Recognizing novel cyanobacterial diversity in marine benthic mats, with the description of Sirenicapillariaceae fam. nov., two new genera, Sirenicapillaria gen. nov. and Tigrinifilum gen. nov., and seven new species

Figures & data

Figs 1–6. Images of Sirenicapillaria glauca (reference strain BLCC-M125).

Fig. 1. Images showing hair-like thalli.

Fig. 2. Microscope images showing variation in filament pigmentation. Scale bar = 100 µm.

Fig. 3. Blue green cell colour. Scale bar = 50 µm.

Fig. 4. Filament diagonal fragmentation Scale bar = 50 µm.

Fig. 5. Yellow apical cell. Scale bar = 50 µm.

Fig. 6. Filament ends without sheath. Scale bar = 50 µm.

Figs 7–15. Microscope images of Sirenicapillaria rigida (reference strain BLCC-M116).

Fig. 7. Rigid filament sheath and bending. Scale bar = 100 µm.

Fig. 8. Clump of hormogonia usually attached to filaments. Scale bar = 100 µm.

Fig. 9. Reduced pigmentation towards the apices. Scale bar = 100 µm.

Fig. 10. Difference between a long filament centre with a sheath and apices without. Scale bar = 50 µm.

Fig. 11. Necridia. Scale bar = 50 µm.

Fig. 12. Filament slight diagonal fragmentation. Scale bar = 50 µm.

Fig. 13. Extensive mucilaginous sheath. Scale bar = 50 µm.

Fig. 14. Coarse and lamellate sheath. Scale bar = 50 µm.

Fig. 15. Hormogonium with calyptra. Scale bar = 50 µm.

Figs 16–26. Images of Sirenicapillaria stauglerae (reference strain BLCC-M121).

Fig. 16. Thallus.

Fig. 17. Microscope image showing filament clumping. Scale bar = 100 µm.

Fig. 18. Microscope image showing filament morphological plasticity. Scale bar = 50 µm.

Fig. 19. Width and colour difference between filament centre and apex. Scale bar = 100 µm.

Fig. 20. Necridia and textured sheath. Scale bar = 50 µm.

Fig. 21. Diagonal fragmentation. Scale bar = 50 µm.

Fig. 22. Rare false branching. Scale bar = 50 µm.

Fig. 23. Filament prior to false branching. Scale bar = 50 µm.

Fig. 24. Hormogonia formation. Scale bar = 50 µm.

Fig. 25. Calyptra. Scale bar = 50 µm.

Fig. 26. Slight conical apical cell shape. Scale bar = 50 µm.

Figs 27–38. Microscope images of species of Tigrinifilum.

Fig. 27. Variation in filament colours: T. floridanum strain BLCC-M51. Scale bar = 50 µm.

Fig. 28. Variation in filament colours: T. floridanum strain BLCC-M119. Scale bar = 50 µm.

Fig. 29. Variation in filament colours: T. floridanum strain BLCC-M118. Scale bar = 50 µm.

Fig. 30. Floccose thalli of T. guerandense (reference strain BLCC-M99). Scale bar = 50 µm.

Fig. 31. Extreme granulation at the crosswalls resembling tiger stripes in T. floridanum. Scale bar = 50 µm.

Fig. 32. Lack of granulation at apical crosswalls of T. floridanum. Scale bar = 50 µm.

Fig. 33. Lack of granulation at apical crosswalls of T. floridanum. Scale bar = 50 µm.

Fig. 34. Extensive granulation at crosswalls and lack of granulation in apices in T. guerandense. Scale bar = 50 µm.

Fig. 35. Extensive granulation at crosswalls and lack of granulation in apices in T. guerandense. Scale bar = 20 µm.

Fig. 36. Calyptra of T. floridanum. Scale bar = 50 µm.

Fig. 37. Filaments growing past each other of T. floridanum. Scale bar = 50 µm.

Fig. 38. Rare false branching of T. floridanum. Scale bar = 50 µm.

Figs 39–48. Microscope images of Capilliphycus. Scale bars = 50 µm.

Fig. 39. Yellow apical region of C. flaviceps (reference strain BLCC-M137).

Fig. 40. Extended sheath at the apices in C. flaviceps (reference strain BLCC-M137).

Fig. 41. Diagonal fragmentation in C. flaviceps (reference strain BLCC-M137).

Fig. 42. False branching in C. flaviceps (reference strain BLCC-M137).

Fig. 43. Filaments growing past another in C. flaviceps (reference strain BLCC-M137).

Fig. 44. Spiral morphology of some filaments of C. flaviceps (reference strain BLCC-M137).

Fig. 45. Yellow apical cells of C. guerandensis (reference strain BLCC-M76).

Fig. 46. Facultative sheath in C. guerandensis (reference strain BLCC-M76).

Fig. 47. Diagonal fragmentation in C. guerandensis (reference strain BLCC-M76).

Fig. 48. Tapering of apices in C. guerandensis (reference strain BLCC-M76).

Table 1. Morphological characteristics of known species of Sirenicapillaria gen. nov., Tigrinifilum gen. nov. and Capilliphycus. Measurements (μm) are represented as min–max and mean ± st. dev

Genus	Sirenicapillaria	Sirenicapillaria	Sirenicapillaria	Tigrinifilum	Tigrinifilum	Capilliphycus	Capilliphycus
Species	rigida	stauglerae	glauca	floridanum	guerandense	flaviceps	guerandensis
Thallus	rigid and loose, tangled knots	flexuous and erect	loose, tangled knots	entangled, mat-like	floccose	entangled, mat-like	entangled, mat-like
Filament width (µm)	28.0–83.0(–94) 47.6 ± 17.2	12.5–33(–35) 19.5 ± 4.6	18.4–25.2(–28) 22.4 ± 2	(9–)10.2–19.0 14.2 ± 1.5	-	14–18.9 16.5 ± 1.4	18.5–29.2 22.3 ± 3.1
Sheath	hyaline, thick, lamellate	hyaline, thin to thick	hyaline, thin	hyaline, facultative	no sheath	hyaline, facultative	thick, facultative
Trichome	progressive attenuation	progressive attenuation	slight attenuation	no attenuation	no	no	no
Cell shape	discoid	discoid	discoid	discoid	discoid	discoid	discoid
Cell length (µm)	1.8–3.9 2.9 ± 0.5	1.0–2.4 1.5 ± 0.3	1.3–2.8 1.8 ± 0.4	1.0–2.7 1.7 ± 0.3	1.5–2.9 2.2 ± 0.4	1.4–2.4 1.7 ± 0.2	1.0–2.2 1.6 ± 0.3
Cell width (µm)	22.8–63.3 37.9 ± 11.9	10–26.8 15.2 ± 3.9	(14–)15–20(–21) 17.2 ± 1.8	(7–)8.0–19.2 11.2 ± 1.4	5.3–6.2 5.8 ± 0.3	10.7–14 12.4 ± 1.1	15.8–24.9 19 ± 2.5
Apical cell	bluntly rounded	bluntly rounded	bluntly rounded	bluntly rounded	conical to bluntly rounded	conical	conical
Cell content	homogeneous	homogeneous	homogeneous	homogenous	homogeneous	homogenous	homogenous
Cell colour	red, light to dark brown	light to dark brown	forest green	green, blue green, grey, brown, gold,	blue green, green	green to greenish yellow, yellow at apices	green, yellow at apices
Constriction	yes	yes	yes	yes	no	slightly	no
Cross-wall	facultative granulation	facultative granulation	facultative granulation	very granulated	very granulated	granulated	no
Reproduction	release of single cells, hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells	release of single cells, hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells	release of single cells, hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells	hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells	trichome fragmentation, helped or not by necridic cells	hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells	hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells
Occurrence	marine and benthic in seagrasses	marine and benthic in seagrasses	marine and benthic in seagrasses	marine and benthic and epipsammic	hypersaline, marine planktonic	marine and benthic and epipsammic	hypersaline, benthic

Fig. 49. Maximum likelihood and Bayesian inference, showing Bayesian topology of the phylogenetic relationship of the 16S rRNA gene sequence of the 30 cyanobacterial strains presented in this work and 141 cyanobacterial taxa including Gloeobacter violaceus PCC 7421 (NR074282) as outgroup. Bootstrap support and posterior probabilities ≥50 and 0.5 are shown at consensus nodes of both inferential analyses, respectively. (*) represents the reference strains for the species; triangle indicates the branch where the family Sirenicapillariaceae is delimited; stars denote the type species of the genus.

Table 2. Mean p-distance (percentage of genetic similarity) between and within (last column) established cyanobacterial families, based on 16S rRNA gene sequences. Values are based on an alignment of 91 taxa and 7 established cyanobacterial families including the novel family Sirenicapillariaceae with eight genera: Affixifilum, Capilliphycus, Lyngbya, Limnospira, Limnoraphis, Neolyngbya, Sirenicapillaria and Tigrinifilum. Taxonomic family classifications were based on Guiry & Guiry (2021) and Hauer & Komárek (2021)

		1	2	3	4	5	6	7	Within
1	Sirenicapillariaceae								97.20
2	Oscillatoriaceae sensu stricto	92.62							98.35
3	Laspinemataceae	94.39	93.65						98.41
4	Microcoleaceae	94.51	93.98	94.77					98.68
5	Desertifilaceae	93.13	91.83	91.69	92.11				95.25
6	Vermifilaceae	93.96	92.43	92.82	94.67	91.63			98.01
7	Spirulinaceae	92.97	91.58	92.81	93.05	92.33	91.87		94.66
8	Coleofasciculaceae	94.54	93.56	93.64	94.54	93.63	93.61	93.87	99.80

Table 3. Morphological characteristics of the genera Affixifilum, Neolyngbya, Limnoraphis, Capilliphycus, Sirenicapillaria gen. nov. and Tigrinifilum gen. nov. within the family Sirenicapillariaceae fam. nov.

Characteristics	Affixifilum	Neolyngbya	Limnoraphis	Capilliphycus	Sirenicapillaria gen. nov.	Tigrinifilum gen. nov.
Thallus	thick compact mats, rarely solitary	small green mats or large brown fasciculate mats	filaments solitary, free-floating, or growing in small aggregations	extensive fasciculate mats or small clusters	rigid and loose, tangled knots	floccose or entangled, mat-like
Filament	8.1–29.8 µm diameter, filaments straight, or slightly curved to slightly waved	6.9–26.7 µm diameter, filaments straight	5–25 µm diam.; straight or slightly curved	(8–)12–30(–46) µm diam.; straight	12.5–83(–94.0); straight and rigid	(9–)10.2–19.0; straight
Sheath	hyaline or laminate, thin or thick	hyaline, thin or thick, sometimes lamellated	firm, colourless, hyaline, thin, or slightly thick	hyaline or bright yellow to yellow-brown, firm, thin or thick, sometimes lamellated	hyaline or gelatinous, lamellate at times, thin to very wide	facultative, or not present
Trichome width	10.1–32.4 µm diameter	7.8–24.7 µm diameter	15–19 µm diameter	(6.6–)10–21(–24) µm	10–63 µm	5–20 µm
Cell content	contains gas vesicles and numerous cyanophycin granules	containing numerous gas vesicles	gas vesicles facultative	containing numerous gas vesicles usually forming aerotopes	heavily pigmented	homogeneous
Constriction	slightly constricted	not or slightly constricted	not or slightly constricted	not or slightly constricted	slightly constricted	facultative
Cross-wall	heavily granulated	frequently granulated	not informed	often granulated	granulated	very heavily granulated
Attenuation	attenuation present	trichomes not attenuated towards end	not attenuated	not or slightly attenuated towards ends	slight attenuation across filament length towards ends
Cells	short discoid, 0.95–3.8 µm long	short, 1.3–4.5 µm long	cylindrical, short, always wider than long	short, 1.4–5.6(–6) µm long, 1.2–11 times larger than long	discoid, 1–4 µm long, 6–21 times wider than long	discoid, 1–3 µm long, twice as wide
Apical cell	usually rounded or conical-rounded, calyptra common, attenuation when forming hormogonia, may form many at once	usually rounded or conical-rounded, thickened rare, without calyptra	without clear calyptra	rounded, conical, conical-rounded, flat-rounded, truncate, rarely capitate, sometimes with thickened, without calyptra	bluntly rounded, at times slightly conical	conical to bluntly rounded
Reproduction	hormogonia formed within trichomes by straight fragmentation with or without necridia. Terminal hormogonia formed by attenuation of the apices and formation of several hormogonia.	hormogonia formed by diagonal, straight, or irregular fragmentation pattern of the trichome	intense hormogonia production	hormogonia formed by straight or diagonal fragmentation of the trichome, helped or not by necridic cells	release of single cells, hormogonia formed by straight or diagonal fragmentation of the trichome, helped or not by necridic cells	hormogonia by straight or diagonal trichome fragmentation, helped or not by necridic cells
Occurrence	epilithic or epipsammic mats in tropical marine environments	epilithic or epipsammic in tropical to subtropical environments	planktic in mesotrophic waters in Lake Atitlán, Guatemala, Florida freshwater canal	marine and halophilic, and in inland saline biotopes	marine and benthic in seagrasses	marine/hypersaline and benthic, planktonic
Reference	Lefler et al. (2021)	Caires et al. (2018a)	Komárek et al. (2013)	Caires et al. (2018b); this paper	this paper	this paper

Table 4. Lengths of identifiable domains in the 16S–23S rRNA Internal Transcribed Spacer (ITS) regions of strains of Capilliphycus, Limnoraphis, Sirenicapillaria gen. nov. and Tigrinifilum gen. nov. Duplicated strain numbers indicate different ITS patterns (with or without tRNAs). Multiple values indicate variable operon length within species and/or strains. A dash (-) denotes not observed

Species/Strain(s)	Leader	D1-D1ʹ helix	Spacer + D2 + Spacer	D3 + Spacer	tRNA^Ile	Spacer+ V2+ Spacer	tRNA^Ala	Spacer	Box-B Helix	Spacer	BoxA	D4 to ITS end
Capilliphycus flaviceps BLCC-M53	8	58	39	22	-	-	-	-	25	17	11	57
C. flaviceps BLCC-M137	8	58	39	26	-	-	-	-	25	17	11	51
C. flaviveps BLCC-M137	8	58	39	13	74	24	73	83	28	17	11	51
C. guerandensis BLCC-M76; BLCC-M92	8	60	39	22	74	24	73	66	40	17	11	55
C. guerandensis BLCC-M76	8	60	44	20	-	-	-	-	40	17	11	55
C. tropicalis	8	58	39	22	-	-	-	-	24	17	11	53
C. salinus	8	61	39	22	-	-	-	-	34	17	11	45
Sirenicapillaria stauglerae	8	62	75	30	-	-	-	-	27	17	11	47
S. stauglerae BLCC-M121	8	62	45	10	74	17	73	40	27	17	11	47
S. rigida BLCC-M116; BLCC-M134	8	63	41	13	74	18	73	40	31	17	11	47
S. rigida BLCC-M134	8	63	73	33	-	-	-	-	28/31	17	11	47
S. glauca BLCC-M125	8	66	73	30	-	-	-	-	29	17	11	47
Limnoraphis sp. BLCC-F19; BLCC-F23	8	58	38/39	62	-	-	-	-	38	17	11	54
Limnoraphis sp. BLCC-F23	8	58	39	13	74	24	73	61	38	17	11	54
Tigrinifilum floridanum	8	62/63	38	12	74	15	73	70	40	18	11	62
Tigrinifilum guerandense BLCC-M99	8	62	38/39	12	74	15	73	99	34	22	11	56

Fig. 50. The 16S–23S rRNA ITS sequence secondary structures of the D1-D1ʹ of four cyanobacterial genera (Capilliphycus, Limnoraphis, Sirenicapillaria, Tigrinifilum) presented in this work. Nonbolded taxa represent previously published data. A: C. guerandensis BLCC-M76 & BLCC-M92. B: C. guerandensis BLCC-M79 & BLCC-M92 with tRNA. C: C. flaviceps BLCC-M53. D: C. flaviceps BLCC-M137 with and without tRNA. E: C. salinus. F: C. tropicalis. G: Limnoraphis sp. BLCC-F19 & BLCC-F23 with and without tRNA. H: S. glauca BLCC-M125. I: S. rigida BLCC-M116 & BLCC-M134 with and without tRNA. J: S. stauglerae BLCC-M121, BLCC-M122, BLCC-M123, & BLCC-M138. K: S. stauglerae BLCC-M121 with tRNA. L: T. floridanum BLCC-M48, BLCC-M50 clone A, BLCC-M57 clone C, BLCC-M66 clone B, BLCC-M77 clone C, BLCC-M87 clone C, BLCC-M107, BLCC-M118 clone B, BLCC-M119 clone B, & BLCC-M120 clones A & B, with tRNA. M: T floridanum BLCC-M50, BLCC-M57 clones A & B, BLCC-M66 clones A & C, BLCC-M73, BLCC-M87 clones A & B, BLCC-M118 clones A & C, BLCC-M119 clone C, & BLCC-M120 clone C, with tRNA. N: T. guerandensis BLCC-M99, with tRNA.

Fig. 51. The 16S–23S rRNA ITS sequence secondary structures of the Box-B of the four cyanobacterial genera (Capilliphycus, Limnoraphis, Sirenicapillaria, Tigrinifilum) presented in this work. Nonbolded taxa represent previously published data. A: C. flaviceps BLCC-M53. B: C. flaviceps BLCC-M137. C: C. flaviceps BLCC-M137 with tRNA. D: C. guerandensis BLCC-M76 & BLCC-M92 with and without tRNA. E: C. salinus. F: C. tropicalis. G: Limnoraphis sp. BLCC-F19 & BLCC-F23 with and without tRNA. H: S. glauca BLCC-M125. I: S. rigida BLCC-M116 & BLCC-M134 with and without tRNA. J: S. stauglerae BLCC-M121, BLCC-M122, BLCC-M123, & BLCC-M138 with and without tRNA. K: T. floridanum BLCC-M48, BLCC-M50, BLCC-M57, BLCC-M66, BLCC-M73, BLCC-M77, BLCC-M87, BLCC-M107, BLCC-M118, BLCC-M119, & BLCC-M120, with tRNA. L: T. guerandensis BLCC-M99, with tRNA.

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