Capilliphycus tropicalis

Berthold, David E., Lefler, Forrest W. & Laughinghouse, H. Dail, 2022, 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, Phycologia 61 (2), pp. 146-165 : 150-158

Capilliphycus tropicalis T.A. Caires, Sant’Anna & J.M. Nunes

COMMENT: Our material fits the original description of this species in Caires et al. (2018b) and clusters together with Capilliphycus tropicalis in the 16S rRNA gene sequence phylogenetic tree.

SPECIES FEATURES: Thallus green and entangled, mat-like. Filaments straight, 11.8–16.8 µm in diameter. Sheath hyaline. Cells light to dark green. Cells discoid, wider than long, 8.4–12 µm wide, 1–2.1 µm long. This is the first report of this species outside of Brazil.

MATERIALS ANALYSED: BLCC-M 106 ( US 227681).

Cyanobacterial isolation and morphological characteristics

A total of 30 unicyanobacterial isolates were used in this study ( Table S1). All 30 cyanobacterial strains contained discoid cells with or without a sheath. Sampling of marine cyanobacteria from seagrasses, lagoons and marine coastal waters from nine Florida localities revealed floating, epipsammic and epiphytic mats. From Florida BCMs, a total of 24 unicyanobacterial strains were isolated, of which 21 isolates represent two novel cyanobacterial genera, Sirenicapillaria and Tigrinifilum . In addition, three novel cyanobacterial isolates of Capilliphycus and Tigrinifilum from France (Guérande) are described, as well as the phylogenetic information (16S rRNA gene and 16S–23S rRNA ITS) of three isolates of Limnoraphis from freshwater canals in Florida.

Morphological analyses of the 24 Floridian marine cyanobacterial strains demonstrated cyanobacteria with cell dimensions ranging between 10.2 and 83 µm for filament width and discoid cells ranging between 5.3–63.3 and 1.0–3.9 µm for both trichome cell width and length ( Table 1). Sirenicapillaria has cyanobacteria with the largest cell size and sheath, especially S. rigida .

Morphological analyses demonstrated that Sirenicapillaria is composed of taxa with very long thalli (c. 20 cm), filaments with facultative sheaths, and three species: S. glauca ( Figs 1–6 ), S. rigida ( Figs 7–15 ), and S. stauglerae ( Figs 16–26 ). In a healthy lab environment, this genus has concentrated pigments and appears dark ( Figs 1 , 16 ), whereas in the field, in high light, or with unfavourable conditions, it turns light brown to gold colours ( Fig. S2 ). Sirenicapillaria filaments are very long (up to 10 cm), with a thick sheath at the middle of the filament ( Fig. 10 ) and a gradual attenuation of the filament towards the ends with a thin sheath or none at all ( Figs 6 , 9 ). The largest species in terms of filament width is S. rigida , often observed with a lamellate or thickened gelatinous sheath ( Figs 13, 14 ). Compared to its sister species, S. rigida has extremely tough filaments ( Fig. 7 ), forming an intertwined, rigid and net-like mat; this is potentially the justification for the misidentification of Sirenicapillaria as Microseira [ Lyngbya ] in local reports.

Sirenicapillaria stauglerae and S. glauca overlap in both cell width and length. Although S. stauglerae and S. glauca have similar dimensions, these species are easily differentiated based on thallus colour and cell colour; S. stauglerae is generally brown and golden brown while S. glauca maintains a greyish, blue-green pigmentation ( Fig. 3 ). The thallus of Sirenicapillaria is reminiscent of that of Lyngbya majuscula Harvey ex Gomont and Dapis , where a very long thalli resembles fine hair underwater ( Figs 1 , 16 ). Unlike L. majuscula that was originally described as black hair from the coast of England ( Gomont 1892), Sirenicapillaria is more gold to brown and was discovered from the Florida Gulf coast through the Florida Keys. While the morphology of S. rigida overlaps with L. majuscula , the latter reaches larger widths and, together with their different type regions, we do not suggest this to be L. majuscula .

The genus Tigrinifilum is somewhat comparable to previously described cyanobacterial taxa in Lyngbya , including L. aestuarii F. Liebman ex Gomont and L. salina Kützing ex Starmach ( Komárek & Anagnostidis 2005) . Shared morphological features between T. floridanum and L. aestuarii include rare false branching, pronounced cross wall granulation and a marine habitat. However, T. floridanum and L. aestuarii differ considerably in climatic conditions of the type locality, which for L. aestuarii is the North Sea off the coast of present day Friesland in northwestern Germany. Furthermore, there is a large difference in filament and cell sizes between T. floridanum and L. aestuarii , with the latter being up to 10 μm wider. Tigrinifilum floridanum also resembles L. salina in having similar filament width and cell length, as well as in the marine habitat. However, the type locality for L. salina is a thermal spa in Kissingen, Germany, and morphological differences including the presence of a layered and stratified sheath and a lack of constriction between cells within L. salina suggest that T. floridanum is novel. Additionally, T. floridanum demonstrates a calyptra while L. salina does not.

16S rRNA phylogeny, intergeneric and family p -distance analyses

BLASTN analysis of the 16S rRNA gene sequence of the 30 marine cyanobacterial isolates shows them as closely related to the genera Affixifilum , Capilliphycus , Limnoraphis , Limnospira and Neolyngbya with 97% or lower identity. BLASTN analysis of isolates from Tigrinifilum indicated 99.4% or lower identity to uncultured cyanobacteria found in coastal sediments of the Mediterranean Sea ( Païssé et al. 2010) and in sulfidic springs of western USA ( Headd & Engel 2014). Analysis of the 16S rRNA gene sequence of Sirenicapillaria isolates indicated high similarity to sequences of ‘cf. Lyngbya sp. BAN TS02 ʹ (99.2%; HQ419195 ) from Australia and 97.1% or lower similarity to Limnoraphis hieronymusii ( JN854140 ), Capilliphycus tropicalis ( MF190468 ), cyanobacteria found in the guts of marine fish ( Jones et al. 2018), and uncultured cyanobacteria ‘cf. Lyngbya ’ or ‘cf. Limnoraphis ’ from the freshwater Clear Lake in California ( Kurobe et al. 2013) and from Lake Atitlán, Guatemala ( Rejmánková et al. 2011; Komárek et al. 2013), respectively.

BI and ML phylogenetic analyses of the 16S rRNA gene sequence of the 30 isolates demonstrated a large clade containing other taxa including Affixifilum , Capilliphycus , Limnoraphis , Limnospira and Neolyngbya with high support (BS: 89%; PP: 1.0) (triangle in Fig. 49 ). The large Sirenicapillariaceae clade is sister to the Laspinemataceae clade (BS: –; PP: 0.99) and closely related to the Lyngbya confervoides clade (BS: –; PP: 1.0). Within Sirenicapillariaceae , Sirenicapillaria (BS: 88%; PP: 1.0) and Tigrinifilum (BS: 76%; PP: 1.0) formed distinct clades ( Fig. 49 ). The BI and ML analyses also showed clades of described genera including Capilliphycus (BS: –; PP: 1.0) and Limnoraphis (BS: 100%; PP: 1.0). Within Capilliphycus , C. flaviceps (BS: 97%; PP: 1.0) as well as C. guerandensis (BS: 99%; PP: 1.0) formed separate subclades. Within Sirenicapillaria , three separate clades formed including S. glauca (BS: 88%; PP: 1.0), S. rigida (BS: 77%; PP: 0.93) and S. stauglerae (BS: 91%; PP: 1.0). The clade that included Tigrinifilum had two subclades of T. floridanum (BS: 100%; PP: 0.66) and T. guerandense (BS: 74%; PP: 1.0).

Intergeneric pairwise distances of the 16S rRNA gene sequence of the genera within the family Sirenicapillariaceae revealed genetic distances with 93.62%–97.97% among Affixifilum , Capilliphycus , Limnospira , Limnoraphis , Neolyngbya , Sirenicapillaria and Tigrinifilum ( Table S2). Sirenicapillaria and Tigrinifilum showed ≤97.03% and ≤96.08% genetic identity to all other genera within Sirenicapillariaceae , respectively ( Table S2).

The genetic similarity analyses of the 16S rRNA gene sequence across analysed established homocytous cyanobacterial families demonstrated ≤94.5% similarity for the novel family Sirenicapillariaceae ( Table 2). Sirenicapillariaceae is closely related to Coleofasciculaceae sensu stricto (94.5%), Microcoleaceae (94.5%) and Laspinemataceae (94.4%), but comparatively distant to Oscillatoriaceae sensu stricto (92.6%). The within family genetic similarity of Sirenicapillariaceae was 97.2% while the within similarities of the established families were comparatively lower for Spirulinaceae (94.7%) and Desertifilaceae (95.3%) and ≥98.0% for the remaining families including Microcoleaceae , Oscillatoriaceae , Laspinemataceae , Vermifilaceae and Coleofasciculaceae . Morphological descriptions of the genera within Sirenicapillariaceae are summarized in Table 3.

16S–23S rRNA internal transcribed spacer (ITS) secondary structure and p -distance analyses

Lengths of the identifiable domains in the 16S–23S rRNA ITS sequence of all genera can be found in Table 4. Within Capilliphycus , the 16S–23S rRNA ITS secondary structure of both the D1-D1 ʹ ( Fig. 50 ) and the Box-B ( Fig. 51 ) showed contrasting lengths and structures among both proposed species. Two D1-D1 ʹ helices of C. guerandensis were recovered, tRNA dependent. Both helices were comparable in length (60 nucleotides) and were characterized by a six-nucleotide clamp (5 ʹ - GACCUA—UAGGUC -3’) followed by a unilateral bulge on the 3 ʹ side. A unilateral bulge before the terminal loop caused by a single nucleotide on the 5 ʹ side was seen in the helix from the operon lacking tRNA. In the helix for the operon with tRNA, this unilateral bulge is present as a bilateral bulge (5 ʹ - GA—A -3’). Capilliphycus flaviceps BLCC-M53 had a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3’) followed by a unilateral bulge on the 3 ʹ side. A bilateral bulge (5 ʹ - AG—A -3’) occurs in the main stem before the large terminal loop. Capilliphycus flaviceps BLCC-M137 possessed two operons, with and without tRNAs, but only one D1-D1 ʹ helix, which had a six-nucleotide clamp (5 ʹ - GACCUA—UAGGUC -3’) followed by a unilateral bulge on the 3 ʹ side. A bulge of unmatched base pairs (5 ʹ - A—G -3’) occurs before a bilateral bulge (5 ʹ - AA—A -3’), which is followed by the large terminal loop.

The Box B helices of all Capilliphycus species had a four-nucleotide clamp (5 ʹ - AGCA—UGCU -3 ʹ), although the helices varied in length and structure. The Box B of C. guerandensis is 40 nucleotides with a unilateral bulge caused by a single nucleotide on the 3 ʹ side as well as a bilateral bulge (5 ʹ - GA—A -3 ʹ) before the terminal loop. The Box B of C. flaviceps BLCC-M53 is shorter (25 nucleotides) with a unilateral bulge (5 ʹ - C—AA -3 ʹ) in the main stem. Capilliphycus flaviceps BLCC-M137 contained two, tRNA dependent, Box B helices characterized by a unilateral bulge caused by a single nucleotide on the 3 ʹ side and a terminal loop. The Box B helix of the tRNA operon was longer, 28 nucleotides compared to 25 nucleotides. No V3 helices could be identified.

Within Sirenicapillaria , the 16S–23S rRNA ITS secondary structure of both the D1-D1 ʹ and the Box-B showed different lengths and structures among all proposed species. Sirenicapillaria glauca had a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A bulge of unmatched base pairs occurs in the stem after the first bulge (5 ʹ - GA—GA -3 ʹ) and again (5 ʹ - C—A -3 ʹ) before a unilateral bulge on the 5 ʹ side before the terminal loop. Although operons with and without tRNAs were recovered, Sirenicapillaria rigida only possessed one D1-D1 ʹ helix with a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A bulge of unmatched base pairs occurs in the stem after the first bulge (5 ʹ - GA—GA -3 ʹ) and again (5 ʹ - C—A -3 ʹ) before a unilateral bulge on the 5 ʹ side. This is followed by a unilateral bulge on the 3 ʹ side before a small terminal loop. Sirenicapillaria stauglerae possessed two operons, with and without tRNAs. The operon without tRNAs had a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A bulge of unmatched base pairs occurs after the first bulge (5 ʹ - A—A -3 ʹ). Two unilateral bulges on the 5 ʹ side before the terminal loop. The D1-D1 ʹ helix of the operons with tRNAs was similar to that of the helix without differing only at nucleotide 44, although this did not affect the structure. The helix is characterized by a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A bulge of unmatched base pairs occurs after the first bulge (5 ʹ - A—A -3 ʹ). Two unilateral bulges on the 5 ʹ side before the terminal loop.

The Box B helices of all Sirenicapillaria species contained a four-nucleotide clamp (5 ʹ - AGCA—UGCU -3 ʹ), although the helices varied in length and structure. The Box-B of Sirenicapillaria glauca BLCC-M125 was 29 nucleotides long with a bilateral bulge after the clamp (5 ʹ - GCAC—AA -3 ʹ), causing a rightward lean. Only one BoxB helix of Sirenicapillaria rigida was recovered, tRNA independent, it contained a five base pair clamp (5 ʹ - AGCAG—CUGCU -3 ʹ) followed by a unilateral bulge after the clamp (5 ʹ - CAA—A -3 ʹ), causing a rightward lean, and a large terminal loop. Only one Box B helix from Sirenicapillaria stauglerae was recovered, tRNA independent. This BoxB helix was the shortest at 24 nucleotides and a unilateral bulge after the clamp (5 ʹ - C— AA -3 ʹ), causing a leftward lean, and a small (3 nucleotide) loop. V3 helices were recovered for all Sirenicapillaria species (data not shown).

Within Tigrinifilum , the 16S–23S rRNA ITS secondary structure of both the D1-D1 ʹ and the Box-B showed dissimilar lengths and structures between both proposed species. Tigrinifilum floridanum had two D1-D1 ʹ helices, which were 62 nucleotides and 63 nucleotides. The longest D1-D1 ʹ helix is characterized by a five-nucleotide clamp (5 ʹ - GACCU— AGGUC -3 ʹ) followed by a large unilateral bulge on the 3 ʹ side. A unilateral bulge caused by a single nucleotide on the 3 ʹ occurs followed by two unilateral bulges by a single nucleotide on the 5 ʹ side before the terminal loop, there were four variable bases throughout the helix, although they had no effect on the structure. The shorter D1-D1 ʹ helix possessed a shorter, four base pair, clamp (5 ʹ - GACC—GGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A pair of unmatched nucleotides occurs after the first bulge (5 ʹ - U—C -3 ʹ). Both helices were recovered from different clones of the same strains in several isolates. Tigrinifilum guerandense contained one D1-D1 ʹ helix characterized by a four-nucleotide clamp (5 ʹ - GACC—GGUC -3 ʹ) followed by a unilateral bulge on the 3 ʹ side. A pair of unmatched nucleotides (5 ʹ - A—A -3 ʹ) occurs before a unilateral bulge by a single nucleotide on the 5 ʹ side. A unilateral bulge of three nucleotides on the 3 ʹ side occurs before a small terminal loop.

Each species of Tigrinifilum possessed one Box B helix. Tigrinifilum floridanum had a short three nucleotide clamp (5 ʹ - AGC—GGU -3 ʹ) followed by a unilateral bulge on the 3 ʹ side caused by a single nucleotide. A bilateral bulge (5 ʹ - GA—A -3 ʹ) occurs within the helix before the small terminal loop. The Box B helix of T. guerandense was shorter than that of T. floridanum (34 vs 40 nucleotides). Tigrinifilum guerandense contained a four-nucleotide clamp (5 ʹ - UCCU—AGGA -3 ʹ) with a bilateral bulge (5 ʹ - CAA—GTT -3 ʹ). A unilateral bulge on the 5 ʹ side caused by a single nucleotide occurs before the short terminal loop. The V3 helix of T. guerandense was recovered (data not shown) but no V3 helix was recovered from T. floridanum .

We also present the first full 16S–23S rRNA ITS sequence of Limnoraphis sp. from Florida, USA. Operons were recovered with and without tRNA but did not affect the D1-D1 ʹ nor BoxB helices. The D1-D1 ʹ helix is 58 nucleotides long and possessed a five-nucleotide clamp (5 ʹ - GACCU—AGGUC -3 ʹ) with a unilateral bulge caused by a single nucleotide on the 3 ʹ before the terminal loop. The Box B helix is 38 nucleotides long and had a four-nucleotide clamp (5 ʹ - AGCA—UGGU -3 ʹ) followed by a unilateral bulge on the 3 ʹ side caused by a single nucleotide. There is a bilateral bulge before the terminal bulb (5 ʹ - C—AA -3 ʹ); a V3 helix was not recovered.

The 16S–23S rRNA ITS sequence dissimilarity between species of Capilliphycus (without tRNA) including C. tropicalis , C. salinus T.A. Caires, Sant’Anna & J.M. Nunes, C. flaviceps and C. guerandensis was ≥14.8% ( Table S3). The sequence dissimilarity between species of Sirenicapillaria including S. glauca , S. rigida and S. stauglerae was ≥11.6% ( Table S4). The 16S–23S rRNA ITS region (with tRNAs) dissimilarity between the two species of Tigrinifilum , T. floridanum and T. guerandense , was ≥5.8% (Table S5).

Figures

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.

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.

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.