Cyanobacterial diversity in alkaline marshes of ...

Algological Studies 117 (Cyanobacterial Research 6)

265-278

Stuttgart, October 2005

Cyanobacterial diversity in alkaline marshes of northern Belize (Central America) By Jiří KOMAREK' 4 , STEFANO VENTURA2 , SILVIA TURICCHIA 2 , JAROSLAVA KOMARKOVA3 ,4 , CRISTINA MASCALCHI2 and ELISA SOLDATI 2

Institute of Botany AS CR, Třeboii, Czech Republic 2 CNR-Institute for Ecosystem Study, Section of Firenze, Sesto Fiorentino, Italy 3 Institute of Hydrobiology AS CR, Č eské Budějovice, Czech Republic 4 University of South Bohemia, Faculty of Biological Sciences, Č eské Budějovice, Czech Republic I

With 4 figures and 6 tables in the text Abstract: Cyanobacterial mats are important ecosystem components of oligotrophic alka-

line marshes of northern Belize. They initially develop as benthic communities forming thick carpets on the bottom and on submerged plant stems (mainly Eleocharis spp.), and later rise to the surface as floating mats. Rich diversity of cyanobacterial morphospecies was found in these communities, with dominating oscillatorialean (filamentous, non-heterocytous) types. However, the coccoid species were the most diversified, and few heterocytous types were present in larger amount in limited periods of mats seasonal development. The diversity was evaluated both by phenotype criteria and by the genetic analysis including complete sequence of the 165 rRNA gene and TGGE. According to the traditional taxonomy, 45 coccoid, 27 oscillatorialean and 15 heterocytous morphospecies were recognized, of which only less than 70 % were identifiable according to the available literature, and about 60 % of the described species have been known only from tropical regions. These conclusions proved that the cyanoprokaryotic microflora from these unique habitats is highly specialized, and contains numerous adapted forms for this habitat and possibly endemic in this region. The genotype diversity study confirmed the novelty of the endemic form found with the phenotype study. From the comparison with the other studied Central-American habitats it follows that similar specialized cyanobacterial assemblages are characteristic of alkaline marshes in the whole Caribbean district (Florida-Everglades, Puerto Rico, SW coastal regions.of Cuba, Jamaica, islands off Venezuela, southern states of Mexico). Key words:

Cyanobacteria, Cyanoprokaryota, phenotype diversity, genetic diversity, tropical ecology, alkaline marshes, benthic mats, periphyton, Belize, Central America, Caribbean region.

0342-1120/05/0159-265 $ 3.50 © 2005 E. Schweizerbart'scheVerlagsbuchhandlung, D-70176 Stuttgart Algological Studies 117 = Arch. Hydrobiol. Suppl. 159

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Introduction Alkaline marshes in flat coastal areas of northern Belize represent a unique wetland ecosystem. They are spread over many km 2 , and play an important role in the countryside ecology. They are shallow (maximally 2 m deep) and some of them dry almost completely during the late dry season (late May to early July). The dominant wetland vegetation is mainly represented by Eleocharis cellulosa, E. interstincta and Cladium jarnaicense, which occupy the majority of marshes (Fig. 1). However, this vegetation is usually sparse and does not prevent the penetration of solar radiation down to the bottom, thus allowing the development of dense mats of cyanobacteria. These mats are often several cm thick and contain a remarkable diversity of cyanobacterial morphospecies. Similar mats develop on the submersed parts of Eleocharis stems. At a later developmental stage, the mats detach from the substrate, and float to the water surface (Fig. 2).

Fig. 1. Selected localitites from northern Belizean marshes (comp. Tab. 1): a Quiet, b Deep, c — Little Belize, d Chan Chen, e Chan Chen (deeper places with —

—

Typha domingensis).

—

—

Cyanobacterial diversity in alkaline marshes of northern Belize

267

a ts

Fig. 2. Details of cyanobacterial mats, developing in benthos and on submerged stems of Eleocharis spp., and later floating on the surface (b c). —

The cyanobacterial mats are therefore the unique and important component of the marsh habitat, and investigation of their diversity was one of the important goals during the complex investigation of the ecological significance of these marshes. The description and characterization of studied marshes is included in papers of REJmANKovšk et al. (1996a and 2004), and first results about the cyanobacterial biodiversity were presented in articles of REJmANKovA et al. (1996b, 2004). In the present study, the cyanobacterial natural morphospecies were identified according to the traditional taxonomy and the diversity in communities was simultaneously evaluated by the molecular procedures (TGGE).

Methods

th

The localities and habitats were described in details in the previous papérs of REJMÁNKOVÁ et al. (1996a, 2004). The material was collected and studied in the years 2001 to 2004. The main characteristics of sampling locations are summarised in Table 1. Twelve selected marshes, as described in cited papers, were studied in more detail. The species composition was found to be similar at all locations and the gradient of salinity was the main feature controlling the dominance of various species at locations of different conductivity. The samples were studied alive under optical microscope, documented by drawings and microphotos, and measured. For isolation of cyanobacterial strains, smashed portions of samples and their decimal dilutions were plated on solid media, BG-11 0 (12IPPKA et al. 1979) and BG-11 with half salt

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concentration, using agarose (0.65 %, w/v) instead of agar. Cycloheximide (100 mg .1-1) was added to avoid the growth of eukaryotic contaminants. The cultures were incubated at room temperature under natural light. In addition, enrichment cultures in liquid media were set up, inoculated with a small portion of a sample. Incubation lasted about 4 weeks under the conditions described above. Colonies or filaments observed under the microscope on solid media were transferred in liquid media and incubated in the same conditions. The liquid cultures were microscopically controlled and, if new cyanobacterial morphotypes were found, they were isolated by plating. The cultivation of isolated strains was performed on agarplates or in liquid medium BG-11 or BG-110. Altogether, 171 strains were isolated, that have been used for molecular evaluation and taxonomic comparison by traditional phenotype, and molecular identification of taxonomic units. Nucleic acid extraction, PCR, and TGGE of natural samples Total community genomic DNA from samples was extracted with the Fast DNACDSPIN® KIT (Q.BIO gene). The cyanobacterial 16S rRNA was amplified by a nested PCR using in the first step the primers CYA359F (MBEL et al. 1997) and 16S1494R (5'-GTA CGG CTA CCT TGT TAC GAC-3') and, in,the second step, the cyanospecific primers described by NOBEL et al. (1997). The two reverse primers 16S781R (a) and 16S781R (b) were used separately in the amplification reaction as suggested by TATON et al. (2003). The TGGE technique was set up with a TGGE MAXI system (Biometra) following the manufacturer's instructions. The temperature gradient was 42 °C-52 °C. Sequencing of the TGGE bands Individual TGGE bands were excised (WATANABE et al. 1998) and reamplified with the same primers used for the TGGE, except that primer 16S359F was partially modified taking out the clamp (GC-rich 5' end). TGGE band sequences were determined as for 16S rDNA sequences, using the same primer used for band reamplification. Sequences were made by Genome Express, Grenoble, France. Sequences were imported in ARB and analysed as described below for complete 16 5 rDNA sequences.

Table 2003.

1. Main characteristics of 12 studied marshes; data from the period Aug. (After RETMÁNKOVA et al. 2004)

Location Frank Big Snail South Deep Hidden Cane Buena Vista New Quiet Eli's Doubloon Little Belize Chan Chen

Water depth [range, cm]

pH

140-0 135-33 96-7 88-2 95-3 70-4 140-3 129-5 155-31 190-5 35-1 127-15

'7.8 8.8 8.1 7.9 7.5 7.8 7.9 7.6 7.7 8.1 8.0 8.4

Conductivity

[1.1S.cm-1 ] 211.0 151.3 112.0 172.0 121.0 1424.5 2132.5 1153.5 962.5 5602.5 4420.0 3347.5

2001

to Febr.

SRP NH4-N N/P SO4-2 Cl p.p.m. ppm. p.p.b. p.p.b.

1.0 1.9 3.8 1.0 2.9 121.0 585.6 319.7 192.0 864.0 117.1 2400.0

3.9 19.5 8.5 6.0 4.3 62.7 276.5 156.0 106.4 992.6 380.7 329.7

10.4 11.0 14.8 16.5 11.8 17.6 10.7 13.1 13.2 12.9 11.9 14.0

47.0 77.2 97.7 105.8 95.9 63.1 48.3 43.3 72.3 43.8 91.6 76.8

7.3 8.4 12.6 16.3 13.7 4.2 5.3 3.1 7.0 5.7 9.7 9.1

Cyanobactefial diversity in alkaline marshes of northern Belize

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Nucleic acid extraction, PCR, and 16S rDNA sequence analysis of isolated cyanobacterial strains To obtain DNA, cells were treated with lysozyme, proteinase K and SDS and then extracted with a mixture of phenol, chloroform and isoamyl alcohol as described by BEARD et al. (1999). After addition of 0.1 ml of 3 M NaC1, nucleic acids were precipitated in isopropyl alcohol, resuspended in TE (10mM Tris-HC1 pH 8; 0.1 mM EDTA) and purified with Nucleotrap PCR purification kit (CLONTECH). Sequences of the 165 rRNA gene were obtained from a longer DNA fragment amplified with primers 16S27F and 23S30R (TAToN et al. 2003). Sequences of the full-length 16S rRNA gene were made by Genome Express, Grenoble, France, using sequencing primers 16S979F, 16S544R and 16S1092R (HRouzEK et al. 2005). For each complete sequence, three chromatogram files received from Genome Express were analysed with the gauntlet PHRED PHRAP CONSED (University of Washington) which estimates a quality value for each base call and determines the consensus sequence on the basis of such quality values. Assembled sequences (contigs) automatically produced by PHRED and PHRAP modules were visually checked and manually edited using the CONSED sequence editor when needed. 165 rDNA sequences obtained in this study were imported in ARB and aligned with other 165 rDNA cyanobacterial sequences longer than 1400 nucleotides from the public domain. For the phylogenetic analysis, distance trees were built with the Neighbor-joi ňing method (SArrou & NEI 1987), using 16S rDNA sequences longer than 1400 nucleotides. The evolutionary distances in the distance matrix were calculated using the correction of Jukes and Cantor. A few shorter sequences and TGGE band sequences were inserted using a maximum parsimony criterion in order to keep the previous topology. Microcystis sp. was used as outgroup.

Results

1. So far, 87 cyanobacterial morphospecies participating in the formation of massive benthic and later free-floating mats have been recognized in alkaline marshes of northern Belize. Other cyanobacterial morphotypes were found in littoral regions, subaerophytic habitats above water level, and in soils on the shore. The composition of species was similar in all marshes, slight differences in dominancy of several different species were revealed only in dependence on salinity gradient (cf. data in Table 1), and during different phases of the growing season. The lists of species from different localities are presented in Tables 2-4. 2. The dominant (and most abundant) species were filamentous oscillatorialean types, mainly belonging to the genus Leptolyngbya. Heterocytous fonns were more abundant in fully developed mats, and in the later phases of vegetation season (before the dry period). The widest morphological diversity was found in coccoid types, which, however, participated only rarely in dominance in cyanobacterial assemblages (an exception was Aphanothece bacteriosa, the dominant species in primary benthic communities of the oligotrophic location BSS — Big Snail South). We provide the photodocumentation of Chroococcusmorphotypes as an example of one of the most diversified genus of coccoid cyanobacteria (Fig. 3). 3. Cyanobacterial microflora of Belizean marshes has to be considered as specific, and apparently well adapted to this habitat. Of the whole 87 recognized

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Species (* = species known only from Belizean marshes)

Q.

a.)

L)

Buena Vista

B ig Snail South

Table 2. List of coccoid cyanobacterial morphospecies.

=

V

.-6. ..

•.-.=

.._, —,

a

III

x

X

X x

XX

2

0o= •=0

..ca) U = ..=Uas

Aphanocapsa circumtecta Kom. et KOM.-LEGN.* A. cf. intertexta GARDNER A. venezuelae SCHILLER Aphanothece bacilloidea GARDNER A. bacteriosa Kom. et KOM.-LEGN.* A. comasi KOM.-LEGN. et TAVERA A.

X X X

XX XX X XX X

X

XXX XX XX XX X XX xx

granulosa (GARDNER) Kom. et

Xx

x x x

XX

XX

X

XX

XX XX

XX XX

XX XX

X

X

XX

xx

XX

x

xx

X

x

XX

X

xx xx x x

x x

KOM.-LEGN. A. hardersii SCHILLER A. subsalina Kom. et KOM. -LEGN.* A. variabilis (SCHILLER) Kom. A. sp.* Asterocapsa belizensis Kom. et

X

X

xx

X

xx

xx xx x x

XX

X

Xx

x xx

KOM.-LEGN.*

radiata (GARDNER) Kom. et KOM.-LEGN. A. stagnina Kom. et KOM.-LEGN.* A. sp.* A.

X

X

X

X

x

x

x

XX

X

Chlorogloea cuauhtemocii Kom. et MONTEJANO C. gardneri Kom. et KOM.-LEGN. C. gessneri SCHILLER Chroococcidiopsis sp. Chroococcus maior Kom. et KOM.-LEGN.* C. mediocris GARDNER C. cf. minutus (KÜTZINIG) NAGEL! C. mipitanensis (WOLOSZ.) GEITLER C. occidentalis (GARDNER) Kom. et KOM.-LEGN. C. Cf. occidentalis GARDNER C. pulcherrimus WELSH C. cf. subsphaericus GARDNER Coelomoron microcystoides Kom. Cyanobacterium sp. 1* Cyanobacterium sp. 2*

X

XX XX

X

XX XX X

XX XX XX XX XX

X X X X XX X XX x x x X

X

x

X

x x

X

X

XX

X

xX

X X X x X X X X X

XX

xx xx xx xx

x xx xx

XX

X X

X

XX x xx xx

X

X

XX X

x xx x x

xx x x Cyanobium sp.* XX Cyanosarcina sp. * x x x x Eucapsis densa 4,-.7EvEDo et al. X XX XX XX X XX XX Gloeothece cf. interspersa GARDNER XX XX X G. cf. opalothecata GARDNER XX G. cf. parvula GARDNER XX XX X G. cf. zulanirae WERNER et SANT'ANNA Gomphosphaeria semen-vitis Kom. X X Johannesbaptistia pellucida (DICKIE)

XX XX XX XX

xx xx x x x x x X X X X X X X X X X X

X

XX XX XX X

TAYL. et DROUET

Lemmermanniella uliginosa Kom. et

X

xx

KOM.-LEGN.*

Merismopedia punctata MEYEN Onconema paludosum Kom. et

X X

X

X X X X X X

X

KOM.-LEGN.*

Rhabdogloea yucatanensis Kom.

et

X

Synechococcus aggregatus Kom. et

X

x

x

x

KOM.-LEGN. KOM.-LEGN. * S. sp. * X

—

rare occurrence, xx — common, xxx — dominant

xx x XX X x

X

XX XX XX

x x


271

Geitlerinema sp. * ("serpens") G. splendidum (GREv. ex GOM.) ANAGN. Halomicronema sp.?* Komvophoron sp. 1* (larger) K. sp. 2* (smaller)

Leibleinia sp.* Leptolyngbya angustissima (W. et G. S. WEST) ANAGN. et Kom. L. mucosa (GARDNER) ANAGN. et Kom. L. sp. 1* ("eliskae") L. sp. 2* ("subsalina") Lyngbya cf. intermedia GARDNER L. cf. martensiana MENEGH. ex Gom. L. minor (GARDNER) comb. nov. L. cf. ocreata GARDNER L. cf. splendens GARDNER Oscillatoria jenensis SCHMED O. levis (GARDNER) comb. nov. O. miniata [ZANARD.] HAUCK ex Gom. O. sp.* ("pseudookenii") Phormidium granulatum (GARDNER) ANAGN. P. sp. Planktothrix sp. Pseudanabaena sp. 1*

Little Belize

Buena Vista


B ig SnailSouth

Tab. 3. List of oscillatorialean morphospecies.

xx x x x

x xx x x x

(i)

U O as

-C

U

xx xx xx

X

x x

xx x

x xxx xx x x xxx xxx xxx xx xx

xxx xx xx xx xx

x xx xx

x x x x xx xx x xx xx xx x

xxx xxx xx xx

xx xx xx

xxx xxx xxx xxx xx xxx xx x xxx xx xxx xx xxx x xx xxx xxx X

xx

x x xx xx xx xx

xx xx xx

X

XX

X

x

X

X

X

XX

x x

x

x

x

x x x x xx xx xx x

x xx

x

xx xx xx xx

x x xx xx x xx xx xx xx xx XX

x

x

xx xx

xx xx xx

x x x x x x

x

x

xx

x xx

x

xx

x

x xx

("apiculato-flexuosa") P. sp. 2* ("belizensis") x x x x Schizothrix sp. 1* S. sp. 2* (brown sheaths) x xx xx x x Spirulina tenerrima KÜTZ. ex Gom. x X

—

rare

xx x

xx xx xx x xx x

xx xx xx x x xx xx xx

occurrence, xx — common, xxx — dominant

morphospecies, only 6 (7) % were found to have a wider, more or less "cosmopolitan" distribution. 20 % of species is pantropical, and 40 % of species are known from similar biotopes (alkaline habitats) only from the Caribbean region. More than 30% of species were not identifiable according to the available literature (Table 5) and should be taxonomically defined. 4. A distinct similarity of species composition was found between alkaline Belizean marshes, and ecologically corresponding habitats in the Caribbean region, namely with the Everglades in Florida — USA (VYmAzAL et al. 2002, our original data), coastal areas in SW Cuba (including Zapata-peninsula and Ciénaga de Lanier — Isla de Pinos/de la Juventud; orig. data), Los Ayes Islands near Venezuelan Coast (SCHILLER 1956) and Jamaica (CRONBERG 1983). Several specific names that could be used for morphological and ecological identification of some morphotypes from Belizean marshes were described from limestone habitats in the Caribbean region (GARDNER 1927, SCHILLER 1956, KomiiREK & NOVELO 1994).

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Chan Chen

Litt le Belize

Buen a Vista


B ig Sn a il South

Tab. 4. List of heterocytous morphospecies.

Cylindrospermum bourrellyi Kom. C. breve WELSH

Fortiea monilisporaKm. F. sp. * ("ciliosa") Gloeotrichia sp.* ("aurantiaca") Hapalosiphon sp.?* Hassallia sp.1* ("clavata") H. sp.2* ("maya") Microchaete sp. * ("articulata") Nostoc sp. Scytonenta sp.1* ("belizensis") S. sp. 2* Stigonema sp.* ("eliskae") Tolypothrix willei GARDNER Trichormus cf. luteus GARDNER X

X

X

x

x

x

xxxx

XXXX x xx xx X x

xx x x xx x xx x x x x x x x xx xx xx xx

x xx xx

xx

xx

xx x XX XX

X

XX

xx X

XX XX

X

X

X

— rare occurrence, xx — common, xxx — dominant

5. An assessment of cyanobacterial diversity using a molecular approach was performed on selected Belizean marshes. The study involved the characterisation of isolated strains and of natural samples from Big Snail South (BS), Buena Vista (BV), Cane (CA), Chan Chen (CC) and Little Belize (LM) marshes. Moreover, to extend the study of cyanobacterial diversity in the area, samples from subaerophytic (OW) and littoral (PL) habitats were also examined. Out of a total of 171 isolated strains, 27 complete 16S rDNA sequences from filamentous strains have been included in the analysis up to date; they are reported in Table 6. Sequencing work on unicellular strains has not yet been completed. Among the 2'7 available sequences, 25 come from marsh habitats, two from subaerophytic habitats. The phylogenetic analysis of Belizean marsh filamentous cyanobacterial strains is presented in Fig. 4. To build the tree, reference sequences from the public domain have been added. 6. The cyanobacterial community composition of Belizean marshes was studied with Temperature Gradient Gel Electrophoresis (TGGE) of partial sequences of cyanobacterial 16S rDNA from four marshes. Additional samples from subaerophytic and littoral habitats were also studied. Separate experiments using primers 16S781R (a) and 16S781R (b) were performed as explained. Obtained bands per primer and per habitat are given in Table 6. After separation by TGGE, bands were extracted and sequenced and the sequences obtained from Belizean marsh samples were added to the tree in Fig. 4.


273

Fig. 3. Example of morphological diversity of Chroococus-types from Belizean marshes: a — Chroococcus major, b C. mediocris, c C. minutus, d C. mipitanensis, e — C. occidentalis, f — C. cf. occidentalis, g — C. pulcherrimus, h — C. subsphaericus. —

—

—

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J. KOMAREK et al.

Table 5. Numbers and percentage of cyanobacterial species in benthic communities in alkaline marshes in N Belize. Number of morphospecies

87

Species with cosmopolitan distribution Pantropical species Species known from Caribbean region Unidentifiable species

6 18 34 29

7% 20% 40% 33%

Discussion The cyanobacterial assemblages from Belizean inland marshes were found highly diverse, when evaluated by traditional phenotype taxonomy (REJm.ÁNKovik et al. 2004). Species richness of cyanoprokaryotic organisms followed the conductivity gradient (from 4000 p.S cm -i). From our results, it follows that the cyanobacterial microflora from such special habitats as alkaline marshes is specialized, containing numerous adapted forms, possibly endemic for this habitat. Of the 87 recognized morphospecies, only 6(7)% has more or less common ("cosmopolitan") distribution, 20 % of morphospecies are pantropical (or more widely distributed in tropical regions), and about 40 % are known only from similar habitats in Central America. This ecologically unique cyanobacterial microflora is very sensitive to nutrient enrichment and the species richness decreases dramatically as a result of eutrophication (REJmANKovA & KomARKov4k 2000). Similar cyanobacterial communities were found at corresponding locations in USA (Florida-Everglades), Puerto Rico, Cuba (SW coastal areas, Zapata peninsula, Ciénaga de Lanier), Venezuela (Los Ayes Islands), Jamaica, and S. Mexico (S Veracruz, Tabasco, S Yucatán) (VYMAZAL et al. 2002, GARDNER 1927, KOMAREK 1989a, 1989b, 1995, SCHILLER 1956, KOMAREK & NOVELO 1994, CRONBERG 1983, etc.). The alkaline marshes in the Caribbean district in Central America represent therefore a specialized habitat with numerous morpho- and ecospecies, developed only in this ecosystem type. The preliminary list of 87 taxa from N-Belizean marshes was presented in RESMANKOVA et al. (2004). In comparison to this review, further studies were performed. Revision of numerous other samples yielded material for re-evaluation of coccoid species; the resulting revision will be published in the paper by KOMAREK & KomÁRKovi, (in press). Our list contains the proposed revised names. Further changes in determination are expected in both trichal groups (oscillatorialean and heterocytous types), for which the corrections according to molecular approach will be available. Of 87 cyanobacterial taxa observed in natural samples, 171 strains were isolated, mainly from the dominant group of filamentous non-heterocytous (oscillatorialean) genera. 27 available filamentous strains were subjected to a polyphasic characterization, including morphological identification and characterisation (still to be completed) and phylogenetic affiliation determined by 165 rDNA se-


275

r_i_ Aphanizomenon Anabaena spiroldes, PMC9702 sp., PCC7905 Anabaena Iemmermannli, 202A2 Nodularla harveyana, Lukesova18-1994 Tolypothrlx sp., 3BDO2S02 Anabaena sp., PCC7108 Cylindrospermum sp., PCC7417 Nostoc muscorum, NMB11 —I Nostoc sp., PCC73102 Anabaena sp., PCC73105 Cyllndrospennopsis raciborskli, AWT205 1 Fischerella sp., 30W05S02 ' FIscherella sp., 30W05803 Flscherella muscicola, PCC 7414 ChloroglceopsIs sp., PCC 6718 Belizean marsh TGGE band, 3BSO4G1L1A Belizean marsh TGGE band, 313V01G1L1A Belizean marsh TGGE band, 3LMO3G1L2A Belizean marsh TGGE band, 3CCO2G1L2A Belizean marsh TGGE band, 3LMO3G1L3AR -I- Belizean marsh TGGE band, 3LA404G1L1AF Belizean marsh TGGE band, 3L8803G1L1A Belizean marsh TGGE band, 3BSO3G1L2A Belizean marsh TGGE band, 3BSO3G1L5A Belizean marsh TGGE hand, 3BSO3G1L3A Belizean marsh TGGE band, 3BSO3G1L4A Belizean marsh TGGE band, 36SO3G1L1A I Calothrlx desertica", PCC 7102 Calothrix sp., PCC7714 Scytonerna hofmanni, PCC 7110 Stigonernatacean type, 38S06304 Stigonematacean type, 313806805 Belizean marsh TGGE band, 3CCO2G1L1A Belizean marsh TGGE band, 3BSO1G1L1A Belizean marsh TGGE band, 38V0801L2A [ Belizean marsh TGGE band, 3CCO1G1L1A Geitleribactron sp., 181314809 Synechococcus sp., PCC7902 OscillatoriatLyngbya, 3BV01S06 Phormidium sp., 3LM05804 — Belizean marsh TGGE band, 3CCO2G2L28 Phormidiurn cf. granulatum, 3CC03501 Lyngbya sp. thick, 3CC503 OscillatoriaiLyngbya, 3CAS03 Lyngbya sp., 3CAS02 Lyngbya sp. thick, 3CASOI i Lyngbya cf. martensiana, 3CCO3SO4 ' Oscillatoria sp., 3CC03502 Phormidium ambiguum, M-71 1--- Belizean marsh TGGE band, 3LMO3G1L3AF Belizean marsh TGGE band, 3LMO481L1AR Pseudanabaena sp., PCC 7367 r— Leptolyngbya, 08824812 Pseudanabaena, 015819519 Belizean marsh TGGE band, 3HMO1G2L1B F Belizean marsh TGGE band, 31550182U8 1— Belizean marsh TGGE band, 38S06G2L28 ; Belizean marsh TGGE band, 38S0682L18 Komvophoron sp., 38V01S15 ; Leptolyngbya sp., 31805311 Leptolyngbya, 18808805 marsh TGGE band, 3 881KG2L1 8 423eliziean B inzaebar2 marsh TGGE band, 313S0162L3B ena sp., 38V01S013 1 Pseudea Schizothrix sp., 38V01807 Komvophoron sp., 31805306 Pseudanabaena/Komvophoron, 3BV01504 Belizean marsh TGGE band, 3BV02G1L1A Belizean marsh TGGE band, 3BV03G1L1A Pseudanabaena sp., 3CCO4S01 F Leptolyng bya foveolarum, Komarek 1964111 Phorrnldium sp., M-99 Komvophoron sp., 3CCO1S08 Leptolyngbya sp., 3CC04305 Leptolyngbya, 0131319512 Leptolyngbya, 08830502 Oscillatoria sp., Zwart Leptolyngbya sp., PCC 7375 Plectonema sp., F3 Pseudanabaena/Komvophoron, 3LM05S05 Prochlorococcus marinus subsp. pastoris, PCC 9511 Pseudanabaena/Komvophoron, 3LM502 Belizean marsh TGGE band, 3CCO1G2L1B Belizean marsh TGGE band, 3CCO282L113 Be pi ean 7us marsh TGGE band, 3LMO3G2L1B sein PCC Lyrigbya sp., PCC 7419 Oscillatoria sp., CYA126 Oscillatoria/Phonnidium, 3CCS02 Phorrnidium pseudo-okenil, 3CC303 Phormidium sp., 3LMO5S01 Halothece sp., MP! 96P605

H

1

J-

eas

E

re re re es

e.al a-

2, sus

ednema sp.,Í

in

)n. py )s-

.

{-11

MicroLcYn ole gubrsp s.P; P OE N CC IH7340290 Belizean marsh TGGE band, 3CC03G2L4B Spirulina major, 08822509 Synechococcus sp., PCC7117 Synechocystis sp., PCC6805 Belizean marsh TGGE band, 3CC03G2L315 Microcystis sp., PCC7941

oo-

.a;ic Dn ;e-

Fig. 4. Neighbour joining phylogenetic tree of 16S rDNA sequences of Belizean marsh cyanobacteria. The tree includes complete 16S rRNA sequences of isolated cyanobacterial strains and partial 16S rDNA sequences of TGGE fragments obtained from the cyanobacterial community DNA. Sequences not coming from Belizean samples have been retrieved from public sources. Codes referring to Belizean cyanobacteria: CA — Cane marsh; CC Chan Chen marsh; BV Buena Vista marsh; LM Little Belize marsh; BS Big Snail South marsh; HM Hidden marsh; OW subaerophytic habitat. —

—

—

—

—

—

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et al.

Table 6. Cyanobacterial strains which 16S rRNA has been sequenced and analysed; TGGE bands obtained and sequenced subdivided per site. Site Sequenced cyanobacterial strains CA

OW

BS BV

LM

Lyngbya sp. thick Lyngbya sp. Oscillatoria / Lyngbya Fischerella sp. Fischerella sp. Stigonematacean type Stigonematacean type Pseudanabaena / Komvophoron Oscillatoria/Lyngbya Schizothrix type Pseudanabaena sp. Komvophoron? Phonnidium sp. 1 Pseudanabaena / Komvophoron Phormidium sp. 2 Pseudanabaena / Komvophoron Komvophoron? Leptolyngbya?

Code 3CAS01 3CA502 3CAS03 30W05S02 30W05S03 3BS06SO4 3BS06S05 3BV01SO4 3BV01S06 3BV01S07 3BV01S13 3BV01S15 3LMO5S01 3LMS02 3LM05SO4 3LM05S05 3LM05S06 3LMO5S11

PL

CC

Lybgbya sp. thick Oscillatoria / Phormidium Phormidium pseudookenii Komvophoron? Phormidium granulatum Oscillatoria sp. Lyngbya cf. martensiana Pseudanabaena sp. Leptolyngbya?

3CCS01 3CCS02 3CCS03 3CC01508 3CCO3S01 3CC03S02 3CC03SO4 3CCO4S01 3CC04S05

TGGE bands

CYA781R(b)=7 CYA781R(a)=6 CYA781R(b)=6 CYA781R(a)=7 CYA781R(b)=0 CYA781R(a)=4

CYA781R(b)=6 CYA781R(a)=5

CYA781R(b)=2 CYA781R(a)=0 CYA781R(b)=9 CYA781R(a)=3

quence analysis. Among these strains, only two are heterocytous, while the other

are oscillatorialean cyanobacteria. The latter group is spread in all the principal branches of the phylogenetic tree in which the polyphyletic oscillatorialean cyanobacteria are subdivided. No publicly available sequence was found to share a high homology with any Belizean sequence. Strains from the same marsh can be found in different tree branches but in one case a large subcluster housing only Belizean marshes sequences was made; this cluster has a low relatedness with the sequence of Phormidium ambigU um strain M71 (the more or less thermophilic morphospecies). Morphological characterization of the isolated cyanobacterial strains and determination of the complete sequence of their 16S rRNA gene is still in progress. In parallel to this study, fingerprints of the cyanobacterial community have been obtained with TGGE profiling of a fragment of the 16S rRNA gene amplified from environmental DNA. From 32 TGGE bands analyzed, 19 different


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cyanobacterial genotypes were found up to now, representing major cyanobacterial lineages in the marshes. In several cases, the genotype units coincide with morphologically identified taxa, but at the present stage of the work, genotypic diversity recognized through the study of isolated strains (and compared with natural morphotypes) is definitely larger than the diversity found from the analysis of environmental DNA. Work is still in progress to target the less represented members of the cyanobacterial community and to obtain representative TGGE fingerprints of the community. Our results confirmed that the cyanobacterial flora is very diverse in different habitats over the world and is strictly related to specialized ecologies. Tropical and extreme biotopes are especially rich in not yet known types, whose designation by names of known species from habitats with different ecology and from temperate zones is problematic. The investigation of tropical cyanoprokaryotes should be therefore enhanced. The relationships between morphological and genetic diversity is indisputably the necessary methodological approach to their study, yet its reliability has to be improved. Our contribution clearly confirmed the close relations among different moiphotypes and their genetic characterization. Acknowledgments The study was supported by NSF grant no 0089211, the Czech grants Kontakt MŠMT no. ME-653, GA AS CR no. IAA6005308 and KSK6005114. We thank particularly Prof. Dr. Eliška REDAANKovii (Univ. of California, Davis) for the invitation to this project, friendly collaboration and all the help.

References BEARD S. J., HANDLEY B. A., HAYES P. K. & WALSBY A. E. (1999): The diversity of gas vesicle genes in Planktothrix rubescens from Lake Zürich. — Microbiology 145: 2757-2768. CRONBERG G. (1983): Plankton of the Negril and Black River Morasses, Jamaica. — App. III to BJÖRK, S.: Environmental Feasibility Study of Peat Mining in Jamaica, 129 pp. GARDNER N. L. (1927): New Myxophyceae from Porto Rico. — Mem. New York Bot. Gard. 7: 1-144. HROUZEK P., VENTURA S., LUKEšOVA A., MUGNAI M. A., TURICCHIA S. & KOMAREK J. (2005): Diversity of soil Nostoc strains: phylogenetic and phenotypic variability. — Arch. Hydrobiol./Algol. Stud. 117 (Cyanobacterial Research 6): 251-264. KOMAREK J. (1989a): Studies on the Cyanophytes of Cuba 4-6. — Folia Geobot. Phytotax., Praha, 24: 57-97. — (1989b): Studies on the Cyanophytes of Cuba 7-9. — Folia Geobot. Phytotax., Praha, 24: 171-206. — (1995): Studies on the Cyanophytes (Cyanoprokaryotes) of Cuba 10. New and little known chroococcalean species. — Folia Geobot. Phytotax., Praha, 30: 81-90. KOMAREK J. & NOVELO E. (1994): Little known tropical Chroococcus species. — Preslia, Praha, 66: 1-21. KOMAREK J. & KomiaKovii J. (in press): Taxonomic evaluation of cyanobacterial microflora from alkaline marshes of northern Belize. 1. Phenotypic diversity of coccoid morphotypes. — Nova Hedwigia.

278

J. KOMAREK et a1.

NOBEL U., GARCIA-PICHEL F. & MUYZER G. (1997): PCR primers to amplify 16S rRNA genes from Cyanobacteria. Appl. Environm. Microbiol. 63: 3327-3332. REnviANKovii E., KOMAREK J. & Komialcovi, J. (2004): Cyanobacteria a neglected component of biodiversity: patterns of species diversity in inland marshes of northern Belize (Central America). Diversity and Distributions 10: 189-199. RanviANKovii E. & KomARKovii J. (2000): Function of cyanobacterial mats in phosphorus — limited tropical wetlands. — Hydrobiologia 431: 135-153. REJmANKovis. E., POPE K. O., POST R. & MALTBY E. (1996a): Herbaceous wetlands of the Yucatan Peninsula: Communities at extreme ends of environmental gradients. — Intern. Rev. ges. Hydrobiol. 81(2): 223-252. REIMANKOVA E., ROBERTS D. R., MANGUIN S., POPE K. O., KOMAREK J. & POST R.A. (1996b): Anopheles albimanus (Diptera: Culicidae) and cyanobacteria: An example of larval habitat selection. — Popul. Ecology 25: 1058-1067. RIPPKA R., DERUELLES J., WATERBURY J. B., HERDMAN M. & STANIER R. Y. (1979): Generic assignments, strain histories and properties of pure cultures of cyanobacteria. — J. gen. Microbiol. 111: 1-61. SAITOU N. & NEI M. (1987): The neighbour-joining method: a new method for reconstructing phylogenetic trees. — Mol. Biol. Evol. 4: 406-425. SCHILLER J. (1956): Die Mikroflora der roten Tümpel auf den Koralleninseln "Los Ayes" im Karibischen Meer. — Ergebn. Dtsch. limnol. Venezuela-Exped. 1952, 1: 197-216. TATON A., GRUBISIC S., BRAMBILLA E., DE WIT R. & WiLmoTrE A. (2003): Cyanobacterial diversity in natural and artificial microbial mats of Lake Fryxell (McMurdo Dry Valleys, Antarctica): a morphological and molecular approch. — Appl. Environm. Microbiol. 69: 5157-5169. VYMAZAL J., KOMARKOVA J., KUBEČKOVÁ K. & KAšTOVSKÝ J. (2002): [Periphyton composition change along the phosphorus gradient in the Florida Everglades.] — Czech Phycology, Olomouc, 2: 83-92. WATANABE K., TERAMOTO M., FUTAMATA H. & HARAYAMA S. (1998): Molecular detection, isolation, and physiological characterization of functionally dominant phenol-degrading bacteria in activated sludge. — Appl. Environm. Microbiol. 64: 4396-4402. —

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The authors'addresses: Prof. Dr. Jikf KOMÁREK, Academy of Sciences CR, Institute of Botany, Dukelská 135, CZ-379 82 Třeboři, Czech Republic Dr. STEFANO VENTURA, Dr. SILVIA TURICCHIA, Dr. CRISTINA MASCALCHI, Dr. ELISA SOLDATI, CNR-Institute for Ecosystem Study, Section of Firenze, via Madonna del Piano, 1-50019 Sesto Fiorentino F1, Italy Dr. JAROSLAVA KOMARKOVA, Academy of Sciences CR, Institute of Hydrobiology, Na Sádkách 7, CZ-370 05 České Budějovice, Czech Republic