would like to encourage you to re submit in this journal or elsewhere a manuscript that will include detailed environmental data of samples, microscopic observation of environmental samples, more isolate from Signy islands as well as strains available in culture collections (BCCM/ULC; CCALA). Such a paper will be needed as there is still a lack of information about the OUT Leptolyngbya/ Nodosilinea which appeared to be one of the most dominant taxa present under these latitudes. It is also possible to look for location of all sequences that are similar by BLAST (ncbi).

Finally, there are few points for which I think the authors must not draw conclusions based on the data presented. Besides the fact that the sample size is not enough, differences of ITS sequences are not relevant here as D1 D1’ secondary structure is more relevant for this purpose. Genetic variation in ITS region can be very high in the same lineage it is observed in different taxa. Also, I think you can not emit conclusion on morphological characterization as presence of granulation as the other strains might not have been cultured with the same parameter and observed at a different time in their life cyles, as well as no conclusion could be made on the attenuation of the apex as it was not characterized in other strains.

Also, I regret that sequence was not available for reviewing purposes.

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Reviewer #1: No

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Submitted filename: PONE-D-19-15186 -reviewed.pdf

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11 Sep 2019

PLOSOne – revised manuscript by (11/9/2019)

Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from soil collected on Signy Island, South Orkney Islands, Antarctica

Response to reviewers’ comments

Reviewer’s comment 1: So far, the definition of bacterial ‘species’ is still debated. So, the combination of molecular and morphological characterization method is a good approach and a good choice. Nevertheless, my main concern about this manuscript is more about the material. Indeed, all the description of this ‘new’ taxa is based on the characterization of only one isolate in culture. This is not enough to define a new species under botanical or bacteriological codes. Also, the characterization of a new lineage requires the characterization of the organism in multiple environmental samples from diverse location, as well as the characterization of different strains isolated from diverse samples.

> Re: We thank the reviewer for the stimulating comment. Although theoretically we agree with the need to have more than one strain to describe the intraspecific variability, we would like to highlight three reasons that led us to describe this strain as new:

1) The significant morphological, ecological and molecular distinctiveness of this strain as compared to the other seven species previously described for the genus Nodosilinea. We believe that these three independent lines of evidence are a strong indication that our strain represents a new species.

2) The logistical difficulty to sample Signy Island makes it highly unlikely that we can return there in the near future, while the stochastic nature of field collections also provides no guarantee of any specific target taxon being present in any collections made. We wish to emphasize that we have surveyed 20 distinct collections in the initial examination of field samples, and this taxon was not present in any of these. However, upon establishing cultures of the field material from the 20 sites, we found the taxon growing well in plates inoculated only with the sample from Robin Peak. The restricted distribution (only occurring in 1 out of 20 sites) suggests that it is rare. Genetic data strongly supports the identity of the specimen being that a new species as proposed.

3) We also think that in the face of the ongoing, rapid environmental change on Signy Island (warming climate, impact of enlarged fur seal population and on-going human visitors) that it is important to document and describe this distinctive species of Nodosilinea in the literature, for the use of other researchers. As it may be a species that could be threatened by these changes.

We hope that our study will serve as a reference for additional investigations on Antarctica cyanobacteria that might encounter similar strains elsewhere in the region.

Reviewer’s comment 2: My second concern is about the fact that lineages belonging to Nodosilinea genus are very common in Antarctica. Thus, small non heterocytous filamentous cyanobacteria forms conspicuous mats in Antarctica and are well described in the literature (including strains) (eg. Taton et al 2006, Komarek et al 2007). So far, Letpolyngbya antarctica was reported as the most dominant taxa observed in mats. Recently, it was found that one of the clusters composing this polyphyletic taxon (firstly described by Taton et al. 2006) was the most abundant in saline lakes and was related to Nodosilinea (see Pessi et al. 2018). This taxon was also observed and strains were isolated from sample of sub Antarctic islands. They may be related to your isolate, and the latitude may partly explain their relatedness. For examples I know some strains from James Ross islands that are placed more or less in the same phylogenetic position. To my knowledge there are at least 5 strains available in collection culture which belong to this cluster from both maritime and continental Antarctic. I suggest you include the analysis of theses strains in your study.

> Re: We fully agree with the reviewer that the comparison with Leptolyngbya antarctica is critical to determine whether our strain represents a new species or merely a population of L. antarctica. For that, we compared the 16S rRNA sequences of L. antarctica, which were provided by Taton et al. (2006) to describe this species. We convincingly demonstrate that our species is genetically distinct from L. antarctica (the similarity between the two species is below 98%). We agree with the reviewer that L. antarctica, as currently identified in the field, is a species complex and it is closely related to the genus Nodosilinea (and suggest that it should be transferred to this genus as Nodosilinea antarctica). The differences between the two were also supported by phenotypic evaluation of L. antarctica. The morphological characterization of L. antarctica as presented in Komarek (2007) showed no resemblance with our strain in having thinner and longer cells that are not constricted at the cross walls. The occurrence of nodules was also not reported for this species (Komarek, 2007).

We have included more sequences from thin and simple filamentous as suggested.

Reviewer’s comment 3: I would like to encourage you to re submit in this journal or elsewhere a manuscript that will include detailed environmental data of samples, microscopic observation of environmental samples, more isolate from Signy islands as well as strains available in culture collections (BCCM/ULC; CCALA). Such a paper will be needed as there is still a lack of information about the OUT Leptolyngbya/ Nodosilinea which appeared to be one of the most dominant taxa present under these latitudes. It is also possible to look for location of all sequences that are similar by BLAST (ncbi).

>While we do agree with the value of this type of information, we have to emphasize that this is simply not possible, and has never been achieved in other ‘survey’ type studies, where multiple field samples are collected for laboratory processing at multiple specific locations and habitats differing widely in their micro environmental characteristics (see description and discussion in Convey et al. 2018 Polar Biology to emphasize this point). Similarly, this study did not (and could not) set out to make micro-environmental or microscopic descriptions of each of the many habitats sampled.

Reviewer’s comment 4: Finally, there are few points for which I think the authors must not draw conclusions based on the data presented. Besides the fact that the sample size is not enough, differences of ITS sequences are not relevant here as D1 D1’ secondary structure is more relevant for this purpose. Genetic variation in ITS region can be very high in the same lineage it is observed in different taxa.

> Re: We agree with the reviewer that differences in ITS sequences are not apparent in this study. However, this is a standard approach used in many studies (and failure to do so is itself often a source of reviewer criticism. For instance, see the studies of Perkerson et al. (2011) and Vázquez-Martínez et al. (2018), who used this region to distinguish within the Nodosilinea species. Comparison of the D1 D1’ made for all the species in the genus showed that seven out of eight previously species have identical secondary structure containing a unilateral bulge and basal stem, notwithstanding different sequences in every helix. Our findings are consistent with these previous studies (Perkerson et al. 2011, Vázquez-Martínez et al. 2018), which indicated the conserved feature of D1-D1’ structure in most members of the genus, including the newly proposed species N. signiensis

Also, I think you cannot emit conclusion on morphological characterization as presence of granulation as the other strains might not have been cultured with the same parameter and observed at a different time in their life cyles, as well as no conclusion could be made on the attenuation of the apex as it was not characterized in other strains. Also, I regret that sequence was not available for reviewing purposes.

> Re: Concerning the granulation, we only briefly mentioned the lack of granulation in the present specimen. We agree with the reviewer that this feature is not a diacritical characteristic unless there is a distinct pattern that is conserved under a range conditions in culture and at different ages in culture. Following normal practice, the sequences will be deposited in the GenBank should the manuscript be accepted for publication.

We thank the reviewer for these critical comments.

Submitted filename: Nodosilinea signiensis sp. nov._PLOSOne Reviewer response.docx

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14 Oct 2019

Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from soil collected on Signy Island, South Orkney Islands, Antarctica

PONE-D-19-15186R1

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23 Oct 2019

PONE-D-19-15186R1

Nodosilinea signiensis sp. nov. (Leptolyngbyaceae, Synechococcales), a new terrestrial cyanobacterium isolated from mats collected on Signy Island, South Orkney Islands, Antarctica

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