Phyllonema Aviceniicola Gen. Nov., Sp. Nov. and Foliisarcina Bertiogensis Gen

International Journal of Systematic and Evolutionary Microbiology (2016), 66, 689–700 DOI 10.1099/ijsem.0.000774

Phyllonema aviceniicola gen. nov., sp. nov. and Foliisarcina bertiogensis gen. nov., sp. nov., epiphyllic cyanobacteria associated with Avicennia schaueriana leaves Danillo Oliveira Alvarenga,1 Janaina Rigonato,1 Luis Henrique Zanini Branco,2 Itamar Soares Melo3 and Marli Fatima Fiore1

Correspondence 1University of Saõ Paulo, Center for Nuclear Energy in Agriculture, Avenida Centena´rio 303, Marli Fatima Fiore 13400-970 Piracicaba, SP, Brazil fi[email protected] 2Saõ Paulo State University, Institute of Bioscience, Languages and Exact Sciences, 15054-000 Saõ Jose´ do Rio Preto, SP, Brazil 3Embrapa Environment, Laboratory of Environmental Microbiology, 13820-000 Jaguariuńa, SP, Brazil

Cyanobacteria dwelling on the salt-excreting leaves of the mangrove tree Avicennia schaueriana were isolated and characterized by ecological, morphological and genetic approaches. Leaves were collected in a mangrove with a history of oil contamination on the coastline of Saõ Paulo state, Brazil, and isolation was achieved by smearing leaves on the surface of solid media or by submerging leaves in liquid media. Twenty-nine isolated strains were shown to belong to five cyanobacterial orders (thirteen to Synechococcales, seven to Nostocales, seven to Pleurocapsales, one to Chroococcales, and one to Oscillatoriales) according to morphological and 16S rRNA gene sequence evaluations. More detailed investigations pointed six Rivulariacean and four Xenococcacean strains as novel taxa. These strains were classified as Phyllonema gen. nov. (type species Phyllonema aviceniicola sp. nov. with type strain CENA341T) and Foliisarcina gen. nov. (type species Foliisarcina bertiogensis sp. nov. with type strain CENA333T), according to the International Code of Nomenclature for Algae, Fungi, and Plants. This investigation shows some of the unique cyanobacteria inhabiting the phyllosphere of Avicennia schaueriana can be retrieved by culturing techniques, improving current taxonomy and providing new insights into the evolution, ecology, and biogeography of this phylum.

Phyllosphere, the external surface of plant leaves, is a habi- the plant, and are subject to ﬂuctuations in temperature, tat that has traditionally received low attention in microbial UV radiation, wind, moisture and relative humidity vary- ecology, with most of the initial research being primarily ing in scales ranging from seconds to hours (Hirano & focused on the study of plant–pathogen interactions in cul- Upper, 2000; Schreiber et al., 2004). The phyllosphere tures of economic interest (Lindow & Brandl, 2003; Belkin from Avicennia mangroves presents even more unique con- et al., 2010). Though still lagging behind rhizosphere ditions. To maintain their osmotic balance, these trees studies, phyllosphere research has been a subject of eliminate up to 90 % of the salt absorbed from seawater increased interest in recent years (Vorholt, 2012; Rastogi through a transpiration current carrying it from the roots et al., 2013). to glands on the abaxial epidermis of leaves, which in turn release it on the leaf surface, sometimes resulting in Micro-organisms in the phyllosphere face several chal- crystals visible to the naked eye (Drennan & Pammenter, lenges. They are in direct contact with the cuticle, a barrier 1982; Fitzgerald et al., 1992). Moreover, microbial commu- for the release of water, ions and nutrients to the exterior of nities in the Avicennia phyllosphere may be subjected to volatile organic compounds produced by aerial parts The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA of the hosts, including some with antimicrobial activity gene sequences of strains CENA315–CENA348 are KT731136– (Bobbarala et al., 2009; Junker & Tholl, 2013). KT731164, respectively. Two supplementary ﬁgures are available with the online Supplementary In spite of the unfavourable conditions frequently found in Material. these habitats, a number of micro-organisms are capable of

Downloaded from www.microbiologyresearch.org by 000774 G 2015 IUMS Printed in Great Britain 689 IP: 186.217.236.64 On: Thu, 27 Jun 2019 18:43:57 D. O. Alvarenga and others tolerating environmental stress in the phyllosphere and either smeared and placed on the surface of solid media establishing diverse and complex microbiomes, with sev- contained within Petri dishes, or were submerged into eral consequences for the host plants and their ecosystem liquid media within 125 ml Erlenmeyer flasks. Five repli- (Gau et al., 2002; Pen˜uelas & Terradas, 2014). Up to 107 cates of each medium were used. The Petri dishes and bacterial cells per cm2 can be detected in the leaf surface Erlenmeyer flasks were kept at a temperature of 25¡1 8C of some plants, and many of them pertain to taxa which with 14 : 10 h light/dark cycles using fluorescent light 2 2 have not been studied yet and which may possibly present (20 mmol photons?m 2?s 1). unique adaptations to survival on this hostile habitat (Lindow & Leveau, 2002). Survival of bacterial commu- Growth of inoculated material was constantly monitored nities in the phyllosphere depends mainly on carbon, nitro- with an Axiostar Plus light microscope (Zeiss). After con- gen and essential inorganic nutrients released on the leaf firmation of cyanobacterial growth, colonies were purified surface (Leveau & Lindow 2001; Miller et al. 2001). How- by constant transfers to fresh sterile solid media and incu- ever, as cyanobacteria usually have lower nutritional bated under the aforementioned conditions, until each requirements due to their ability to fix atmospheric culture was free of other cyanobacteria and eukaryotic organisms. Cycloheximide (Sigma-Aldrich) was added to carbon and (in some taxa) nitrogen, they are less depen- ? 21 dent on the plant exudates. Cyanobacteria have been media at a final concentration of 75 mg ml to inhibit found to be the main nitrogen-fixing epiphytes in the phyl- growth of eukaryotes. Whenever possible, the leaf side of losphere of some tropical vascular plants, promoting a origin of the isolate was noted. Cyanobacterial isolates significant input of bioavailable nitrogen into these envir- were studied under the Axioskop 40 light microscope onments (Freiberg, 1998; Fu¨rnkranz et al., 2008). This (Zeiss) for the evaluation of morphological features of trait constitutes a significant ecological advantage that taxonomic interest and comparison to previously described ´ ´ also facilitates the establishment of heterotrophic and taxa (Komarek & Anagnostidis, 1998, 2005; Komarek, non-diazotrophic organisms in these habitats. 2013). Detailed descriptions of novel genera and species were produced and their taxonomic placement was deter- Mangroves host several cyanobacteria with important eco- mined according to the classification system proposed by logical roles, including a considerable number of unde- Koma´rek et al. (2014). Isolates were photographed by an scribed taxa (for a review, see Alvarenga et al., 2015). Olympus BX53 optical microscope equipped with differen- A study using culture-independent methods to assess the tial interference contrast and imaging systems (Olympus). diversity of cyanobacteria inhabiting leaf surfaces of man- Afterwards, cells were fixed with 1 ml modified Karnovsky grove trees observed the phyllosphere of Avicennia solution for 64 h at 4 8C (Karnovsky, 1965) and post-fixed schaueriana was colonized by a unique cyanobacterial com- with 1 % osmium tetroxide for 1 h at room temperature. munity (Rigonato et al., 2012). In order to investigate these Fixed cells were subjected to pre-staining with 2.5 % ura- findings further and to access unknown taxa, the present nile acetate for 18 h at 4 8C, followed by dehydration study was undertaken with the purposes of isolating and with acetone solutions at increasing concentrations. Spurr characterizing cyanobacteria inhabiting the phyllosphere resin (Electron Microscopy Sciences) was used for the infil- of Avicennia schaueriana trees from a Brazilian mangrove tration and polymerization of samples (Spurr, 1969). Resin forest. blocks were cut into 600–1000 mm ultrathin sections in a Porter Blum MT-2 ultramicrotome (Sorvall Instruments), Leaves of three adult trees identified as Avicennia schaueri- which were collected with 200-mesh copper grids covered ana Stapf & Leechman were collected at a trunk height of with 5 % colodium. After staining with uranile acetate approximately 1.75 m on 25 March 2008 (early Autumn) and lead citrate, the samples were observed and photo- from trees at the margin of the Iriri river (238 539 50.40 S 8 9 0 graphed in a Zeiss EM 900 electron transmission micro- 46 12 30.6 W), along the Bertioga channel, on the coast- scope at 50 kV. line of Saõ Paulo, Brazil. After detachment from the trees, leaves were packed in sterile plastic bags and kept at 4 8C DNA extraction was carried out according to Fiore et al. until the moment of processing. The Bertioga mangrove (2000). The extracted DNA was used for PCR amplification is close to a seaside resort, and hence it is subject to the of the 16S rRNA gene under previously described con- influence of human activities. In addition, the Iriri river ditions using the primers 27F1 and 1494Rc (Neilan et al., mangrove was impacted by an accident during the con- 1997) in a Techne TC-412 thermocycler (Bibby Scientific). struction of the SP-55 road on 14 October 1983 when it Amplicons were ligated into pGEM-T Easy Vector Systems received a large volume of crude oil from a broken pipeline, (Promega), inserted into Escherichia coli DH5a chemo- an event which added further complexity to its conditions. competent cells, and plated for blue-white colony screening The isolation of cyanobacteria from the surfaces of the (Sambrook & Russell, 2001). Sequencing according to the sampled leaves was achieved using the culture medium method of Sanger et al. (1977) was performed in the ABI BG-11 (Allen, 1968) and three modifications of this PRISM 3100 Genetic Analyzer (Life Technologies). Con- medium: BG-110 (Stanier et al., 1971), lacking nitrogen; sensus sequences were generated with the Phred/Phrap/ SWBG-11 (Castenholz, 1988), simulating salt water; and Consed software package (Ewing & Green, 1998; Ewing SWBG-110 (nitrogen-free SWBG medium), lacking nitro- et al., 1998; Gordon et al., 1998) and only gene regions gen and simulating salt water. Individual leaves were with base-calling qualities over Phred 20 were considered.

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Multiple sequence alignments were performed by CLUSTAL not be determined. According to morphological criteria W (Thompson et al., 1994) and evolutionary models were and the cyanobacterial classification system proposed by estimated with jModelTest 2.1.7 (Guindon & Gascuel, Koma´rek et al. (2014), the strains represented five different 2003; Darriba et al., 2012). Reconstruction of phylogenetic orders: thirteen belonged to Synechococcales, seven to Nos- trees by the maximum-likelihood algorithm (Felsenstein, tocales, seven to Pleurocapsales, one to Chroococcales and 1981) was performed with RaxML 8.2.3 (Stamatakis one to Oscillatoriales, which were distributed among six et al., 2005), which was tested by a bootstrap value of families. Eleven strains had their genera identified as Gloeo- 1000 (Felsenstein, 1985). Bayesian inference (Mau et al., capsopsis, Nodosilinea, Microcoleus, Phormidesmis or Brasi- 1999) was performed with MrBayes 3.2.5 (Ronquist & lonema. Identification of the remaining 18 strains was Huelsenbeck, 2003) using two separate runs, four chains feasible at the family level, but their genera could not be and 5 000 000 Markov Chain Monte Carlo generations. determined. Overall, phylogenetic analyses showed agree- The tree was visualized with Figtree 1.4.2 (http://tree.bio. ment with morphological identifications (Fig. 1). ed.ac.uk/software/figtree) and edited with Inkscape 0.48.5 Seven unicellular baeocyte-producing cyanobacteria were iso- (https://inkscape.org). lated in this study (CENA315, CENA331, CENA333T, Twenty-nine cyanobacterial strains were isolated. The CENA337, CENA345, CENA346 and CENA348) and ident- majority of the strains (25) were obtained using liquid ified as members of the family Xenococcaceae. This family media, while only four strains grew on solid media encompasses genera whose morphology is usually not infor- (Table 1). Seven strains were obtained with the BG-11 mative since their morphological traits are unstable and medium, nine with SWBG-11, four with BG-110, and their genetic diversity exceeds the described morphological nine with SWBG-110. Eleven strains were isolated from diversity (Ishida et al., 2001). As expected, phylogenetic recon- the adaxial side of leaves and eight from the abaxial side, struction showed several genera from this cyanobacterial but no correlation between taxon and leaf side was family are polyphyletic and require revision (Fig. 1A). observed; the location for the ten remaining strains could Although some of these strains seem related to cyanobacteria

Table 1. Cyanobacterial strains isolated from the leaf surface of Avicennia schaueriana

Strain Family Genus Isolation medium Leaf side

CENA315 Xenococcaceae – liquid SWBG-11 adaxial

CENA316 Leptolyngbyaceae Phormidesmis liquid SWBG-110 unidentiﬁed CENA317 Leptolyngbyaceae Phormidesmis liquid SWBG-110 unidentiﬁed CENA318 Leptolyngbyaceae Phormidesmis liquid SWBG-11 adaxial CENA319 Leptolyngbyaceae – liquid SWBG-11 adaxial CENA320 Leptolyngbyaceae – liquid BG-11 adaxial CENA321 Leptolyngbyaceae – liquid BG-11 abaxial

CENA322 Leptolyngbyaceae Nodosilinea liquid SWBG-110 unidentiﬁed CENA323 Leptolyngbyaceae Nodosilinea liquid SWBG-110 abaxial CENA324 Rivulariaceae Phyllonema gen. nov. liquid BG-11 unidentiﬁed

CENA325 Rivulariaceae Phyllonema gen. nov. solid BG-110 abaxial CENA326 Rivulariaceae Phyllonema gen. nov. liquid SWBG-110 unidentiﬁed CENA327 Chroococcaceae Gloeocapsopsis liquid SWBG-110 abaxial CENA328 Rivulariaceae Phyllonema gen. nov. liquid BG-11 abaxial CENA330 Rivulariaceae Phyllonema gen. nov. liquid SWBG-11 adaxial CENA331 Xenococcaceae Foliisarcina gen. nov. liquid BG-11 abaxial CENA332 Leptolyngbyaceae Phormidesmis solid SWBG-11 adaxial CENA333T Xenococcaceae Foliisarcina gen. nov. solid SWBG-11 adaxial CENA335 Leptolyngbyaceae Phormidesmis liquid SWBG-11 unidentiﬁed

CENA337 Xenococcaceae Foliisarcina gen. nov. liquid SWBG-110 abaxial CENA339 Leptolyngbyaceae Phormidesmis liquid SWBG-11 unidentiﬁed

CENA340 Leptolyngbyaceae – liquid BG-110 adaxial T CENA341 Rivulariaceae Phyllonema gen. nov. solid BG-110 abaxial CENA342 Leptolyngbyaceae – liquid BG-11 unidentiﬁed CENA344 Microcoleaceae Microcoleus liquid SWBG-11 adaxial

CENA345 Xenococcaceae – liquid SWBG-110 unidentiﬁed CENA346 Xenococcaceae Foliisarcina gen. nov. liquid SWBG-110 unidentiﬁed CENA347 Scytonemataceae Brasilonema liquid BG-110 adaxial CENA348 Xenococcaceae – liquid BG-11 adaxial

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92/100 A 60/96 --/100 B

56/96 --/95 --/51 C 92/100 72/100 D

100/100 90/100 E Gloeobacter violaceus PCC 7421 (AF132790) 100/100 Gloeobacter violaceus PCC 8105 (AF132791) Escherichia coli K12 (NC_000913)

0.08

Xenococcaceae CENA348 (KT731164) A 99/100 C Pleurocapsa minor SAG 4.99 (AJ344564) 69/99 sp. CALU 1126 (DQ293994) 78/100 Pleurocapsa /55 sp. LEGE 06174 (HQ832924) Chroococcidiopsis 100/100 Microcoleus sp. CENA344 (KT731160) sp. PCC 7319 (AB039006) 61/99 Pleurocapsa Microcoleus sp. DAI (EF654029) --/67 Xenococcus sp. PCC 7307 (AB074510) 99/100 100/89 Microcoleus sp. HTT-U-KK5 (EF654070) 53/94 sp. CCMP1489 (AJ344556) Chroococcidiopsis Microcoleus sp. PCC 7113 (NR_102467) Dermocarpella incrassata SAG 29.84 (AJ344559) 83/100 100/100 --/76 Microcoleus sp. SAG 2212 (EF654075) Xenococcaceae CENA315 (KT731136) --/58 80/55 Coleofasciculus chthonoplastes MEL (EF654038) Xenococcaceae CENA345 (KT731161) 99/100 Microcolesu chthonoplastes SAG 2209 WW7 (EF654055) 57/99 sp. PCC 7301 (AB039009) Stanieria Coleofasciculus chthonoplastes WW3 (EF654052) 84/100 sp. PCC 7516 (X78681) --/51 Pleurocapsa 63/91 Coleofasciculus chthonoplastes WW6 (EF654054) 100/100 Pleurocapsa sp. PCC 7314 (AB074511) 91/76 Microcoleus vaginatus SNM1-KK1 (EF654077) 98/100 sp. PCC 7312 (AJ344561) Myxosarcina 100/100 Microcoleus vaginatus CSU-U-KK1 (EF667962) 96/100 sp. PCC 7325 (AJ344562) Myxosarcina Microcoleus vaginatus SRS1-KK2 (EF654078) Foliisarcina bertiogensis CENA331 (KT731151) T 100/100 Foliisarcina bertiogensis CENA333 (KT731153) --/63 100/100 Foliisarcina bertiogensis CENA337 (KT731155) Foliisarcina bertiogensis CENA337 (KT731162) 74/100 Chroococcidiopsis sp. PCC 6712 (AB039004) 61/100 Xenococcus sp. PCC 7305 (AF132783) Cyanobacterium sp. CENA 169 (KC695862) Stanieria cyanosphaera PCC 7437 (AF132931) 92/100 98/97 Gloeocapsa sp. KO38CU6 (AB067575) 95/51 Leptolyngbya boryana IAM M-101 (AB245143) 64/100 Gloeocapsa sp. KO20B5 (AB067578) D 100/100 Leptolyngbya boryana UTEX-B-485 (AF132793) 100/100 Gloeocapsa sp. KO30D1 (AB067579) 100/100 Leptolyngbya sp. CENA 104 (EF088333) 96/100 Cyanothece sp. ATCC 51142 (AF132771) Pseudophormidium sp. ATA2-1-CV21 (KC311898) 97/100 Aphanocapsa sp. HBC6 (EU249123) 98/100 Pseudophormidium sp. ATA5-5-1-CV6 (KC3119011) Gloeothece sp. KO68DGA (AB067580) 75/74 Pseudophormidium sp. ATA5-5-1-DP06 (KC3119161) 56/99 Phormidesmis priestleyi ATA5.LG2.4 (AY493580) --/66 81/100 Phormidesmis priestleyi ATA5.66.1 (AY493581) 100/100 sp. IAM M-262 (AB093483) sp. WJT36-NPBG45 (KC525089) Scytonema 100/100 Phormidesmis B 100/100 Scytonema sp. U-3-3 (AY069954) 60/89 Phormidesmis priestleyi ANT.L61.2 (AY493582) /61 Scytonema hofmanni PCC 7110 (AF132781) 99/100 Phormidesmis priestleyi CYN71 (JQ687335) 70/92 Symphyonemopsis sp. VAPOR1 (AJ544085) Phormidesmis sp. WJT24-NPBG9 P23C (KC525086) 100/100 Iphinoe spelaeobios LO2 B1 (HM748317) 72/99 Phormidesmis sp. WJT36-NPBG27 P5A (KC525090) 97/100 sp. CENA347 (KT731163) Phormidesmis sp. WJT24-NPBG20 P5A (KC525091) 98/100 Brasilonema Brasilonema octagenarum UFV-E1 (EF150854) 100/100 Leptolyngbya frigida ANT. MANNING.1 (AY493573) 98/98 Brasilonema octagenarum UFV-OR1 (EF150855) 72/100 99/100 Leptolyngbya frigida ANT. LH70.1 (AY493574) 64/99 59/90 Brasilonema octagenarum HA4186-MVI (HQ847562) Leptolyngbya sp. 1T12c (FR798935) 74/99 Brasilonema sp. CENA114 (EF117246) 64/100 Leptolyngbya sp. VP3-07 (FR798933) Brasilonema terrestre CENA116 (EF490447) Leptolyngbyaceae CENA340 (KT731157) --/65 Tolypothrix sp. IAM M-259 (AB093486) Leptolyngbya sp. CENA112 (EF088337) sp. PCC 7415 (AM230668) --/97 CENA321 (KT731142) 74/10099/100 Tolypothrix Leptolyngbyaceae sp. UAM 332 (HM751847) 100/100 CENA342 (KT731159) Tolypothrix 100/100 Leptolyngbyaceae 100/66 sp. UAM 337 (HM751851) JSC-1 (FJ788926) 66/100 Tolypothrix Oscillatoriales Tolypothrix sp. PCC 7504 (AM230669) Leptolyngbya sp. CENA103 (EF088339) 98/100 Tolypothrix sp. TOL328 (AM230706) 100/100 Cylindrospermum stagnale PCC 7417 (AF132789) 60/99 Cylindrospermum alatosporum SAG 43.79 (GQ287650) 82/100 Calothrix sp. PCC 7507 (NR_102891) --/81 64/100 Nostoc calcicola VI (AJ630448) 99/100 Nostoc commune UTEX 584 (AY218833) SAG 57.79 (DQ185254) 54/100 Nostoc commune 97/100 Petalonema sp. ANT.GENTER2.8 (AY493624) 100/100 Anabaena cylindrica NIES19 (AF247592) 100/100 Anabaena cylindrica UTAD_A212 (GQ443447) TFEP1 (AF320093) E 100/100 Halomicronema excentricum Anabaena augstumalis SCMIDKE JAHNKE/4a (AJ630458) Halomicronema sp. SCyano39 (DQ058860) --/75 78/99 Trichormus variabilis HINDAK 2001/4 (AJ630456) 95/100 Halomicronema sp. PCyano40 (DQ058890) --/77 --/72 GREIFSWALD (AJ630457) 90/100 Trichormus variabilis Leptolyngbya sp. 0BB24S04 (AJ639893) KCTC AG 10180 (DQ234832) 92/100 Trichormus variabilis 73/100 Leptolyngbya sp. 0BB32S02 (AJ639894) 75/100 sp. XP6A (EF568902) 51/99 100/100 Anabaena --/94 Nodosilinea nodulosa UTEX 2910 (EF122600) Anabaena sp. BECID20 (EF583858) --/89 sp. 0BB19S12 (AJ639895) /57 Leptolyngbya Anabaena sp. BECID23 (EF583859) 100/100 ANT.ACE.1 (AY493588) --/84 Leptolyngbya antarctica 55/100 Gloeotrichia schinulata PYH6 (AM230703) 68/100 Leptolyngbya antarctica ANT.ACEV6.1 (AY493589) 100/100 Gloeotrichia schinulata PYH14 (AM230704) /72 Leptolyngbya sp. LEGE 07298 (HM217044) sp. HA4356-MV2 p8GH (JN385288) Calothrix 59/96 100/100 Nodosilinea sp. CENA323 (KT731144) Calothrix sp. BECID33 (AM230683) sp. CENA144 (KC695838) --/55 81/100 Nodosilinea 88/100 Calothrix desertica PCC 7102 (AM230699) 64/98 sp. CENA167 (KC695860) 98/100 Nodosilinea 100/100 Calothrix sp. MU27 UAM-315 (EU009152) 78/100 Nodosilinea epilithica Kovacik 1998/7 (HM018677) Calothrix sp. PCC 7103 (AM230700) Leptolyngbya sp. CENA322 (KT731143) sp. XSP25A (AM230665) 83/100 63/80 Rivularia Leptolyngbya sp. CENA155 (KC695849) 100/100 Rivularia sp. XP3A (AM230672) 59/100 sp. CENA156 (KC695850) 53/100 Leptolyngbya Rivularia sp. BECID10 (AM230673) 69/98 CENA320 (KT731141) 83/100 Leptolyngbyaceae sp. CCAP 1410/14 (HF678500) 63/100 67/100 Calothrix Leptolyngbya sp. CENA 134 (HQ730083) CENA324 (KT731145) Phyllonema avicenniicola Halomicronema hongdechloris C2206 (JX089399) CENA326 (KT731147) --/100 Phyllonema avicenniicola Leptolyngbya sp. ANT.LH52 (AY493584) 67/54 CENA328 (KT731149) Phyllonema avicenniicola 61/96 Phormidium pristleyi ANT.PROGRESS2.6 (AY493585) 100/100 Phyllonema avicenniicola CENA330 (KT731150) sp. ANT.PENDANT.3 (AY493587) T Pseudophormidium CENA341 (KT731158) 57/98 Phyllonema avicenniicola 95/99 Phormidesmis sp. CENA316 (KT731137) CENA325 (KT731146) 90/100 Phyllonema avicenniicola 90/100 Phormidesmis sp. CENA317 (KT731138) 5.2 s.c1 (AY380791) 100/100 Chroogloeocyctis siderophila Phormidesmis sp. CENA318 (KT731139) 100/100 sp. CENA327 (KT731148) Gloeocapsopsis 99/100 Phormidesmis sp. CENA332 (KT731152) 100/100 LEGE 06123 (FJ589716) Gloeocapsopsis crepidinum Phormidesmis sp. CENA335 (KT731154) sp. BB79.2 (AJ344552) 80/99 Chroococcidiopsis Phormidesmis sp. CENA339 (KT731156) sp. CC2 (DQ914864) Chroococcidiopsis Phormidesmis priestleyi ANT.LACV5.1 (AY493586) 82/100 sp. CC1 (DQ914863) Chroococcidiopsis Leptolyngbyaceae CENA319 (KT731140)

Fig. 1. Phylogenetic tree reconstructed by Bayesian inference. Strains isolated in the present work are highlighted in bold. Values.50 for bootstraps observed in maximum-likelihood topologies followed by Bayesian posterior probabilities are illus- trated in the tree knots. Bar, 0.08 substitutions per nucleotide position.

Downloaded from www.microbiologyresearch.org by 692 International Journal of Systematic and Evolutionary Microbiology 66 IP: 186.217.236.64 On: Thu, 27 Jun 2019 18:43:57 Epiphyllic cyanobacteria from Avicennia schaueriana identiﬁed in previously described genera, the current polyphy- maximum-likelihood (100 % bootstrap) and Bayesian infer- letic state of a considerable number of Xenococcacean genera ence (100 % posterior probability) trees (Fig. 1A). Ultrastruc- allowed identiﬁcation only at the family level. Strains tural analyses of strain CENA333T revealed typical CENA331, CENA333T, CENA337 and CENA346 had mor- Xenococcacean thylakoid arrangements (Fig. 3a, b). Simi- phological traits typical of the family Xenococcaceae (Fig. 2 larities between 16S rRNA gene sequences from these strains and Fig. S1, available in the online Supplementary Material), and their closest relatives are found in Table 2. The combi- and grouped in a cluster close to, but phylogenetically distinct nation of ecological, morphological, ultrastructural and mol- from, Xenoccocacean genera with high support from both ecular data of these four strains allowed the proposal of

(a) (b)

(e) (f)

Fig. 2. Photomicrographs of Foliisarcina bertiogensis gen. nov., sp. nov. strains CENA337 (a, d), CENA333T (b), CENA331 (c), and CENA346 (e, f). Bars, 100 mm (a), 20 mm (b, c, e, f), 10 mm (d). A coloured version of this ﬁgure is available as Fig. S1 in the online Supplementary Material.

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(a) (b)

Fig. 3. Ultrastructures from strains CENA333T (a, b) and CENA341T (c, d) cells, highlighting thylakoid arrangements typical of the family Xenococcaceae and the order Nostocales, respectively, as proposed by Hoffmann et al. (2005). Bars, 1 mm (a, b, d), 2 mm(c).

Foliisarcina gen. nov. with type species Foliisarcina bertiogensis Type species: Foliisarcina bertiogensis. gen. nov., sp. nov., according to the International Code of Nomenclature for Algae, Fungi, and Plants. Description of Foliisarcina bertiogensis sp. nov. Etymology: Foliisarcina bertiogensis (ber.ti.o.gen9sis N.L. Description of Foliisarcina gen. nov. fem. adj. bertiogensis from the Bertioga municipality, Saõ Etymology: Foliisarcina (Fo.li.i.sar.ci9na L. neut. n. folium a Paulo, Brazil). leaf; L. fem. n. sarcina a package; N.L. fem. n. Foliisarcina a Description: Solitary cells or, more commonly, micro- package-like organism from a leaf; referring to the colony scopic colonies (stratified) with several cells (up to 64 or morphology of this cyanobacterium). more), forming multicolonial groups, irregular to squarish in outline, up to 44.2 mm (longer axis: mean 27.1 mm; Description: Cells isolate or forming regular (young, with n534), with dense, more or less regularly arranged cells, few cells) or irregular (older, with more cells) colonies clusters of four cells are recognizable within the older colo- with variable number of cells (usually up to 64 cells), some- nies or groups. Colonial envelope sometimes visible, thin, times several colonies grouped; colonial envelope thin, usually tightly surrounding the aggregates, hyaline. Cells firm, colourless; cells usually close to each other within rounded when isolate, hemispherical when in pairs, the colony, variable in shape; cell division by binary fission, nearly squarish when in dense colonies (mutual pressure) in two or three planes in successive generations; reproduc- or irregular when dividing or forming baeocytes. Isolate tion by baeocyte formation. cells 2.1–5.7 mm diameter without sheath (mean 3.7 mm;

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Table 2. Similarity between 16S rRNA gene sequences obtained from strains of Foliisarcina bertiogensis gen. nov, sp. nov. and the closest relatives deposited in the NCBI GenBank database

Strain Size (bp) Closest sequence (NCBI access number) Sequence similarity (%) Coverage (%)

CENA331 1416 Unidentified cyanobacterium GI-1 (JN202625) 98 99 CENA333T 1418 Unidentified cyanobacterium GI-1 (JN202625) 98 99 CENA337 1418 Unidentified cyanobacterium GI-1 (JN202625) 98 99 CENA346 1418 Unidentified cyanobacterium GI-1 (JN202625) 98 99 n537), 3.6–7.1 mm diameter with sheath (mean 5.4 mm; taxon, CENA341T, exhibited thylakoid arrangements typi- n537), cells in colonies 1.3–5.2 mm diameter. Cell content cally observed in the order Nostocales (Fig. 3c, d). Similarities usually brownish, sometimes bright green, with few large between 16S rRNA gene sequences from these strains and granules or finely granular. Baeocytes are short cylindrical their closest relatives are found in Table 3. Therefore, the com- rounded or more or less spherical, from 1.7 mm diameter bination of ecological, morphological, ultrastructural and when young, with thin, hyaline sheath when a little older, molecular data allowed the proposal of the new Rivulariacean content is brownish to light green. genus Phyllonema gen. nov. with the type species Phyllonema aviceniicola gen. nov., sp. nov., according to the International Holotypus: SP 429275, dried material deposited at the her- Code of Nomenclature for Algae, Fungi, and Plants. barium of the Saõ Paulo Institute of Botany, Saõ Paulo, Brazil. Description of Phyllonema gen. nov. Type strain: CENA333T. Etymology: Phyllonema (Phyl.lo.ne9ma Gr. neut. n. phyllon GenBank accession number for 16S rRNA gene sequence of leaf; Gr. neut. n. nema thread; N.L. neut. n. Phyllonema a type strain: KT731153. thread from a leaf; reference to the typical habitat of this filamentous cyanobacterium). Filamentous false-branched and heterocyted strains CENA324, CENA325, CENA326, CENA328, CENA330 and Description: Filaments in groups not surrounded by abun- CENA341T grouped in a well-supported cluster in both phy- dant mucilage or gelatin, without radial disposition, logenetic analyses (100% bootstrap from maximum-likeli- entangled, long; branches rare; sheath present, usually hood and 100% posterior probability from Bayesian thin and hyaline, opened at the apex; trichomes heteropo- inference), clearly distinguished from clusters composed of lar, usually constricted; cells of variable length, shorter or already described genera (Fig. 1B). These cyanobacterial longer than wide; heterocytes basal or intercalary, single strains, although morphologically identified as Rivulariaceae, or in pairs, variable in shape; necridia present; akinetes did not cluster with known genera of this family. Further- absent; phyllosphere inhabitant. more, they do not present terminal hairs, even in salt-less Type species: Phyllonema aviceniicola. media (Figs 4 and S2). Although terminal hairs are not universally found in genera of the family Rivulariaceae,they Description of Phyllonema aviceniicola sp. nov. are a common characteristic for members of this family. 9 Since the terminal hair structures may be inhibited not only Etymology: Phyllonema aviceniicola [a.vi.cen.ni.i co.la N.L. by phosphatase activity, but also by high salinity, as observed fem. n. Avicennia a tree genus; L. suff. -cola (from L. n. in a Cuban mangrove strain (Mahasneh et al., 1990), it is incola), inhabitant dweller; N.L. n. avicenniicola inhabiting Avicennia trees; in reference to the host of the species]. possible their absence in the isolated strains is the result of an adaptation to the leaf surface of Avicennia schaueriana. Description: Filaments in groups (culture), up to 535 mm The ultrastructural analysis of a representative of this novel long, 3.2–13.0 mmdiameter(mean6.49mm; n5112),

Table 3. Similarity between 16S rRNA gene sequences from strains of Phyllonema aviceniicola gen. nov., sp. nov. and the closest relatives

Strain Size (bp) Closest sequence (NCBI access number) Sequence similarity (%) Coverage (%)

CENA324 1414 Uncultured bacterium clone YF930 (KF037928) 94 100 CENA325 1415 Calothrix sp. 336/3 (CP011382) 94 100 CENA326 1414 Calothrix sp. 336/3 (CP011382) 94 100 CENA328 1401 Scytonema sp. ‘Coccocarpia sp. kj30 cyanobiont’ (KF359679) 94 96 CENA330 1414 Calothrix sp. 336/3 (CP011382) 94 100 CENA341T 1414 Calothrix sp. 336/3 (CP011382) 94 100

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(a) (b)

(e) (f)

Fig. 4. Photomicrographs of Phyllonema aviceniicola gen. nov., sp. nov. strains CENA326 (a), CENA324 (b), CENA328 (c), CENA340 (d), and CENA341T (e, f). Bars, 100 mm (a), 20 mm (b, d), 10 mm (c, e), 5 mm (f). A coloured version of this ﬁgure is available as Fig. S2 in the online Supplementary Material.

commonly narrowed towards the ends, rarely branched. hairs. Cells 1.7–8.1 mm long (mean 4.6 mm; n5138), 4.1– Sheath usually thin (widened and lamellate sheaths occasion- 8.5 mm diameter (mean 6.1 mm), length/diameter ratio ally found), ﬁrm, colourless, lamellate, opened at the apex of from 0.3 to 1.3 at swallowed region; 1.5–6.0 mmlong the adult ﬁlament. Trichomes long and sharply narrowed (mean 3.0 mm), 3.5–6.1 mmdiameter(mean4.7mm), towards the ends in older trichomes, short but not so evi- length/diameter ratio from 0.3 to 1.2 at the middle of tri- dently attenuated in young ones (cylindrical trichomes are chomes. Cell content blue-green to light green, centroplasm not rare), distinctly constricted at cross-walls, without and chromatoplasm evident, large granules present.

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Heterocytes basal, single or in pairs, occasionally in the similarities, Foliisarcina differs from Cyanosarcina due to middle of the trichome (when intercalary and in pairs poss- the production of baeocytes, since Cyanosarcina does not ibly a trichome fragmentation spot), very variable in shape form baeocytes or nanocytes. The distinction between and size, usually conical, but also rounded, hemispherical Foliisarcina and Chroococcidiopsis is more complicated or or cylindrical, 3.6–9.1 (–11.8) mmlong,4.1–9.0mm diameter even not possible by using morphological markers alone, at the longer axis. considering that the characteristics of colonies and cells, growth patterns and reproduction strategies are quite simi- Holotypus: SP 429276, dried material deposited at the her- lar between species of both genera. Although there are barium of the Sa˜o Paulo Institute of Botany, Sa˜o Paulo, several coincidences in morphology, there is a strong back- Brazil. ground for erecting Foliisarcina as a new genus based on Type strain: CENA341T. ecological and molecular characteristics.

GenBank accession number for 16S rRNA gene sequence of Morphologically, Phyllonema is very close to Hassalia Ber- type strain: KT731158. keley ex Bornet et Flahault. These genera share features Strains CENA319, CENA320, CENA321, CENA340 and such as short cells, firm, conspicuous and sometimes lamel- CENA342 presented morphological traits similar to those late sheath, and filaments that are not very long and slightly traditionally described for Leptolyngbya, which are usually attenuated to the apex. The two genera differ by the fre- characterized for their morphological simplicity. Notwith- quency of branching, which is more frequent in Hassalia standing, this simplicity makes it difficult to assure their than in Phyllonema; however, considering that Phyllonema identification in this genus since Leptolyngbya is a polyphy- is described based on cultured material, this characteristic letic genus (Taton et al., 2006; Johansen et al., 2011). cannot be assured in samples obtained directly from Sequences of Leptolyngbya-like strains were scattered in nature. Once again, DNA sequences are determining in the phylogenetic tree, far from the Leptolyngbya boryana the distinction between Phyllonema and other genera. clade (Fig. 1D, E), the type species of this genus. The most divergent sequence observed was CENA319, which A previous study reporting the cyanobacterial diversity in behaves as an outgroup to the Nodosilinea, Leptolyngbya, the phyllosphere of Avicennia schaueriana and other man- and Phormidesmis clades (Fig. 1E). These observations led grove trees using culture-independent analyses uncovered a us to maintain identification at the family level for these considerable number of unknown cyanobacteria in these strains. habitats (Rigonato et al., 2012). This finding was furthered in the present study by methods providing direct access to Phylogenetic analyses indicate it is possible that at least part of this diversity, as evidenced by the isolation of Phyl- some of the strains isolated in this work whose genera lonema aviceniicola and Foliisarcina bertiogensis strains, could not be determined (CENA315, CENA319, CENA herein described for the first time. Therefore, this study 320, CENA321, CENA340, CENA342, CENA345 and confirmed some of the unique cyanobacteria inhabiting CENA348), might also be representatives of novel cyano- the phyllosphere of Avicennia schaueriana can be retrieved bacterial taxa. However, most of these strains are not clo- by the use of culturing techniques, a fact that raises several sely related. With single representatives, the description new possibilities for cyanobacterial taxonomy and man- of novel taxa is more difficult, and therefore these strains grove microbiological research. will be further explored elsewhere. To our knowledge, with the exception of Avicennia, only Historically, cyanobacterial systematics has been through two other trees with salt-excreting leaves have had several modifications, which were intensified after the their phyllosphere microbial communities studied to adoption of polyphasic approaches. In this approach, clas- date, Atriplex halimus (Simon et al., 1994) and Tamarix sical morphological analyses are supplemented by other sp. (Qvit-Raz et al., 2008, 2012; Finkel et al., 2011). How- methods that provide alternative views on variation ever, no cyanobacteria were detected in the samples evalu- between organisms, and a consensus between them is ated. The results of culturing and culture-independent achieved (Vandamme et al., 1996; Koma´rek, 2006; studies on Avicennia schaueriana trees suggest this absence Koma´rek et al., 2014). This approach allowed to assert could be a consequence of lower abundance or methodo- with higher confidence, the relationship between the mem- logical bias, since cyanobacteria are able to withstand and bers of the phylum Cyanobacteria and to produce more thrive on the conditions found in at least some salt-rich solid taxonomic revisions. The present work describes leaf surfaces. two novel cyanobacterial genera that, despite presenting subtle morphological variation when compared to pre- Bacteria are found mainly at the epidermal cell-wall junc- viously described genera, are clearly distinguished from tions, the stomata and the base of trichomes known taxa when additional factors are also taken into (Whipps et al., 2008). The diversity of microbial commu- consideration. nities in the phyllosphere may also sometimes be positively correlated with herbivory by insects (Humphrey et al., Foliisarcina morphologically resembles Cyanosarcina Kova´- 2014). Usually, higher bacterial abundance in the phyllo- cˇik and Chroococcidiopsis Geitler. Despite morphological sphere is found in contact with the abaxial leaf surface

Downloaded from www.microbiologyresearch.org by http://ijs.microbiologyresearch.org 697 IP: 186.217.236.64 On: Thu, 27 Jun 2019 18:43:57 D. O. Alvarenga and others due to a higher density of stomata and trichomes, and to 2009). Most likely, the combination of the environmental the presence of a thinner cuticle in this leaf side characteristics of mangrove ecosystems, the physiological (Whipps et al., 2008). Although Avicennia schaueriana characteristics of the host plant and the natural conditions excretes a higher amount of salt on the abaxial leaf epider- of the phyllosphere provide a unique scenario for the mis due to its higher concentration of salt glands (Fitz- occurrence of cyanobacterial taxa. Despite the usual low gerald et al., 1992), it seems the salt concentration did plant diversity, mangroves are an important area of study not inﬂuence the distribution of cyanobacteria on the for cyanobacterial diversity, and trees such as Avicennia leaves. However, this observation may not be accurate schaueriana may host yet-unknown strains that can provide since the origin of some strains could not be determined new insights into the evolution, ecology and biogeography and due to the known bias inherent to cultivation tech- of this phylum. niques (Ward et al., 1998; Furtado et al., 2009).

The use of at least two different media for the isolation of Acknowledgements cyanobacteria from extreme habitats is recommended – one with characteristics emulating the conditions naturally This study was supported by grants from the Saõ Paulo Research found in the studied environment, and another with mod- Foundation (FAPESP) to I. S. M. (BIOTA 2004/13910-6) and to erate characteristics (Waterbury et al., 2006). In this study, M. F. F. (2013/09192-0), and from the National Council for the main variable tested was salinity, and a significant vari- Scientific and Technological Development (CNPq) to M. F. F. (471898/2007-4). D. O. A. was supported by FAPESP and CNPq ation of isolated genera was not verified between isolations graduate fellowships (grants 2008/52556-4 and 132494/2010-8, performed in media with and without the addition of sodium, respectively). J. R. was supported by the Brazilian Federal Agency magnesium and potassium. The division of micro-organisms for the Support and Evaluation of Graduate Education (CAPES) from saline environments into specialist/stenohaline (with National Postdoctoral Program. M. F. F. would like to thank CNPq a narrow range of tolerance to alteration of salinity for a research fellowship (306607/2012-3). We thank Professor Dr levels) and generalist/euryhaline (tolerant to wide ranges Neusa de Lima Nogueira and Moˆnica Lanzoni Rossi for helping of salt concentrations) has been proposed (Golubicˇ, with the preparation of samples for transmission electron microscopy, and Professor Dr Francisco Andre´ Ossamu Tanaka, Professor Dr 1980). Hence, the isolated cyanobacteria most likely are Elliot Watanabe Kitajima and their team at NAP-MEPA/ESALQ- generalists. USP for permission to use the transmission electron microscope and assistance with the equipment. We would also like to thank Dr Similar micro-habitats separated by obstacles that hinder Armando Cavalcante Franco Dias and Joaõ Luiz da Silva for collecting dispersion may be considered islands, and they may samples, and Watson A. Gama Jr and Professor Dr Ce´lia Leite favour the survival of organisms that would otherwise be Sant’Anna for the deposit of reference samples at the Saõ Paulo Insti- eliminated by ecological interactions in richer environ- tute of Botany. ments (Simberloff, 1974). Thus, microbial dynamics in the phyllosphere could be understood in the framework References of island biogeography, in which the leaf community is viewed as analogous to communities in oceanic islands Allen, M. M. (1968). Simple conditions for growth of unicellular blue- (Kinkel et al., 1987). Plant specificity may have a major green algae on plates. J Phycol 4, 1–4. influence on the composition of the phyllosphere microbial Alvarenga, D. O., Rigonato, J., Branco, L. H. Z. & Fiore, M. F. 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