Fottea 11(1): 127–140, 2011 127

Tapinothrix clintonii sp. nov. (Pseudanabaenaceae, ), a new species at the nexus of five genera

Markéta Bo h u n i c k á 1, Jeffrey R. Jo h a n s e n 2, and Karolina Fu č í k o v á 3

1Faculty of Science, University of South Bohemia, Branišovská 31, České Budějovice, CZ–370 05, Czech Republic; e–mail: [email protected] 2Department of Biology, John Carroll University, University Heights, OH 44118, U.S.A. 3University of Connecticut, Department of Ecology and Evolutionary Biology, 75 North Eagleville Road, Storrs, CT 06269–3043, U.S.A.

Abstract: A new species was isolated and characterized from Grand Staircase–Escalante National Monument. This taxon shares morphological characteristics with five different genera, Ammatoidea, Homoeothrix, Leptolyngbya, Phormidiochaete, and Tapinothrix. An argument could be made to place it in any of these genera, however, we consider the most taxonomically correct genus for our pseudanabaenalean species to be Tapinothrix, and we accordingly describe it as T. clintonii sp. nov. Phylogenetically, the taxon is closest to Leptolyngbya sensu stricto, but it has very distinctive morphology and 16S–23S ITS sequence and secondary structure that support our conclusion to recognize Tapinothrix as a genus separate from Leptolyngbya.

Key words: Ammatoidea, Homoeothrix, Leptolyngbya, Phormidiochaete, Tapinothrix, Cyanobacteria, taxonomy, 16S rRNA, 16S–23S ITS, rRNA secondary structure

2005). Tapinothrix bornetii was transferred into Introduction Homoeothrix bornetii (Sa u v a g e a u ) Ma b i l l e (1954), and Tapinothrix was forgotten for nearly In the course of study of the algal flora of half a century. In the most recent treatment of Grand Staircase–Escalante National Monument the Oscillatoriales, Ko m á r e k & An a g n o s t i d i s (Kr a u t o v á 2008), we isolated a distinctive non– (2005) recognized that Homoeothrix was likely heterocytous, filamentous strain of cyanobacteria. polyphyletic, with the type species, H. juliana This strain is characterized by having isopolar (Bo r n e t et Fl a h a u l t ) Ki r c h n e r clearly belonging filaments which taper at the apices and fragment in to the Oscillatoriaceae, while the majority the center to form heteropolar tapering trichomes. of described taxa apparently belong in the The resulting “basal” portion of the heteropolar Pseudanabaenaceae. They concluded that these filament forms a mucilage pad, which likely serves pseudanabaenacean species belong in Tapinothrix, to cement the widened trichome end to the substrate. but they did not make any taxonomic transfers. The cell contents are rich in phycoerythrin, giving In looking for species conforming the trichomes a reddish coloration, with mature morphologically to our isolate, the closest species sheaths blackish. Cell organization (thylakoid is actually Phormidiochaete fusca (St a r m a c h ) Ko- arrangement and mode of cell division) is typical m á r e k in An a g n o s t i d i s (2001). Phormidiochaete of the Pseudanabaenaceae. was split out of Homoeothrix to accommodate the Within the Pseudanabaenaceae there is species within Homoeothrix that have a mode of a single genus with tapering trichomes whose cell division and thylakoid structure consistent species are attached to the substrate at the base with the Phormidiaceae (An a g n o s t i d i s 2001). The – Tapinothix Sa u v a g e a u . Only two species were type species of Phormidiochaete, P. nordstedtii described in this genus, T. bornetii Sa u v a g e a u (Bo r n e t et Fl a h a u l t ) Ko m á r e k et An a g n o s t i d i s , (1892) and T. mucicola Bo r g e (1923). Another clearly fits into the Phormidiaceae, as does P. tapering genus, Homoeothrix (Th u r e t ex Bo r n e t balearica (Bo r n e t et Fl a h a u l t ) Ko m á r e k et et Fl a h a u l t ) Ki r c h n e r (1898) was subsequently An a g n o s t i d i s . This genus is in the subfamily described, and many species were described Ammatoideoideae. However, P. fusca has much within this taxon (Ko m á r e k & An a g n o s t i d i s smaller diameter than the vast majority of 128 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Phormidiaceae, and it may have been mistakenly was completed under 16:8 light:dark cycle and placed in this genus. Interestingly, a feature of our corresponding day and night temperatures of 15 °C taxon which separates it from Phormidiochaete and 10 °C. Colonies were picked 4–6 weeks later. The is the common occurrence of isopolar trichomes isolate discussed in this paper was a black cyanobacterial tapering towards both apices that bend acutely in colony, and was isolated onto a Z8 slant. Morphological variability of the culture was determined by examining the center. Such isopolar forms are placed in the the culture as it aged from exponential growth phase genus Ammatoidea We s t et G.S. We s t (1897), through senescence (a period of about three months). which includes mostly species with typical Characterization was completed using an OLYMPUS phormidiacean features as well as the apparently BMAX 60 microscope with high resolution Nomarski pseudanabaenacean marine species A. murmanica DIC optics, equipped with a Spot digital camera. The Pe t r o v (1961). cell dimensions were repeatedly measured using an Finally, we must mention the genus ocular micrometer. Leptolyngbya. This genus has become the home for a plethora of pseudanabaenacean species Molecular characterization. Tapinothrix was scraped transferred from Lyngbya, Plectonema, and from slants, and genomic DNA isolation was performed Phormidium because of their narrow, untapered using the UltraClean Microbial DNA Isolation Kit from trichomes forming simple sheaths. This genus MO BIO (Carlsbad, CA, USA). DNA was eluted and stored at –20 °C. has been said to be a polyphyletic assemblage A PCR product of 1600 bps containing the 16S– (Al b e r t a n o & Ko v á č i k 1994; Tu r n e r 1997; 23S ITS was generated using primers 1 and 2 (Bo y e r Wi l m o tt e et al. 1997; Ca s t e n h o l z 2001; et al. 2002). Fifty microliter reactions were performed Wi l m o t t e & He r d m a n 2001; Ta t o n et al. 2003; in a Bio–Rad DNA Engine PTC200 (Hercules, CA, Ca s a m a tt a et al. 2005; Ko m á r e k & An a g n o s t i d i s USA). Final concentrations of reagents in the reactions 2005; Jo h a n s e n et al. 2008), but no revisionary were 1X Taq polymerase buffer (USB, OH, USA), work recognizing the separate clades as separate 1.5 mM MgCl2, 2.5 pmol per μl of each primer, 1 μl genera has been completed. of template DNA (100–200 ng total), 0.2 mM dNTPs Our isolate is at the nexus of these five (USB), and 1.25 units Taq polymerase (USB).Cycling genera. It fits no described species in any genus, conditions were 35 cycles of 94 °C for 45 sec, 57 °C for 45 sec, and 72 °C for 135 sec. A 5 min extension and some justification could be given to place it at 72 °C was performed, and the reactions were held in any of the five genera discussed above. The at 4°C indefinitely. All PCR products were analyzed intent of this paper is to describe our isolate as on 1% agarose gels before being TA–cloned into the a new species in the genus which we consider pSC–amp/kan plasmid of the Stratagene Cloning Kit to be most correct (Tapinothrix), determine the (La Jolla, CA, USA). Putative clones were isolated phylogenetic position of the isolate, and discuss using QIAprep Spin kits (Qiagen, Carlsbad, CA, USA) the systematic and taxonomic ramifications of with elution in 50 μl of sterile water. The presence of the discovery and characterization of this species, an insert was confirmed byEco R I digestion. including reassignment of problematic species to Four 16S rRNA and 16S–23S ITS plasmids what we consider to be the correct genera. While were sequenced by Functional Biosciences, Inc. (Madison, WI, USA) using the M13 forward and this may seem like an unusual case, it is likely M13 reverse primers. Additionally, the 16S–23S representative of many similar stories that will be ITS–containing plasmids were sequenced with internal told as the revisionary process of the cyanobacteria primers 5 (5’–TGTACACACCGCCCGTC–3’) continues. and 8 (5’–AAGGAGGTGATCCAGCCACA–3’) (Fl e c h t n e r et al. 2002). Sequences were aligned and proofread using Sequencher software (version 4.8, Materials and Methods Ann Arbor, MI, USA). Only one 16S–23S ITS operon was identified, the one that encodes both tRNAIle and Isolation and cultivation. A sample was collected tRNAAla. Subsequently, the folded secondary structures in August 2006 from a sandstone seep wall adjacent were determined using Mfold version 3.2; the default to the cascade at Lower Calf Creek Falls in the Great conditions were used in this analysis except for draw Staircase–Escalante National Monument, Utah, mode: untangle with loop fix. Conserved domains USA (37°49’44.77” N, 111°25’12.58” W) and kept within the 16S–23S ITS were identified through refrigerated until further processing in laboratory. employment of comparative analysis with the ITS of A small, unmeasured portion of the sample other Pseudanabaenales, particularly with respect to was dilution plated onto agar–solidified Z8 universal the basal portions of each helix. Structures were drawn algal culture medium (Ko t a i 1972). Enrichment in Adobe Illustrator CS4 prior to publication. Fottea 11(1): 127–140, 2011 129

Phylogenetic Analyses. In the first round of analyses & Ro n q u i s t 2001, Ro n q u i s t & Hu e l s e n b e c k 2003) was reported in this paper, we selected a large group of non– used for Bayesian inference. A Dirichlet (1,2,1,1,2,1) heterocytous taxa to examine the phylogenetic position prior was used for the substitution rate parameters, of our newly sequenced strain. A total of 272 sequences a Dirichlet (2) prior was used for base frequencies, including representatives of Pseudanabaenaceae, Phor- and a uniform (0,1 prior) was applied to the pinvar midiaceae, and Oscillatoriaceae were used in the first parameter. An exponential (1.0) prior was set on analysis. Sequences were aligned using CLUSTAL–W, branch lengths and gamma shape parameters. Two and then proofed manually by eye. Secondary structure runs were run for 107 generations, using one cold chain was consulted to align indels. Space does not permit and three heated chains and sampling every 100 trees. listing the accession numbers of all strains used in this The first 40,000 samples of each run were discarded analysis, but single taxa shown in figures and tables as a burn–in phase. The stability of model parameters are given with accession numbers. Based on the results and the convergence of the two runs were confirmed of distance and parsimony analyses of this large data using Tracer v1.4.1 (Ra m b a u t & Dr u m m o n d 2003) set, a smaller subset of taxa was selected to exclude and AWTY (Wi l g e n b u s c h et al. 2004). A maximum taxa outside of the Pseudanabaenales as well as some likelihood (ML) analysis was performed using Garli isolates of uncertain affiliation (not identified at least (Zw i c k l 2006). A GTR+I+gamma model was applied to genus level). The final set for phylogenetic analysis and bootstrap support values obtained from 100 contained 114 OTU’s. Descriptions of the analyses pseudoreplicate data sets. Additionally, an unweighted using this data set are given below. maximum parsimony analysis was carried out using The GTR+I+gamma model was selected PAUP* 4.0b10 (Sw o ff o r d 2002), and bootstrap under the Akaike Information Criterion using PAUP* supports were obtained from 1000 pseudoreplicate 4.0b10 (Sw o ff o r d 2002) and Modeltest 3.7 (Po s a d a data sets. Trees were prepared for publication using & Cr a n d a l l 1998) and was used for all model–based Adobe Illustrator CS4. analyses. The software MrBayes 3.1.2 (Hu e l s e n b e c k

Figs 1–3. Morphological variability within Tapinothrix clintonii: (1) young tapered filament with thin sheath and mucilaginous pad at the base; (2) young filaments connected with common mucilaginous pad; (3) mature filaments with firm structured sheath and mucilaginous pad at the base. Scale bar 10 μm. 130 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Results

Tapinothrix clintonii Bo h u n i c k á et Jo h a n s e n , sp. nov. (Figs 1–29)

Diagnosis: Phormidiochaete fuscae maxime simile, a quo trichomate morina ad septum constricta, diametro latiore trichomae, habitatione paritum irrugarum in desertis differt. Homoeothrix violaceae (Bo r n e t et Fl a h a u l t ) Ko m á r e k et Ka n n simile, a quo diametro latiore trichomae, cellulis apicalibus elongatis et habitatione aquae dulcis differt. Most similar to Phormidiochaete fusca, from which it differs by the purplish color of the trichomes, the distinctly constricted crosswalls, the wider trichome diameter, and habitat preference of desert seep walls. Also similar to Homoeothrix violacea, from which it differs by the wider trichome diameter, the elongated cells in the apical region, and habitat preference of freshwater.

Descriptio: Colonia junior badia morinescens expansa. Filamento juniora isopolaria et ad apices ambo Figs 4–6. Development of heteropolar filaments with decrescentia, in centro rumpens heteropolarescentia, mucilaginous pad at the base in Tapinothrix clintonii: (4) ad maturitatem tumida ad basim ad apices decresentia, young filament tapered towards both ends, bent in the middle muco basali incolorato, interdum pseudoramosa. part; (5) filament breaks in the middle part, mucilaginous pad Vagina firma aspera incolorata vel morina nigrescens, forms; (6) two young heteropolar filaments with mucilaginous pads. Scale bar 10 μm. 0.5–1 μm lata. Trichomae ad basim et ad partem mediam constrictae ad septum, ad apices nonconstrictae ad septum, 3–4 μm latae ad basim, ad apices 0.75–1 Holotype here designated: BRY37705 (unialgal μm latescentes, in pilum terminantes. Cellulae ad basim latitudone breviora, cytoplasmate granuloso population preserved in 4% formaldehyde), badio, thylakoidibus parietalibus, saepe granulato Herbarium of Nonvascular Cryptogams, Monte centrali amplo, 0.75–1.5 μm longae. Cellulae ad L. Bean Museum, Provo, Utah. Isotype materials apices cylindricae, sine vagina, pallide bruneorosae, included in the accession to BRY consisted of latitudone longiora, 2–3 μm longae. dried culture populations on filter paper (three Colony brown when young, becoming mulberry– filter papers, BRY37705). black, spreading. Filaments isopolar and tapering Etymology: Tapinothrix clintonii = Tapinothrix to both apices when young, fragmenting centrally named in honor of President William Jefferson to become heteropolar with maturity, swollen at Clinton, in recognition of his efforts to provide the base, tapered towards the ends, with colorless protection for the ecosystems and landscapes mucilaginous pad at the base of mature filaments, now set aside in the Grand Staircase–Escalante occasionally pseudobranched. Sheath firm, rough, National Monument. colorless to blackish–purple to black, 0.5–1 μm Reference strain: Tapinothrix GSE–PSE06–07G thick. Trichomes constricted at cross–walls in (UTEX temporary no. B ZZ872, CCALA 940). basal and median regions, not constricted towards the apices, 3–4 μm wide at the base, tapering to Life cycle 0.75–1 μm wide at the apices, ending in a hair. Young filaments were observed to be tapered Cells at the base shorter than wide, with brownish towards both ends, with a width of around 3.5 granulated content, parietal thylakoids, often with μm in the central, thickest part. These isopolar a large central granule, 0.75–1.5 μm long. Cells at filaments apparently break in the middle to form the apices cylindrical, not enclosed in sheath, pale two filaments attached to each other at the base by brownish–pink, longer than wide, 2–3 μm long. the mucilaginous disk (Figs 3, 4–6, 18, 20, 28). Fottea 11(1): 127–140, 2011 131

Figs 7–15. Tapinothrix clintonii, young filaments: (7) short young filaments breaking in the middle part; (8) young filaments swollen at the base, with no clear sheath; (9) very young Leptolyngbya–like filaments, not tapered; (10) middle part of long young filaments, central granules visible; (11) young filament breaking in the middle part; (12) middle part of long filament with irregular cells; (13) false branching; (14) tapering of young filaments; (15) young tapered filament. Scale bar 10 μm.

This process of fragmentation leads to formation upright filaments on hard surfaces (rocks). They of star–like colonies, with many swollen ends are clearly members of the Pseudanabaenales in in the center and tapering ends extending out of subclass Synechococcophycidae, based on cell the clump (Figs 22, 23). Young filaments have diameter and peripheral thylakoid arrangement. colorless or faintly purplish sheaths. The sheath Many are associated with a basal community of becomes blackish–purple to black as the culture Chroococcus or Pleurocapsa species, and this ages (Fig. 29). Colony on agar is brown to black. association led to the incorrectly conceived and Comparisons to other taxa now abandoned genus Amphithrix (e.g. Amphithrix Most Tapinothrix taxa taper only very slightly janthina Bo r n e t et Fl a h a u l t ). Most species of (often not at all), and form grass–like turfs of Tapinothrix were originally described as members 132 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Figs 16–29. Tapinothrix clintonii, mature filaments: (16–21) mature filaments swollen at the base, with thick, structured, blackish sheath; (22–23) star–like colonies, with many swollen ends in the center and tapering ends extending out of the clump; (24–25) long mature filaments with thick, structured, colored sheath; (26–27) old, mat–like colonies; (28) two mature filaments connected with one mucilaginous pad; (29) very old colony with black sheaths. Scale bar 10 μm. of the genus Homoeothrix, but the type species and likely had the typical heteropolar, basally of that genus, Homoeothrix juliana (Bo r n e t attached filaments in nature. However, we did not et Fl a h a u l t ) Ki r c h n e r , clearly belongs in the see Tapinothrix clintonii in the natural material, Oscillatoriaceae in subclass Oscillatoriophycidae and suspect that it was very rare in the original (based on cell diameter and irregular thylakoid sample. Most species of Tapinothrix are pale arrangement). greyish blue–green in color, and the brownish and Our strain was growing on a rock surface, purplish colors this strain possesses even in culture Fottea 11(1): 127–140, 2011 133 are fairly distinctive. Homoeothrix violacea is are polyphyletic, including Synechococcus one of the purplish species previously described, (Clades F and H), redekei (Clades F but is narrower at the base and has shorter cells and H), Pseudanabaena (Clades D and H) and in the terminal regions. Most importantly, H. Leptolyngbya (Clades A, C, D, E, F, G). The strain violacea is a marine taxon. Homoeothrix fusca identified as Phormidium (Clade F) is certainly can be yellowish or blackish, but has blue–green incorrectly identified. trichomes, is not constricted or at most weakly constricted at the crosswalls, and is also narrower. ITS structural comparisons Ecological separation for this taxon is also strong; In cyanobacteria, the D1–D1’ helix is the most H. fusca is reported to be xeno– to oligosaprobic conserved structure of the 16S–23S ITS region in clear mountain waters, while T. clintonii was (Jo h a n s e n et al. 2008). In all Pseudanabaenales found in a warm desert seep wall associated with examined to date, this helix contains a 5 bp basal a stream flowing through cattle country (i.e., not helix (GACCU:AGGTC), a unilateral bulge of oligosaprobic!). 5–8 nucleotides on the 3’ side, followed by a 3 bp Tapinothrix is likely most easily confused helix (ACC:GGU – except Pseudanabaena GSE– with species of , which are PSE20–05C and Leptolyngbya appalachiana heteropolar, attached at the base, but never GSM–SFF–MF60 which differ in sequence). A tapering. The latter genus also seems consistently bilateral bulge or unilateral bulge comes next, with epiphytic rather than epilithic. Neither genus has the rest of the helix being too variable to recognize been sequenced up until this time, and almost a common pattern (Figs 31–43). Apart from these certainly confusion will surround these taxa similarities, we did find further consistencies until collection and analysis of sequence data is within the various LPP clades. completed. There are no Heteroleibleinia species The clade consisting of 12 OTU’s in that could be confused with T. clintonii. Leptolyngbya sensu stricto includes Leptolyngbya boryana, L. tenerrima, and a number of Phylogenetic placement unidentified species/strains. Of these 12, we have Tapinothrix clintonii and Leptolyngbya sensu stri- ITS sequences for eight strains, and all were cto (the group of taxa associated with Leptolyng- markedly similar in secondary structure of the bya boryana, the type species of the genus) belong D1–D1’ helix to the two strains chosen to represent to the same clade (Clade A, Fig. 30). A strain this group (Figs 31, 32). This group of taxa all has possessing the morphology of Leptolyngbya, sequences of 51 nucleotides, with a terminal loop GSE–PSE30–01B may be sister to Tapinothrix, of four unpaired nucleotides, a small irregularity but there is poor support for this putative caused by an adenosine residue that does not pair, relationship. A number of Pseudophormidium and a basal unilateral bulge of only 5 nucleotides strains belong to Clade B (Fig. 30). These (Figs 31, 32). Leptolyngbya crispata which fell strains are wider than most Leptolyngbya, and outside of Leptolyngbya sensu stricto, had a demonstrate multiple trichomes in a sheath with markedly similar D1–D1’ helix to this clade (Fig. frequent false branching. Other clades contain 33). The desert soil strains of Leptolyngbya had mostly putative Leptolyngbya (Clades C, D, E more variability in structure in this region than and G). Clade F (Fig. 30) contains many taxa of L. sensu stricto, and differed in several regards: uncertain placement in the LPP groups, along with a basal bilateral bulge of 7 nucleotides, the loss some well established species and genera, such of the mid–helix bulge, and a larger terminal loop as Halomicronema excentricum Ab e d , Ga r c i a – (Figs 34, 35). Pi c h e l et He r n á n d e z –Ma r i n é , Prochlorothrix The taxon sister to T. clintonii according hollandica Bu r g e r –Wi e r s m a , St a l et Mu r and to the phylogenetic analysis, Leptolyngbya GSE– Leptolyngbya nodulosa Li et Br a n d . Finally, PSE30–01B, had a strikingly different D1–D1’ Clade H contains a mixture of strains assigned to helix (Fig. 36). T. clintonii was similar to this Pseudanabaena (10 OTU’s), Limnothrix redekei strain in the basal part of the helix (Fig. 37), (3 OTU’s), and Synechococcus (1 OTU). particularly with respect to the adenosine residue If the identification of the strains in this tree inserted opposite the unilateral bulge on the 3’ part were made correctly (and we are not confident of the helix (a feature so far unique for this clade). that they all were made consistent with Ko m á r e k The more terminal part of this helix was similar & An a g n o s t i d i s 2005), then a number of genera in length to those of Leptolyngbya sensu stricto, 134 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Fig. 30. Bayesian analysis of 113 OTU’s of Pseudanabaenales plus Gloeobacter violaceus from the Gloeobacteriales. Node support is indicated as Bayesian posterior probabilities/bootstrap support from parsimony analysis/bootstrap support from likelihood analysis; an * means 1.0 or 100%, a – means support less than 0.50 or 50%. Major clades are indicated by letters (A–H). Black dots at the end of OTU names or sets of OTU’s indicate that ITS structures were available and are shown in Figs 31–65. but the sequence was markedly different in both Leptolyngbya appalachiana had an unusually the helix and terminal loop. The D1–D1’ helix long D1–D1’ helix (Fig. 43), and differed from in strains more distantly related to Leptolyngbya all other taxa in the Pseudanabaenales sequenced sensu stricto had larger terminal loops, or a terminal thus far. loop subtended by a bilateral bulge (Figs 38–42). The Box–B antiterminator helix in Tapino- Fottea 11(1): 127–140, 2011 135

Figs 31–43. Secondary structure of the D1–D1’ helix from the 16S–23S ITS region in several OTU’s representing various Pseudanabaenalean clades, arranged in the order (top to bottom) they appear in the Bayesian analysis (Fig. 30): (31–32) Leptolyngbya sensu stricto, including the type species, Leptolyngbya boryana. This structure was seen in nearly all strains in this clade; (33–35) Leptolyngbya spp. from desert soils in New Mexico; (36) Leptolyngbya sp. with untapered, reddish colored trichomes from wet wall in Grand Staircase–Escalante National Monument; (37) Tapinothrix clintonii; (38–40) Leptolyngbya spp. from clade E (with cells longer than wide, see center, Fig. 30); (41) Leptolyngbya nodulosa UTEX2910, representing the L. nodulosa clade and clade F (Fig. 30); (42) Pseudanabaena sp. representing a clade of 10 OTU’s (clade H, Fig. 30); (43) Leptolyngbya appalachiana, a species with exceptionally long helices in all conserved helices of the 16S–23S ITS (clade D, Fig. 30). thrix clintonii was distinct from all other Box–B when the 23S–5S ITS structure is sequenced helices. This region is more variable than the D1– and available. We did have this sequence for D1’ helix, as evidenced by the different lengths Tapinothrix so we are confident in this V3 structure and different placement of unilateral and bilateral (Fig. 60). The V3 for all taxa in Leptolyngbya bulges (Figs 44–56). The basal structure is more sensu stricto (including L. crispata SEV4–3–c6) conserved, and this is the region different in were identical in sequence and structure (Fig. 57). Tapinothrix (Fig. 50). Most strains have a basal Surprisingly, the V3 helix for L. crispata SEV4– bilateral bulge above the initial helix (A–CC), 3–c2 (Fig. 58) was very different than L. crispata whereas Tapinothrix has a unilateral double uracil SEV4–3–c6 despite their close relationship insert on the 5’ side of the helix. Leptolyngbya inferred from the 16S rRNA phylogeny (Fig. 30). GSE–PSE28–08A and Leptolyngbya appala- It seems possible this is due to sampling different chiana had distinctively long helices that were ribosomal operons. Tapinothrix has a unique V3 also different than the Box–B regions of other among all taxa characterized (Fig. 60), with the strains. base of the helix (the most conserved part of most The V3 helix is the most difficult structure helices) very distinct (5’GGU–ACC3’) from the to determine because its length is dependent on the typical base (5’UGUC–GACA3’). Its putative length of the D4 and D5 helices. These latter two sister taxon, Leptolyngbya GSE–PSE30–01B, helices can only be determined with confidence also had a unique structure for the V3 among 136 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Figs 44–56. Secondary structure of the Box–B helix from the 16S–23S ITS region in several OTU’s representing various Pseudanabaenalean clades, arranged in the order (top to bottom) they appear in the Bayesian analysis (Fig. 30).

Figs 57–65. Secondary structure of the V3 helix from the 16S–23S ITS region in several OTU’s representing various Pseudanabaenalean clades, arranged in the order (top to bottom) they appear in the Bayesian analysis (Fig. 30). Fottea 11(1): 127–140, 2011 137

the sampled strains (Fig. 59). The remaining V3 Tapinothrix calcarea (Li) Bo h u n i c k á et helices were all unique (Figs 61–65), save for that Jo h a n s e n , comb. nov. of Leptolyngbya nodulosa UTEX2910. We have Basionym: Homoeothrix calcarea Li (1994, p. 70). several sequences of strains in this clade, and they Tapinothrix crustacea (Vo r o n i c h i n ) Bo h u n i c k á all have the same V3 helix (data not shown). et Jo h a n s e n , comb. nov. Basionym: Homoeothrix crustacea Vo r o n i c h i n (1923, Nomenclatural revision of Homoeothrix species p. 115). We recommend that all of the Homoeothrix species Synonyms: Homoeothrix globulus Vo r o n i c h i n (1932), clearly belonging to the Pseudanabaenaceae Homoeothrix woronochinii Ma r g a l e f (1953); non Homoeothrix crustacea (Bo r z ì ) Ma r g a l e f (1953). be transferred to Tapinothrix. While Ko m á r e k & An a g n o s t i d i s (2005) recommended that Tapinothrix fusca (St a r m a c h ) Bo h u n i c k á et this revision occur, they felt that further work Jo h a n s e n , comb. nov. (cytological, morphological, and molecular) Basionym: Homoeothrix fusca St a r m a c h (1934, p. should be completed. The present work supports 294). the hypothesis that these taxa are not in the Synonym: Phormidiochaete fusca (St a r m a c h ) Ko m á r e k et An a g n o s t i d i s . Oscillatoriaceae, and further confusion will be experienced until the taxonomy is complete. Tapinothrix gloeophila (St a r m a c h ) Bo h u n i c k á Revisionary work is still needed in this genus, as et Jo h a n s e n , comb. nov. many of the species overlap in morphology, and Basionym: Homoeothrix gloeophila St a r m a c h (1960, p. 227). differentiation and identification are problematic. Currently, no living strains exist for any species of Tapinothrix gracilis (Ha n s g i r g ) Bo h u n i c k á et Tapinothrix or Homoeothrix except T. clintonii. Jo h a n s e n , comb. nov. Based on its morphology, Phormidiochaete Basionym: Leptochaete crustacea var. gracilis fusca (= Homoeothrix fusca) is the likely Ha n s g i r g (1892b, p. 138). Synonyms: Leptochaete gracilis (Ha n s g i r g ) Ge i t l e r sister taxon of T. clintonii. It has similar cell (1925), Homoeothrix gracilis (Ha n s g i r g ) Ko m á r e k & measurements, but differs in the amount of Ko v á č i k (1987). constriction at the crosswalls, the trichome color, Tapinothrix janthina (Bo r n e t et Fl a h a u l t ) and habitat preference. This taxon should surely Bo h u n i c k á et Jo h a n s e n , comb. nov. be in Tapinothrix, and the transfer is made below. Basionym: Amphithrix janthina Bo r n e t et Fl a h a u l t A few of the Homoeothrix species (1886–1888, ser. 3, p. 344). do not belong in the Pseudanabaenaceae or Synonym: Homoeothrix janthina (Bo r n e t et Fl a h a u l t ) Oscillatoriaceae, and therefore correspond best St a r m a c h (1959). with Phormidiochaete in the Phormidiaceae. H. Tapinothrix minuta (Se c k t ) Bo h u n i c k á et margalefii Ko m á r e k et Ka l i n a has trichomes up to Jo h a n s e n , comb. nov. 7 µm wide at the base and filaments up to 8.5 µm Basionym: Homoeothrix minuta Se c k t (1921, p. 425). wide. This species is transferred below as well. Tapinothrix poljanskii (Mu z a f a r o v ) Bo h u n i c k á et Jo h a n s e n , comb. nov. Phormidiochaete crustacea (Bo r z ì ex Bo r n e t et Basionym: Homoeothrix poljanskii Mu z a f a r o v (1952, p. 84). Fl a h a u l t ) Bo h u n i c k á et Jo h a n s e n , comb. nov. Synonym: Homoeothrix schizotrichoides Mu z a f a r o v Basionym: Leptochaete crustacea Bo r z ì ex Bo r n e t et (1952). Fl a h a u l t (1886–1888, ser. 3, p. 342). Synonyms: Homoeothrix crustacea (Bo r z ì ex Bo r n e t et Tapinothrix rivularis (Ha n s g i r g ) Bo h u n i c k á et Fl a h a u l t ) Ma r g a l e f (1953), Homoeothrix margalefii Jo h a n s e n , comb. nov. Ko m á r e k et K a l i n a (1965), non Homoeothrix crustacea Basionym: Leptochaete rivularis Ha n s g i r g (1892a, p. Vo r o n i c h i n (1923). 54). Tapinothrix articulata (St a r m a c h ) Bo h u n i c k á Synonym: Homoeothrix rivularis (Ha n s g i r g ) Ko m á r e k et Ka n n (1973). et Jo h a n s e n , comb. nov. Basionym: Homoeothrix articulata St a r m a c h (1960, Tapinothrix simplex (Vo r o n i c h i n ) Bo h u n i c k á et p. 231). Jo h a n s e n , comb. nov. Tapinothrix batrachospermorum (Sk u j a ) Basionym: Homoeothrix simplex Vo r o n i c h i n (1932, p. 309). Bo h u n i c k á et Jo h a n s e n , comb. nov. Basionym: Homoeothrix batrachospermorum Skuja Tapinothrix stagnalis (Ha n s g i r g ) Bo h u n i c k á et (1964, p. 68). Jo h a n s e n , comb. nov. 138 Bo h u n i c k á et al.: Tapinothrix clintonii sp. nov.

Basionym: Leptochaete stagnalis Ha n s g i r g (1892a, p. considerable study and revision. These taxa are 54). actually fairly common in unpolluted streams Synonym: Homoeothrix stagnalis (Ha n s g i r g ) Ko m á r e k with rocky benthos, forming tufted fascicles of et Ko v á č i k (1987). filaments. Tapinothrix subtilis (Sk u j a ) Bo h u n i c k á et Leptolyngbya GSE–PSE30–01B appeared Jo h a n s e n , comb. nov. to be the sister taxon to Tapinothrix clintonii. Basionym: Homoeothrix subtilis Sk u j a (1964, p. 69). However, the two strains did not share high Tapinothrix varians (Ge i t l e r ) Bo h u n i c k á et sequence similarity in the 16S rRNA gene (P Jo h a n s e n , comb. nov. = 91.1 %), did not have similar 16S–23S ITS Basionym: Homoeothrix varians Ge i t l e r (1927, p. secondary structures, and were dissimilar in 801). morphology. We assume that this may be a case of Synonyms: Homoeothrix simplex var. elegans long–branch attraction. Tapinothrix actually has Vo r o n i c h i n (1932), Homoeothrix simplex f. elegans highest sequence similarity to Leptolyngbya sensu (Vo r o n i c h i n ) El e n k i n (1949). stricto (P = 92.4–93.6 %), although this is still too Tapinothrix violacea (Bo r n e t et Fl a h a u l t ) low for it to be considered to belong to that genus Bo h u n i c k á et Jo h a n s e n , comb. nov. since < 95 % similarity is often considered to be Basionym: Amphithrix violacea Bo r n e t et Fl a h a u l t the cut–off for indication of different genera. (1886–1888, ser. 3, p. 344). This paper reports the first sequence Synonym: Homoeothrix violacea (Bo r n e t et Fl a h a u l t ) attributed to the genus Tapinothrix, a taxon fairly Ko m á r e k et Ka n n (1973). common in clean to slightly enriched mountain streams (Ko m á r e k & Ka n n 1973). While common, Discussion the genus is not easy to obtain in culture as it often appears in an epilithic mat with many other taxa, Tapinothrix clintonii shares characteristics with and likely requires flowing water to develop its five different genera. To our knowledge, the characteristic heteropolar, tapering morphology. unusual feature of forming trichomes which taper In examination of other Tapinothrix in North towards both apices is restricted to Ammatoidea America, Jo h a n s e n et al. (2011) found three species of Tapinothrix: T. varians, T. janthina, (Ko m á r e k & An a g n o s t i d i s 2005). However, this taxon is based on a type species (A. normannii and a species new to science, T. ozarkiana Jo h a n s e n et Ře h á k o v á . They noted that species of We s t et G.S. We s t ) clearly belonging to the Phormidiaceae. Phormidiochaete possesses Tapinothrix were difficult to differentiate and that heteropolar trichomes that taper from the more species might indeed be represented in the attached base to the apex, but it too belongs in the populations which they examined. Some species Phormidiaceae. The source of confusion between of Tapinothrix taper very little, and consequently Tapinothrix and these two genera is that some resemble the epiphytic genus, Heteroleibleinia. species belonging to the Pseudanabaenaceae The phylogenetic relationships of these taxa need apparently have been transferred into these determination. It seems possible that more than genera erroneously. While T. clintonii has features one tapering genus may be present within the similar to these taxa, it clearly belongs in neither Pseudanabaenaceae. because of its phylogenetic placement in the Pseudanabaenales. Phormidiochaete can rarely Acknowledgements form isopolar trichomes that are Ammatoidea– We thank the Bioinformatics Facility in the University like, and these taxa may not be well separated. of Connecticut Biotechnology Center for use of their Homoeothrix is a relatively species–rich computing cluster and Terry Tolbert, Liliya Khaibullina, taxon with a long nomenclatural history. No and Lira Gaysina for help and support during the representative of Homoeothrix juliana has collection field trip. The project was supported by been sequenced to date, but it possesses the MSM 600 766 5801 and GAJU 135/2010/P grants. dimensions, cell–division pattern, and thylaloid arrangement consistent with the Oscillatoriaceae. The pseudanabaenalean taxa in Homoeothrix are References likely very phylogenetically distant from the type species. We have placed the majority of these Al b e r t a n o , P. & Ko v á č i k , L. (1994): Is the genus taxa in Tapinothrix, a genus which still needs Leptolyngbya (Cyanophyta) a homogenous Fottea 11(1): 127–140, 2011 139

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