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Morphological and molecular characterization of Stigonema jureiensis sp. nov. (Nostocales, Cyanobacteria) from the Atlantic Rainforest, São Paulo, Brazil

Tropical regions are considered hotspots of biodiversity, and the genus Stigonema has been underexplored in all world, mainly due to the difficulties of culturing. In this study, we sequenced the 16S rRNA gene of a natural Stigonema population from the tropical Atlantic Rainforest (São Paulo, Brazil) and describe S. jureiensis sp. nov. based on phylogenetic and morphological data. For the molecular analysis, the genomic DNA was isolated from aliquots of the population, which were previously manually cleaned under a microscope, using a point–tapered Pasteur pipette to remove other organisms and debris. For morphological analysis, 30 individuals were analyzed and photographed using microscope. Both maximum likelihood and Bayesian phylogenies support S. jureiensis as a new species, and morphological analysis revealed the presence of a special type of mosaic ornate sheaths.


Introduction
The nostocalean genus Stigonema Bornet et Flahault is abundantly distributed worldwide, but phylogenetic studies on it are scarce compared to other cyanobacterial genera. This is mainly due to difficulties on isolating and culturing this taxon, and consequently, until recently, taxonomic studies on Stigonema were based solely on morphology. In a study on the diversity of true-branched Cyanobacteria from the Brazilian Atlantic Rainforest, Ferreira et al. (2013) published the first combined molecular and morphological analysis of a Stigonema population (S. ocellatum Bornet et Flahault). Using the glass-capillary isolation technique, Mareš et al. (2015) were able to sequence the 16S rRNA gene of several morphologically characterized Stigonema presenting a robust 16S rRNA gene phylogeny for the genus. In the same year, Song et al. (2015) described S. dinghuense Song et Li based on both morphological and molecular data. These two latter studies support the monophyly of the genus Stigonema.
After these initial publications, a large number of 16S rRNA gene sequences of Stigonema were deposited in public DNA sequence databases (GenBank). Thus, the current situation of the genus is: (1) there are 164 Stigonema 16S rRNA gene sequences in GenBank; (2) most of them are not associated to a published manuscript, thus lack morphological data; (3) there is a lack of information on 16S-23S rRNA Internal Transcribed Spacer (ITS) sequences and secondary structures; (4) as with other cyanobacteria, the infrageneric phylogenetic relationships are uncertain; and (5) there are few Stigonema strains in culture collections, making the access to the 16S rRNA gene of these populations difficult. Thus, most of the taxonomic studies of Stigonema are restricted to morphological data presented in classic works, such as Bornet & Flahault (1889), Gardner (1927), Frémy (1930, Geitler (1933), and Komárek (2013).
The genus Stigonema is abundant in the Brazilian Atlantic Rainforest, being studied by Silva & Sant'Anna sequences and all sequences with less than 1297 nucleotides length. In order to study our populations at the species level, we constructed trees using only those Stigonema sequences associated to articles with morphological data. To do this, we searched GenBank for the publications of all 164 Stigonema sequences available on 08 March 2019. Then, we selected the published sequences, excluded the clones, and performed the alignment for phylogenetic analysis.
For all generated trees, the sequences were aligned using ClustalW and the GTR+G+I evolutionary model was selected by running Modeltest in MEGA 7.0 (Kumar et al. 2016). The robustness of ML trees was estimated by bootstrap, using 1000 replications in MEGA 7.0 (Kumar et al. 2016). The Bayesian trees were constructed in two independent runs, with four chains each for 5 × 10 5 generations, using Mr. Bayes (Ronquist & Hueksenbeck 2003). The outgroups used were Gloeobacter violaceus AF132791, Chroococcidiopsis sp. FR798923 and Chroococcidiopsis sp. AB039005.
A similarity matrix (p-distance) was also generated to compare Stigonema jureiensis with the closest phylogenetically related taxa. The 16S rRNA gene sequence of S. jureiensis was deposited in GenBank (NCBI) under the accession Uncultured Stigonema JUR147 (MK471331).

Results
While studying the cyanobacterial flora of tropical and subtropical areas of the Atlantic Rainforest (São Paulo, Brazil), an interesting new terrestrial stigonematacean taxon was observed. The taxon fit very well into the morphological circumscription of Stigonema, featuring true branches (T-type) and multiseriate trichomes. However, it differed from all the currently described species of Stigonema, due to unique mosaic-like sheaths that had not been previously described for the genus (Figs 1-7). Thus, we propose a new species, Stigonema jureiensis sp. nov., supported by morphological and molecular data.
The first step of the 16S rRNA gene phylogeny (total of 170 OTUs with 752 informative sites) confirmed that our proposed new species was placed in the strongly supported Stigonema clade (ML = 100%, BA = 1) (tree not shown). In the second step, the ML and BA trees (63 OTUs with 1325 informative sites) showed similar topology, presenting two main clades with strong phylogenetic support (Fig. 8). In the basal position of Nostocales, the cluster containing Brasilonema, Chlorogloeopsis, Iphinoe, Rivularia, and Scytonema (ML = 32%, BA = 0.99) is sister to the cluster of Coleodesmium, Dapisostemon, Hassallia, and Stigonema (ML = 99%, BA = 1.0). Although Stigonema is monophyletic (ML = 99%, BA = 1.0), the relationships among its species are unresolved. In the Stigonema clade, the uni-and multiseriate species are intermixed (Fig. 8). Furthermore, some infrageneric clades were common by different species with strong phylogenetic support.These were the cases of S. spectabile KT867140 (multiseriate) and S. elegans KT867212 (uniseriate), which are in the same clade with ML support of 100% and BA of 1.0; the clade of S. cornutum KT867200 and S. turfaceum KT867159 (1988), who described S. gracile Silva et Sant'Anna from Ilha do Cardoso (Atlantic Rainforest) and Silva & Sant'Anna (1996), who revised the genus for São Paulo State, Brazil. The most recent study on Stigonema from the Atlantic Rainforest described three new species and presented four other morphotypes commonly found in the biome (Sant'Anna et al. 2013). Although there has been a large diversity of Stigonema reported for the Atlantic Rainforest, the genus is still poorly studied in this biome, with all papers, except for Ferreira et al. (2013), restricted to morphological analysis.
In this study, we sequenced the 16S rRNA gene from a natural population of Stigonema collected in the Atlantic Rainforest (São Paulo State, Brazil). The population presented a unique type of mosaic-like sheath and based on both phylogenetic and morphological data, we describe and propose the new species, Stigonema jureiensis sp. nov.

Material and Methods
A Stigonema population was collected in August 2011 by scraping biofilms growing on an igneous rocks in the tropical Ecological Station Juréia-Itatins (24º24'36''S and 47º01'15''W) area of the Brazilian Atlantic Rainforest. The material was stored and dried using paper bags, and later in the laboratory the dried material was rehydrated for 20 hours in distilled water for morphological analysis. At least 30 individuals were analyzed and photographed using a Zeiss Axioplan 2 light microscope, equipped with a Zeiss Axiocam MRc digital camera. These images were compared to known Stigonema species in Komárek (2013). A subsample was preserved in formaldehyde (4%) and deposited in the Herbarium of the Institute of Botany São Paulo, Brazil (SP), under the number SP 427311. Subsequently to the morphological inspection, for the 16S rRNA gene analysis, the natural population was cleaned using a point-tapered Pasteur pipette under a microscope to remove organisms and debris attached to the filaments. Afterwards, 50 individuals were transferred and washed sequentially into five new droplets of sterile distilled water. Total genomic DNA was extracted from these individuals using a DNeasy UltraClean Microbial Kit (Qiagen) and the 16S rRNA gene was amplified using the 27F and 1494Rc PCR primers (Neilan et al. 1997). For amplification and sequencing of our material, we followed the methods in Fiore et al. (2007) and Hentschke et al. (2016). Phylogeny was inferred based on Maximum Likelihood (ML) and Bayesian (BA) trees, which were constructed in several steps (Hentschke et al. 2016). To position our species in the Nostocales tree, first we aligned our sequence with sequences In order to connect morphological and phylogenetic data, the third step of the ML and BA trees (Fig  9) were built only with strains that contained published morphological data. In these trees, S. jureiensis formed again a unique branch separated from other Stigonema species, and it was sister to S. hormoides and S. tomentosum Hieronymus (ML = 85%, BA = 0.98). The similarity (p-distance) values between S. jureiensis and these strains range from 97% with S. hormoides KM047007 to 99.5% with S. tomentosum KM047014.

Branches
With perpendicular hormogonia. Erect, uniseriate, commonly in fascicles, with the same diameter of the main filament or slightly narrower.
Branches identical to the main filament, bending toward the apex.
Compressed or sphaerical cells.
Sheaths abundant, hyaline or yellowish, mosaic-like sculptured with irregular margins.
Subaerophytic on wet wood and rocks.
Aerophitic, on an igneous rock in the Atlantic rainforest. species are morphologically distinct. S. tomentosum is uniseriate and has lamellate sheaths, while S. jureiensis is multiseriate and has a unique type of mosaic sculptured sheaths, making the new species morphologically distinct from any other species of the genus (Table 1). This hypothetical genetic relationship was very likely influenced by the relatively short length of S. tomentosum KM047014 sequence (1075 bp), and the low number of informative sites in the phylogenetic analysis (965 bp). Also, our phylogenies show that S. jureiensis is separated from other species within the Stigonema clade. Thus, we propose and describe our novel species following criteria  environment such as high temperature and herbivory. In addition to the description of S. jureiensis, we corroborate previous studies (Mareš et al. 2015;Song et al. 2015) showing that uni-and multiseriate species of Stigonema are intermixed in phylogenies. These characters have most likely arisen more than once in the genus Stigonema. Our phylogenies also highlight the problem of species delimitation in Cyanobacteria, since the same species is found in more than one clade (i.e. Stigonema hormoides and S. ocellatum). In this case we agree with mareš et al. (2015) that molecular and ecological divergent populations of these morphospecies will likely be further separated with more molecular data.
We also found that different morphospecies are grouped in monophyletic clades, as for example, the multiseriate S. spectabile WYS16 and the uniseriate S. elegans WYS54 (ML = 100%, BA = 1.0). Considering the species concept of Mishler & Theriot (2000), adapted by Johansen & Casamatta (2005), which states that species should be "monophyletic clusters of strains or natural populations that are diagnosable by some unique combination of traits, those traits being any combination of morphological, biochemical, molecular, or other outlined in Johansen & Casamatta (2005). Our results agree with the first criterion of new species description presented by the authors, which requires evidence of genetic separation to existing species. The mosaic-sculptured sheaths are a strong evidence of genetic separation.
Our results also follow the authors' second criterion, with S. jureiensis separated from other phylogenetic clades. According to those, the morphological feature of S. jureiensis can be used to separate the species, and reinforces the phylogenetic data, strongly indicating that our population is a new species of Stigonema. The special kind of sheath in S. jureienesis has previously been described in  (Hentschke & Komárek 2014), but we did not observe this for S. jureiensis. Probably, this character evolved independently in different lineages as an adaptation to the harsh conditions found in this characteristics", we think that in our trees, the clustering of different morphospecies in monophyletic clades is primarily due to the poor resolution of the 16S rRNA gene at the species level and also by the scarce number of available 16S sequences of Stigonema populations.
Other factors that negatively influence species delimitations in Stigonema are that most of the sequences of morphologically studied Stigonema populations are short, causing polytomies in phylogenies (Mareš et al. 2015;Song et al. 2015), and that there is a lack of 16S-23S rRNA ITS data. The longer and more useful Stigonema sequences in GenBank are from populations that have not yet been published, thus cannot be used in deeper discussions of species delimitations.

Conclusion
In this paper, we propose a new species Stigonema jureiensis, and conclude that the delimitations of species of Stigonema remain unclear. The current lack of ecological and morphological data from morphotypes which have sequences available in GenBank coupled with the short sequences from those morphologically well-studied, make the discussion at the species level difficult. Also, access to more molecular markers, such as the 16S-23S rRNA ITS is urgently needed to improve the resolution of monophyletic clades in the genus.