Discovery of novel Nodosilinea species (Cyanobacteria, Nodosilineales) isolated from terrestrial habitat in Ryukyus campus, Okinawa, Japan
Article information
Abstract
Terrestrial cyanobacteria are extremely diverse. In urban areas, they can be found as black stains on the surface of building walls, stone monuments, or man-made structures. Many of the terrestrial cyanobacteria are still understudied. To expand knowledge of terrestrial cyanobacterial diversity, a polyphasic characterization was performed to identify 12 strains isolated from campus of University of the Ryukyus, Okinawa, Japan. Multigene phylogenetic analyses based on 16S rRNA gene and 16S-23S rRNA internal transcribed spacer (ITS) region showed that the isolated strains formed two independent subclades within Nodosilinea, and were distantly related to all described Nodosilinea species. The 16S-23S rRNA ITS secondary structures showed variations for D1-D1′ and Box B domain, while V3 domain was almost identical among entire species of Nodosilinea, including the studied strains. In addition, a unique morphological character, i.e. forming nodule or spiral shape, was also observed in certain studied strains. According to polyphasic characterization, Nodosilinea coculeatus sp. nov. and Nodosilinea terrestrialis sp. nov., were proposed as two new species of terrestrial cyanobacteria from Okinawa.
INTRODUCTION
Cyanobacteria are among the most geographically widespread oxygenic photosynthetic organisms (Whitton and Potts 2000). They can be found in various habitats, such as terrestrial, freshwater, marine, or even in extreme environments (Dvořák et al. 2015, Sciuto and Moro 2016, Kim et al. 2022). Cyanobacteria play crucial ecological roles in providing carbon, oxygen, and nitrogen fixers in global ecosystems (Charpy et al. 2010, 2012, Gaydon et al. 2012, Brocke et al. 2018). Despite their wide distributions and essential ecological contributions, taxonomic classification of cyanobacteria is extremely complex. Numerous cryptic taxa have been incorrectly classified. Traditional identification, solely based on morphology without considering genetic differences has led to taxonomic confusion because of the similarity in morphological characteristics among different taxa (Komárek 2018). It has been reported that the morphological classification does not accurately correspond to the actual evolutionary history of cyanobacteria (Zammit et al. 2012, Mühlsteinová et al. 2018). Recently, integration of traditional morphological and modern molecular identification, known as polyphasic characterization, has emerged as the most effective approach to correctly classifying cyanobacterial taxa. It has now become fundamental framework for taxonomic classification of cyanobacteria (Hoffmann et al. 2005, Komárek 2016, Caires et al. 2018).
In the last century, cyanobacterial taxonomic classification has massively changed (Komárek et al. 2014). The most recent taxonomic revision, based on multigene phylogeny by Strunecký et al. (2023), proposed ten new orders and 15 new families to achieve monophyly in all taxonomic ranks. One of the newly proposed orders is Nodosilineales. This order consists of filamentous cyanobacteria with isodiametric or two times longer than wider cells, very thin cell width (<2 μm), and parietal thylakoids. Due to lack of morphologically diagnostic features, many strains of Nodosilineales were incorrectly assigned to Leptolyngbya sensu stricto, which is one of the most problematic genera in cyanobacteria. The most diverse genus in Nodosilineales is Nodosilinea, containing at least 10 described species (Perkenson et al. 2011, Heidari et al. 2018, Vázques-Martínez et al. 2018, Radzi et al. 2019, Davydov et al. 2020, Cai et al. 2022). Nodosilinea species have capability to form nodules in their filaments under low light conditions, a unique morphological character that can be used to distinguish them from Leptolyngbya species and other species in the Pseudanabaenaceae (Perkenson et al. 2011). Despite numerous taxonomic studies on Nodosilinea, some species of Nodosilinea remain undescribed. Therefore, increased surveys from unexplored regions are still necessary to uncover the true diversity of this genus.
Okinawa is located in a subtropical region influenced by high temperature, humidity, and intense sunlight exposure. This climate condition can promote proliferation of terrestrial microbial biofilms (Hoffmann 1989, Lewin 2006) which are usually found in residential or public areas, forming blackened stains on the surface of old building, stone monuments, or other man-made structures. The current main campus of Ryukyus University located in Nishihara, Okinawa has been utilized since 1975. Nowadays, various types of microbial biofilms have grown in the campus areas, including on building walls, the university monument, or limestone stairs. These microbial biofilms constitute a complex ecosystem, harboring extremely diverse microorganisms, including subaerial cyanobacteria. Surprisingly, the diversity of the cyanobacteria from Ryukyus campus is extraordinarily diverse, however, the specific status of many strains is still undescribed (Nguyen et al. 2017). Based on this information, we conducted more detailed identification for the undescribed cyanobacterial species from Nishihara campus of University of the Ryukyus. As the result, two potential novel species from genus Nodosilinea (order Nodosilineales) have been discovered. Furthermore, two new species name, Nodosilinea coculeatus sp. nov. and Nodosilinea terrestrialis sp. nov., are proposed as new members of Nodosilinea under the provision of the International Code of Nomenclature for Algae, Fungi and Plants (ICN).
MATERIALS AND METHODS
Culture conditions
A total 12 terrestrial cyanobacterial strains from our culture collections were analyzed for the taxonomic identification of Nodosilinea species. The strains were previously isolated from surfaces of building walls, stairs, monuments, or roadside walls within Ryukyus campus (Nguyen et al. 2017). All strains were maintained using BG-11 with pH 7.4, and kept under 14 : 10 (L : D) cycle using light intensity of approximately 40 μE m−2 s−1 at 25 ± 1°C. In addition, experiment using a nitrogen-depleted medium was performed to observe nodule formation in all isolated strains. Nodosilinea strains were examined under the light microscope after four weeks incubation.
DNA extraction and polymerase chain reaction amplifications
Genomic DNA was extracted using DNeasy plant mini kit (Qiagen, Hilden, Germany) following manufacturer’s protocol. Two genetic markers, 16S rRNA gene and 16S-23S rRNA internal transcribed spacer (ITS), were amplified for molecular identification of the targeted strains. Two pairs of primers, 27F (Neilan et al. 1997) with CYA1371R(1+2+3) (Murakami et al. 2004) and CYA781F (Nübel et al. 1997) with 23S30R (Taton et al. 2003), were used to amplify 16S rRNA gene and 16S-23S ITS region, respectively. The amplification of 16S rRNA and 16S-23S rRNA ITS region was performed following protocols outlined in Nuryadi and Suda (2022). Subsequently, the quality of polymerase chain reaction products was checked using 1% agarose gel. The amplicons with a single bright DNA band were sent to Macrogen Japan Corp. (Tokyo, Japan) for sequencing process in forward and reverse directions using the same pairs of primers for amplification.
Molecular analyses of the 16S rRNA gene and the 16S-23S ITS region
Phylogenetic analyses were performed based on sequences of 16S rRNA gene and 16S-23S rRNA ITS region. Sequences of studied strains were aligned with sequences of various cyanobacterial species obtained from GenBank, including Nodosilinea nodulosa UTEX 2910 (EF122600) as the type species of Nodosilinea. The alignment was performed separately for each gene using MUSCLE algorithm (Edgar 2004) in MEGA X software (Kumar et al. 2018), then, the alignment result was manually corrected. Both phylogenetic trees, 16S rRNA gene and 16S-23S rRNA ITS region, of Nodosilinea and related taxa were constructed using both maximum likelihood (ML) and Bayesian inference (BI). Appropriate substitution models (Table 1) were separately selected for each gene using jModelTest 2.1.6 (Darriba et al. 2012, Guindon and Gascuel 2023) through CIPRES science gateway (Miller et al. 2010). The ML phylogenetic tree was constructed using RaxMLGUI 2.0 (Edler et al. 2021) with 1,000 bootstrap replications. The BI analysis was performed using MrBayes 3.2.7a (Ronquist and Huelsenbeck 2003) through CIPRES science gateway, with four Markov chain Monte Carlo simulations, 107 generations, resampling every 100 generations, and 0.25 burnin rate. A similar setting was applied for 16S rRNA genes and 16S-23S rRNA ITS region. A matrix of genetic similarity among all Nodosilinea species, including strains from Okinawa, was estimated based on calculation of p-distance in MEGA X.
Best-fit models for nucleotide substitution used in phylogenetic analyses
The 16S-23S rRNA ITS secondary structures of each strain were constructed using Mfold version 2.3 with default parameters (Zucker 2003). The identifiable secondary structures were compared between studied strains with described species of Nodosilinea. All predicted structures of Nodosilinea species were identified following Iteman et al. (2000), Perkerson et al. (2011), Heidari et al. (2018), Vázques-Martínez et al. (2018), Radzi et al. (2019), Davydov et al. (2020), and Cai et al. (2022). All sequences of Nodosilinea strains from Okinawa were deposited to GenBank under the accession numbers LC731253–LC731264.
Morphological observation
Morphological characters were observed using an inverted microscope (CKX31; Olympus, Tokyo, Japan) and light microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) equipped with Spot Idea 5 MP camera (Diagnostic Instrument, Sterling Heights, MI, USA). Morphological features such as filament shape, sheaths, cell shape, cell dimensions, cross-walls, apical cells, and nodule formation were observed from a total 50 filaments for morphological characterization. Subsequently, all isolated strains were morphologically identified according to Komárek and Anagnostidis (2005), Perkerson et al. (2011), Heidari et al. (2018), Vázques-Martínez et al. (2018), Radzi et al. (2019), Davydov et al. (2020), and Cai et al. (2022).
RESULTS
Phylogenetic analyses
Both ML and BI phylogenetic trees demonstrated almost similar topologies. The ML phylogenetic tree was used to describe phylogenetic relationship among Nodosilinea with other related cyanobacterial taxa. Based on the 16S rRNA phylogeny, two new species, Nodosilinea coculeatus H. Nuryadi, S. Sumimoto and S. Suda and Nodosilinea terrestrialis H. Nuryadi, S. Sumimoto and S. Suda, were described from 12 strains isolated from terrestrial habitat within Ryukyus University areas. Each new species formed highly supported monophyletic subclade within Nodosilinea clade, and they were genetically distant to other described species (Fig. 1). According to the 16S rRNA gene phylogenetic inference, the closest related species to N. coculeatus and N. terrestrialis were N. signiensis and N. bijugata, respectively. The genetic similarity between strains of N. coculeatus and N. signiensis were 99.1–99.2%, whereas strains of N. terrestrialis shared 98.8% similarity to N. bijugata (Table 2). Strains of N. coculeatus shared 99.9–100% similarity one another, and N. terrestrialis was 100% identical among all strains. N. coculeatus shared 97.7–97.8% similarity to N. terrestrialis. The 16S-23S rRNA ITS also showed that both N. coculeatus and N. terrestrialis were clearly distinct from other described species of Nodosilinea (Fig. 2). All strains of N. coculeatus were sister to Nodosilinea sp. IkpPStn44 with a genetic similarity of 93%, while N. terrestrialis shared a 96.5% genetic similarity to N. bijugata (Table 3).
The 16S rRNA maximum likelihood phylogenetic relationship of Nodosilinea spp. with other related cyanobacterial taxa. Two new species from terrestrial habitat in Ryukyus campus Okinawa are highlighted in gray. Numbers at the nodes indicate Bayesian posterior probabilities / bootstrap values of maximum likelihood. Only support values >0.50 and >50% are shown for posterior probabilities and bootstrap values, respectively.
Genetic similarity of Nodosilinea species based on 16S rRNA sequences
Phylogenetic inferences based on the 16S-23S internal transcribed spacer region sequences of Nodosilinea spp. Support values on the nodes are shown as Bayesian inference / maximum likelihood. Posterior probabilities >0.5 and bootstrap values >50% are shown. Gray highlight indicates new species of Nodosilinea isolated from terrestrial habitat in Ryukyus campus, Okinawa.
Genetic similarity of all species of Nodosilinea calculated from sequences of 16S-23S ITS region
16S-23S rRNA ITS secondary structures
Three informative domains of the 16S-23S rRNA ITS region: D1-D1′, Box B, and V3 were successfully identified in 12 strains from Okinawa. The structure length was nearly consistent for each structure, except for Box B domain (Table 4). The structure of D1-D1′ was composed of 61 to 62 bp of nucleotide sequences. Despite having almost similar length, this domain exhibited variation in structure form among species (Fig. 3). The bases of the D1-D1′ structures of every species had almost identical shape. They were characterized by a conserved 5 bp of basal stem (5′-GACCU–AGGUC-3′) followed by 6 bp of unilateral bulge in 3′ side. Two 1 bp unilateral bulges were found on the 3′ and 5′ side in long stem helix. Some variations in nucleotide sequences were identified in this long stem helix. A distinct shape was clearly observed in the top of structure. Three species: N. coculeatus, N. ramsarensis, and Nodosilinea sp. IkpPStn44 had a 1 : 4, 1 : 3, and 1 : 3 bilateral bulge, respectively, followed by 4 bp terminal loop with a short stem at the top of structure. One species, N. epilithica Kovacik 1998/7, presented a 1 : 1 bilateral bulge before 10 bp of terminal loop. The remaining species, including N. terrestrialis showed a 16 bp of terminal loop with slight differences in nucleotide composition. Furthermore, both tRNAIle and tRNAAla were present in all species of Nodosilinea (secondary structures were not shown), while structure of V2 domain was absent in all species of Nodosilinea.
Identifiable domains in the 16S-23S rRNA ITS region from species of Nodosilinea
Structures of D1-D1′ domain of Nodosilinea spp., including strains isolated from Okinawa. Red and blue color indicate nucleotide sequence variation in identical structures.
In contrast to D1-D1′ domain, variations in length and shape were observed in the Box B domain in some species of Nodosilinea (Fig. 4). The length of Box B structure ranged from 39 to 47 bp. In terms of structure shape, all species shared similar conserved 4 bp of basal stem (5′-AGCA–UGCU-3′), followed by varied structure shapes in certain species. N. coculeatus and N. nodulosa had two unilateral bulges 5′ and 3′ side before 4 bp terminal loop with a long stem. Another new species, N. terrestrialis was almost identical with N. bijugata. The structure showed two bilateral bulges after conserved basal stem. However, 4 and 5 bp of terminal loops were found in N. terrestrialis and N. bijugata, respectively. The V3 domain showed the most conserved structure in every species of Nodosilinea (Fig. 5). All species had 5 bp of basal stem with nucleotide substitution U and C in fifth bases (5′-CAUUU/C–GAGUG-3′). A 4 : 2 bilateral bulge followed by a 4 or 5 bp of terminal loop with a 3 bp of stem was identified in the top of structure.
Structures of Box B domain among species of Nodosilinea. Red and blue color are variable nucleotide sequences between two identical structures.
Nearly identical structures of V3 domain among strains of Nodosilinea. Variation in nucleotide sequences is in red colors.
Morphological identification
A total 12 strains were isolated from several sampling sites in the university area. The filaments of 12 strains comprising from two species, N. coculeatus (Fig. 6) and N. terrestrialis (Fig. 7), exhibited identical morphological characteristics. In general, all strains were characterized as wavy filaments, blue-green coloration, with almost isodiametric cells or mostly longer than wide, distantly constricted at the cross-walls, rounded apical cells without calyptra and enclosed with thin colorless sheaths. Under a nitrogen-depleted condition, filaments of N. coculeatus formed spiral shape (Fig. 6E & F), while N. terrestrialis showed presence of nodule formation (Fig. 7E & F). No nodule or spiral shape were observed in the filaments of two new species cultured in medium containing nitrogen source (Figs 6C & 7D). Moreover, heterocytes or other specialized cells were not observed in any of the studied strains. The morphological characteristics of all species of Nodosilinea, including new species from Okinawa, are presented in Table 5.
Morphological characteristics of Nodosilinea coculeatus. (A) Culturable strains of N. coculeatus forming blue-green fasciculate, thin layer mats in laboratory conditions. (B & C) Filament appearances of N. coculeatus with different magnifications. (D) A colorless thin sheath in filaments of N. coculeatus (arrow). (E & F) Filaments of N. coculeatus formed spiral shapes in low nitrogen condition (arrows). Scale bars represent: B–F, 20 μm.
Morphological appearances of Nodosilinea terrestrialis. (A) Green to dark green fasciculate, thin mats of N. terrestrialis in laboratory conditions. (B & C) Filaments characteristics observed in N. terrestrialis with different magnifications. (D) Trichome of N. terrestrialis enclosed with thin and colorless sheath (arrow). (E & F) Nodules formed by filaments of N. terrestrialis under nitrogen-depleted condition (arrows). Scale bars represent: B–F, 20 μm.
Morphological comparison among two new species of Nodosilinea from Okinawa and described species of Nodosilinea
Nodosilinea coculeatus H. Nuryadi, S. Sumimoto et S. Suda sp. nov
Description
Thallus forms blue-green fasciculate, thin layer mats in culture conditions. Filament typically very thin, straight or sometimes curvy. Sheath generally thin, colorless. Trichome blue-green, composed from barrel shaped-cells or sometimes almost isodiametric. Cell dimension usually longer (1.4–2.7 μm) than wide (1.2–1.5 μm), cells well separated by cross-walls, not granulated, heterocytes or other specialized cells absent. Apical cells rounded without calyptra. Forming spiral shape in nitrogen-depleted condition. Forming hormogonia by straight trichome fragmentation without necridic cell in reproduction process.
Holotype
A metabolically inactive dried specimen from reference strain Ru1-11, RYU A0060 in the RYU (Herbarium of the Faculty of Science, University of the Ryukyus, Okinawa, Japan) sampled from building walls at campus of University of the Ryukyus, Nishihara, Okinawa, Japan.
Reference strain
Nodosilinea coculeatus Ru1-11 (GenBank accession No. LC731255).
Etymology
coculeatus from Latin meaning “spiral”, referring to the spiral shape which often found from filaments of this species.
Diagnosis
Forming blue-green fasciculate thallus in laboratory condition. Capable to form spiral shape in their filaments. Species delimitation based on phylogeny of 16S rRNA gene and 16S-23S rRNA ITS region, as well as distinct ITS secondary structures of D1-D1′ and Box B domains.
Habitat
Microbial biofilms growing on the surface of building walls.
Nodosilinea terrestrialis H. Nuryadi, S. Sumimoto et S. Suda sp. nov
Description
Thallus forms green to dark green fasciculate, thin mats in culture conditions. Filament typically very thin, straight sometimes curvy. Sheath generally thin, colorless. Trichome pale green, cells often almost isodiametric 1.2–2.2 μm width and 1.2–1.7 μm long, cells completely separated by ungranulated cross-walls, lacking heterocytes or other specialized cells. Apical cells rounded without calyptra. Forming nodules in poor nitrogen condition. Hormogonia formed by straight fragmentations with no formation of necridia.
Holotype
A metabolically inactive dried specimen from reference strain Ru4-25, RYU A0066 in the RYU (Herbarium of the Faculty of Science, University of the Ryukyus, Okinawa, Japan) sampled from man-made structure at campus of University of the Ryukyus, Nishihara, Okinawa, Japan.
Reference strain
Nodosilinea terrestrialis Ru4-25 (GenBank accession No. LC731263).
Etymology
terrestrialis refers to habitat of the species.
Diagnosis
Forming green to dark green fasciculate thallus in laboratory condition. Capable to form nodules in the filaments. Species delimitation based on phylogeny of 16S rRNA gene and 16S-23S rRNA ITS region, as well as distinct ITS secondary structures of D1-D1′ and Box B domains.
Habitat
Forming blackened microbial biofilms on the surface of man-made structure.
DISCUSSION
Nodosilinea has been reclassified under the newly described order Nodosilineales Strunecký and Mareš ordo.nov. and family Nodosilineaceae Strunecký and Mareš fam. nov. (Strunecký et al. 2023). This genus has relatively high species diversity, consisting at least ten described species from various environments. However, many of the taxa within this genus are still undescribed or incorrectly classified. In this study, we have described two new species of Nodosilinea, N. coculeatus and N. terrestrialis. The 16S rRNA phylogeny demonstrated that clades of Okinawan strains were genetically distant from other described Nodosilinea species. The new species, N. terrestrialis, was a sister group of N. bijugata. However, sequences of these two species were not completely identical, and they do not cluster within a single clade. Therefore, N. terrestrialis can be described as different species from N. bijugata. The genetic similarity of 16S rRNA gene sequences between new species and closely related species was >98.7% (dissimilarity <1.3%), which does not satisfy the threshold criteria for differentiating bacterial species as suggested by Stackebrandt and Ebers (2006) as well as Yarza et al. (2014). However, it is suggested that sequences of 16S rRNA gene are often insufficient for species delimitation in cyanobacteria (Bohunická et al. 2015, Bravakos et al. 2016, Akagha et al. 2023, Jusko and Johansen 2023). Recently, more variable region, such as 16S-23S rRNA ITS region, is considered as reliable genetic marker for identifying cyanobacterial species (Boyer et al. 2001, Perkerson et al. 2011, Martins and Branco 2016, Mai et al. 2018, Vázquez-Martínez et al. 2018, Martins et al. 2019, Pietrasiak et al. 2019, Lefler et al. 2021, Berthold et al. 2022). Therefore, analysis of 16S-23S rRNA ITS region is required to support speciation in cyanobacteria.
The phylogeny of 16S-23S rRNA ITS region also demonstrated genetic differences between Okinawa strains and described species of Nodosilinea. The genetic similarity was lower than 97% (dissimilarity >3%), which has exceeded criterion species differentiation established by González-Resendiz et al. (2019). In addition to phylogeny, secondary structures of informative domains in 16S-23S rRNA ITS region were constructed to provide more robust evidence for speciation. The secondary structures of 16S-23S rRNA ITS region have been widely considered as important characters for cyanobacterial taxonomic classification (Gugger et al. 2005, Johansen and Casamatta 2005, Casamatta et al. 2006), especially for describing both new genus and species (Řeháková et al. 2007, Siegesmund et al. 2008, Johansen et al. 2011, Mühlsteinová et al. 2014, Vaz et al. 2015, Brito et al. 2017, Nuryadi and Suda 2022). Despite having similar length of sequence, D1-D1′ domain exhibited variation in the structure form. Furthermore, some nucleotide substitutions can be found within identical structure. The Box B domain also exhibited diverse structure among species, both in term of sequence length and structure form. In contrast, V3 domain presented a nearly identical structure in all species. Considering variation in the secondary structure configuration, the D1-D1′ and Box B domains are appropriate for identifying two new species from Okinawa. Overall, variations in secondary structures may reflect species divergence within this genus.
Morphologically, it is very difficult to differentiate two new species with phylogenetically closely related species because they have very identical morphological features. However, they are ecologically found from different environments. Two new species are isolated from terrestrial area in subtropical region, while N. bijugata and N. signiensis are found in freshwater habitat (Perkenson et al. 2011) and extremely cold environment in Antarctica (Radzi et al. 2019), respectively. In general, the two new species from Okinawa morphologically resemble Leptolyngbya species. The morphological features frequently overlap with many species of Leptolyngbya. For instance, N. coculeatus and N. terrestrialis share similar morphological characteristics with Leptolyngbya ectocarpi (Gomont) Anagnostidis et Komárek 1988, however, the trichome color differs between them. L. ectocarpi has pinkish to pale purple trichomes, whereas both new species have blue-green trichomes. Nodule or spiral shape formation can be found in the filaments of new species but is absent in L. ectocarpi. Moreover, L. ectocarpi is a marine species, while the new species from Okinawa are found from terrestrial habitats. The morphological and ecological differences can serve as diagnostic traits, supporting molecular genetic results in the classification of N. coculeatus and N. terrestrialis as new species of cyanobacteria.
In this study, combination of some analyses, including morphology, multigene phylogeny of 16S rRNA and 16S-23S rRNA ITS region, genetic similarity, and secondary structure identification, has been applied to identify terrestrial cyanobacterial strains from Okinawa. The results of these analyses have provided compelling evidence for the description of two novel species, N. coculeatus and N. terrestrialis. The discovery of these new species has revealed high species diversity in genus Nodosilinea. Some Nodosilinea taxa are still classified as undescribed species. Further studies are necessary to define specific status of these taxa. Moreover, many locations outside campus areas have yet to be explored for terrestrial cyanobacteria. A broad exploration in various environments can lead to the discovery of new cyanobacterial species shortly.
ACKNOWLEDGEMENTS
Part of this work was supported by JSPS KAKENHI Grant (#19K06090).
Notes
The authors declare that they have no potential conflicts of interest.