'kopara' microbial mats in Rangiroa, Tuamotu Archipelago, French Polynesia

22 Pages·2006·1.27 MB·English

by  Laurent Richert

e
sit
b
e
W
her EVoulruompee 4a1n, NJoumurbnear l3 o/Afu Pghusytc 2o0l0o6g :y 2 59-279 ArchimArechr ivhett pIn:/s/wtitwutwio.infrneemllee rd.fer/ dl’oIfcreemleecr/
s
bli http://dx.doi.org/10.1080/09670260600804868
u © 2006, MetaPress
p
e
h
n t The original publication is available at http://www.tandf.co.uk/journals/
o
e
bl
a
ail
v
a
s Cyanobacterial populations that build ‘kopara’ microbial mats in
n i
o Rangiroa, Tuamotu Archipelago, French Polynesia
si
r
ve
ed Laurent Richert1, Stjepko Golubic2,*, Roland Le Guédès3, Annie Hervé3 And
at Claude Payri1
c
nti
he 1 Laboratoire d’Ecologie Marine, Université de la Polynésie Française, Papeete, Polynésie Française
ut 2 Biological Science Center, Boston University, 5 Cummington Str., Boston, MA 00215, USA
er-a 3 IFREMER, Centre de Nantes, Laboratoire VP/PBA, BP 21105, 44311, Nantes, France
h
s
bli
u
p
e *: Corresponding author : Correspondence to: Stjepko Golubic, Tel.: [phone]. Fax.:
v [phone]. e-mail: [email protected]
niti
efi
d
e
h
T
w.
e
vi Abstract:
e
er r
e Cyanobacterial populations, the primary producers and builders of ‘kopara’ microbial mats were
p
g studied in four selected ponds along the rim of the Rangiroa Atoll, French Polynesia using a
n
wi polyphasic approach. Seven isolates were maintained in uni-cyanobacterial cultures, characterized
o
oll morphotypically and phylogenetically by evaluating sequences of the 16S rRNA gene of about 620
n f base pairs in length. Cyanobacteria in natural populations were analyzed microscopically,
atio characterized morphotypically, and compared with cultured strains. Three of the isolates were
c identified in the field samples: Lyngbya aestuarii, Johannesbaptistia pellucida and Chroococcus
ubli submarinus were present in the mats only as minor components, whereas the species of Schizothrix
p
or that dominated the mat community could not be cultured. The sequence of Johannesbaptistia
d f pellucida is published for the first time. The phylogenetic and taxonomic relations are discussed on the
e basis of a reconstructed phylogenetic tree in relation to morphotypic characters. Sequences of Kopara
pt
e isolates plot separately from those cultured from the lagoon of the neighbouring atoll Tikehau,
c
ac indicating a narrow niche differentiation of benthic cyanobacterial taxa. The results support the
cle application of a polyphasic approach to characterization, ecology and diversity of cyanobacteria.
arti
n
a
of
F Keywords: culture, cyanobacteria, diversity, kopara, microbial ecology, microbial mats, phylogeny,
D
P polyphasic approach, 16S rRNA
d
e
c
u
d
o
pr
or-
h
ut
a
n
a
s
s i
hi
at t
h
e t
ot
n
e
s
a
e
Pl 1
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
Eur. J. Phycol., (2006), 41(3): 1–21
Cyanobacterial populations that build ‘kopara’ microbial mats in
Rangiroa, Tuamotu Archipelago, French Polynesia
5 LAURENT RICHERT1, STJEPKO GOLUBIC2, ROLAND LE GUE´ DE` S3, ANNIE HERVE´ 3 AND
CLAUDE PAYRI1
1Laboratoire d’Ecologie Marine, Universite´ dela Polyne´sie Franc¸aise, Papeete,Polyne´sie Franc¸aise
2Biological Science Center,Boston University, 5 Cummington Str.,Boston, MA 00215, USA
3IFREMER, CentredeNantes, Laboratoire VP/PBA, BP 21105, 44311, Nantes,France
10 (Received 24December 2005; accepted 16May2006)
Cyanobacterial populations, the primary producersand buildersof ‘kopara’microbial matswere studiedin fourselected
pondsalongthe rim ofthe Rangiroa Atoll, French Polynesia usinga polyphasic approach. Seven isolateswere maintained
inuni-cyanobacterialcultures,characterizedmorphotypicallyandphylogeneticallybyevaluatingsequencesofthe16SrRNA
geneof about 620base pairs inlength. Cyanobacteria in natural populations were analysedmicroscopically, characterized
15 morphotypically, andcomparedwith culturedstrains. Threeof the isolates wereidentified in the field samples: Lyngbya
aestuarii,JohannesbaptistiapellucidaandChroococcussubmarinuswerepresentinthematsonlyasminorcomponents,whereas
the speciesof Schizothrixthat dominated the mat communitycould notbecultured. Thesequence ofJohannesbaptistia
pellucidaispublishedforthefirsttime.Thephylogeneticandtaxonomicrelationsarediscussedonthebasisofareconstructed
phylogenetictreeinrelationtomorphotypiccharacters.SequencesofKoparaisolatesplotseparatelyfromthoseculturedfrom
20 the lagoonof the neighbouring atollTikehau, indicating a narrow niche differentiation ofbenthic cyanobacterial taxa.
Theresults support the applicationof a polyphasic approachto characterization, ecology anddiversity of cyanobacteria.
Key words: culture, cyanobacteria, diversity, kopara, microbial ecology, microbial mats, phylogeny, polyphasic approach,
16SrRNA
25 Introduction 2001; Ru¨tters et al., 2002; Abed et al., 2003a, b),
estuaries (Mir et al., 1991), hypersaline ponds
Microbial mats are stratified communities
(Krumbein et al., 1977), hot springs (Ward et al.,
arranged along steep vertical gradients of light,
1994; Lopez-Cortes, 1999a), and desert soils
oxygen, sulphide, Eh and pH, with each stratum
(Campbell et al., 1989; Garcia-Pichel et al., 2001). 50
dominatedbyspecializedguildsofmicroorganisms
A variety of shallow ponds on land rims of
30 (see Riding & Awramik, 2000; Krumbein et al.,
South Pacific atolls offer good conditions for
2003). The illuminated surface layers are domi-
development of thick microbial mats called
nated by microbial oxygenic and anoxigenic
‘kopara’ by cyanobacteria. This system of ponds
phototrophs, separated by a sharp oxic-anoxic
with variable water supply and a wide range of 55
boundary (Potts; 1980; Stal et al., 1985; Nicholson
salinities provides a good model to study environ-
35 etal.,1987;Golubic,1991),withchemolithotrophs
mentalpreferencesanddifferentiationofmicrobial
distributed across this boundary (Jørgensen &
communities. The study is relevant to our under-
Gallardo, 1999). Under stress conditions, such as
standing of similar ancient microbial systems.
fluctuations in temperature, water supply and
Possible fossil counterparts of ‘kopara’-type mats 60
salinity, cyanobacteria often remain unchallenged
were recognized in Jurassic strata (Tribovillard
40 as the principal primary producers of microbial
et al., 2000) and fossil microbial mats comprise
mats (Golubic, 1994; Golubic et al., 2000).
a significant part of the stromatolite fossil record,
Microbial mat communities are encountered
which dominated early Earth history.
world wide in a variety of marine, freshwater and
The biogeochemical properties of ‘kopara’ have 65
terrestrial environments, including marine subtidal
been extensively studied with respect to relation-
45 and intertidal ranges (Golubic, 1985; Pearl et al.,
ships between organic compounds and mineraliza-
tion processes (Trichet, 1967; De´farge et al.,
Correspondenceto:StjepkoGolubic, Tel.:[phone].Fax.:
[phone].e-mail:[email protected] 1985, 1996; De´farge & Trichet, 1990, 1993;
ISSN0967-0262print/ISSN1469-4433online/06/030001–21(cid:1)2006BritishPhycologicalSociety
DOI:10.1080/09670260600804868
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
L. Richert et al. 2
70 Trichet & De´farge, 1997; Trichet et al., 2001). The
role of proteinaceous compounds and amino acid
composition in controlling calcification was stu-
died in deeper layers of ‘kopara’ mats (Gautret &
Trichet, 2005) and other microbialites (Gautret
75 et al., 2004). The formation of phosphorite
deposits in ‘kopara’ was studied by Jehl &
Rougerie (1995) and Rougerie et al. (1997).
Exopolysaccharides were studied by Rougeaux
et al. (2001) and recently, in conjunction with the
80 present investigation, by Richert et al. (2005). The
ultrastructureofaccumulatedexopolymers(mostly
Fig. 1. Rangiroa Atoll, Tuamotu Archipelago, French
polysaccharides) was studied using Cryoscan scan-
Polynesia with the locations of the studied ‘kopara’
ning electron microscopy (De´farge et al., 1996),
ponds. The neighbouring Tikehau Atoll is seen on the
but only a few studies dealt with the microbial
left. Modified from the NASA image. Scalebar: 10km.
85 composition of ‘kopara’ mats using light micro-
scopy (De´farge et al., 1994a, b; Mao Che et al.,
2001). Thesurfacelayersof‘kopara’ areorange,darkblueish-
This study deals with polyphasic characteriza- green or greyish-black depending on the prevailing
microorganisms,which,inturn,respondtothedegreeof
tion of cyanobacterial populations of ‘kopara’
wetness. Deeper layers are reddish-orange alternating 130
90 microbial mats as the principal primary producers
with white layers encrusted with carbonate (De´farge
in four selected ponds in the north-western part of
et al., 1985, Gautret & Trichet, 2005).
the Rangiroa Atoll, by combining field observa-
tions, microscopic analysis of natural populations
and cultured isolates from these populations. Sampling
95 Itexplorestheapparentandcrypticcyanobacterial
The current study explored microbial composition in
diversity in a series of tropical ponds. The mats in four ponds located in the northwestern part of 135
characterization assesses morphotypic and geno- theRangiroaAtolldesignatedasR2,R59,R60andR63
typic properties, the latter based on partial (Fig. 1). Pond R2, located on the islet Pavete, was
(620 base pair [bp]) sequences of 16S rRNA gene. studiedearlierbyDe´fargeetal.(1994a).MaoCheetal.
(2001) studied ponds R2 and R59 and characterized
their physical and chemical properties. The other two 140
100 Materials and methods ponds,R60andR63werestudiedforthefirsttime.This
studywasinitiatedbytwofieldtrips,inNovember2000
Environmental setting
and November 2001. Pond R59 was sampled during
Rangiroa Atoll in the Tuamotu Archipelago, French both trips and the other three ponds only in 2001.
Polynesia is the second largest atoll in the world In each of the ponds studied, several sampling sites 145
covering 1,763km2 (Fig. 1). It consists of a narrow were identified and selected by colour, texture and
105 landrimsurroundingthecentrallagoon.Thelandrimis degree of wetness of the mats. The basic environmental
segmentedbyshallowchannels,locallycalled‘hoa’,into parameters (pH, salinity and temperature) were mea-
islets called ‘motu’. Most ‘hoa’ are shallow passages, suredandrecordedasapartofthesamplingprocedure.
which link the ocean and the lagoon. Along the atoll’s Square pieces of mats ((cid:1)50cm2) were cut, placed in 150
rim there are numerous isolated shallow ponds, mostly sterile plastic boxes and stored at ambient temperature
110 less than 1m deep, varying in size from tens to several overnight.Theywerethenstoredat20(cid:2)Cinthedarkfor
hundredsofsquaremetres.Thesepondsoriginatedfrom 48h before the isolation procedure. Subsamples were
thecombinedactionoferosionandsedimentation.Parts fixed in 5% formaldehyde solution in environmental
ofthelandrimdamagedduringmajorstormsproduced water and stored in the dark in 5-ml vials for 155
depressions, which were subsequently closed by accu- microscopic analysis.
115 mulatingreefsandandgravelsediments.Thepondsare
fed by rain and, during heavy swells, by seawater from
Isolation and maintenance of cultures
the ocean and from the adjacent lagoon. They support
luxuriousgrowthofmicrobialmats,whichcontributeto A small piece (c. 1g) of mat was excised under sterile
particular organic-rich sediment build-up. The salinity conditionsandhomogenizedinautoclavedenvironmen-
120 in these ponds is quite variable, ranging from nearly tal water; 0.1ml of the suspension was plated on solid 160
freshwater to hypersaline conditions. Majorstorms and Conwaymedium(agar1.3%).TheConwaymediumisa
swells may re-set the salinity to the values prevailing in modified version of the Walne medium (Walne, 1966).
the ocean and/or lagoon, i.e. between 34 and 36 ø. In 1l of natural sea water: NaNO , 100.00mg;
3
A typical structure of the ‘kopara’ mat consists of a Na EDTA, 45.00mg; H BO , 33.60mg; NaH PO -
2 3 3 2 4
125 succession of gelatinous laminae, each a few mm thick, 2H O, 26.00mg; FeCl -6H O, 1.28mg; MnCl -4H O, 165
2 3 2 2 2
forming layered organic-rich sediment 20–50cm thick. 0.36mg; trace metal solution, 1ml; vitamins solution,
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
Cyanobacteria in ‘kopara’ microbial mats 3
50ml; Trace metal solution: ZnCl , 1.05g; CoCl -6H O, culture was centrifuged for 3min at 9,000g, the pellet 225
2 2 2
1.00g; (NH )6Mo O -4H O, 0.45g; CuSO -5H O, rinsed twice with 500ml of H O and re-suspended in
4 7 24 2 4 2 2
1.00g; H O, 50ml; vitamins solution: vit. B1, 400mg; 500ml of 1(cid:4)PCR buffer (Eurogentec). The samples
2
170 vit. B12, 20mg; H O, 100ml. The Petri dishes were were stored at 4(cid:2)C overnight prior to PCR.
2
incubated at 25(cid:2)C with permanent artificial lighting The PCR amplifications were performed with a PTC
(70mmolm(cid:3)2s(cid:3)1) for 1–3 weeks. 100 Programmable Thermal Controller (MJ Research 230
During incubation, the cultures were frequently Inc., Waltham, USA). Fifty picomoles of each primer,
observed under light microscopy for colony picking. 25 nmol of each deoxynucleoside triphosphate, 5ml of
175 The apparently monospecific colonies of cyanobacteria 10(cid:4)PCRbuffer(Eurogentec),0.5mlofTaqpolymerase
and microalgae were picked using micromanipulation (Eurogentec), and 2ml of prepared culture were com-
techniques (Rippka, 1988). The small colonies were binedwithH Otoavolumeof50mlina100-mltesttube 235
2
transferred into liquid medium in plates with 96 wells – and overlaid with two drops of mineral oil (Sigma
each well contained about 200ml of Conway medium. ChemicalInc.,SaintLouis,USA).After10minat94(cid:2)C,
180 Theplateswereincubatedunderthesameconditionsas 35 cycles were carried out as follows: 94(cid:2)C for 1min,
for the Petri dishes. 60(cid:2)C for 1min and 72(cid:2)C for 1min. The amplification
The emerging colonies were checked for monospeci- wasfinishedwiththetubeskeptat72(cid:2)Cfor6minbefore 240
ficity using an inverted microscope, subcultured and storage at 4(cid:2)C. The PCR products were purified using
transferred for maintenance to 30ml of fresh liquid the StrataPrep PCR Purification Kit (Stratagene, CA,
185 medium in 50-ml flasks containing 30ml of double- USA) and sequenced commercially in both directions.
spiked(2(cid:4)conc.)Conwaymediumat22(cid:2)Cundercool- The forward and reverse-complementary of the reverse
white fluorescent light (20mmolm(cid:3)2s(cid:3)1) with a 12:12h sequences obtained were aligned against each other in 245
light-dark cycle. No attempt was made to make the order to check the quality of the sequences. The seven
isolates axenic. Five ml of the stock cultures were consensus sequences were then deposited at EMBL
190 transferred into 25ml of fresh Conway medium every under the accession numbers: AJ621832 to AJ621838.
month and pictures were taken using a Donpisha-
TriCCD (Sony) digital camera mounted on a Leitz
Phylogenetic affiliations
Diaplanmicroscope.Aliquotsofthestockcultureswere
fixed in 3% formaldehyde and stored in darkness at The sequences were first used for a BLAST search 250
195 room temperature for later microscopic comparative
(Altschul et al., 1997) to check whether they were
analysis.
cyanobacterialinorigin.Theywerethenaddedtoatext
file (graciously provided by Annick Wilmotte) contain-
ing 143 complete and 163 partial (>600bp) cyanobac-
Microscopy and morphometry
terial sequences of the 16S rRNA gene. The 16S rRNA 255
Light-microscopic analysis of fixed field samples and sequences of E, coli (J01859), Bacillus subtilis
cultures was carried out by using an AxioStar micro- (AJ276351), and Agrobacterium tumefaciens (D14500)
200 scope(Zeiss,Germany)equippedwithtransmittedlight, were used as the outgroup. The file was edited with
phase contrast and Differential Interference Contrast BioEditv5.0.9software(Hall,1999).Allthesesequences
(DIC, Nomarski) illumination. Additional information were multiple-aligned using ClustalX v1.81 program 260
was obtained by autofluorescence using blue and green (Thompson et al., 1997) prior to construction of the
light excitation. The findings were photodocumented phylogenetic trees with MEGA v2.1 (Kumar et al.,
205 using an AxioCam digital camera attached to the 2001). Phylogenetic trees were constructed using
microscope. The measurements were performed with neighbour-joining and maximum parsimony methods.
a Zeiss Universal microscope equipped with an in-scale The distance matrix was computed using Kimura 2- 265
camera lucida. The projections of cell dimensions were parameter model for dissimilarity values, followed by
scanned and subjected to morphometric analysis using the construction of a tree with the neighbour-joining
210 Sigma-Scan measuring software (Jandel Scientific, method (Saitou & Nei, 1987); these calculations were
Sausalito CA). The dimensions are given as mean (cid:5) subjected to bootstrap analysis with 1,500 replicates.
standard deviation (number of measurements). The Themaximumparsimonycalculationwasalsosubjected 270
species dominance is expressed numerically based on to bootstrap analysis (500 replicates).
percentage coverage in compressed microscopic pre-
215 parations,assumingthesameproportionalityinvolume.
This estimate follows botanical practice as modified for Morphotype identification
microorganisms (Golubic, 1967).
The collected and cultured cyanobacteria were char-
acterized morphotypically and identified to the species
level, using traditional phycological determination 275
PCR amplifications
manuals of Gomont (1892), Bornet & Flahault
PCR was carried out without DNA extraction to (1886–88), Tilden (1910), Geitler (1932), Kossinskaya
220 amplify a 16S rRNA gene fragment of about 650bp in (1948), Desikachary (1959) and Umezaki (1961).
length using two of the cyanobacteria-specific primers Generic assignments were made in consultation with
designed by Nu¨bel et al. (1997), CYA106F and newer phycological (Anagnostidis & Koma´rek, 1985; 280
CYA781R corresponding to equivalent positions of Koma´rek & Anagnostidis, 1989, 1999, 2005; Koma´rek,
the E, coli genome. One ml of exponentially growing 1994) and bacteriological systems (Rippka et al., 1979;
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
L. Richert et al. 4
Fig. 2. ‘Kopara’ microbial mat
in pondR59 onthe NWrim
of RangiroaAtoll. Note the
colour changes fromthe wet
centreto the dryermargins
of the pond.
Castenholz et al., 2001). Distinct populations of species Incontrast,pondsR2(Fig.3)andR63exhibited
that could not be related to published and named taxa consistently low salinity correlated with their 320
285 were identified to the genus level with a species isolated position within the land rim of the atoll
designation marked provisionally by a number.
(Table 1). De´farge et al. (1994b) noticed a rain-
dependent decrease of salinity in pond R2 from
Results 8.9 ø in 1983 to 1.0 ø in 1992. We measured a
salinityof7.8øinNovember2001.PondR63was 325
The ‘kopara’ ponds as microbial habitats
a well protected depression separated from both
The ‘kopara’ ponds studied could be divided into ocean and the lagoon and surrounded by land
290 twogroupsonthebasisoftheirprevailingsalinity. vegetation. It was in part deeper than 1m and fish
Ponds R59 (Fig. 2) and R60 are characterized by were observed to live in this pond. The ‘kopara’
fluctuating salinity with periodic returns to sea mat was well expressed at the edges of this pond. 330
water values, following recharging during storm Polygon formation and colour distribution was
events. Prolonged calm and dry periods introduce similar to those observed in ponds R59 and R60.
295 hypersaline conditions in these ponds, whereas Pond R2 was larger than pond R63 and the mat
they turn brackish in rainy periods. Pond R60 is was very well developed. The colour of large areas
located in a protected tortuous branch of a ‘hoa’, of the mat surface, however, was different from 335
connected with the lagoon at high tide. The other ponds: itwas greentopale orange inthe wet
‘kopara’ mat of this pond is submerged by parts. The familiar coloration gradient was here
300 lagoonal water only when the tide is relatively compressed against the shore, showing a narrow
high. During extended periods of exposure, the orange zone followed by an equally narrow dark
mat surface dries out, hardens, turns dark grey zone with polygons along the shoreline (Fig. 3). 340
to black in colour and cracks into polygons. Natural populations of cyanobacteria were
A gradient becomes established with relative analysed by light microscopy for species composi-
305 moisture increasing from the edge towards the tion and abundance in ponds R2 and R59, which
centre of the pond. The wet part of the mat is soft were selected as representative local end members
and orange-brown in colour. Pond R59 is larger withrespecttotheobservedsalinitygradient.Pond 345
than pond R60 and is well delimited as it is in an R2, located within the atoll’s rim maintained very
enclosed depression on the land rim of the atoll. It low salinity levels, whereas pond R59 fluctuated
310 is separated from the ocean by several hundred from hypo- to hypersaline conditions relative
metres of land with terrestrial vegetation and from to the surrounding ocean and the nearby lagoon.
the lagoon in places only by a beach ridge The samples from all four ponds were used as a 350
comprised of coral rubble. The ‘kopara’ mat source of inocula for culturing.
covers a larger surface in this pond and the wet-
315 to-dry gradient is better expressed. The cracks are
Field populations of cyanobacteria
deeper and the polygons thicker than in pond R60
and the wet parts in the protected areas are bright The microscopic analysis of field populations
orange. focused on actively growing surfaces of the mat,
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
Cyanobacteria in ‘kopara’ microbial mats 5
Fig. 3. Microbial matzonation
at the margin ofpondR2.
Dried, polygonally cracked
mats in the frontof the picture
are dark(black whenwet) due
to highconcentration of
UV-protective scytonemin pig-
ment. The zonewith bumpy
surface in the centreof the
picture is regularly wettedand
bright orange in colourdue to
highconcentration ofcarote-
noid pigments. Agerminating
coconutserves as a scale.
Table 1. Watertemperature,salinityandpHinfourponds Thick, dark brown sheaths with upward diverging
studiedon RangiroaAtoll. layering surround 8–10-mm wide trichomes. Cells
areshortandcompactedinthemeristematicapical 385
Pond R59 R60 R2 R63
regions becoming longer and torulose in the older,
Temperature((cid:2)C) 33.7–40.0(cid:6) 31.1 32.0 36.6 mature trichome segments. Dry habitats in the
Salinity(ø) 12.0–49.0(cid:6) 27.3 7.8 6.1
ponds with higher salinities (R59-8) were occupied
pH 9.5–8.3(cid:6) 8.4 7.7 8.2
byCalothrixsp.(Figs7,8)andtoalesserextentby
*MeasuredNovember2000. Lyngbya aestuarii (Mertens) Liebman (R59-5). 390
Both taxa are protected by thick, layered,
scytonemin-stained sheaths. Lyngbya aestuarii was
355 lessthan1cmindepth.Samplesineachpondwere successfully transferredintoculture(seebelow).
collected along a gradient of varying water supply Microbial mats in the slightly wetter zone inside
from dry toward wet, as identified by surface the peripheral dark rim around the ponds were 395
colour, wetness, consistency and internal texture intensive yellow-orange in colour due to a high
and by species composition of the mat. concentrationofintracellularcarotenoidpigments.
360 The species composition of the actively growing They were observed in all ponds, but were
surfacelayerofmicrobialmatswasanalysedinfive particularly well developed in Pond R59, where
sets of samples, two from Pond R2 and three the salinity fluctuated between brackish and 400
from Pond R59. The results are presented in hypersaline levels. Scanning electron microscopy
Table 2. Cell dimensions characterizing particular (SEM) of critical-point dried preparations of these
365 populations are listed as cell width(cid:4)length mats showed that they comprised intertwined
(shorter(cid:4)longer cell dimension for coccoid filaments of variable diameter (Figs 9–11). Light
species). Species determination was carried out microscopy revealed that these mats were built by 405
wherever a relationship to published descriptions multitrichomous filaments of two species of
couldbeestablished.Theabundancewasestimated Schizothrix. In pond R59, they were distributed
370 as% coverage (see Golubic, 1967). in a mosaical pattern, in which the larger one,
Differences in species composition and domi- identified as S, splendida (Golubic, 1973) formed
nancewereconspicuouswithineachpondalongthe cushionsupto5cmindiameterelevatedupto1cm 410
gradientofwatersupplyandretention.Thechange abovethewaterlevel(Figs12,13).Thedepressions
in species composition along this desiccation around these cushions were overgrown by the
375 gradient was related with the observed change in smaller species designated here asSchizothrix sp. 2
colour and texture of the mat (Fig. 3). Microbial (Fig. 16; Table 2). S, splendida achieved complete
mats along the margins of ‘kopara’ ponds dominance in dryer portions of the mat (samples 415
were often polygonally cracked by desiccation. R59-5, R59-8). A third species, designated here as
Scytonema cf. myochrous (Dillwin) Agardh Schizothrix sp. 1 (Figs 14, 15) was dominant only
380 (Figs 4–6) occurred in ponds with relatively low inthesemi-drysamplesfrompondR2.Noneofthe
salinity(R2-4).Thisisalargecyanobacteriumwith Schizothrix species encountered in ‘kopara’ mats
25.9(cid:5)2.1 (16)mm wide falsely branched filaments. grew in culture. 420
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
L. Richert et al. 6
Table 2. Cell dimensions andabundance of cyanobacteria in ‘kopara’ponds.
Abundanceineachpondb
Taxon Cellwidth(cid:4)length(mm)a R2-1 R2-4 R59-5 R59-2 R59-8
Schizothrixsplendida. 2.03(cid:5)0.37(196)(cid:4)4.59(cid:5)0.98(175) 1 5 5
Schizothrixsp.1 1.35(cid:5)0.15(52)(cid:4)2.66(cid:5)0.52(52) 2
Schizothrixsp.2 0.88(cid:5)0.17(51)(cid:4)4.13(cid:5)0.90(47) 3 þ þ
Scytonemacf.myochrous 9.02(cid:5)0.87(15)(cid:4)5.11(cid:5)1.02(35) 2
Calothrixsp. 5.89(cid:5)0.98(7)(cid:4)2.61(cid:5)0.58(14) 1 2
Lyngbyaaestuarii 8.92(cid:5)0.37(11)(cid:4)1.82(cid:5)0.44(11) 1
Oscillatoriaspp. na 1 2 2 1 1
Leptolyngbyaspp. na 1 1 1 1 1
Spirulinaspp. na 1 2 1
Johannesbaptistiasp.1 4.93(cid:5)0.63(67)(cid:4)2.74(cid:5)0.65(62) 1 1
Johannesbaptistiasp.2 3.02(cid:5)0.36(227)(cid:4)1.06(cid:5)0.22(223) 1 1
Chroococcussp.1 9.02(cid:5)3.77(5)(cid:4)12.74(cid:5)1.21(5) 1 1 1
Chroococcussp.2 6.22(cid:5)1.70(14)(cid:4)8.08(cid:5)0.81(14) 1 1 1
Chroococcussp.3 3.92(cid:5)0.64(58)(cid:4)5.16(cid:5)0.69(58) 1 1 1
Aphanocapsasp.1 2.42(cid:5)0.31(146)(cid:4)2.83(cid:5)0.33(146) 1 1
Aphanocapsasp.2 2.91(cid:5)0.31(12)(cid:4)2.52(cid:5)0.42(12) 1
Aphanocapsasp.3 4.70(cid:5)0.37(16)(cid:4)5.24(cid:5)0.22(16) 1
Aphanothecesp.1 3.86(cid:5)0.32(107)(cid:4)5.64(cid:5)0.88(105) 1
Aphanothecesp.2 3.82(cid:5)0.36(15)(cid:4)7.74(cid:5)1.39(16) 1
Aphanothecesp.3 2.12(cid:5)0.24(35)(cid:4)2.84(cid:5)0.64(29) 1
Gloeocapsadeusta 2.50(cid:5)0.44(7)(cid:4)3.52(cid:5)0.41(7) þ
Gloeocapsasp.1 3.24(cid:5)0.78(20)(cid:4)4.24(cid:5)0.62(20) 1
Gloeothecesp.1 2.95(cid:5)0.26(24)(cid:4)4.98(cid:5)0.48(16) 1
Gloeothecesp.2 4.58(cid:5)0.61(46)(cid:4)6.71(cid:5)1.06(44) 1
Xenococcussp. na 1
aThesedimensionsareexpressedasmean(cid:5)SD(numberofmeasurments);na¼notapplicable.
b Abundance codes: 5¼75–100% coverage; 4¼50–75% coverage; 3¼25–50% coverage; 2¼10–25% coverage; 1¼<10% coverage, but
frequent;þ¼rare.
Microbial mats in pond R2, with lower and (Figs 27–29). The identification as cyanobacteria
stablesalinity,weresignificantlymorediversethan needed to be confirmed by autofluorescence to 450
those in pond R59. The colour ranged from pale distinguish them from flexibacteria that were also
orange to blueish-green. Populations of different present in the mat (e.g. Fig. 30, upper right).
425 Spirulina species and cyanobacteria with extremely Several small filamentous cyanobacteria occurred
narrow trichomes classified as Leptolyngbya spp. intermingled in the mat as free trichomes. These
alternated in frequency of occurrence without could be classified within the genus Geitlerinema 455
prominent dominance. The group of species listed (Oscillatoria, sensu Geitler).
in Table 2 as Spirulina spp. refers to the following Coccoidcyanobacteriacommonlyoccurredinter-
430 identified morphospecies: S, tenerrima Ku¨tzing, spersed among filamentous members of the mat.
S, subtilissima Ku¨tzing, S, subsalsa Oersted and They included colonial forms with division in one
S,labyrinthiformis(Meneghini)Gomont.Themost planerepresentingdifferentspeciesofAphanothece 460
common were S, tenerrima with helices 1.32(cid:5)0.13 (Figs 31–33), and those dividing in two or three
(69)mm wide and 9–10 windings per 10mm length planes such as Aphanocapsa (Figs 34, 35) and
435 of helix (Figs 17–21) and S, subtilissima with Gloeocapsa(Fig.36),Atleastthreedifferentspecies
helices 2.49(cid:5)0.2mm wide and 6–7 windings per ofChroococcuswereobservedasminorconstituents
10mm length. Several sheathed cyanobacteria with ofthemats,occurringinsmallclusters(Figs37–41). 465
narrow trichomes, classified as Leptolyngbya, less These populations were distinct, with hardly any
than 1mm wide were present in these mats in overlap in size (Table 2) and were considered
440 considerable densities. Some of these morphotypes different species. The largest, Chroococcus sp.1.
are characterized by aerotopes (intracellular clus- (Figs 37, 38), was most likely the source of the
ters of gas vesicles; Figs 22, 23), as determined by cultured strain C, cf. submarinus strain BM. 470
the useofDifferential Interference Contrast (DIC) The two smaller taxa, Chroococcus sp. 2 and 3 did
microscopy. Others have gas vesicle clusters at the not correspond to any known species and may
445 tip of their terminal cells (Figs 24–26). These represent new species. Clusters of cells with
observations need to be confirmed by electron variable dimensions (Fig. 42) possibly belong to
microscopy. Some of these filaments are distin- cyanobacteria with multiple fission, such as 475
guished by their extremely small cell dimensions Xenococcus spp.
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
Cyanobacteria in ‘kopara’ microbial mats 7
Figs. 4–8. Filamentous, heterocystous (N-fixing), falsely branched cyanobacteria responsible for dark colouration of the
mat along the margins of ‘kopara’ ponds. These relatively large microorganisms are embedded into a matrix of
finerfilamentous cyanobacteriaof Leptolyngbyamorphotype.Fig. 4.Filamentsof Scytonemacf.myochrousfrom pondR2,
with dark scytonemin pigment invested in the sheaths. Fig. 5. Club-shaped ‘meristematic’ growth-zone at the tip of a
Scytonema trichome, characterized by compact stacks of short cells. Fig. 6. Central portion of the filament with torulose
trichome cells and a bipolar intercalary heterocyst. Fig. 7. Calothrix cf. pulvinata from pond R59. Fig. 8. Detail of false
branching in Calothrix with differentiated basal unipolar heterocysts. Scale bars: 100mm (Fig. 4), 50mm (Fig. 7) and 10mm
(Figs 5,6, 8).
Two different morphotypes of Johannesbaptistia but most cultured cyanobacteria were identified in
pellucida (Dickie) Taylor et Drouet (listed in natural populations as minor components. 490
Table 2 as Johannesbaptistia sp. 1 and 2) occur The isolation and culturing lead to establishment
480 in separate populations with distinctive size ranges of seven uni-cyanobacterial cultured strains,
(see below). Both morphotypes co-occurred in three of coccoid and four of filamentous
pond R2, but only the larger one was found cyanobacteria:
in pond R59. One strain of this species has been
(1) Chroococcus cf. submarinus (Hansgirg) 495
successfully isolated and grown in culture.
Kova´cˇik – strain BM;
(2) Johannesbaptistia pellucida (Dickie) Taylor et
485 Phylogenetic and morphotypic characterization
Drouet – strain GC;
of cultured isolates (3) Rhabdoderma cf. rubrum (A˚ lvik) Koma´rek et
None of the dominant forms of cyanobacteria Anagnostidis – strain CH; 500
observed in natural samples grew in our cultures, (4) Geitlerinema (Oscillatoria) sp. – strain FE;
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
L. Richert et al. 8
Figs.9–16. Schizothrixspeciesaredominantmat-buildingcyanobacteriain‘kopara’ponds,comprisingmostofthebiomass
of the bumpy brightly orange mats. Fig. 9. SEM of critical point-dried fabric of multitrichomous filaments of Schizothrix
splendidaforming1–2-cmhighcushionsonsurfaceofmat.Figs10,11.Detailsoffabric.Figs12–16.DIC(Nomarskicontrast)
light micrographs. Fig. 12. Multitrichomous filaments of Schizothrix splendida from pond R59. Fig. 13. Filament of
Schizothrixsplendida withsingle trichome andwidetexturedandbirefringent sheath. Fig.14. Trichomeof Schizothrixsp.1,
frompondR2withconicalterminalcell,surroundedbywavy,externallydiffluentsheath.Fig.15.Abundleoffilamentsof
Schizothrix sp.1. Fig. 16. Multitrichomous filament of Schizothrix sp.2 from pond R59. Scale bars: 100mm (Fig. 9), 20mm
(Fig.11) and10mm Figs (10, 12–16).
properties and phylogenetically by analysing
(5) Lyngbya aestuarii (Mertens) Liebman –
partial, ((cid:1)620bp) 16S rRNA sequences. Their
strain LY;
phylogenetic affiliation was first assessed through
(6) Leptolyngbya cf. golenkiniana (Gomont)
theBLASTsearch,whichrelatedthemtocomplete
505 Koma´rek & Anagnostidis – strain FF;
or partial 16S rRNA gene sequences published 515
(7) Pseudophormidium cf. battersii (Gomont)
in the data banks (Genbank, EMBL, DDBJ). The
Koma´rek & Anagnostidis – strain GF.
closest affiliations varied between 99% and less
These organisms were characterized phenotypi- than 92%. The seven sequences obtained were
510 cally by their microscopic morphological then multiple-aligned with 143 complete and
NewXMLTemplate(2006) [15.6.2006–10:36pm] [1–21]
{TANDF_FPP}TEJP/TEJP_A_180430.3d (TEJP) [FirstProof]
Cyanobacteria in ‘kopara’ microbial mats 9
Figs. 17–30. Small filamentous cyanobacteria distributed within the matrix of the ‘kopara’ mats. Figs 17–21. Spirulina
tenerrima occurred in small groups among fine filamentous cyanobacteria. Figs 22, 23. A sheathed Leptolyngbya-type
cyanobacteriumwithcoarseintracellulargasvesicleclusters.Notethesheathedtrichomesonthetopofbothpicturesandthe
hood-like gas vesicle clusters in the terminal cells (also in Fig. 21, upper right). Figs 24–26. A submicron-sized sheathed
‘Leptolyngbya’ morphotype with light-refracting terminal gas vesicle clusters. Figs 27–29. Fine, filamentous ‘Leptolyngbya’
formswithshorter,clearlyseparatedcells.Fig.30.Alarger‘Leptolyngbya’formwiththicksheaths,withoutgasvesicles.Scale
bar: 10mm.
520 163 partial (>600bp) known sequences of the There are at present no 16S rRNA gene
cyanobacterial 16S rRNA gene. The interrelation- sequences available in data banks for the genus
ships were expressed by their positions in the Chroococcus,althoughtwoChroococcusstrainsare
constructedphylogenetictrees.Thetopologyofthe maintained in culture collections, identified as
trees is the same with the two methods used, Chroococcus turgidus PCC 7946 and Chroococcus 550
525 neighbour-joining and maximum parsimony. Only prescottii Drouet and Daily CCAP 1412/4. The
the distance tree is presented here as a simplified sequence of our Chroococcus cf. submarinus strain
outline in Fig. 43.The isolates are markedby bold BM plots distant from all published sequences.
lines and lettering. This strain occupies a position close (8.1%
The total number of nucleotides compared was divergence) to Chroococcus turgidus PCC 9340 in 555
530 600 after elimination of non-informative portions. thephylogenetictreepublishedinBergey’smanual,
Fifteen shorter partial ((cid:1)450bp) sequences of i.e. between clusters VII and VIII of Wilmotte
mat-forming cyanobacteria from the lagoon of & Herdman (2001).Thenextclosestsequencewith
theneighbouringatollTikehau(Abedetal.,2003b) 8.8% divergence is Cyanothece PCC 7424 (isolated
were added for comparison (TK, bold). by P.A. Roger from a rice field, Senegal), which is 560
535 1. Chroococcus cf. submarinus strain BM currently the PCC reference strain for the
(Figs 44–47) is characterized by cells dividing by Cyanothece group, subcluster 1.1.
cleavage in three planes, surrounded by 2. Johannesbaptistia pellucida strain GC
firm gelatinous envelopes. Cells are isodiametric, (Figs 48,49) isacoccoidcyanobacterium, dividing
sometimes mutually flattened at contact, in one plane forming series of cells, often in pairs 565
540 elongated in the course of the division with after division, embedded into a thick smooth
shorter to longer dimensions: 7.78(cid:5)1.09 gelatinous sheath. Cells of the cultured strain are
(59)mm(cid:4)10.35(cid:5)1.28 (59)mm. The cultured wider than long, 10.87(cid:5)1.94 (122) mm wide and
strain occupies by its size an intermediate position 6.12(cid:5)2.14 (116)mm long. They are significantly
between the natural populations of Chroococcus largerthanthecellsinnaturalpopulations(Fig.50; 570
545 sp. 1 and 2. (Figs 37, 38; Table 2). Table 2). As in the case of Chroococcus, there are