Scytonema schmidtii Gomont

Hentschke, Guilherme Scotta, Sant’Anna, Célia Leite & Vasconcelos, Vitor, 2025, Phylogenetic insights into the genus Scytonema Bornet et Flahault (Cyanobacteria): A 16 S rRNA gene review with the taxonomic assessment of S. schmidtii Gomont, Acta Botanica Brasilica (e 20240044) 39, pp. 1-11 : 3-8

Scytonema schmidtii Gomont

( Fig. 1 A-F)

In nature, filaments 9–21 µm wide, creep on substrate forming mats. Filaments intensely entangled, frequently double false branched ( Fig. 1A, B ). Sheaths irregularly striated, wide, yellowish when older ( Fig.1C, E ). Trichomes cylindrical, constricted ( Fig. 1C ), or rarely not constricted ( Fig. 1D, F ). Cells quadratic or rarely slightly shorter than wide ( Fig. 1 C-F), 7–18 µm wide. Heterocytes with the same shape and size as vegetative cells. In culture, filaments are thinner, 7–15 µm wide, and usually colorless.

Phylogenetic analysis

Our search resulted in 218 sequences of strains identified as ‘ Scytonema ’, and other sequences labeled as Tolypothrix Bornet & Flahault (1), Desikacharya Saraf et al. (1), Hapalosiphon Bornet & Flahault (1), Iningainema (2), Scytonematopsis Kisseleva (3), Brasilonema (11) and Heteroscytonema (19), totalizing 256 sequences. All of these results were included in our study after the application of the exclusion criteria ( Figure S1 ).

The tree constructed using the FastTree method ( Fig. 2 ) exhibited 16 distinct clades, which may represent different genera and include strains labeled as ‘ Scytonema’. Among these clades, in addition to the clade where the type of Scytonema ( S. hofmanni ) was recovered (clade 5), five clades correspond to previously described genera, such as Brasilonema , Streptostemon Sant’Anna et al., Iningainema , Camptylonemopsis Desikachary , and Heteroscytonema ( Fig. 2 ). The remaining ‘ Scytonema ’ sequences were dispersed among other Scytonemataceae genera, and their classification deserves further investigation, as they may represent potential new cyanobacterial supraspecific taxa, which is discussed further below. The clades containing ‘ Scytonema ’ sequences are highlighted in different colors in Figure 2 .

An overall examination of our FastTree analysis ( Fig. 2 ) revealed that the majority of sequences labeled as ‘ Scytonema ’ are located within what is referred to here as the core of Scytonemataceae , which comprises two primary lineages. The first lineage includes the Brasilonema clade (clade 4), the true Scytonema sensu stricto clade (clade 5), ‘S. mirabile’ clade (clade 2), Iphinoe Lamprinou & Pantazidou , and Symphyonemopsis Tiwary & Mitra. The second lineage is primarily formed by ‘ S. hyalinum ’ strains (clades 6 and 7 ( S. hyalinum cluster II)) and Symphyonema Jao. More details on these two lineages are provided in the text below. Based on the presence of these true branched genera within the Scytonemataceae cluster, the morphological delimitation of the Scytonemataceae family must be revised.

The clade containing the type species of the genus, S. hofmanni (clade 5), represented by the reference strain S. hofmanni PCC 7110 ( AF132781 ), contains other Scytonema species, such as S. javanicum Bornet & Flahault ( S. javanicum U41-MK36 ( HF911525 )), S. pachmarhiense Saraf et al. ( S. pachmarhiense 10A1_PS ( MH260366 )), S. bilaspurensis Singh ( S. bilaspurensis 10C-PS ( KT222810 )), S. singhii Singh ( S. singhii 1F-PS ( KT935473 )), and S. foetidum Tawong et al. ( S. foetidum NUACC 06 ( LC633742 ). However, this clade has low phylogenetic support (ML = 86%), and the 16S rRNA gene similarity among the sequences within this clade can be very low. For instance, in the case of the comparison between S. javanicum ( HF911525 ) and Scytonema sp. ( KX951410 ), they exhibit only 91.7% similarity. These findings indicate that this clade must undergo further investigation and may or may not represent different genera to be recognized in the future.

It is also possible to observe that there are two additional large clusters, we named ‘ S. hyalinum cluster I (clade 1) and II (clade 7)’, both containing S. hyalinum strains, along with other strains identified as S. arcangelii . Regarding these clusters, some strains present in cluster I were also found in cluster II as was the case for S. hyalinum WJT 9-NPBG6A, for example. There are two 16S rRNA gene sequences for this strain, Genbank accession KY 365488 in S. hyalinum cluster I and KY 365451 in cluster II.

Following the FastTree analysis, we identified clades within the sister clade to the Scytonema sensu stricto clade ( Fig. 2 ). The ‘ S. cf. mirabile ’ clade (clade 2) is sister to the other three clades. Our findings indicate that this clade is phylogenetically distinct from the Scytonema sensu stricto, and the 16S rRNA gene sequence similarity between these clades ranges from 92.4% to 94.4%. The other recognized clades are the Scytonema sp. CCIBT3568 (clade 3), which is sister to the Iphinoe / Symphyonemopsis and the Brasilonema (clade 4) clades, these latter sister to each other.

The ML and BI trees ( Fig. 3 ), recovers Scytonema sp. CCIBt3568 not phylogenetically close to the Brasilonema type species, S. bromeliae SPC 951 ( EF117246 ), nor to the Iphinoe and Symphyonemopsis type strains I. spelaeobios LO 2-B1 ( HM748317 ) and Symphyonemopsis sp. VAPOR1 (J544085). Although morphologically identified as Scytonema , the strain presents low 16S rRNA gene similarity with the Scytonema sensu stricto clade, with values ranging from 93.1% to 93.7% ( Table 1 ), and therefore is not phylogenetically closely related to the type species, S. hofmanni PCC 7110 ( Fig. 3 ).

The 16S rRNA gene analyses revealed high similarity between Scytonema sp. CCIBt3568 and the Brasilonema bromeliae clade, ranging from 95.3% to 97.1%, and also in comparison to Iphinoe and Symphyonemopsis (95.6% to 96%) ( Table 1 ). However, we found that the similarity between the Brasilonema clade and the Iphinoe / Symphyonemopsis clade ranges from 95.7% to 97.3%, and they present very different morphological characters. Brasilonema is a scytonematoid genus, with markedly fasciculate thalli and Iphinoe and Symphyonemopsis present true branching. Scytonema sp. CCIBt3568 is also a scytonematoid type, similar to Brasilonema , but differs by never presenting fascicles. We analyzed nature populations and the cultured Scytonema sp. CCIBt3568 and found that the thallus is intensely entangled ( Fig. 1 ).

Other clades in our FastTree analysis also encompass ‘ Scytonema ’ strains found in our search. In the big cluster containing Tolypotrichaceae, Rivulariaceae , Hapalosiphonaceae , Nodulariaceae , Aphanizomenaceae, Leptobasaceae , and Nostocaceae genera, is placed the strain Scytonema crispum U55-MK38 ( HF911526 ) as a sister clade of the Scytonemataceae genus Ewamiania (clade 8), with low bootstrap support (ML=89). However, assigning this strain to the genus is not possible, because they share only 92.8% to 93.5% of 16S rRNA gene similarity. Consequently, Scytonema crispum U55-MK38 ( HF911526 ) should be further investigated and can be possibly described as a new genus in further studies. It’s worth noting that the genus Ewamiania , placed outside the core Scytonemataceae ( Fig. 2 , clades 2-7), may undergo taxonomic reevaluation in the future and be moved from this family.

The strains Scytonema sp. , ‘ Coccocarpia sp. kj30 cyanobiont’ ( KF359679 ), Scytonema sp. ‘ Coccocarpia sp. kj16’ ( KF359678 ) and Scytonema sp. ‘ Coccocarpia sp. kj38’ ( KF359680 ) are recovered within the genus Streptostemon (KJ5669461) (clade 9) with strong phylogenetic support (ML=99) and share more than 97.8% of 16S rRNA gene similarity with this genus. Consequently, these strains can be possibly assigned as a new species Streptostemon in further studies. These strains are from a cyanobiont population of the lichen Coccocarpia Pers. , while the type Streptostemon capitatus Sant’Anna et al., grows forming mats on soils and is not associated with lichens ( Sant’Anna et al., 2010).

Within the Iningainema clade (ML=100) (clade 12) ( Fig. 2 ), there are three strains labeled as Scytonema stuposum P15-MK34 ( HF911528 ), S. stuposum P13-MK35 ( HF911527 ) and S. myochrous SERB 29 ( KM982578 ), which should be assigned to Iningainema . Related to this clade are observed two clades (clades 11 as sister to it and 10 as sister to 11 + 12) containing ‘ Scytonema’ strains labeled as S. stuposum M10-F15A ( KY411153 ), S. cf. chiastum UCFS 19 ( JN565280 ), S. cf. chiastum GSE-NOS-MK14-07B ( KY411157 ), S. cf. chiastum M32-F26III ( KY411154 ), S. cf. chiastum F04-MK25 ( KY411155 ). These strains share more than 91.9% of 16S rRNA gene similarity with Iningainema , and probably are new genera, which should be described in the future. As well as Ewamiania , the current Scytonemataceae genus Iningainema is out of the core Scytonemataceae , and might be moved from this family in the future.

The strain S. mirabile SAG 83.79 ( KM019943 ) (clade 13) is within the Camptylonemopsis clade (ML = 96) and should be assigned to this genus.The strain presents 97.6% of 16S rRNA gene similarity with Camptylonemopsis sp. HA4241-MV5 ( HQ847564 ). The strains S. cf.crispum UCFS 16 ( JN565276 ), S. cf. crispum UCFS 17 ( JN565277 ), S. cf. crispum UCFS 21 ( JN565278 ), Scytonema sp. Ft11 ( MG549309 ), Scytonema sp. UCFS10 ( HM629428 ), Scytonema sp. F 12 ( MG549312 ), S. cf. crispum UCFS 15 ( JN565279 ) are recovered within the Heteroscytonema clade (clade 14), indicating that these strains most likely belong to Heteroscytonema . The 99% 16S rRNA gene similarity observed among the sequences of this clade further corroborates these findings.

The strain S. bohnerii SAG 255.80 ( KM019923 ) (clade 16) is sister to Goleter , with low bootstrap support (ML=69), but presents high 16S rRNA gene similarity (98.3%) with the strain Goleter apudmare HA 4340-LM2 ( KF417425 ), and so far, should be assigned to this genus, until further studies are conducted. As a sister clade to Gloeotrichia Bornet & Flahault are observed the strains Scytonema sp. HAN3/2 ( KP701039 ) (clade 15), Scytonema sp. F 18 ( MG549319 ), which share between 93.5% to 96.8% of 16S rRNA gene similarity with the reference strain Gloeotrichia echinulata PYH 14 ( AM230704 ). Based on that, more studies are needed to verify if these strains identified as ‘ Scytonema ’ are a new genus or should be assigned to Gloeotrichia .

Figures

Figure 1. Scytonema sp. CCIBt3568. A. Original biomass collected. B. Natural population with intensely entangled filaments. C. Detail of filament from nature, featuring yellowish and rugulose sheaths. D.Detail of filament from nature. E. Filament from nature with false branching, yellowish, and rugulose sheaths. F.Photo with phase contrast of cultured filament. Magnification: Fig.1A = 4x; Fig. 1B = 10x; Figs. 1C-F = 40x.

Figure 2. FastTree phylogeny with 383 OTUs and 800 nucleotide positions analyzed. The 16 different clades containing ‘Scytonema’ sequences are ranged in different colors. The outgroup used was Gloeobacter violaceus PCC 8105 (AF132791). Table S1 lists all strains included in the phylogenetic tree.

Figure 3. Maximum Likelihood phylogeny of 16S rRNA gene sequences, with 97 OTUs and 971 nucleotide positions analyzed. Bootstrap values are presented at the nodes, along with the Bayesian Inference posterior probabilities.The average standard deviation of split frequencies of BI is <0.01.

Tables

Table 1. Similarity (p-distance) among Scytonema schmidtii (CCIBt3568) and closest related clades according to Fig. 8. Values are represented by percentage.

	1	2	3	4	5	6	7	8
Brasilonema	96.1-100
Scytonema hyalinum cluster II	94.2-95.9	99.2-100
Symphyonema	93.3-94.4	95.4-95.9	–
“ S. mirabile ” cluster	92.2-95	92.8-95.2	91.8-92.6	96.7-99.9
Scytonema sensu stricto	92.9-94.9	92.9-94.5	91.7-92.5	92.2-94.2	94.7
S. schmidtii (CCIBt3568)	95.3-97.1	95.6-96.1	93.1	93.3-94.3	93.1-93.7	–
Iphinoe	95.7-97	94.1-94.6	94.6	92.6-94.1	93.4-93.5	95.6	–
Symphyonemopsis	95.7-97.3	94.4-94.6	94.8	92.6-93.7	93.2	96	99.3	–