Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 Potamolinea gen. nov. (Oscillatoriales, Cyanobacteria): a phylogenetically and ecologically coherent cyanobacterial genus Mari�ellen Dornelles Martins and Luis Henrique Zanini Branco Correspondence Luis Henrique Zanini Branco branco@ibilce.unesp.br Zoology and Botany Department, IBILCE/UNESP, S~ao Paulo State University, Rua Cristóv~ao Colombo, 2265 – BR15054-000, S~ao Jos�e do Rio Preto (SP), Brazil Phormidium Kützing ex Gomont, a common genus of the Cyanobacteria, is widely known as a problematic group. Its simple morphology is not congruent with its genetic heterogeneity and several new generic entities have been described based on 16S rRNA gene sequence analyses from populations with similar morphology. During a study of the diversity of Phormidioideae (Phormidiaceae, Oscillatoriales) in Brazil, ten Phormidium-like strains from south-eastern and mid-western regions were isolated in monospecific cultures and submitted to polyphasic evaluation (morphological, ecological and molecular studies). The populations studied presented homogeneous morphology (trichomes straight, not attenuated and apical cell rounded or obtuse), differing mainly in cell length from the type species of the genus Phormidium (Phormidium lucidum Agardh ex Gomont) and occurring as three morphotypes. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the populations studied, with European Phormidium aerugineo-caeruleum (Gomont) Anagnostidis & Kom�arek strains, were placed together in a very distinctive and highly supported clade. Thus, the set of characteristics of the strains resulted in the recognition of the new genus Potamolinea Martins et Branco with two species: Potamolinea magna as the type species (strains 47PC and 48PC) and Potamolinea aerugineo-caerulea (Gomont) Martins et Branco (strains 1PC, 2PC and 38PC). These two species plus one still undetermined lineage, Potamolinea sp., are morphologically and genetically distinguishable, whereas the secondary structures of the D1-D1¢, box-B and V3 regions were conserved within each one. The generic name and specific epithets of the new taxa are proposed under the provisions of the International Code of Nomenclature for algae, fungi and plants. Introduction To build a classification system that reflects the evolutionary history of Cyanobacteria, many changes have been occur- ring in the delimitation of orders, families, genera and spe- cies. The classification system recently proposed by Kom�arek et al. (2014), which is based on 16S rRNA gene sequence analyses, has brought considerable changes and new taxonomic groups have been described. Taxonomic studies based on polyphasic approach are changing the arrangement of Cyanobacteria and the value of characters used in the reconstruction of its phylogeny. In the order Oscillatoriales, the heterogeneous nature of which has been emphasized by many authors (Teneva et al., 2005; Casamatta et al., 2012; Engene et al., 2012, 2013; Kom�arek et al., 2013; McGregor & Sendall, 2015; Martins et al., 2016), Phormidium is considered a taxonomi- cally complex genus, due to its morphological simplicity and large number of described species (about 200, accord- ing to Kom�arek & Anagnostidis, 2005), and is widely known to be polyphyletic. Several genera have been recently described from species previously classified under the genus Phormidium, such as Phormidesmis (Turicchia et al., 2009), Wilmottia (Strunecký et al., 2011), Roseofilum (Casamatta et al., 2012), Ammassolinea (Hašler et al., 2014), Kampto- nema (Strunecký et al., 2014), Cephalothrix (Malone et al., 2015) and Ancylothrix (Martins et al., 2016). Abbreviations: BI, Bayesian inference; ITS, internal transcribed spacer; ML, maximum-likelihood; NJ, neighbour-joining. The GenBank/EMBL/DDJB accession numbers for the 16S–23S rRNA gene sequences of the strains reported in this study are KX001786, KX001787, KX001788, KX001789, KX001790, KX001791, KX001792, KX001793, KX0017943 and KX001795. Two supplementary tables are available with the online Supplementary Material. 3632 001243 ã 2016 IUMS Printed in Great Britain International Journal of Systematic and Evolutionary Microbiology (2016), 66, 3632–3641 DOI 10.1099/ijsem.0.001243 http://dx.doi.org/10.1601/nm.624 http://dx.doi.org/10.1601/nm.624 http://dx.doi.org/10.1601/nm.624 Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 Morphologically, the genus Phormidium is characterized by uniseriate, cylindrical, isopolar, non-branched trichomes, without differentiated cells. The apical part of trichomes can present wide morphological variation and Kom�arek & Anagnostidis (2005) used that criterion to organize the Phormidium species into eight groups. According to these authors, group V is formed by species with cylindrical tri- chomes along their whole length and the apical cell is widely rounded; this group comprises most of Phormidiumspecies. During an investigation about Phormidioideae diversity in Brazil, ten Phormidium-like strains, isolated from nine Brazilian streams and morphologically belonging to the above mentioned group V, were investigated. The taxo- nomic positioning of these and European strains, also iso- lated from streams, was defined based on a polyphasic evaluation (morphological, ecological and molecular analy- ses) and resulted in the proposition of a new genus, Potamo- linea gen. nov. Methods Origin of the strains and cultivation. Ten environmental samples, growing in nine stream bottoms in Brazil, were collected (Table 1). Each cyanobacterial strain was isolated from a single trichome grown on BG11 medium (Rippka et al., 1979). Trichomes of each mat were repeatedly separated from others and successively transferred to clean drops of deionized water using a Pasteur pipette under an inverted light microscope (Leica DMIL LED). The sole trichomes were then inoculated in tubes with BG11 growth medium for the establishment of unicyanobacterial cultures. Strains were cultured and maintained under 20±1 � C, 50 µmol photons m�2 s�1 of irradiance and a 14 : 10h light–dark cycle in the culture collection of IBILCE/UNESP. Morphological characterization and identification. Morphologi- cal variability of populations was evaluated from fresh field material and from cultured samples by using an Olympus BH2 microscope. Taxonomic features, such as cell width, cell length, attenuation of tri- chomes and apical cell shape and dimensions, were analysed in at least 30 trichomes for each sample. Representatives of each species studied were deposited in Herbarium SJRP (IBILCE/UNESP), Brazil. Molecular analyses. Biomass for DNA extraction was obtained from non-axenic unicyanobacterial cultures by repeated centrifugations. Dur- ing the centrifugation process, the filaments were washed several times with sterile deionized water to remove or reduce mucilage and growth medium substances. DNA was extracted using the PowerSoil DNA Iso- lation kit from MO BIO Laboratories according to the manufacturer’s protocol. The 16S rRNA gene and 16S–23S internal transcribed spacer (ITS) markers were amplified by polymerase chain reaction (PCR) using the primers 16S27F and 23S30R (Taton et al., 2003), which was performed in a Techgene TC-512 thermal cycler using 25 µl reaction volumes con- taining 5 µl 10� PCR buffer, 2 µl 50 mM MgCl2, 1 µl 10 mM dNTP mix, 1.25 µl of each primer (5 pmol), 14.2 µl Milli-Q water, 1.5 U Plati- num Taq DNA polymerase (Life Technologies) and 10 ng genomic DNA. Thermal cycling was 94 � C for 5 min, followed by ten cycles of 94 � C for 45 s, 57 � C for 45 s and 72 � C for 2 min; 25 cycles of 92 � C for 45 s and 54 � C for 45 s; and one final cycle of 72 � C for 7 min. The PCR products were analysed on 1% agarose gels stained with GelRed 0.6� (Biotium) and viewed on a ‘Mini Bis Pro’ transilluminator (Micro Pho- tonics). The positive products were cloned using the pGEM-T Easy Vec- tor System I (Promega) according to the supplier’s manual. Competent Escherichia coli DH5a cells were transformed by heat shock and recom- binant plasmids were isolated using the ‘GeneJET Plasmid Miniprep’ kit (Thermo Fisher Scientific). Sequencing was performed on an ABI 3130 sequencer, using a ‘BigDye Terminator v3.0 Cycle Sequencing Ready Reaction’ kit (Applied Biosystems) as the manufacturer’s protocol. Primers M13F and Sp6R correspond to the vector sites and the internal primers 357F, 704R, 1114F and 1494R (Neilan et al., 1997) were used for sequencing. The DNA fragments were assembled into contigs using the Phred/Phrap/Consed software (Ewing & Green, 1998; Ewing et al., 1998; Gordon et al., 1998), and only bases with quality higher than 20 were considered. The nucleotide sequences obtained in this study have been deposited in the GenBank database under accession numbers: KX001786, KX001787, KX001788, KX001789, KX001790, KX001791, KX001792, KX001793, KX0017943 and KX001795. Alignment and phylogenetic analyses The sequences obtained were compared with sequences previously published in NCBI (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/) by BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) and the closest related were selected to compose the database. The alignment was car- ried out using the software CLUSTAL W v1.8 (Thompson et al., 1994) in MEGA 6.06 (Tamura et al., 2013) and inspected and refined manually. The unicellular cyanobacteria Gloeobacter violaceus PCC 7421 and G. violaceus PCC 8105 (GenBank accession numbers NC005125 and AF132791, respectively) were designed as the evolutionary outgroup. Phylogenetic analyses were performed based on partial 16S rRNA gene sequences. The appropriate nucleotide substitution models were selected in jModelTest 2.1.1 (Darriba et al., 2012). Bayesian inference (BI) analy- sis was performed in MrBayes 3.1.2 software (Huelsenbeck & Ronquist, 2001), run with GTR+G+I model (rate matrix with six different substi- tution types, number of rate categories = 4, and with the nucleotide fre- quencies, shape parameter and pINVAR estimated from the data). BI analysis comprised two runs of four Monte Carlo Markov chains, each with 10 000 000 generations and sampling every 100 generations. The initial 10 000 generations were discarded as burn-in. Neighbour-joining (NJ) and maximum-likelihood (ML) inferences were performed using MEGA 6.06 (Tamura et al., 2013) and the GTR model was applied in the last method assuming heterogeneous substitution rate and gamma sub- stitution of variable sites. Bootstrap resampling was performed on 1000 replicates. Sequence similarity matrix/nucleotide divergence was calcu- lated from the alignment in BioEdit (Hall, 1999) from all positions including gaps. Alignment of the 16S–23S ITS region was made using a combination of CLUSTAL_W v1.8 (Thompson et al., 1994) in MEGA6.06 (Tamura et al., 2013) and manual alignment utilizing secondary structure of conserved domains. The tRNA sequences were identified with tRNAscan-SE 1.21 (Lowe & Eddy, 1997). D1-D1¢, box-B and V3 ITS regions were identi- fied and their secondary structures were determined using Mfold 3.2 (Zuker, 2003) and re-drawn in Macromedia Fireworks 8.0. Results Morphological evaluation The ten populations were observed forming benthic, mac- roscopic, dark green mats in streams. All the strains, belonging to three different morphotypes (Potamolinea magna, P. aerugineo-caerulea and Potamolinea sp.), pre- sented a morphology corresponding to species included in Phormidium group V, according to Kom�arek & Anagnosti- dis (2005), but different from the type species of the genus Phormidium (P. lucidum Agardh ex Gomont). http://ijs.microbiologyresearch.org 3633 Potamolinea gen. nov. http://dx.doi.org/10.1601/nm.3093 KX001786 KX001787 KX001788 KX001789 KX001790 %20KX001791 KX001792 KX001793 KX0017943 KX001795 http://www.ncbi.nlm.nih.gov/ http://www.ncbi.nlm.nih.gov/BLAST/ http://dx.doi.org/10.1601/nm.643 http://dx.doi.org/10.1601/nm.643 NC005125 AF132791 Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 One morphotype (strains 47PC and 48PC) presented densely entangled filaments, 13.2–16.8 µm wide; sheaths facultative, attached to the trichomes, firm, thin, colourless; trichomes cylindrical, not attenuated, not constricted to slightly constricted at the ungranulated or slightly granu- lated cross-walls, 9.6–16.8 µm wide; cells 0.4–0.9 time longer than wide, 4–12.8 µm long; cell content blue–green, homogeneous to finely granulated; apical cell rounded, without calyptra (Fig. 1a–d, Table 2). Strain 47PC was selected as representative of the populations studied and was deposited in Herbarium SJRP (IBILCE/UNESP), Brazil, under voucher number SJRP 31642. Table 1. Sampling sites for the Potamolinea strains studied Strains: 1PC, 2PC and 38PC, P. aerugineo-caerulea; 32PC, 33PC, 34PC, 35PC and 36PC, Potamolinea sp.; 47PC and 48PC, P. magna. MT, Mato Grosso State; SP, S~ao Paulo State. Strain Locality Latitude (S) Longitude (W) Habitat 1PC Guarant~a do Norte stream/ MT 9 � 46¢ 07† 54 � 39¢ 09† On rocky bottom of an oligotrophic, partially shaded stream in a pasture with remnant marginal vegetation at a disturbed Brazilian Amazon area 2PC Santos Reis stream/MT 9 � 45¢ 23† 54 � 34¢ 46† On rocky bottom of an oligotrophic, partially shaded stream in a pasture with remnant marginal vegetation at a disturbed Brazilian Amazon area 32PC Felicidade stream/SP 20 � 47¢ 55† 49 � 18¢ 41† On clay bottom of an oligotrophic, open stream in a pasture at a disturbed semi-deciduous seasonal forest area 33PC Picinguaba Station - Serra do Mar State Park/SP 23 � 22¢ 16† 44 � 49¢ 50† On sandy-clay bottom of an oligotrophic, shaded stream in a preserved Atlantic Rainforest area 34PC Picinguaba Station - Serra do Mar State Park/SP 23 � 22¢ 47† 44 � 49¢ 21† On sandy-clay bottom of an oligotrophic, shaded stream in a preserved Atlantic Rainforest area 35PC & 47PC Jacar�e stream/SP 20 � 51¢ 39† 49 � 36¢ 54† On sandy bottom of a partially shaded stream in a pasture area with remnant marginal vegetation at a disturbed semi-deciduous seasonal forest area 36PC Barra Funda stream/SP 20 � 38¢ 45† 49 � 24¢ 13† On sandy-clay bottom of an oligotrophic, partially shaded stream in a pasture area with remnant marginal vegetation at a disturbed semi-deciduous seasonal forest area 38PC Euclides Brentino stream - Furnas Bom Jesus State Park/SP 20 � 15¢ 15† 47 � 27¢ 23† On sandy bottom of an oligotrophic, shaded stream in a preserved Brazilian savanna area 48PC Preto river at S~ao Roberto waterfall/SP 20 � 11¢ 10† 49 � 41¢ 06† On sandy-clay bottom of an oligotrophic partially shaded stream in a pasture area Table 2. Morphological comparison among Potamolinea strains Dimensions (µm) represent full ranges observed. L/W, cell length/width ratio; �, absence; +, presence. Strains: 1PC, 2PC and 38PC, P. aerugineo- caerulea; 32PC, 33PC, 34PC, 35PC and 36PC, Potamolinea sp.; 47PC and 48PC, P. magna. Strain Granuled cross-walls Filament width* Trichome width* Cell length† Apical cell width* Apical cell length* L/W 1PC � 6–8.4 5–7.2 5.6–7.6 6–7.2 7.6–9.6 0.7–1.1 2PC � 6–9 5.4–7.5 5–7.2 6–7.5 6.8–10.4 0.7–1.1 32PC � 10.4–13.6 9.6–13.6 5.6–14.4 9.6–13.6 8–16.8 0.4–1.5 33PC � 6.8–13.6 6.4–13.6 4.8–13.6 6.4–13.6 7.2–14.4 0.5–1.5 34PC � 7.2–10.4 6–10.4 4.8–14.4 6–10.4 6.8–15.2 0.4–1.5 35PC � � 7–11.2 5–13.6 7–11.5 8–12.8 0.5–1.3 36PC � � 9.6–10.8 4.8–12 9.6–10.8 7.2–12 0.4–1.2 38PC + � 5.4–7.2 3.2–6.4 6.4–8 4–6 0.4–0.9 47PC +/� 13.2–16.8 9.6–16.8 4–12.8 9.2–16.8 6.4–15.2 0.4–0.8 48PC +/� 13.6–17.6 12.8–16 5.6–12 12.4–16 8–11.2 0.4–0.9 *n=30. †n=300. 3634 International Journal of Systematic and Evolutionary Microbiology 66 M. D. Martins and L. H. Z. Branco Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 The second morphotype, represented by strains 1PC, 2PC and 38PC, presented filaments densely entangled, 6–9 µm wide; sheaths facultative, attached to the trichomes, firm, thin, colourless; trichomes cylindrical, not attenuated, not constricted at the ungranulated cross-walls, 5–7.5 µm wide; cells 0.7–1.1 time longer than wide, 5–7.6 µm long; cell con- tent blue–green, homogeneous to finely granulated, some- times with scattered larger granules; apical cell rounded, without calyptra (Fig. 1e–h, Table 2). Strain 1PC was selected as representative of the populations studied and it was deposited in Herbarium SJRP (IBILCE/UNESP), Brazil, under voucher number SJRP 31634. The third morphotype, represented by strains 32PC, 33PC, 34PC, 35PC and 36PC, presented filaments densely entangled, 6.8–13.6 µm wide; sheaths facultative, attached to trichomes, firm, thin, colourless; trichomes cylindrical, not attenuated, not constricted to slightly constricted at the ungranulated cross-walls, 6–13.6 µm wide; cells 0.4–1.5 time longer than wide, 4.8–14.4 µm long; cell content blue–green, homogeneous to finely granulated; apical cell rounded or truncate, without calyptra (Fig. 1i–l, Table 2). Strain 35PC was chosen as representative of the populations studied and it was deposited in Herbarium SJRP (IBILCE/UNESP), Brazil, under voucher number SJRP 31636. Molecular analyses and phylogeny – 16S rRNA gene The comparison among partial 16S rRNA gene sequences of the ten studied strains and sequences from GenBank showed similarities above 95.3% with six Spanish (a) (g) (h) (i) (j) (k) (l) (b) (c) (d) (e) (f) Fig. 1. Photomicrographs of Brazilian strains of Potamolinea: (a–d) P. magna; (e–h) P. aerugineo-caerulea; (i–l) Potamolinea sp. Bars, 10 µm. http://ijs.microbiologyresearch.org 3635 Potamolinea gen. nov. Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 Phormidium aerugineo-caeruleum strains (MNZ-37, MNZ- 32, MNZ-31, MED-28, MED-31, MED-17; Loza et al., 2013) and below 94% with other strains. The tree obtained with BI analysis contained more well- supported nodes than those obtained with ML and NJ analyses and it is consequently the tree topology reported herein. Analyses of the 16S rRNA gene sequences showed a clear separation of the group comprising the studied popu- lations and Spanish Phormidium aerugineo-caeruleum strains from the remaining strains (clade I; Fig. 2), and especially from the true Phormidium clade that is, according to Sciuto et al. (2012), represented by Phormidium cf. irrig- uum CALA 759 and P. irriguum f. minor ETS-02. Similarity scores based on the comparison of 16S rRNA gene sequences of strains of clade I with sequences of strains pertaining to other genera, such as Phormidium, Oscillato- ria, Lyngbya, Kamptonema, Microcoleus and Coleofasciculus, were lower than 93.8%. The tree topology showed a close phylogenetic relationship among members of clade I with Wilmottia and Phormidium sp. B-Tom, highly supported by posterior probabilities, and the divergence among the three groups was higher than 5.6%. The clade I was highly sup- ported in ML, NJ and BI analyses (99%, 100% and 1, respectively; Fig. 2) and the similarity scores based on 16S rRNA gene sequences within this cluster were higher than 95.3% (Table S1, available in the online Supplementary Material). The strains of clade I clustered into three subgroups, labelled A, B and C, which were well supported by posterior probabilities (Fig. 2) and each of the subgroups corre- sponded to one of the three morphotypes previously cited. Similarity scores within each subgroup were from 97.3 to 99.9% in subgroup A, 99.4 to 100% in subgroup B and 99.5% in subgroup C (Table S1). Molecular analyses – 16S–23S ITS Analysis of the 16S–23S ITS region for all ten strains studied was informative and supported both morphological and molecular (16S rRNA gene sequences) data. All strains had operons only with the tRNAIle gene and the length of the 16S–23S ITS region ranged from 372 to 406 bp (Table 3). Six of the 12 ITS regions had the same length in all strains and the more variable-length regions among the strains were the spacers preceding box-B and V3 helices. The V2 region was not identified, as it is located between tRNAIle and tRNAAla and this latter gene was absent in the studied strains. The basal sequences of the helices were mostly con- served and the secondary structure could be determined (Fig. 3). Similarity scores based on 16S–23S ITS sequences within each subgroup were higher than 98.6% (Table S2) but lower than 74% when compared among subgroups of the genus. The D1-D1¢ helix presented small differences in nucleotide sequences among the strains (Fig. 3a–d). Strains 1PC and 2PC presented identical nucleotide sequences and, conse- quently, the same secondary structure. The D1-D1¢ sequence and secondary structure of strain 38PC were very similar to those of strains 1PC and 2PC, but not identical (Fig. 3a, b). Strains 32PC, 33PC, 34PC, 35PC and 36PC had identical nucleotide sequence and secondary structure for the D1-D1¢ helix, differing from the others (Fig. 3c). Strains 47PC and 48PC also showed identical D1-D1¢ nucleotide sequence and secondary structure, but distinct from other strains (Fig. 3d). The studied strains presented four different box-B helix sec- ondary structures (Fig. 3e–i). Although strains 1PC and 2PC had box-B sequences distinct in one nucleotide, they presented the same secondary structure (Fig. 3e, f). Strain 38PC also presented box-B nucleotide sequence very similar to those of strains 1PC and 2PC, differing in only one Table 3. Lengths of 16S–23S ITS regions (number of nucleotides) in the analysed Potamolinea strains Strains: 1PC, 2PC and 38PC, P. aerugineo-caerulea; 32PC, 33PC, 34PC, 35PC and 36PC, Potamolinea sp.; 47PC and 48PC, P. magna. Strain Complete ITS Leader D1–D1¢ helix D2 with spacer D3 with spacer tRNAIle gene Pre-box- B spacer Box-B helix Post- box-B spacer Box- A D4 V3 with spacer D5 1PC 372 6 63 33 19 74 17 49 19 12 7 51 22 2PC 372 6 63 33 19 74 17 49 19 12 7 51 22 32PC 384 6 63 33 18 74 17 41 19 12 7 73 21 33PC 384 6 63 33 18 74 17 41 19 12 7 73 21 34PC 384 6 63 33 18 74 17 41 19 12 7 73 21 35PC 384 6 63 33 18 74 17 41 19 12 7 73 21 36PC 384 6 63 33 18 74 17 41 19 12 7 73 21 38PC 372 6 63 33 19 74 17 48 19 12 7 51 22 47PC 407 6 63 33 19 74 33 48 18 12 7 72 22 48PC 407 6 63 33 19 74 33 48 18 12 7 72 22 3636 International Journal of Systematic and Evolutionary Microbiology 66 M. D. Martins and L. H. Z. Branco http://dx.doi.org/10.1601/nm.703 http://dx.doi.org/10.1601/nm.703 http://dx.doi.org/10.1601/nm.698 http://dx.doi.org/10.1601/nm.700 Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 0.02 Gloeobacter violaceus PCC 7421 (NC005125) Phormidesmis priestleyi ULC147 (HM101225) Leptolyngbya foveolarum VP1-08 (FR798945) Leptolyngbya boryana PCC 6306 (EF429290) Phormidesmis priestleyi ANT.L 61.2 (AY493582) Synechococcus elongatus PCC 6301 (NC006576) Pseudanabaena sp. PCC 6903 (AB039017) Pseudanabaena sp. Sai011 (GU935357) Gloeobacter violaceus PCC 8105 (AF132791) Trichodesmium erythraeum (AF013030) Trichodesmium contortum (AF013028) Okenia plumata NAC8-45 (GU724196) Okenia plumata NAC8-55 (GU724208) Oscillatoria acuminata (AB039014) Oscillatoria acuminata PCC 6304 (NR102463) Oxynema thaianum CCALA 960 (JF729323) Planktothricoides raciborskii OR1-1 (AB045964) Planktothricoides raciborskii NSLA3 (AB045962) Phormidium cf. irriguum CCALA 759 (FN813343) Phormidium irriguum f. minor ETS-02 (FN813342) Phormidium ambiguum IAM M-71 (AB003167) Aerosakkonema funiforme Lao26 (AB686261) Annamia toxica HOs24 (HQ658457) Wilmottia murrayi KGI28 (HQ873481) Wilmottia murrayi CYN76 (JF925320) Wilmottia murrayi ANT.LPE.2 (AY493598) Phormidium sp. B-Tom (EU196618) Desertifilum tharense PD2001/TDC17 (FJ158997) Desertifilum tharense PD2001/TDC4 (FJ158994) Coleofasciculus chthonoplastes SAG 2209 (EF654055) Coleofasciculus chthonoplastes CCY9603 (GQ402020) Moorea producens NAC8-48 (GU724200) Moorea producens PNG6-221 (FJ356669) Moorea bouillonii PNG5-198 (FJ041298) Symploca atlantica CCY9617 (GQ402026) Symploca atlantica PCC 8002 (AB039021) Planktothrix pseudagardhii HAB1347 (FJ184442) Planktothrix agardhii HAB209 (FJ184408) Planktothrix sp. PCC 7811 (GQ351564) Limnoraphis robusta CCALA 966 (JN854138) Limnoraphis hieronymusii N-929 (JN854140) Lyngbya aestuarii PCC 7419 (AB075989) Arthrospira fusiformis AB2002/01 (AY575923) Arthrospira platensis PCC 9223 (DQ393285) Phormidium autumnale SAG 35.90 (EF654081) Phormidium setchellianum CCALA 144 (JN230343) Phormidium cf. amoenum BW1 (EU196636) Microcoleus vaginatus ISBAL M31 (KC633983) Microcoleus vaginatus CCALA 152 (KC633969) Kamptonema animale SAG 1459-6 (EF654087) Kamptonema formosum P010 (JQ712613) Kamptonema formosum P001 (JQ712611) Potamolinea sp. 35PC (KX001794) Potamolinea magna 47PC (KX001789) Potamolinea magna 48PC (KX001790) Potamolinea aerugineo-caerulea 38PC (KX001788) Potamolinea sp. 36PC (KX001795) Potamolinea sp. 34PC (KX001793) Potamolinea sp. 33PC (KX001792) Potamolinea sp. 32PC (KX001791) Potamolinea aerugineo-caerulea 1PC (KX001786) Potamolinea aerugineo-caerulea 2PC (KX001787) Phormidium aerugineo-caeruleum clone MED-28 (JN382236) Phormidium aerugineo-caeruleum clone MED-31 (JN382235) Phormidium aerugineo-caeruleum clone MNZ-37 (JN382237) Phormidium aerugineo-caeruleum clone MNZ-32 (JN382239) Phormidium aerugineo-caeruleum clone MNZ-31 (JN382238) Phormidium aerugineo-caeruleum clone MED-17 (JN382234) 100/100/1 78/77/1 95/97/1 _/_/0.99 92/94/1 86/94/_ _/_/0.98 100/100/1 97/100/1 99/100/1 100/100/1 _/_/0.91 _/_/1 99/100/1_/_/1 _/_/1 100/100/1 92/97/1 99/100/1 100/100/1 _/_/1 91/100/0.99 100/100/1 86/83/1 _/_/1 99/100/1 99/100/1 91/100/1 73/89/1 _/_/0.96 _/_/0.99 _/_/0.99 _/89/1 99/100/1 100/100/1 _/90/0.99 73/77/0.9 100/100/1 96/93/1 98/100/1 99/100/1_/_/1 99/100/1 71/_/1 99/100/1 91/100/1 96/97/1 _/_/1 _/_/1 99/100/1 100/100/1 _/75/0.92 72/78/1 80/84/0.99 96/100/1 97/100/1 A B C C la d e I = P o ta m o li n e a Fig. 2. BI tree based on the 16S rRNA gene sequences of oscillatorialean cyanobacteria. The clade comprising Potamolinea gen. nov., consisting of Brazilian (in bold) and European strains, is highlighted. A bootstrap test involving 1000 resamplings was performed. Bootstrap values (>70%) and probabilities (>0.7) obtained from ML/NJ/BI methods, respectively, are dis- played at the relevant nodes. GenBank accession numbers are shown in parentheses. Bar, 0.02 substitutions per nucleotide position. http://ijs.microbiologyresearch.org 3637 Potamolinea gen. nov. Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 nucleotide, and its secondary structure of box-B helix also was distinct (Fig. 3g). Box-B helix sequences and secondary structures of strains 32PC, 33PC, 34PC, 35PC and 36PC were identical (Fig. 3h). Box-B helix sequence and second- ary structure of strains 47PC and 48PC were identical between them and different from other strains (Fig. 3i). Finally, the V3 helix of studied strains revealed three different secondary structures (Fig. 3j–n). Strains 1PC, 2PC and 38PC had identical secondary structure (Fig. 3j–l), although they had two to three divergent nucleotides. Strains 32PC, 33PC, 34PC, 35PC and 36PC presented the same nucleotide sequence and secondary structure (Fig. 3m). Strains 47PC and 48PC showed the same sequences and secondary structures, but these were different from the other strains (Fig. 3n). Discussion Morphology has been the basis for the taxonomic identifi- cation and traditional classification systems of cyanobacte- ria, such as by Gomont (1892), Geitler (1932) and Kom�arek & Anagnostidis (2005). However, polyphasic approach, mainly after the inclusion of molecular data, has revealed that morphological characters are insufficient to delimit species and understand the evolutionary history of Cyano- bacteria. Recent taxonomic studies of members of the Oscil- latoriales have shown that phenotypically similar organisms can exhibit considerable genotypic diversity (Siegesmund et al., 2008; Kom�arek et al., 2013; Hašler et al., 2014; Stru- necký et al., 2014; McGregor & Sendall, 2015; Martins et al., 2016). The failure of morphological characters to reflect (a) (g) (h) (i) (j) (k) (l) (m) (n) (b) (c) (d) (e) (f) Fig. 3. Secondary structure of conserved regions of 16S–23S ITS of Potamolinea strains studied. (a–d) D1-D1¢ helices: (a) 1PC and 2PC; (b) 38PC; (c) 32PC, 33PC, 34PC, 35PC and 36PC; (d) 47PC and 48PC. (e–i) Box-B helices: (e) 1PC; (f) 2PC; (g) 38PC; (h) 32PC, 33PC, 34PC, 35PC and 36PC; (i) 47PC and 48PC. (j–n) V3 helices: (j) 1PC; (k) 2PC; (l) 38PC; (m) 32PC, 33PC, 34PC, 35PC and 36PC; (n) 47PC and 48PC. 3638 International Journal of Systematic and Evolutionary Microbiology 66 M. D. Martins and L. H. Z. Branco http://dx.doi.org/10.1601/nm.624 http://dx.doi.org/10.1601/nm.624 Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 biodiversity has been increasingly evident with the many new taxonomic entities that have been described based mainly on molecular data (16S rRNA gene and 16S–23S ITS) in the last few years. Many of these were found in less well-studied geographical locations, such as tropical habitats (Fiore et al., 2007; Genu�ario et al., 2015; Malone et al., 2015; Vaz et al., 2015; Martins et al., 2016). The polyphyly of Phormidium, previously reported by many authors (Turicchia et al., 2009; Strunecký et al., 2011, 2014; Casamatta et al., 2012; Malone et al., 2015), was also cor- roborated in this study, indicating the heterogeneity of the genus and reinforcing questions about the fragility of the morphological data in delimitation of taxa. The phylogenetic analyses presented indicate that the group comprising Brazilian and European strains (Phormidium aerugineo-caeruleum MNZ-37, MNZ-32, MNZ-31, MED- 28, MED-31, MED-17) defines a new genus, hereafter named Potamolinea, with strong support (99% ML, 100% NJ and 1 BI) and distant from ‘true Phormidium’, according to Sciuto et al. (2012). The high similarity of 16S rRNA gene sequences among Potamolinea strains (95.3–99.9%) and low similarity between Potamolinea and other genera (<93.8%) corroborate that they belong to a consistent and distinct generic entity. Morphological and ecological data also confirm Potamolinea as a well-delimited genus, as strains that comprise it have similar morphology of apex of trichomes (not attenuated, with rounded or obtuse apical cells) that corresponds to Phormidium group V (Kom�arek & Anagnostidis, 2005) and were found in freshwater ben- thos of lotic ecosystems. Considering morphological and ecological characteristics, it is reasonable to consider that other species of Phormidium group V of Kom�arek & Anagnostidis (2005) that are found in the same type of environment belong to this genus: Phormidium granulatum (Gardner) Anagnostidis, P. retzii (Agardh) Gomont ex Gomont, P. taylorii (Drouet et Strick- land) Anagnostidis and P. tergestinum (Kützing) Anagnosti- dis et Kom�arek. However, this hypothesis will need to be supported by molecular data, and these are missing for the cited species. Similarity scores of 16S rRNA gene sequences among strains of the same subgroup were high, indicating that this analysis was sufficient to separate the different subgroups and each corresponds to a species of Potamolinea: A, P. aerugineo- caerulea comb. nov; B, Potamolinea sp.; and C, P. magna sp. nov. Potamolinea aerugineo-caerulea showed greater varia- tion in the 16S rRNA gene, but this is probably related to its geographical distribution, as European strains were closer to each other (98.5–99.7% similarity) than to Brazilian lineages. The ITS sequences presented little infraspecific variation and significant distinction among species, corroborating analyses based on 16S rRNA gene sequences. The secondary structures of D1-D1¢, box-B and V3 regions were conserved within each species, with small variations in D1-D1¢ and box-B of Potamolinea aerugineo-caerulea strains, which were not sufficient to separate them into different species. Thus, the results confirm ITS as an important marker, adequate for species-level recognition/distinction, as observed by Boyer et al. (2001, 2002), Bohunick�a et al. (2011), Johansen et al. (2011), Perkerson et al. (2011) and Malone et al. (2015). Although morphological data are not considered to be phy- logenetically informative (Casamatta et al., 2003; Muhlsteinov�a et al., 2014; Osorio-Santos et al., 2014; Martins et al., 2016), our results showed that they are important for species identification in Potamolinea. The species can be distinguished based on cell content (granular in Potamolinea aerugineo-caerulea and homogeneous in the others), characteristics of cross-walls (inconspicuous in P. aerugineo-caerulea and conspicuous in the others, granu- lated only in P. magna) and morphology of the apical cell (truncate in Potamolinea sp. and rounded in the other spe- cies). Although overlapping in P. aerugineo-caerulea and Potamolinea sp., filament and trichome width are clearly wider in P. magna. Sheaths are facultative in all populations studied and cell length showed a wide and overlapping vari- ation and therefore these characters are not relevant for spe- cies distinction. Brazilian populations of Potamolinea aerugineo-caerulea are morphologically very similar to populations of Phormidium aerugineo-caeruleum described in several classic taxonomic studies (Gomont, 1892; Geitler, 1932; Kom�arek & Anagnostidis, 2005) and to Spanish populations described by Loza et al. (2013). They are also similar according to their occurrence habitat: lotic ecosystems. The 16S rRNA gene sequences of Spanish populations allowed to compare them at the genetic level to Brazilian material, which revealed a very close relationship. Although the two geo- graphical/climatic regions are distinct, a number of conver- gent characteristics suggest that all populations pertain to the same species. It is possible and reasonable to consider that some genotypes enable individuals of the same species to adapt to a wide range of environmental conditions (eury- tolerant) if these are not extreme. The similarities found are robust in different markers, justifying the transfer of Phor- midium aerugineo-caeruleum to the genus Potamolinea. Potamolinea sp. strains presented morphological and eco- logical characteristics that are in agreement with those cited for Phormidium retzii by Gomont (1892), Geitler (1932) and Kom�arek & Anagnostidis (2005). Besides all morpho- metric characters, these included the formation of truncate apical cells and the occurrence in flowing water for all the populations studied. However, they were not identified as Phormidium retzii because they were collected in streams of a tropical region that can be considered very different from the type locality (streams of southern Sweden). Despite the high number of occurrences of this species reported around the world (Sheath & Cole, 1992; Branco et al., 1999; Kom�arek & Anagnostidis, 2005; McGregor, 2007), there are no 16S rRNA gene sequences available in GenBank, http://ijs.microbiologyresearch.org 3639 Potamolinea gen. nov. Downloaded from www.microbiologyresearch.org by IP: 186.217.236.60 On: Mon, 20 May 2019 19:00:23 which should be an imperative to confirm the identification (or not) of Potamolinea sp. as Phormidium retzii and to effectively proceed to transfer it to the new genus. Never- theless, based on the results of the present study for Pota- molinea aerugineo-caerulea, this is highly likely when molecular data of European strains of Phormidium retzii become available. The phylogenetic relationships of cyanobacteria based on analyses of molecular markers have been accepted as the decisive criteria for their classification (Kom�arek et al., 2014). The 16S rRNA gene is most frequently used for generic definition and a combined analysis with ITS is used to achieve a higher taxonomic resolution. However, other characters must also be considered, mainly in species identi- fication. Thereby, Potamolinea is shown to be very similar based on morphology, ecology and molecular data, and is probably widely distributed, occurring in similar environ- mental conditions around the world. Description of the genus and species Potamolinea Martins et Branco gen. nov. Diagnosis: Thal- lus gelatinous, mucilaginous, attached to the substrate, dark blue–green. Filaments densely entangled, trichome motility present. Sheaths facultative, attached to trichomes, firm, thin, colourless, hyaline. Trichomes isopolar, cylindrical along their whole length, not constricted or slightly con- stricted at the cross-walls, not attenuated toward the apex, 6–16.8 µm wide. Cells isodiametric or shorter or longer than wide. Cell content finely granular or with scattered larger granules. Apical cell rounded, without calyptra. Het- erocytes and akinetes missing. Reproduction by disintegra- tion of trichomes by necridic cells into hormogonia. Etymology: Potamolinea (Po.ta.mo.li¢ne.a. Gr. n. potamos river; L. n. linea line; N.L. fem. n. Potamolinea filament from a river). Type species: Potamolinea magnaMartins & Branco sp. nov. Potamolinea magna Martins & Branco sp. nov. Thallus mucilaginous, dark blue–green. Filaments densely entangled, 13.2–16.8 µm wide. Sheaths facultative, attached to trichomes, firm, thin, colourless. Trichomes motile, cylindrical, not attenuated, not constricted to slightly con- stricted at the ungranulated or slightly granulated cross- walls, 9.6–16.8 µm wide. Cells 0.4–0.9 time longer than wide, 4–12.8 µm long. Cell content blue–green, homoge- neous to finely granulated. Apical cell rounded, without calyptra. Etymology: magna (mag¢na. L. fem. adj. magna large, refer- ring to the greater diameter of trichomes). Type locality: Jacar�e stream. Municipality of Neves Paulista, S~ao Paulo State, Brazil; 20 � 51¢ 39† S 49 � 36¢ 54† W. Habitat: stream bottoms. Holotype: formaldehyde-fixed sample of strain 47PC depos- ited in Herbarium SJRP (IBILCE/UNESP), Brazil, voucher number SJRP 31642. Potamolinea aerugineo-caerulea (Gomont) comb. nov. Basionym: Lyngbya aerugineo-caerulea Gomont, Ann. Sci. nat. 7, Bot. 16 : 146, 1892. Acknowledgements We thank the S~ao Paulo Research Foundation (FAPESP 2010/09686- 4, 2012/19468-0, 2013/08207-3) for financial support. We are grateful to Dr Ji�ri Kom�arek (Academy of Sciences of the Czech Republic, Institute of Botany, T�rebo H , Czech Republic) for his valu- able contributions that greatly improved the paper and to Dr Orlando Necchi Júnior (IBILCE/UNESP, S~ao Paulo State University) for collecting some of the samples included in this study. References Bohunick�a, M., Johansen, J. 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