3. Results
We were able to isolate over 30 cyanobacterial strains from Yellowstone National Park, most of which represented thin filamentous non-heterocytous forms formerly in the Synechococcocales, as defined in [
21], but which are now recognized as belonging to three different orders, the Leptolyngbyales, Oculatellales, and Nodosilineales [
22]. Some cultures were lost subsequent to sequencing, but appear in our phylogeny, which contains representatives from all three orders listed, as well as outgroup taxa from the Synechococcales, Prochlorotrichales, Acaryochloridales, Pseudanabaenales, Thermostichales, and Gloeobacteriales. Ten species are recognized, two in the new genus Copelandiella, the rest in pre-existing genera, including
Kovacikia Miscoe, Pietrasiak & J.R.Johansen [
35],
Leptolyngbya Anagnostidis & Komárek [
36] (
Figure 2),
Albertania Zammit [
37],
Oculatella Zammit, Billi & Albertano [
38] (
Figure 3), and
Nodosilinea Perkerson Casamatta [
39] (
Figure 4). These species are almost all morphologically cryptic, in genera that are morphologically similar. However, they are phylogenetically distinct and evolutionarily separated based on both 16S rRNA similarity data and 16S–23S ITS dissimilarity thresholds. They are additionally ecologically separable from other species in their genera, due to their presence in thermal waters, and biogeographically separate from these other species as well. Evidence for lineage separation for all ten taxa will be given in the descriptions of each species, while a discussion of these mostly recently described genera will follow in the discussion.
Taxonomic descriptions and diagnoses
Phylum: Cyanobacteria
Class: Cyanophyceae
Order: Leptolyngbyales
Family: Leptolyngbyaceae
Copelandiella Johansen, J.R., Kaštovský, J. & Akagha, M.U., gen. nov.
Thallus formed of blue-green or green filaments, entangled in macroscopic mats. Filaments long, straight, or flexuous, without false branching. Sheaths thin, colorless. Trichomes not attenuated, constricted at cross walls, without necridic cells, motile and with granulation. Apical cells rounded, rarely conical-rounded. Cells mostly longer than wide.
ETYMOLOGY: This genus is named in honor of Joseph J. Copeland (1907–1990), who authored the first and only monograph of cyanobacteria from Yellowstone National Park, USA [
2].
TYPE SPECIES: Copelandiella yellowstonensis Johansen, Kaštovský & Akagha
Copelandiella yellowstonensis Johansen, J.R., Kaštovský, J. & Akagha, M.U. spec. nov, Figure 5. DIAGNOSIS: Cryptic species, morphologically similar to other
Leptolyngbya-like taxa. Distinguished by its isolated phylogenetic position in the Leptolyngbyaceae (
Figure 2), its low 16S rRNA similarity to its closest sister taxa in other genera (mean = 93.2%, see
Table 1), and its low 16S rRNA similarity to
C. thermalis spec. nov. (<97.8 in all comparisons,
Table 1). It is further separated from
C. thermalis by its high dissimilarity to that taxon in 16S–23S ITS sequence (>20%,
Table 2). ITS conserved domains are longer than in
C. thermalis, with the D1-D1′ helix 12 nt longer, the Box-B helix 3 nt longer, and the V3 helix 6 nt longer (
Figure 6). A variable V2 helix is present in
C. yellowstonensis but absent in
C. thermalis (
Figure 6). The D1-D1′, Box-B, and V2 helices among populations of
C. yellowstonensis were variable in sequence among populations of this species, but structural differences only occurred in the V2 helix. The V3 helix was absent in all cases.
DESCRIPTION: Colony a mat, with entangled filaments, blue-green or green. Filaments long, straight, or flexuous, without false branching, 1.2–1.5 µm wide. Sheaths thin, colorless. Trichomes not narrowed toward the end, constricted at cross walls, without necridic cells, with motility, with granulation (conspicuous at the cross walls,
Figure 5A), gray- or blue-green. Apical cells rounded, rarely conical rounded (
Figure 5B,C). Cells mostly longer than wide, (1) 1.1–1.3 µm wide, (1.5) 2.0–3.2 (5.5) µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A184-1!.
TYPE LOCALITY: Black Warrior Lake (Yellowstone National Park, WY, USA), 44.5444544 N, 110.7864528 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský, and Jan Mareš.
HABITAT: Blackish crust under the boardwalk with rushing hot water, slightly submersed in water ~40 °C.
ETYMOLOGY: yellowstonenis = after the locality of the original sample.
REFERENCE STRAIN: YNP81-MA1, available as CCALA 10,292 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred U. Akagha.
GENE SEQUENCES: GenBank accession numbers OR259141-145 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: In addition to the type locality, this species was also found in several other sites: a second site in Black Warrior Lake (YNP83), in hot water in Fairy Meadows (YNP30), and as an atmophyte close to Clearwater Springs (YNP70). These other isolates were grouped together based on high 16S rRNA similarity (>99.6% in all comparisons). ITS dissimilarity was indicative of greater diversity among these strains, with ITS percent dissimilarity being 6.9–7.6%, above the established threshold for distinguishing species. Strains from the type locality were notably different than strains from the other sites. YNP30 differed from YNP70 and YNP83 by 3.6–4.4%, which could indicate species separation for this population as well. However, given the high 16S rRNA similarity, and the morphological and ecological similarity, we cannot rule out the possibility that these differences in ITS sequence are due to multiple ribosomal operons, and we choose to include all strains in this species for the present, until further evidence of lineage separation can be obtained.
Copelandiella thermalis Kaštovský, J., Johansen, J.R. & Akagha, M.U. spec. nov., Figure 7. DIAGNOSIS: Morphologically similar to
C. yellowstonenis. Distinguished by smaller granulation in cytoplasm (none visible at cross walls) and shorter cells. Clear separation was also indicated both in percent similarity of 16S rRNA gene sequence (
Table 1) and percent dissimilarity among 16S–23S rRNA ITS sequence (
Table 2). Furthermore, secondary structures of all conserved domains in the ITS distinctly differ (
Figure 6).
DESCRIPTION: Colony a mat, with entangled filaments, blue green or green. Filaments long, straight, or flexuous, without false branching, 1.3–1.5 µm wide (
Figure 7A). Sheaths thin, colorless (
Figure 7B). Trichomes not narrowed toward the end, constricted at cross walls, without necridic cells, with motility and with small granulation in cytoplasm, gray- or blue-green, rarely brownish-green (
Figure 7C,D). Apical cells rounded, rarely conical-rounded. Cells mostly longer than wide, 1.2–1.3 µm wide, (1.5) 1.7–2.5 µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A185-1!
TYPE LOCALITY: Black Warrior Lake (Yellowstone National Park, Wyoming, USA), 44.5444544 N, 110.7864528 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský and Jan Mareš.
HABITAT: green mats, submersed at the bottom of hot lake.
ETYMOLOGY: thermalis = living in hot water.
REFERENCE STRAIN: YNP80B-MA3, isolate lost. Isolated by Mildred Akagha.
GENE SEQUENCES: GenBank accession numbers OR259139-140 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: Two strains were isolated from sample YNP80B, but unfortunately both were lost. Fortunately, morphological characterization, sequencing, and voucher preparation were completed before isolate failure.
Kovacikia anagnostidisii Kaštovský, J., Johansen, J.R. & Hauerová, R. spec. nov., Figure 8. DIAGNOSIS: Morphologically differs from other
Kovacikia species by constriction at cell walls and wider cells. Only
K. brockii spec. nov. is wider (2.7 µm). Genetically distinguished by both 16S rRNA sequence (
Table 3) and 16S–23S rRNA ITS sequence data (
Table 4). Differing from
K. brockii by having a V2 helix, shorter Box-B helix, and shorter V3 helix. Differing from
Leptothermofonsia sichuanensis Daroch, Thang & Shah [
43] in having D1-D1′ helix 58 nucleotides shorter.
K. muscicola Miscoe, Pietrasiak & Johansen [
35] and
K. minuta Li-Qin Shen, Renhui Li & Qiu [
44] are non-thermal.
DESCRIPTION: Colony a flat mat, with entangled filaments, green to yellow-green. Filaments slightly coiled, sometimes spirally coiled (
Figure 8A–C), without false branching, 1.5–2.5 µm wide. Sheaths thin, colorless, attached to the trichome, scarcely visible. Trichomes not attenuated, distinctly constricted at cross walls, lacking necridia, immotile, granulated, grey-green or blue-green (
Figure 8A–C). Apical cells rounded. Cells mostly shorter than wide or almost isodiametric, (1.3) 1.6–2.0 (2.4) µm wide and 1–2.1 (2.5) µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A186-1!.
TYPE LOCALITY: Clearwater springs (Yellowstone National Park, WY, USA), 44.7886475 N, 110.7392136 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský and Jan Mareš.
HABITAT: atmophytic, in massive ropy crust close to water ca. 85 °C.
ETYMOLOGY
anagnostidisii = in honor of Konstantinos Th. Anagnostidis (1924–1994), Greek phycologist who studied thermal springs [
45].
REFERENCE STRAIN: YS86-RH1, available as CCALA 10,299 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Radka Hauerová.
GENE SEQUENCES: GenBank accession numbers OR236708-709 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: Two additional strains from Clearwater Springs were sequenced, YS89-RH1 and YNP90-MA1.
Kovacikia brockii Johansen, J.R., Kaštovský, J. & Hauerová, R. spec. nov., Figure 9. DIAGNOSIS: Morphologically differs from other
Kovacikia species partly by the greater width of the cells (the other thermal species from Yellowstone,
K. anagnostidisii spec. nov., is maximally 2.4 µm wide, while
K. muscicola,
K. minuta, and
Kovacikia (
Leptothermofonsia)
sichuanensis are all less than 1.7 µm wide). Phylogenetically separated from other species (
Figure 2). Genetically distinguished by both 16S rRNA (
Table 3) and 16S–23S rRNA ITS (
Table 4).
DESCRIPTION: Colony a flat mat, with entangled filaments, grey-green to blue-green. Filaments straight, coiled, sometimes spiral (
Figure 9E), without false branching, 1.3–2.7 µm wide (
Figure 9A,B). Sheaths thin, colorless, attached to the trichome (
Figure 9A). Trichomes not narrowed toward the end, constricted or slightly constricted at cross walls (
Figure 9B,C)., often disintegrating into hormogonia in the absence of necridia (
Figure 9D), lacking motility, often with granulation in the middle of cell, pale blue-green to green. Apical cells rounded. Cells more or less isodiametric in younger filaments, or shorter in old trichomes, (0.9) 1.1–2.5 µm wide, 0.8–2.2 µm long, older trichomes are distinctly wider that young trichomes (
Figure 9A).
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A187-1!.
TYPE LOCALITY: Firehole Lake (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský and Jan Mareš.
HABITAT: atmophytic mats near shore of the hot water.
ETYMOLOGY:
brockii = named after the prominent microbial physiologist Thomas D. Brock, who wrote early works concerning microbial extremophiles in the hot springs of Yellowstone National Park [
4].
REFERENCE STRAIN: YNP74-RH1, available as CCALA 10,294 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Radka Hauerová.
GENE SEQUENCES: GenBank accession numbers OR220391-193, OR236707 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: In addition to the type locality, also found in Mound Spring as an atmophytic crust on the bank close to a stream (strain YS7-RH2).
Leptolyngbya tildenae Kaštovský, J., Johansen, J.R. & Akagha, M.U. spec. nov., Figure 10. DIAGNOSIS: Morphologically similar to several nonthermal
Leptoplyngbya/
Plectolyngbya species, but the combination of morphological features with specific ecology is unique. It differs from its sister species
L. atmophytica spec. nov. partly in size (it is thinner), but there is overlap. Genetically distinguished from
Tapinothrix clintonii clintonii Bohunická & Johansen [
46], the sister taxon to
Leptolyngbya sensu stricto, by having percent similarity of the 16S rRNA gene <93.5% (
Table 5). Differentiated from all described
Leptolyngbya and
Plectolyngbya species by having 16S–23S rRNA ITS sequence dissimilarity >9.0. (
Table 6). Phylogenetic separation from other species was also seen (
Figure 11), with the sister species to
L. tildenae being
L. vaporiphila sp. nov. and an unnamed species from soils of Joshua Tree National Park (WJT66-NPBG17).
DESCRIPTION: Colony a flat mat, with entangled filaments, dark green. Filaments straight or slightly coiled (
Figure 10C), without false branching, 1.6–2.2 µm wide. Sheaths firm, thick, colorless, attached to the trichome or widened at the end (
Figure 10A,B). Trichomes not narrowed toward the end, constricted or unconstricted at cross walls (
Figure 10D), necridic cells not observed, without motility, with granulation, blue-green. Apical cells rounded. Cells mostly shorter than wide or almost isodiametric, 1.4–1.9 (2.2) µm wide, 1.3–1.8 (2) µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A188-1!.
TYPE LOCALITY: Firehole Lake, (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský and Jan Mareš.
HABITAT: Black mat from soil on the bank of hot water lake.
ETYMOLOGY:
tildenae = in honor of Josephine Elisabeth Tilden (1869–1957), American phycologist, who worked on the Yellowstone algal flora [
47].
REFERENCE STRAIN: YNP77-MA3, available as CCALA 10,300 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred U. Akagha.
GENE SEQUENCES: GenBank accession number OR259138 for the 16S rRNA and 16S–23S rRNA ITS genes.
Leptolyngbya vaporiphila Johansen, J.R., Kaštovský, J., & Akagha, M.U. spec. nov., Figure 12. DIAGNOSIS: Morphologically similar to several nonthermal
Leptolyngbya/
Plectolyngbya species, but the combination of morphological features with specific ecology is unique. It differs from its sister species
L. tildenae spec. nov. partly in size (it is wider), although there is some overlap. Differentiated from all sequenced
Leptolyngbya and
Plectolyngbya species by having 16S–23S rRNA ITS sequence dissimilarity >9.0. (
Table 6). Phylogenetic separation from other species was also evident (
Figure 11).
DESCRIPTION: Colony a flat mat, with entangled filaments, dark green. Filaments straight or slightly coiled (
Figure 12A,B), with rare false branching (
Figure 12C), 2–3.2 µm wide. Sheaths firm, thick, colorless, attached to the trichome (
Figure 12A). Trichomes not attenuated, constricted or unconstricted at cross walls, with necridia (
Figure 12A,B), lacking motility, with granulation, blue-green. Apical cells rounded. Cells mostly shorter than wide or almost isodiametric, (1.7) 2.0–3.0 µm wide, 1.6–2.1 µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A189-1!.
TYPE LOCALITY: Clearwater springs (Yellowstone National Park, WY, USA), 44.7886475 N, 110.7392136 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský and Jan Mareš.
HABITAT: atmophytic, in massive ropy crust close to water ca 85 °C.
ETYMOLOGY: vaporiphila = living with constant presence of hot vapor.
REFERENCE STRAIN: YNP69B-MA2, available as CCALA 10,295 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred Akagha.
GENE SEQUENCES: GenBank accession numbers OR259132-137 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: In addition to the type locality, this species was also found as an atmophyte in several places on the bank of Firehole Lake.
Phylum: Cyanobacteria
Class: Cyanophyceae
Order: Oculatellales
Family: Oculatellaceae
Albertania pratii Johansen, J.R., Kaštovský, J. & Akagha, M.U. spec. nov., Figure 13. DIAGNOSIS: Sole thermal species in genus
Albertania. Morphologically similar to both described
Albertania species, cell width is larger than
A. alaskaensis Strunecký, Raabová, Bernardová, Ivanova, Semanova, Crossley & Kaftan [
48] and slightly smaller than
A. skiophila Zammit [
37]. In contrast to
A. skiophila, trichomes of
A. prattii have no necridic cells. Distinguished by its thermal ecology. Pairwise comparisons of 16S rRNA gene sequence with other species and strains are all <98.7% (
Table 5). The percent dissimilarity of the 16S–23S ITS region was high among all strains, >11% (
Table 6).
DESCRIPTION: Colony a flat mat, with entangled filaments, blue-green. Filaments normally straight or slightly waived, without false branching, 1.9–2.3 µm wide. Sheaths thin, colorless, attached to the trichome, often widened at the end (
Figure 13A,B). Trichomes not narrowed toward the end, slightly constricted at cross walls, without necridia, lacking motility, with granulation, pale green. End cells rounded, sometimes with more evident constriction at cross walls, lacking granulation (
Figure 13C–E). Hormogonia few celled, motile (
Figure 13E). Cells mostly isodiametric or slightly longer than wide (1.3) 1.7–2.1 µm wide, (1.7) 2.3–3.2 µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A190-1!
TYPE LOCALITY: Firehole Lake, (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský, and Jan Mareš.
HABITAT: atmophytic near shore, not submersed, brown velvet mat.
ETYMOLOGY:
pratii = in honor of Silvestr Prát (1895–1990), Czech phycologist, who worked on the Yellowstone algal flora [
49].
REFERENCE STRAIN: YNP74-MA3, available as CCALA 10,298 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred U. Akagha.
GENE SEQUENCES: GenBank accession numbers OR244603, OR259146 for the 16S rRNA and 16S–23S rRNA ITS genes.
Oculatella castenholzii Kaštovský, J., Johansen, J.R. & Akagha, M.U. spec. nov., Figure 14. DIAGNOSIS: The only known described thermal species of the whole genus
Oculatella. Phylogenetically most similar to several tropical unnamed strains from Nigeria (N16-MA6, N15-MA5, N14-MA2) and Costa Rica (LLi8) (
Figure 3). Phylogenetically closest to
O. hafneriensis according to 16S–23S rRNA ITS sequence analysis (
Figure 15). Below the 16S rRNA similarity threshold for separation of species (98.7%) for all strains and species except
Oculatella sp. LLi8 collected from a thermal spring in Costa Rica, which has a sequence similarity of 99.1%. It is plausible that the Costa Rican strain and our strain are conspecific, but we do not have 16S–23S ITS data for this strain, so this conclusion is not presently supported, as we cannot include it in our phylogenetic analysis (
Figure 15) or our pairwise comparisons of the 16S–23S ITS dissimilarity. The pairwise comparisons with all strains for which ITS sequence data exist were >5.2%, indicating
O. castenholzii is a separate species from all presently described species.
DESCRIPTION: Colony a flat mat, with entangled filaments, blue green. Filaments long, straight, or flexuous (
Figure 14A,B), without false branching, 1.0–1.2 µm wide. Sheaths thin, colorless, attached to the trichome. Trichomes not narrowed toward the end, slightly constricted at cross walls (
Figure 14C,D), without necridia, without motility, without granulation, pale blue-green. Apical cells conical rounded, often with orange carotenoid inclusion at the tip (
Figure 14A,B,D). Cells longer than wide (0.8) 0.9–1.1 µm wide, 2.0–4.2 (6) µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A191-1!.
TYPE LOCALITY: Firehole Lake, (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský, and Jan Mareš.
HABITAT: atmophytic near shore, not submersed, part of brown velvet mat.
ETYMOLOGY: castenholzii = in honor of Richard W. Casteholz (1931–2018), American phycologist, who worked on Yellowstone algal flora.
REFERENCE STRAIN: YNP74-MA2, available as CCALA 10,293 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred Akagha.
GENE SEQUENCES: GenBank accession number OR259147 for the 16S rRNA and 16S–23S rRNA ITS genes.
Phylum: Cyanobacteria
Class: Cyanophyceae
Order: Nodosilineales
Family: Nodosilineaceae
Nodosilinea calida Johansen, J.R., Kaštovský, J. & Akagha, M.. spec. nov., Figure 16. DIAGNOSIS: One of only two thermal species in the whole genus
Nodosilinea. N. igneolacustris spec. nov. and
N. calida spec. nov. are sister taxa, differing only slightly by length of cells (
N. igneolacustris has a maximum length of cells 2.5 µm,
N. calida can reach 3, exceptionally up to 4, µm). These two thermal species are distinct in both the 16S rRNA phylogeny and the 16S–23S ITS phylogeny. Furthermore, they are <98.7% similar in the ITS region, and >15% dissimilar in their aligned 16S–23S ITS sequences, so by both threshold criteria they meet the standard for being recognized as separate species. In comparison to other strains in the genus, they are closest to GSE-PSE-MK27-15A, ATA11-6B-CV9, and ATA1106K-CV21 (
Figure 4), but these are all unnamed and at the 16S rRNA similarity threshold for species (98.6–99.1% similar). They exceed the ITS dissimilarity threshold (>12% in comparisons with these taxa).
N. calida is separated from
N. igneolacustris according to the 16S–23S ITS phylogeny (
Figure 17), and both taxa have long branches. We lack ITS data for some of the close taxa identified in the 16S rRNA phylogeny (
Figure 4).
DESCRIPTION: Colony a flat mat, with entangled filaments, dark green. Filaments long, straight, or flexuous, sometimes spiral (
Figure 16A,D,E) without false branching, 1.3–1.5 µm wide. Sheaths thin, colorless, attached to the trichome, often almost invisible (
Figure 16B). Trichomes not narrowed toward the end, constricted at cross walls (
Figure 16B,C), without necridia, lacking motility, without granulation, blue-green. End cells rounded. Cells longer than wide or isodiametric (
Figure 16B,C), 1.2–1.4 µm wide, 1.7–3.0 (4) µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS AA192-1!.
TYPE LOCALITY: Firehole Lake, (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský, and Jan Mareš.
HABITAT: submersed or atmophytic calcareous concretions in pool with hot water by the road.
ETYMOLOGY: calida = from a hot environment.
REFERENCE STRAIN: YNP76AMA10, available as CCALA 10,296 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred Akagha.
GENE SEQUENCES: GenBank accession numbers OR259149-150 for the 16S rRNA and 16S–23S rRNA ITS genes.
TAXONOMIC NOTES: This species was found repeatedly in different locations within the type locality, which is quite large and ecologically variable, including a submerged population. Nodule formation, a diacritical character of the genus, was not observed.
Nodosilinea igneolacustris Kaštovský, J., Johansen, J.R. & Akagha, M.U, spec. nov., Figure 18. DIAGNOSIS: Thermal species genetically separated from all other described species (see above).
DESCRIPTION: Colony a flat mat, with entangled filaments, dark green. Filaments long, straight or flexuous, without false branching, with nodules (
Figure 18A), 1.2–1.5 µm wide. Sheaths thin, colorless, attached to the trichome, sometimes widened (
Figure 18B,C). Trichomes not narrowed toward the end, constricted at cross walls, without necridia, lacking motility, without granulation, blue-green. End cells rounded (
Figure 18C). Cells longer than wide or isodiametric, 1.1–1.4 µm wide, 1.5–2.5 µm long.
HOLOTYPE: Dried material preserved in a permanently inactive state at Herbarium for Nonvascular Cryptogams at the Department of Botany, Faculty of Science, University of South Bohemia, Czech Republic), under the code CBFS A193-1!.
TYPE LOCALITY: Firehole Lake, (Yellowstone National Park, WY, USA), 44.5440433 N, 110.7865600 W. Collected on 18 September 2019 by Jeffrey R. Johansen, Jan Kaštovský, and Jan Mareš.
HABITAT: submersed calcareous concretions in pool with hot water by the road.
ETYMOLOGY: igneolacustris = of the fire lake, referring to its occurrence in Firehole Lake.
REFERENCE STRAIN: YNP76AMA5, available as CCALA 10,297 (Culture Collection of Autotrophic Organisms at the Institute of Botany, Třeboň, CZ), isolated by Mildred Akagha.
GENE SEQUENCES: GenBank accession numbers OR244604, OR259148 for the 16S rRNA and 16S–23S rRNA ITS genes.
4. Discussion
Merging of genera
Although the formation of new genera has been much more frequent in recent decades than their merging, merging is not an isolated phenomenon [
50]. Most often, this comes about because a subsequent larger set of molecular data show that the incomplete data available when the new taxon was postulated led to a conclusion that the larger dataset does not support, e.g., the collapsing of
Sphaerocavum [
51] or
Cylindrospermopsis [
52].
The same reasons motivated us to merge the two pairs of genera:
Leptothermofonsia Daroch, Tang & Shah 2022 to
Kovacikia Miscoe, Pietrasiak & Johansen 2016 and
Plectolyngbya Taton, Wilmotte, Smarda, Elster & Komárek 2011 [
53] to
Leptolyngbya Anagnostidis & Komárek 1988.
In addition to respecting the priority rule in both cases (the older name should persist)—according to the International Code of Nomenclature for Algae, Fungi, and Plants [
54]—the situation in the case of
Leptothermofonsia was made even more complicated by the fact that it is not a valid name, due to improper designation of the holotype [
23].
Leptolyngbya has long been a problematic genus, having been used as a catch-all for any thin filamentous taxon with a sheath. Over a hundred species were recognized in [
23], and its polyphyletic character in numerous phylogenies has required extensive revision. All of the taxa covered in this paper would have been placed in
Leptolyngbya 20 years ago. The genus should be narrowed to just those species belonging to the same clade as the type species,
L. boryana (Gomont) Anagnostidis & Komárek [
36]. This species has been sequenced and is quite close to other species in the genus, including
L. angustata Casamatta & Johansen [
55],
L. corticola Johansen, Kováčik, Cassamata, Fučíková & Kaštovský [
56],
L. foveolarum (Gomont) Anagnostidis & Komárek [
36] and
L. tenerrima (Hansgirg) Komárek [
57].
Plectolyngbya is phylogenetically very close to
Leptolyngbya, but was initially recognized for its double false branching. However,
Leptolyngbya sensu stricto has single false branching and similar cell structure (mostly isodiametric to shorter than wide). The two genera are above 94.5% similar in the majority of possible comparisons (
Table S1). Furthermore, the sister taxon to the clade containing both
Plectolyngbya and
Leptolyngbya is
Tapinothrix clintonii, and there is a marked discontinuity in 16S similarity between it and all members of the
Plectolyngbya/Leptolyngbya cluster. Finally, many of the more recently studied
Plectolyngbya do not have the diagnostic feature of double false branching. We conclude that
Plectolyngbya is a later synonym of
Leptolyngbya, and below will give an emended description of
Leptolyngbya and complete the new combinations, so that the species described in that genus will now reside in
Leptolyngbya. The clade that is created by uniting these taxa is
Leptolyngbya sensu stricto. All other sequences occurring outside that clade but designated as
Leptolyngbya require revision.
Leptolyngbya Anagnostidis & Komárek 1988.
SYNONYM: Plectolyngbya A.Taton, A.Wilmotte, J.Smarda, J.Elster & Komárek
EMENDED DESCRIPTION:
Filaments long, solitary or coiled into clusters and fine mats (which are sometimes macroscopic and several cm in diameter), arcuated, waved or intensely coiled, isopolar, thin, fine, 1.5–3.2 µm wide, with simple, thin, usually colorless facultative sheaths opened at the apical end; sheaths joined to the trichomes or slightly distant from them, enveloping only one, very rarely (in short sections) two trichomes; often with false branching. Trichomes fine, cylindrical, usually not attenuated to the ends or slightly attenuated, with rounded or conical apical cells, not constricted or constricted at the cross walls, immotile when encased in sheath. Cells isodiametrical, longer or shorter than wide, cylindrical, with homogeneous content, without aerotopes, rarely with scarce prominent granules, pale blue-green, greyish, olive-green, yellowish or reddish; end cells without thickened cell walls or calyptra. Heterocytes and akinetes absent. Cells divide by symmetrical (rarely asymmetrical) crosswise binary fission, cells grow to original size before next division. Reproduction by motile hormogonia, liberated at trichome ends, fragmented with or without necridic cells.
ECOLOGY: in soils, periphyton and metaphyton of freshwater and halophilous (marine) biotopes, thermal and mineral springs, aerophytic, endogloeic in mucilage of other algae.
TYPE SPECIES: Leptolyngbya boryana (Gomont) Anagnostidis & Komárek 1988: 391
New combinations:
Leptolyngbya hodgsonii (Taton, Wilmotte, Šmarda, Elster & Komárek) Johansen et Kaštovský, comb. nov.
Leptolyngbya koreana (D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee) Johansen et Kaštovský, comb. nov.
BASIONYM:
Plectolyngbya koreana D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee 2022: 7,
Figure 4 and
Figure 5 [
58].
Leptolyngbya salina (D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee) Johansen et Kaštovský, comb. nov.
BASIONYM:
Plectolyngbya salina D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee 2022: 9,
Figure 6 and
Figure 7.
Leptolyngbya terrestris (D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee) Johansen et Kaštovský, comb. nov.
BASIONYM:
Plectolyngbya terrestris D.-H.Kim, N.-J.Lee, E.-C.Yang & O.-M.Lee 2022: 5,
Figure 2 and
Figure 3.
Taxonomically more complicated is the situation in the genus
Leptothermofonsia. This genus has not been validly described [
23]—lacking type material in a physiologically inactive state, [
45], nor can the species be transferred to
Kovacikia, which is the name with priority. However, our results clearly show that this genus is not molecularly distinct from the genus
Kovacikia. It is in a clade with several
Kovacikia species, and it does not meet the similarity thresholds accepted by microbiologists. The occurrence of more thermal species in
Kovacikia indicates that this criterion is not sufficient for recognizing a genus separate from
Kovacikia. Furthermore,
Leptothermofonsia is not morphologically distinct from
Kovacikia. There would be no significant changes in the original description of the genus
Kovacikia even with the inclusion of the species
Leptothermofonsia sichuanensis Daroch, J.Tang & M.R.Shah, nom. inval.; perhaps only the inclusion of the possibility of trichomes unconstricted at the cross walls. If a nomenclaturally valid type were prepared and deposited in a curated collection, then the species epithet
sichuanensis could be used in combination with the genus
Kovacikia. We hope that the curators of these well-characterized strains will validate the species in the near future.
Morphological evaluation with existing species
Although many of the cyanobacterial species mentioned by Copeland were found during our sampling trip, this was not the case with these simple filaments. For the most part, the morphology of the species we described does not match that described (or reported) by Copeland [
2].
The species described within the genus
Phormidium Kützing ex Gomont [
59] could be considered for identity, the other filamentous taxa differ too much in the width of the filament (e.g.,
Oscillatoria Vaucher ex Gomont [
59]) or the greater number of trichomes in the filament (e.g.,
Schizothrix Kützing ex Gomont [
60]).
Many of the species formerly belonging to the genus
Phormidium from localities in Yellowstone National Park have a pointed end cell (
Leptolyngbya laminosa (Gomont) Anagnostidis & Komárek [
36],
L. vesiculosa (Copeland) Anagnostidis [
57],
L. subcapitata (J.B.Petersen) Anagnostidis [
57] and
L. subuliformis (Gomont) Anagnostidis [
57], or even a gradually tapering end as in
L. fragilis (Gomont) Anagnostidis & Komárek [
36],
L. granulifera (Copeland) Anagnostidis [
57] or
L. tenuis (Gomont) Anagnostidis & Komárek [
36], which none of our species have.
The newly described species also differ from
L. cartilagina (Copeland) Anagnostidis [
57], because none of our species have granules on both sides of the cross walls. Other taxa also differ in color—ours are all blue- or grey-green and
Drouetiella lurida (Gomont) Mai, Johansen & Pietrasiak [
61] is brownish-violet.
Leptolyngbya rubra (Tilden) Anagnostidis [
57] is scarlet or pink.
L. purpurascens (Gomont) Anagnostidis & Komárek [
36] is violet.
L. copelandii Anagnostidis [
27] (a synonym of
Phormidum truncatum var.
thermale Copeland [
2]) and
L. ramosa (J.B.Petersen) Anagnostidis & Komárek [
36] are unconstricted at cross walls (our species are minimally at least sometimes constricted).
L. geysericola (Copeland) Anagnostidis [
57],
Phormidium bajahense Copeland [
2] and
Phormidium subterraneum Phillipson [
62] are too small (both with cell width max. 0.6 µm) and create pale salmon or yellowish expanded mats.
The only taxon from Copeland’s list that could be morphologically somewhat similar to some of our species is
Leptolyngbya foveolarum (Gomont) Anagnostidis & Komárek [
36]—1.5 µm width of the cells, 0.8–2.0 µm length, not tapering, rounded apical cell, no granulation, pale blue-green trichomes. However, this description is so general that it can be identified with several different genera in today’s understanding, and its phylogenetic position (within the
L. boryana clade) is different from our strains. Furthermore, its habitat of origin is very different from thermal mineral waters.
Other cyanobacteria of similar morphological appearance are collected in the monograph by [
63]. However, even here we do not find identical thermal species. Contrary to our species
Leptolyngbya gelatinosa (Woronichin) Anagnostidis & Komárek [
36] and
L. thermobia Anagnostidis [
57] have cells shorter than wide,
L. orientalis (G.S.West) Anagnostidis & Komárek [
36] is not constricted and possesses very long cells (to 8 µm).
L. thermarum (Woronichin) Anagnostidis & Komárek [
36] is not constricted and possesses solitary granules at cross walls.
L. phormidioides (Anagnostidis) Anagnostidis & Komárek [
36] has apical cells conically narrowed into a pointed apex and
L. treleasei (Gomont) Anagnostidis & Komárek [
36] is too small (0.4–0.8 µm wide).
We therefore proposed our strains as legitimate new species for science.
The work remaining
This manuscript describes 10 new species of simple filamentous cyanobacteria from Yellowstone National Park. This could certainly be just a beginning. Copeland [
2] reported 81 new species from the hot springs of Yellowstone. He did his work without the benefit of molecular data. We only made isolation efforts in a few of the samples we collected and are certain there would be many more if a more systematic and longer-lasting effort was made. Much of the past work on Yellowstone cyanobacteria was ecophysiological in nature, and a modern taxonomic effort is needed. We had difficulty with some of our cultures, which failed before full characterization. However, based on our sequencing efforts, we know there were five more simple filamentous taxa that could have been described. A unique species from
Elainella cluster was isolated twice, but in both cases became contaminated with another cyanobacterium. Two undescribed genera, one in the
Scytolyngbya/Limnolyngbya cluster in the Leptolyngbyales and one in the Oculatellales cluster sister to
Trichotorquatus Pietrasiak & Johansen [
64]. Finally, we have two new
Stenomitos Miscoe & Johansen [
35] species, which will be described in a separate treatment of that genus. This work demonstrates that there is ample opportunity for discovery of microbial diversity in extreme habitats such as those found in Yellowstone National Park.