Reofilinostoc matlalcueyense, G. S. Hentschke, G. Garduño-Solórzano & M. Martínez-García, 2024

Garduño-Solórzano, Gloria, Martínez-García, Martha, Hentschke, Guilherme Scotta, Solorza, Luis Fernando Miguel & Vasconcelos, Vitor M., 2024, A phylogenetically distant clade of Nostoc-like (Cyanobacteria) taxa with the description of Reofilinostoc matlalcueyense gen. et sp. nov. from an extreme environment, Phytotaxa 655 (2), pp. 125-143 : 128-135

Reofilinostoc matlalcueyense gen. et sp. nov. G. S. Hentschke, G. Garduño-Solórzano & M. Martínez-García ( Figures 1 , 2 )

The material collected in the field had a morphology resembling a mouse ear, of 0.4-1.2 cm in length (n=25) and a firm cartilaginous texture ( Fig. 1C, D ), colonies pale to dark green to brown with a thick peridermal layer yellowish (17-24 µm) where capturing a mass of filaments develop perpendicularly towards the interior of the thallus ( Fig. 2 A, B ). In solid cultures, after 3 to 4 weeks, warty colonies are enclosed in a firm mucilage formed by compacted filaments.

The filaments are moniliform, first uniseriate ( Fig. 2D ) and then non-seriate ( Fig. 2C, I ). Trichomes can reach dimensions greater than 25 isopolar cells ( Fig. 2E ). The terminal and intercalary vegetative cells are barrel-shaped, of 4.5-6.1 µm width and 4.0-6.0 µm length, and 3.3-3.6 µm width and 2.3-4.0 µm length, respectively. It is a population with a polymorphic life cycle. Heterocytes, both terminal and intercalary solitary, demonstrate an apoheterocytic propagation scheme (2F). Reproduction formed by akinetes, ellipsoid, 2.5-3.9 µm width and 3-0-6.0 µm length ( Fig. 2G ), which develop a giant vegetative cell that conceives a four-celled individual through an unequal division ( Fig. 2H ). It is sheath is evident ( Fig. 2F ).

Holotype specimen: Herbarium IZTA 1929 ( IZTA), collected on May 5, 2022, from the stream of the melting snow of Nevado de Toluca , State Mexico, Mexico.

Reference strain: F02 (OR 724089)/ Facultad de Estudios Superiores Iztacala, UNAM.

Type locality: MEXICO. Stream from the melting snow from Protected Natural Area Nevado de Toluca, Estado de Mexico.

Habitat: growing on andesite rock.

Etymology: Reofilinostoc matlalcueyense (matlalcueyense, in nahuatl language, slope of the volcano), Reofilinostoc , Nostoc from stream.

Morphologically, Reofilinostoc matlalcueyense is close to Komarekiella atlantica , Mojavia , Desmonostoc , Halotia and microscopic species of Nostoc , although they differ in some aspects. The new genus has the aggregated filaments of microcolonies like Mojavia and Halotia and stages in it is life cycle resembling some microscopic Nostoc species e.g. N. punctiforme var. populorum . If only isolated stages of the life cycle of Komarekiella were observed, it would be possible to incorrectly assume that those stages were one or more of these other taxa. Comparing the whole life cycle of Komarekiella with other genera, it was observed that this taxon is indistinguishable from Chlorogloeopsis , especially with regard to the unequal germination of the akinetes, which, until this paper, served as the diagnostic feature of the last. A complete summary of these genera illustrates the difficulty of using morphology alone to define them ( Table 1 ).

Molecular Characterization. The first tree, comprising 363 Operational Taxonomic Units (OTUs) with 752 positions analyzed (not shown), was constructed to identify the phylogenetically most related genera to Reofilinostoc . Within this tree, the order Nostocales forms a monophyletic group, and Reofilinostoc matlalcueyense is situated within a clade alongside Minunostoc and Desikacharya ( Nodulariaceae ) (ML=100). This clade is sister to another clade containing various genera from both Nodulariaceae and Nostocaceae families, including Nodularia , Aliinostoc , Pseudoaliinostoc , Amazonocrinis , Atlanticothrix , Cyanocohniella , Halotia , Nostoc , Desmonostoc , Komarekiella , Roholtiella , Mojavia and Violetonostoc .

The ML phylogeny of Nodulariaceae / Nostocaceae (second stage analysis) ( Fig. 3 ) was constructed using 101 OTUs and analyzing 1071 positions. At the base of the tree, the Pseudanabaenales are positioned, followed by the Chroococcidiopsidales clade, the sister group to Nostocales . The Nostocales group is monophyletic (ML=99), and all the genera exhibit strong bootstrap support. However, the relationships among these genera must be better established, as indicated by the low bootstrap values within the Nostocales clade. This effect is likely attributed to the high similarity (p-distance) values shared among the Nostocales genera, as further detailed in this paper.

In this tree, it is possible to divide the Nostocales cluster into three major lineages. At the top of the tree, there’s the cluster containing the core of Nodulariaceae , including the reference strain Nodularia BCNOD 9427, along with other Nodulariaceae genera like Aliinostoc , Pseudoaliinostoc , Purpureonostoc , Cyanocohniella , Chrysosporum , Anabaenopsis , Goleter , Halotia and Atlanticothrix . Forming another lineage sister to this cluster, but with strong bootstrap support (ML=100), there is a lineage ( Nodulariaceae II ), with two genera Minunostoc and Desikacharya . Reofilinostoc is placed within this lineage, in it is clade, and is not closely related to any previously described genus. Furthermore, in proximity to Reofilinostoc , there are other strains misidentified as Nostoc , which must be described as new genera in the future. At the base of these two lineages, two strains are misidentified as “ Amazonocrinis ” alongside the Nostocaceae genera Komarekiella , Violetonostoc , Compactonostoc and Dendronalium .

The third lineage is composed of the core Nostocaceae , including the reference strain Nostoc WY 1KK1, along with other Nostoc species and Desmonostoc . The type strain Amazonocrinis nigriterrae CENA 67 is in a basal position in relation to the Nostocaceae / Nodulariaceae clade, rendering the genus a polyphyletic status.

According to this phylogeny, Reofilinostoc was classified within the Nodulariaceae family due to it is close phylogenetic relationship with Minunostoc and Desikacharya and with the core Nodulariaceae clade.

The BI ( Fig. 4 ) (Average Standard Deviation of Split Frequencies <0,01) and ML ( Fig. 5 ) Nodulariaceae phylogenies (third stage phylogenies), with 56 OTUs and 1284 positions, confirmed the previous results and presented phylogenetic solid support at the backbone. Both trees exhibited identical topologies, consistent with the structure observed in the Nodulariaceae / Nostocaceae tree (second stage). Again, the Nodulariaceae core is distinctly separated from the Desikacharya / Minunostoc cluster, where Reofilinostoc is positioned. This separation of Nodulariaceae in two lineages is strongly supported by BI posterior probabilities (BI=1), which this is also true in the ML tree. Moreover, the genera-level clades presented strong phylogenetic support in both phylogenies.

The phylogenetic relations among genera presented strong statistic support in the BI tree ( Fig. 4 ) but not in the ML tree ( Fig. 5 ), which is Nodulariaceae / Nostocaceae tree constructed. This is probably because of the high similarity (p-distance) values among genera, typical for Nostocaceae and Nodulariaceae .

BI and ML phylogenies confirm Reofilinostoc as a new genus in it is clade among strains misidentified as Nostoc . It cannot include these “ Nostoc ” strains in Reofilinostoc because of their weak phylogenetic relation, evidenced primarily in the ML tree. The closest related genera to new genus are Minunostoc and Desikacharya .

The intrageneric similarity of the 16S rRNA gene (p-distance) analysis within Nodulariaceae genera is presented in Table 2 . It ranged from 97.2% within Aliinostoc , to 100% within Purpureonostoc and Desikacharya . When comparing different genera, the minimum similarity value was 91%, observed between Desikacharya and Halotia . The highest similarity value comparing genera was 97.8%, found in Halotia and Goleter . This value overlapped the lowest intrageneric similarity observed (97.2%). Furthermore, this analysis revealed that some comparisons among Nodulariaceae genera exhibited similarity values exceeding 97%, such as Atlanticothrix vs. Goleter and Cyanocohniella vs. Anabaenopsis . Based on these findings, the results of p-distance analysis also support the proposal of Reofilinostoc , considering that it displayed a maximum similarity of 96.4%, observed when compared to Desikacharya , which is a clearly separated clade. This value (96.4%) is lower than those cited above for other genera comparisons.

The 16S-23S rRNA ITS secondary structures analysis corroborates with the separation of Reofilinostoc in a new cyanobacterial genus ( Figs. 6 , 7 , 8 ). It was compared the D1-D1’, Box B and V3 helix of Reofilinostoc with the closest phylogenetic-related genera, such as Desikacharya nostocoides and Minunostoc cylindricum . Because of the lack of both tRNAs Ile and Ala in the sequence analyzed, it was not possible to retrieve the V2 helix from the Reofilinostoc 16S-23S ITS region ( Table 3 ).

The D1-D1’ helix of Reofilinostoc ( Fig. 6 ) exhibits a distinct nucleotide sequence and structure compared to Desikacharya nostocoides . Both helices initially share a basal stem, commencing with 5’ GACCTACCCA 3’. However, beyond this point, the sequences of the helix diverge completely. Furthermore, Desikacharya features two residues (5’ AC 3’) located opposite the first lateral bulge, while no residues occupy this position in Reofilinostoc . Additionally, D. nostocoides possesses two loops below the terminal loop, whereas Reofilinostoc only has one. Although both genera have terminal loops with five residues, they differ significantly in sequence. The D1-D1’ helix of Minunostoc cylindricum is remarkably different from the others, being much longer and presenting additional stems in the first lateral bulge region.

The Box B helices of Reofilinostoc and Desikacharya ( Fig. 7 ) present the same basal stem, starting with the typical Box B initial sequence 5’AGCA3’, but after that they are very different in sequence and structure. In Desikacharya the helix present three residues forming a central loop, while in Reofilinostoc the structure present only one mismatch in this position. The terminal loops are different considering length and sequence. For Minunostoc cylindricum , the helix is remarkably different in sequence, length and by presenting two loops below the terminal loop.

The V3 helices ( Fig. 8 ) were the most variable among those genera. From Desikacharya nostocoides is remarkably different, being much longer and presenting four loops below the terminal loop. While, Reofilinostoc matlalcueyense F02 has a basal stem with a 13 bp helix followed by terminal loop consisted of four residues (AGAG). Also, the Reofilinostoc differs from Minunostoc cylindricum in length and structure.

Figures

FIGURE 1. Study area: A) pine forest from Nevado de Toluca. B) stream from the melting snow. C) general aspect of growing upper andesite. D) mouse-ear-shaped brown colonies.

FIGURE 2. Micrographs of Reofilinostoc matlalcueyense F02 under the light microscopy (LM). (A–B) longitudinal section showing trichomes with intercalary heterocyte (arrow), and the thick yellow periderm (*), also perpendicular and coiled trichomes from field material. (C) Colonies wrapped by mucilaginous sheath. (D-E) trichomes simple with few cells vegetative, and with more than 20 cells long, respectively. (F) trichomes with terminal heterocyte (arrow). (G) Akinete. (H) unequal division of an akinete intro a two vegetative cell and subsequent division. (I) Coiled trichomes with sheath. (B–I) dyed with cresil blue. (C–I) material in culture BG11. Scale bars A= 0 70 μm, B= 30 μm, C= 10 μm, D–I= 5 μm.

FIGURE 3. Maximum Likelihood (ML) phylogeny of Nodulariaceae and Nostocaceae, based on 1071 nucleotide positions analyzed.

FIGURE 4. Bayesian Inference (BI) phylogeny of Nodulariaceae family based on 1284 nucleotide positions analyzed.

FIGURE 5. Maximum Likelihood (ML) phylogeny of Nodulariaceae and Nostocaceae, based on 1284 nucleotide positions analyzed.

FIGURE 6. Secondary structure of D1-D1’ helix (16S-23S ITS) of Reofilinostoc matlalcueyense, Desikacharya nostocoides, and Minunostoc cylindricum.

FIGURE 7. Secondary structure of Box-B helix (16S-23S ITS) of Reofilinostoc matlalcueyense, Desikacharya nostocoides, and Minunostoc cylindricum.

FIGURE 8. Secondary structure V3 helix (16S-23S ITS) of Reofilinostoc matlalcueyense, Desikacharya nostocoides, and Minunostoc cylindricum.

Tables

TABLE 1. Comparison of morphological and ecological features between Reofilinostoc matlalcueyense and near representatives from the closest related genera in the clades.

	Nodulariaceae			Nostocaceae
	Desikacharya nostocoides	Minunostoc cylindricum	Reofilinostoc	Komarekiella atlantica
	Saraf and Singh et al.	Cai et Li (2019a)	matlalcueyense	Hentschke et al. (2017)
	(2019 a, b)		(study)
Thallus	Macroscopic green mat	Macroscopic,	Macroscopic	Macroscopic, up to 1
		Trichomes with colonies	ear mouse shape, brown a	cm of long; first bright
		wrapped in a diffluent	bright blue-green	blue–green, becoming
		mucilaginous sheath		a darker duller blue–green
				with age
Terminal vegetative cells
Length (μm)	3.4–5.3	3.1–5.4	4.0–6.0	3–5
Width (μm)	3.9–4.0	2.5–3.8	4.5–6.1	3.4–5.5
Shaped	Barrel	Barrel	Barrel	Barrel
Intercalary vegetative cell
Length (μm)	3.8–5.0		2.3–4.0	3–5
Width (μm)	3.4–5.3		3.3–3.6	3.4–5.5
Shape	Barrel		Barrel	Barrel
Sheaths	Thin	Thick, colourless	Thick, firm	Firm or sometimes
				diffluent
Hormogonia	ND		ND	quadratic, isodiametric
				and motil, old inmotil
Width (μm)				3
Filaments	uniseriate	uniseriate	Uniseriate o multiseriate	Uniseriate o multiseriate
Number of division	ND	ND	Two	Two
planes in tricomes
Akinetes	Absent	Absent	Present	Present

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TABLE 2. Matrix comparing the p-distance values of the 16S rRNA gene of the Nodulariaceae genera, based on phylogenetic analysis

		1	2	3	4	5	6	7	8	9	10	11	12	13
1	Aliinostoc	97.2– 99.9
2	Pseudoaliinostoc	94.3– 96.3	98– 98.8
3	Cyanocohniella	95.9– 97.2	95– 95.9	98.6
4	Anabaenopsis	95.5– 96.2	93.9– 94.5	96.3– 97.2	99.6– 99.8
5	Chrysosporum	93.9– 94.9	92.4– 93.5	94.8– 95.6	96.2– 96.5	99.9
6	Nodularia	94.7– 95.9	93.1– 94.2	95.6– 96.6	96.5– 97.3	95.8– 96.6	98.9
7	Goleter	95.2– 96.6	94.4– 95.4	96.7– 97.3	96.5– 97	97.5– 96	95.8– 96.8	98.7
8	Purpureonostoc	95.5– 96.3	94.2– 95	95.6– 95.6	94.9– 95.1	94.6– 94.7	94.6– 95	95.9– 96.8	100
9	Halotia	95.1– 95.9	93.9– 95.5	96.8– 97.3	95.4– 96.6	93.7– 94.8	94.7– 95.9	96.3– 97.8	94.4– 95.8	97.3
10	Atlanticothrix	94.4– 95.4	93.8– 94.5	95.9– 97	96.2– 96.5	94.9– 95	95.1– 95.7	96.8– 97.3	94.5– 94.6	96.7– 97.8	99.8
11	Minunostoc	91.4– 92.8	92.7– 93.8	92.1– 92.4	92.2– 92.5	92.7	92.2– 93.1	92.8– 93.1	92– 92.2	91.7– 91.8	92.8	99.7
12	Desikacharia	91.4– 94.2	91.6– 93.7	92– 93.9	92– 93.9	92.8– 94.5	91.9– 94	92.7– 94.7	92– 93.6	91– 94.1	92.3– 94	93.5– 95.3	98.4– 100
13	Reofilinostoc	94.4– 94.8	94.3– 94.8	95– 95.4	94.9– 95.2	93.4– 93.5	93.6– 94.2	95.4– 95.6	93.8	94– 94.3	94.9– 95	94.9	94.7– 96.4	NA

TABLE 3. Matrix comparing the p-distance values of the 16S rRNA gene of the Nodulariaceae genera, based on phylogenetic analysis

	Strain	D1-D1´	tRNA Ile	V2	tRNA Ala	BoxB	V3	ID NCBI	Reference	Country
Reofilinostoc	F02-IZTA	62	-	-	-	28	30	OR724089	This work	Mexico
matlalcueyense
Desikacharya soli	BHU2-PS	67	-	-	-	65	71	MH036168	Saraf et al.	India
									2019 a, b
Desikacharya	BHU1-PS	63	-	-	-	64	70	MH036167	Saraf et al.	India
nostocoides									2019 a, b
Nodularia sp.	BCNOD9427	65	-	-	-	32	29	AJ224448.2	Barker et	Baltic
									al. 1999	sea
Nodularia spumigena	NODSPU1	65	-	-	-	34	36	KX557267	Silveira et	Brazil
									al. 2017
Minunostoc	CHAB5843	103	74	80	73	50	36	MH919979	Cai et al.	China
cylindricum									2019a
Nodularia spumigena	NODSPU2	65	74	72	73	33	33	KX557268	Silveira et	Brazil
									al. 2017
Nostoc sp.	CENA269	61	74	63	73	49	25	MN551912	Shishido et	Brazil
									al. 2020
Nostoc sp.	CENA296	63	74	74	73	48	36	MN551922	Shishido et	Brazil
									al. 2020
Nostoc commune	WY1KK1	69	74	70	73	39	103	EU586733	Řeháková	Brazil
									et al. 2014a
Komakeriella	CCIBt 3486	65	74	69	73	26	22	KX638489	Hentschke	Brazil
atlantica									et al. 2017
Halotia branconii	CENA186	65	74	76	73	44	44	KC695877	Genuário et	Brazil
									al. 2015