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2017 Cyanomargarita gen. nov. (Nostocales, Cyanobacteria): convergent evolution resulting in Jeffrey R. Johansen John Carroll University, [email protected]
Sergei Shalygin
Regina Shalygina
Nicole Pietrasiak
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Recommended Citation Johansen, Jeffrey R.; Shalygin, Sergei; Shalygina, Regina; and Pietrasiak, Nicole, "Cyanomargarita gen. nov. (Nostocales, Cyanobacteria): convergent evolution resulting in" (2017). 2017 Faculty Bibliography. 39. https://collected.jcu.edu/fac_bib_2017/39
This Article is brought to you for free and open access by the Faculty Bibliographies Community Homepage at Carroll Collected. It has been accepted for inclusion in 2017 Faculty Bibliography by an authorized administrator of Carroll Collected. For more information, please contact [email protected]. CYANOMARGARITA GEN. NOV. (NOSTOCALES, CYANOBACTERIA): CONVERGENT EVOLUTION RESULTING IN A CRYPTIC GENUS1
Sergei Shalygin 2 Department of Biology, John Carroll University, University Heights, Ohio 44118, USA Polar-Alpine Botanical Garden-Institute, Kola Science Center, Russian Academy of Science, Kirovsk-6 184230, Russia Regina Shalygina Institute of Industrial Ecology Problems of the North, Kola Science Center, Russian Academy of Science, Akademgorodok 14a, Apatity 184209, Russia Jeffrey R. Johansen Department of Biology, John Carroll University, University Heights, Ohio 44118, USA Department of Botany, Faculty of Science, University of South Bohemia, Brani sovsk a 31, Ceske Budejovice 370 05, Czech Republic Nicole Pietrasiak Department of Plant and Environmental Sciences, New Mexico State University, 945 College Drive, Las Cruces, New Mexico 88003, USA Esther Berrendero Go mez Department of Botany, Faculty of Science, University of South Bohemia, Brani sovsk a 31, Ceske Budejovice 370 05, Czech Republic Marketa Bohunicka Institute of Botany of the Academy of Sciences of the Czech Republic, Dukelsk a 135, T rebon 379 82, Czech Republic Research and Breeding Institute of Pomology, Holovousy 129, Ho rice 508 01, Czech Republic Jan Mares Department of Botany, Faculty of Science, University of South Bohemia, Brani sovsk a 31, Ceske Budejovice 370 05, Czech Republic Institute of Botany of the Academy of Sciences of the Czech Republic, Dukelsk a 135, T rebon 379 82, Czech Republic Biology Centre of the Academy of Sciences of the Czech Republic, Institute of Hydrobiology, Na S adk ach 702/7, Ceske Budejovice 37005, Czech Republic and Christopher A. Sheil Department of Biology, John Carroll University, University Heights, Ohio 44118, USA
Two populations of Rivularia-like cyanobacteria 16S rRNA and rbcLX phylogenies. We here name the were isolated from ecologically distinct and new cryptic genus Cyanomargarita gen. nov., with type biogeographically distant sites. One population was species C. melechinii sp. nov., and additional species from an unpolluted stream in the Kola Peninsula of C. calcarea sp. nov. We also name a new family for Russia, whereas the other was from a wet wall in the these taxa, the Cyanomargaritaceae. Grand Staircase-Escalante National Monument, a Key index words: 16S rRNA gene phylogeny; 16S-23S desert park-land in Utah. Though both were virtually ITS; cryptic genus; Cyanobacteria; Cyanomargarita; indistinguishable from Rivularia in field and cultured rbcLX phylogeny; Rivularia material, they were both phylogenetically distant from Rivularia and the Rivulariaceae based on both Abbreviations: BA, Bayesian Analysis; DIC, differen- tial interference contrast; ITS, internal transcribed spacer; ML, maximum likelihood; MP, maximum parsimony; RAS, russian academy of sciences
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With the advent of molecular methods, many phy- hemispherical to spherical mucilage investment in a cologists, including those who study cyanobacteria, small, spring-fed, unpolluted stream near the town of began to recognize the existence of cryptic species Apatity in the Kola Peninsula, Russia. It was com- (Boyer et al. 2002, Casamatta et al. 2003, Erwin and pletely consistent with the description of Rivularia Thacker 2008, Joyner et al. 2008, Premanandh et al. C.Agardh ex Bornet & Flahault, the type genus of 2009, Ren~e et al. 2013, Johansen et al. 2014, Rivulariaceae, which contains tapering, heterocytous Muhlsteinov€ a et al. 2014a,b, Patzelt et al. 2014). taxa. This taxon fit no established species in Rivularia, However, while in these papers the existence of and upon sequencing was determined to be phyloge- cryptic species was suggested, the species were not netically distant from all members of that family. A recognized taxonomically. Subsequently, cryptic spe- second species belonging to the same clade as the cies have been named in several algal groups, Russian material was found, and sequenced several including euglenids (Marin et al. 2003, Kosmala years earlier from a wet wall in the Grand Staircase- et al. 2007, Kosmala et al. 2009, Karnkowska-Ishi- Escalante National Monument in Utah, USA. These kawa et al. 2010, Kim et al. 2010, 2013, Linton et al. two populations differ morphologically and ecologi- 2010), eustigmatophytes (Fawley and Fawley 2007, cally, and are described herein as two new species Fawley et al. 2015), chlorophytes (Fawley et al. 2005, based on a modified phylogenetic species concept 2011, Fu c ıkov a et al. 2014), and cyanobacteria (Oso- (Mishler and Theriot 2000, Johansen and Casamatta rio-Santos et al. 2014, Pietrasiak et al. 2014). 2005) in a newly proposed genus, Cyanomargarita gen. Some cyanobacterial systematists have suggested nov. This genus cannot be placed in any family-level the existence of cryptic genera as well (Kom arek grouping of taxa based on the phylogenetic analyses et al. 2014, Dvo r ak et al. 2015a), although very few performed – we place these taxa in a new family to cryptic genera have actually been described. Pinoc- science, the Cyanomargaritaceae fam. nov. chia, which is morphologically identical to Pseudan- abaena, has a phylogenetic position distant from that genus, and therefore was described as a cryptic MATERIALS AND METHODS genus (Dvorak et al. 2015b). Kovacikia, which is Isolation and strain characterization. Both strains of morphologically similar to Phormidesmis but molecu- Cyanomargarita were isolated from natural populations into larly distinct, would also fit the definition of a cryp- unialgal cultures using standard microbiological methods, tic genus, although the authors did not label it as including enrichment plates and direct isolation from the orig- such (Miscoe et al. 2016). There are also a number inal samples, in Z8 medium (Kotai 1972, Carmichael 1986). Cultures were observed under a Zeiss Axioskop photomicro- of pseudocryptic genera (genera defined by mor- scope with both bright field and DIC optics. All morphological phological traits that are minor or phenotypically measurements were obtained using AxioVision 4.8 software plastic, and therefore not always expressed in the provided by Zeiss (Oberkochen, Germany). Living cultures population) as well, such as Nodosilinea, Oculatella, were deposited into the Cyanobacterial Culture Collection at Limnolyngbya, Pantanalinema, and Alkalinema. These John Carroll University, Cleveland, OH, USA. Natural popula- genera belong to the Synechococcales, an order tions of material from which the strain C. melechinii APA-RS9 containing taxa with few morphological characteris- was derived were dried and deposited as an isotype in the Her- barium of the Polar-Alpine Botanical Garden-Institute, Kola tics (simple filamentous forms, variations in tri- Science Centre, RAS, Kirovsk-6, Murmansk Region, Russia, and chome width and sheath characteristics). information about habitat, coordinates and locality can be Outside of the Synechococcales, few cryptic gen- found in the online database Cyanopro (Melechin et al. 2013). era have been recognized. In the Chroococcales, The dried holotype material of this strain was deposited in the Chalicogloea is similar to Gloeocapsa and could be con- Herbarium for Nonvascular Cryptogams in the Monte L. Bean sidered a cryptic genus (Rold an et al. 2013). There Museum, Provo, UT, USA. Liquid materials of both species fixed in 4% formaldehyde, as well as dried materials of C. cal- are likely more cryptic genera in this order, but carea, were also deposited in the Herbarium for Nonvascular identifying them is more problematic because mem- Cryptogams in the Monte L. Bean Museum, Provo, UT, USA. bers of the genus are difficult to grow in culture, Molecular methods. Genomic DNA was extracted following and consequently, fewer sequences are available. In techniques described in Pietrasiak et al. (2014). PCR amplifi- the Oscillatoriales, Ammassolinea is the one of the cation of the 16S rRNA gene was accomplished following described cryptic genera (Ha sler et al. 2014), being Osorio-Santos et al. (2014), with the exception that forward morphologically inseparable from Phormidium,asit primer 8F was used instead of forward primer VRF2, for amplification of a longer sequence, starting near the begin- is presently defined, as well as Moorea, Okeania and ning of the 16S rRNA gene (Perkerson et al. 2011). The 16S Microseira (Engene et al. 2012, 2013, McGregor and rRNA amplicons were cloned to avoid problems in sequenc- Sendall 2015). Within the Nostocales, there is much ing of multiple operons (Siegesmund et al. 2008). PCR greater morphological complexity than the nonhete- amplicons and sequences of rbcLX and rpoC1 genes were rocytous orders. Some pseudocryptic genera have obtained exactly according to Rudi et al. (1998) and Seo and been described, including Mojavia (Reh akov a et al. Yokota (2003), respectively. The nifD gene amplification was completed using a protocol described in Roeselers et al. 2007), Dapisostemon (Hentschke et al. 2016), and (2007). All three protein-encoding genes (rbcLX, rpoC1, nifD) â Pelatocladus (Miscoe et al. 2016). were sequenced after purification procedure using Wizard We recently discovered a population of tapering, SV Gel and PCR Clean-Up System (Promega, Madison, WI, heterocyte-bearing trichomes embedded in a USA); rather than cloned, because they are single-copy genes 764 in the cyanobacterial genome. All sequences obtained in this The final average standard deviation of split frequencies was study were deposited in the NCBI Nucleotide database, under <0.010. The PSRF value for all the estimated parameters in accession numbers – for 16S rRNA gene: KY296602, the BA was 1.000, indicating that convergence of the MCMC KY296603, KY296604, KY296605, KY296606, KY296607, chains was statistically achieved (Gelman and Rubin 1992). KY296608; rbcLX: KY296611, KY296612; nifD: KY296609, P-distance values for all sequences were calculated in PAUP KY296610; rpoC1: KY296613, KY296614. v. 4.02b (Swofford 2002). Graphical representations of the Phylogenetic analyses. All sequences chosen for alignment ITS structures were created in Adobe Illustrator CS5.1 and phylogenetic analyses were obtained from our internal (Adobe Systems Inc., San Jose, CA, USA) based upon sec- set of sequences and relevant sequences (chosen based on ondary structure configurations given by Mfold (Zuker 2003). both BLAST searches and named taxon searches) from the Line drawings. Drawingsweremadebytheseniorauthor NCBI Nucleotide database before September 1, 2016. Riboso- using stippling technique, completed digitally with Wacom Cin- mal (16S) sequences were aligned using MUSCLE in Mega v. tiq 24HD Pen Display (Wacom Technology Corporation, Port- 6.06 (Tamura et al. 2013), and checked manually in Micro- land, OR, USA) utilizing the original photos as templates. soft Word (Microsoft Corp., Redmond, WA, USA) to ensure that alignments supported preservation of secondary struc- ture (Luke sov a et al. 2009, Reh akov a et al. 2014). Protein RESULTS coding genes were aligned as codons using MUSCLE in Mega v. 6.06 (Tamura et al. 2013). Phylogenetic analyses. The 16S rRNA gene phy- The public software jModeltest2 (Darriba et al. 2012) was logeny has posterior probability support on all used to determine the optimal Maximum Likelihood (ML) nodes in the backbone of the BA, with the excep- model, which was GTR+I+G for 16S rRNA genes, and both ML tion of four nodes at the base of the tree marked and Bayesian Analysis (BA) were subsequently run using this with small light gray circles (Fig. 1). Overall topol- type of model. The exact parameters of the substitution models ogy of the tree is consistent with recent studies of were individually estimated from the data during analysis by Nostocales (Berrendero et al. 2011, Hauer et al. MrBayes v. 3.2.6 (Ronquist et al. 2012) and RaxML v. 7.2.8 (Sta- 2014, Ka stovsk y et al. 2014, Berrendero Gomez et al. matakis et al. 2008) software. The jModeltest2, ML, and BA cal- culations were all run on CIPRES (Miller et al. 2012). In the 2016, Leon-Tejera et al. 2016). BA, two runs of eight Markov chains were applied with 10 mil- Cyanomargarita gen. nov. forms a cluster of two termi- lion generations, sampling every 100 generations, with 25% nal OTUs (Operational Taxonomic Units) correspond- burn-in. The sump command in Mr. Bayes was used. The ML ing to two new species: C. melechinii and C. calcarea, analysis was conducted in RAxML v.7.2.8 with 1,000 bootstrap + + with high support (Fig 1). Cyanomargarita is sister to a replicates, using GTR I G substitution model. Parameters for large clade, containing the well-defined monophyletic the models, with rate matrix, base frequencies, and invariant and gamma settings are given in Table S1 in the Supporting Gloeotrichiaceae, as well as the Fortieaceae, Aphani- Information. The Maximum-parsimony (MP) analysis was per- zomenonaceae, Nostocaceae, and Tolypothrichaceae. formed using PAUP v. 4.02b (Swofford 2002) with steepest des- Additionally, Cyanomargarita is also related to the “Scy- cent, the tree bisection and reconnection branch swapping, tonema cf. crispum” group, which requires revision (i.e., and 1,000 bootstrap replicates. it is not Scytonema C.Agardh ex E.Bornet & C.Flahault) Phylogenies utilizing 16S rRNA gene sequences can yield and has an uncertain taxonomic position (incertae ambiguous or unsupported trees, and in such cases a multiple familiae), falling outside of the Scytonemataceae clade loci approach is recommended (da Silva Malone et al. 2015, Song et al. 2015). We treated the rbcLX alignment as codons in defined by the inclusion of the type species, Scytonema the BA calculation (Fawley et al. 2015), using the Ny98 equal hofmannii C.Agardh ex Bornet & Flahault (the basal rate ratio model available in MrBayes for analysis of protein- clade in Fig. 1). Cyanomargarita is found outside of the coding genes (Ronquist et al. 2012). In the ML analysis the Rivulariaceae sensu stricto, despite the similar mor- GTR+I+G model chosen by jModelTest2 was applied. Parame- phology between Cyanomargarita and Rivularia. ters for the models are given in Table S1. We conducted BA cal- The BA in this study and the MP and ML analyses culation of the rbcLX alignment with two runs of eight Markov chains with 20 million generations, sampling every 100 genera- had highly similar topology, although the latter two tions, with 25% burn-in. Additionally, MP and ML analyses analyses had poorer support. The MP analysis dif- were performed for rbcLX gene using the same settings as in fered slightly in the placement of Roholtiella Bohu- the 16S rRNA phylogeny. The tree topology from BA was cho- nick a, Pietrasiak & J.R.Johansen and Gloeotrichia sen for the resulting visualization for both 16S rRNA gene and J.Agardh ex Bornet & Flahault, but these taxa were rbcLX gene phylogenies, as well as for the mapping of support still associated with the Nostocaceae and Aphani- values from MP and ML analyses. zomenonaceae. In the parsimony analysis, Macro- For 16S rRNA gene the BA had an estimated sample size (ESS) exceeding 2,000 for most of the parameters (790–6,877, chaete Berrendero Gomez, J.R.Johansen & Kastovsky average = 2,621 from 13 parameters), well above the average of moved from a position proximate to Calothrix 200 typically accepted as sufficient by phylogeneticists (Drum- C.Agardh ex Bornet & Flahault to a position sister mond et al. 2006). The final average standard deviation of split to Cyanomargarita,butCyanomargarita remained sis- frequencies was <0.016. The potential scale reduction factor ter to the same polyfamily clade as in the BA (PSRF) value for all the estimated parameters in the BA was (Fig. 1). The ML analysis generally had the poorest 1.000, indicating that convergence of the MCMC chains was sta- node support, but had the same topology as the BA tistically achieved (Gelman and Rubin 1992). For rbcLX gene the BA had an ESS exceeding 2,000 for except for a slight change in the position of “S. cf. some of the parameters (79–2,193, average = 345 from 67 crispum.” In all three analyses, Cyanomargarita was parameters), well above the average of 200 typically accepted always a supported clade outside of the Rivulari- as sufficient by phylogeneticists (Drummond et al. 2006). aceae and outside of any other family-level clade. 765
FIG. 1. Phylogeny for Cyanomargarita spp. inferred by Bayesian analysis within Nostocales based on a maximum of 1,495 nucleotides from the 16S rRNA gene (240 OTUs). Branch support values are shown as BA/ML/MP. Support values of 100 in ML and MP, or 1.00 (BA), is displayed as “*,” respectively, nodes with no support are shown as “–.” Two species of Cyanomargarita are highlighted in bold, with sequences representing different operons and clones indicated (OP1, OP2, OP3, etc.), the Rivulariaceae and Cyanomargaritaceae clades are highlighted in dark gray boxes; remaining family-level clades are highlighted with light gray boxes. Drawings of the spherical colonies in the right part of the boxes indicates tapering filaments showing similar morphology between Cyanomargarita and Rivularia. OTUs in quotation marks are strains we consider to either be incorrectly identified or in need of revision. 766
Another piece of evidence supporting the place- Total ment of Cyanomargarita outside the Rivulariaceae is length that representatives of this new genus have ITS lacked regions with only one tRNA gene (tRNAIle) across four different ribosomal operons (Table 1), which is spacer went + ending + different from both the Nostocaceae (with two or of ITS