Endolithic photosynthetic communitieswithinancient and recenttravertine deposits inYellowstoneNational ParkTracy B. Norris & Richard W. Castenholz

Center for Ecology and Evolutionary Biology, University of Oregon, Eugene, OR, USA

Correspondence: Tracy B. Norris, Center for

Ecology and Evolutionary Biology, University

of Oregon, Eugene, OR 97403, USA.

Tel.:11 541 7298815; fax: 11 541 3462364;

e-mail: tbnorris@yahoo.com

Received 12 October 2005; revised 10 February

2006; accepted 12 February 2006.

First published online 12 May 2006.

DOI:10.1111/j.1574-6941.2006.00134.x

Editor: Gary King

Keywords

endolithic; photo-microbial community;

cyanobacteria; travertine; Yellowstone National

Park.

Abstract

Molecular and culture based methods were used to survey endolithic, photosyn-

thetic communities from hot spring-formed travertine rocks of various ages,

ranging fromo10 to greater than 300 000 years. Much of this travertine contained

a 1–3-mm-thick greenish band composed mainly of cyanobacteria 1–5 mm below

the rock surface. The travertine rocks experienced desiccation in summer and

freezing in winter. A total of 83 environmental 16S rRNA gene sequences were

obtained from clone libraries and denaturing gradient gel electrophoresis. Small

subunit rRNA gene sequences and cell morphology were determined for 36

cyanobacterial culture isolates from these samples. Phylogenetic analysis showed

that the 16S rRNA gene sequences fell into 15 distinct clusters, including several

novel lineages of cyanobacteria.

Introduction

Extremophilic microorganisms are by definition those that

live at the outer limits of the physical parameters that define

the boundaries for life on Earth. One niche that encompasses

a number of extreme physical stresses is the endolithic

environment of rocks of hot and cold deserts. Here we are

using the term endolithic as defined by Friedmann et al.

(1967) as meaning ‘occupying a space within the rock tissue

without an apparent connection to the outer rock surface’, in

contrast to chasmolithic (living in fissures in rocks). The

term cryptoendolithic is used by some authors with the same

meaning as the more general term endolithic (e.g. Sun &

Friedmann, 1999; Hughes & Lawley, 2003). In such environ-

ments microorganisms must endure extremes of temperature

(often including freezing), desiccation, low nutrient supply,

and low photon flux. In most regions these communities are

composed primarily of photosynthetic microorganisms

(Friedmann, 1982; Bell, 1993; Gerrath et al., 1995; Sigler

et al., 2003). Cyanobacteria are commonly the principal

phototrophic members of endolithic communities. However,

these communities include heterotrophic bacteria and often

fungi, protolichens, and micro-algae. Studies of hot desert

and Antarctic endolithic communities have demonstrated

that, in general, endolithic communities are of low species

diversity (Friedmann, 1982; Bell, 1993; Nienow & Fried-

mann, 1993; Wessels & Budel, 1995), especially compared

with soil crusts that are subject to similar extremes but are

generally composed of more complex consortia of micro-

organisms (Garcia-Pichel et al., 2001; Redfield et al., 2002).

Many endolithic communities are characterized by a

1–4 mm thick greenish layer of phototrophs that commonly

occurs 1–5 mm below the upper surface of the rock they

inhabit (Fig. 1); however, the depth may vary considerably,

and in some cases the visible green layer may be spread over

more than 5 mm. The depth is presumably dependent on the

penetrance of light mainly (Matthes et al., 2001). Since

endolithic microorganisms inhabit the spaces between the

mineral particles and crystals they are usually found in

relatively porous and somewhat translucent rocks such as

limestone, sandstone, gypsum and dolomite. In addition to

these periodically dry environments, a thermal, active endo-

lithic community has been reported in extremely acidic, but

moist, siliceous sinter and dominated by unicellular red algae

of the order Cyanidiales (Gross et al., 1998; Walker et al., 2005).

Endolithic microorganisms, in general, probably favor

this environment as it allows them to escape competition by

occupying a niche that is uninhabitable for most other

microorganisms, although growth may be extremely slow

(Sun & Friedmann, 1999). Another possible advantage to

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

living in the rock interior is that it would provide an escape

from harsher conditions at the surface, such as high solar

irradiance (including UV radiation), rapid desiccation, and

abrasion by wind-borne elements. The endolithic environ-

ment may act as a refugium, where light, moisture and

nutrient availability form a fragile but sufficient balance for

life. The combination of these three factors surely dictates

the depth to which endolithic communities live within the

rocks.

Although much early work on endolithic communities

has stressed their prevalence in extremely hot and cold

deserts (Friedmann et al., 1967; Friedmann & Ocampo-

Friedmann, 1984), these types of communities are appar-

ently distributed globally and often in temperate climates as

well (e.g. Gerrath et al., 1995; Van Thielen & Garbary, 1999).

However, the ecology of these communities and their species

composition in temperate climates have rarely been studied.

Ancient and recent travertine deposited by hot springs in

the temperate climate of Yellowstone National Park, Wyom-

ing, USA, harbor an apparently unique endolithic commu-

nity. Travertine is a form of calcium carbonate (CaCO3)

deposited by calcareous springs or rivers, but with numer-

ous variations in porosity, crystal forms, and mineralogy

even within the Mammoth area of Yellowstone (see Fouke

et al., 2000). The July high temperature in this area averages

about 28 1C and often exceeds 34 1C but with nighttime lows

of �4–10 1C and a mean monthly precipitation of 2.0 cm.

The January mean low temperature is �15 1C, with ex-

tremes of �45 1C. Relative humidity values in summer may

commonly drop as low as 14%.

The goal of this work is to provide an initial survey of the

photosynthetic microorganisms that inhabit this extreme

environment. Much of the previous work on endolithic

communities has entailed only microscopy and relatively

little cultivation (Friedmann, 1982) or cultivation without

molecular evaluation, thus revealing mostly morphotypic

information. Only recently have molecular techniques been

applied to describe the community diversity of endolithic

communities (Sigler et al., 2003; Taton et al., 2003; de la

Torre et al., 2003; Walker et al., 2005). The present study uses

molecular methods as well as morphotypic characterization

of culture isolates to survey the taxonomic composition. We

believe studies of endolithic microbial communities are key

areas of investigation in furthering the exploration of past

life on Mars and, most certainly, exploration of early life on

the terrestrial Earth that may have occurred as early as

2.6� 109 years before the present (Watanabe et al., 2000).

Materials and methods

Field site and collections

Dry travertine rock samples were collected from six different

sites in or near the northwestern quadrant of Yellowstone

National Park. Replicate samples for molecular work and

culture isolation were collected in 2002. Samples for mole-

cular work were frozen at �20 1C upon returning to the lab

and dry samples to be used for culture isolations were stored

in plastic containers at ambient temperature in the dark.

Additional collections for cultures were made in 2003. All of

the sample sites are located in the Mammoth Upper Terraces

of Yellowstone National Park, except for the ‘Hoodoos’,

which comprise the travertine rockfall of Terrace Mountain

(above the Mammoth Upper Terraces), formed over 350 000

Fig. 1. Photograph of the Hoodoos area near

Mammoth Springs in Yellowstone National Park,

one of six sampling sites used in this study. The

rockfall in the foreground has come from Terrace

Mountain in the background, an ancient traver-

tine terrace formed over 300 000 yr BP. Inset:

close-up of travertine rock sample from the rock-

fall, demonstrating a subsurface green layer of

endolithic phototrophs, the appearance of which

is typical of the majority of endolithic bands

examined from various sites.

FEMS Microbiol Ecol 57 (2006) 470–483 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

471Endolithic photosynthetic microorganisms

years ago (Sturchio et al., 1994), and the ancient Gardiner

Lower Terraces, which are above the city of Gardiner,

Montana, and outside the boundaries of Yellowstone Na-

tional Park. The global positioning system (GPS) coordi-

nates for each collection site are as follows:

(1) Narrow Gauge Lower Terrace (NGL) 44158016600N

Lat., 110142062500W. Long.

(2) Narrow Gauge Upper Terrace (NGH) 44158018800N

Lat., 110142060900W. Long.

(3) Painted Pool Terrace (PAP) 44157090500N Lat., 1101

42080700W. Long.

(4) Narrow Gauge Ancient Terrace (NGA)

44158014800N Lat., 110142056500W. Long.

(5) Gardiner Lower Terrace (GLT) 45102008000N Lat.,

110141048500W. Long.

(6) Hoodoos (HDO) 44156065200N Lat., 110143004400W.

Long.

Abbreviations following site names were used for naming

the environmental clones and denaturing gradient gel

electrophoresis (DGGE) bands. The sampling locations

included travertine of various depositional ages.

DNA purification

DNA purification from travertine samples was done using a

modification of the method of More et al. (1994). The green

layer occurring 1–2 mm beneath the surface of travertine

samples was removed using a sterilized metal file and razor

blade, after abrasive removal of the upper few millimeters of

rock surface. Two aliquots of �1 g filings were collected for

each sample and transferred to 2 mL screw-cap microcen-

trifuge tubes. Next, 0.75 g of 0.1 mm diameter zirconia/silica

beads (BioSpec Products, Bartlesville, OK) were added to

each tube along with 600mL 120 mM sodium phosphate

(pH 8.0) and 400mL lysis buffer (10% sodium dodecyl

sulfate, 0.5 M Tris-HCl (pH 8.0) and 0.1 M NaCl). Cells

were lysed by shaking for 3 min at high speed on a Mini-

Beadbeater (BioSpec Products, Bartlesville, OK) and then

centrifuged for 3 min at 13 000 g. Supernatant (700mL) was

collected and DNA was precipitated on ice using 2 : 5 (v/v) of

7.5 M ammonium acetate and then centrifuged again. The

supernatant was collected and the DNA was isopropanol

precipitated. Finally, the pellet was washed with 70% ethanol

(�20 1C), air dried and resuspended in 100 mL of 10 mM

Tris (pH 8.0).

Clone library construction

PCR amplification from total community DNA was used for

clone library construction and DGGE. The cyanobacterial

specific primers CYA106F and CYA781R (Nubel et al., 1997)

were used to amplify 16S rRNA gene sequences, resulting in

a product of approximately 675 bp. Each PCR reaction

contained 1.5 mM MgCl2, 0.2 mM dNTPs, 0.5mM of each

primer, approximately 10 ng of template DNA, 0.1 mg mL�1

bovine serum albumin, 2.5 U Taq polymerase and 1�PCR

buffer (Promega, Madison, WI) in a total volume of 50mL.

The PCR amplification cycle was as follows: initial denatura-

tion for 5 min at 95 1C, then 30 cycles of 1 min of denatura-

tion at 94 1C, 1 min annealing at 60 1C, and 1 min extension

at 72 1C followed by a final extension of 7 min at 72 1C. PCR

amplicons were cloned into the PCR 2.1 cloning vector

using the TOPO TA cloning kit (Invitrogen, Carlsbad, CA).

Clone libraries of 60–100 clones were created for each site in

the study. For each library, 12–15 random clones were

selected for re-amplification of vector inserts and commer-

cial sequencing using the cyanobacteria-specific primers

CYA359F and CYA781R (Nubel et al., 1997). Sequenced

fragments had a length of approximately 380 bp. A GenBank

BLAST (basic local alignment search tool) search was

performed for each sequence to identify the nearest relatives.

The environmental clone sequences from this study were

submitted under the GenBank accession numbers

AY790390–AY790464.

DGGE

For DGGE analysis, cyanobacterial 16S rRNA gene se-

quences were amplified from total community DNA using

the cyanobacterial specific primers CYA359F and CYA781R.

DGGE analysis was performed using a D-Code system (Bio-

Rad, Hercules, CA) essentially as described by Muyzer et al.

(1993). Reamplified PCR products from bands were se-

quenced commercially and a BLAST search of the GenBank

database was done to identify species or strains of nearest

relatives. The sequences of DGGE bands from this study

were submitted to the GenBank database under the acces-

sion numbers AY790465–AY790472.

Culture methods

Rock samples that were used for molecular analysis were

also used for culture isolations of phototrophs. Fine parti-

culate samples of the green layer in the travertine samples

were removed with sterile file and blade as in the molecular

analyses. Most culture isolates were obtained by the dilution

to extinction method, although direct enrichments were also

used. A small portion of the filings (�0.5 g) was used to

initiate a dilution series in BG11 medium, or in some cases,

ND medium that lacked combined nitrogen (Castenholz,

1988). Cultures of filamentous morphotypes were isolated

from final dilution enrichments by transfer to agar plates.

Filaments (trichomes) were allowed to migrate or grow out

from the source; then single trichomes (clones) on minute

agar blocks were removed with watchmaker’s forceps under

a dissecting microscope, and transferred to liquid medium

in sterile tubes (Castenholz, 1988). To obtain non-filamen-

tous types, the final enrichment was streak-plated and single

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

472 T.B. Norris & R.W. Castenholz

colonies were transferred to liquid medium. In most cases,

these cultures were replated for greater assurance of clon-

ality. In many cases, axenic cultures resulted, but the

presence of heterotrophic bacteria in some cultures still

allowed molecular cyanobacterial identification with the

use of the cyanobacteria-specific primers CYA359F and

CYA781R. Culture isolates of green algal strains were

sequenced using the 18S rRNA gene primers NS1 and NS2

(White et al., 1990), yielding a 555 bp product. The culture

isolate sequences from this study were submitted under the

GenBank accession numbers AY790836–AY790874.

Phylogenetic analysis

The 16S rRNA gene sequences were analyzed for phyloge-

netic affiliation using the BLAST (Altschul et al., 1997)

available on the NCBI website (http://www.ncbi.nlm.nih.-

gov/). The aligned SSU rRNA gene sequences of six known

cyanobacterial relatives were exported from the ARB pro-

gram (Ludwig et al., 2004) into CLUSTAL X (Thompson et al.,

1997) and used as a guide file for profile alignment of both

sequences from this study and additional cyanobacterial

sequences from GenBank. Alignments of partial 16S rRNA

gene sequences corresponded to Escherichia coli sequence

positions 412–807. The program PAUP 4.0b10 was used to

construct a distance tree by the neighbor joining method

(Swofford, 2001). The Kimura two-parameter algorithm was

used with pairwise deletion of gaps and missing data.

Bootstrap values from 1000 resamplings were calculated

and Chloroflexus aurantiacus DSM 637 was used as the

outgroup. The tree includes all of the environmental clones,

DGGE bands and culture sequences from this study as well

as some of the nearest relatives determined by BLAST.

Phylotypes that included more than one sequence, defined

by groups of sequences with greater than 97.5% sequence

identity (i.e. operational taxonomic units, OTUs), were

represented by a single sequence from that group for tree

construction. Parsimony was used as an alternate method of

tree construction to check for robustness of sequence

clusters.

Microscopy

Collections and cultures were examined using an American

Optical research microscope with phase contrast �40 and

�100 objectives. The photomicrography was with a Zeiss

Axioplan microscope, using a �100 plan-neofluor objective

with Nomarski DIC. Photo-images were made with a Nikon

Coolpix 990 digital camera. The taxonomic descriptions and

tentative genus names used in Bergey’s Manual of Systema-

tic Bacteriology (Boone & Castenholz, 2001) were used for

morphotypic identifications.

Results

Field collections

The greenish bands of about 1–2 mm thickness were usually

1–4 mm below the upper surface of the rocks and visible,

especially when the upper surfaces were light colored and

not covered with crustose lichens. Most of the samples were

quite similar to that shown in Fig. 1 (inset) but, in some

cases, vertical fracture lines occurred in the rocks that

apparently allowed light penetration and water to greater

depths (e.g. 5–10 mm). Such communities are termed

chasmolithic. If the rocks were extended as overhanging

ledges, the endolithic green layer was also apparent near the

lower surface. There was no visual evidence in this study of

microorganisms boring into the rocks. Although no quanti-

tative measurements were made, the youngest rocks were the

most porous and easily breakable, whereas the older rocks

were usually of significantly greater hardness. The collec-

tions were made mainly in mid summer. During a typical

warm summer day (25 to 430 1C) with 14–18% relative

humidity immediately above most of the sites, the water

content of the rocks was less than 3%, and even lower

(0.4%) in less porous rocks of the Hoodoos. In one sample

of relatively non-porous travertine, with a maximum noon-

time rock surface temperature of 435 1C, a temperature of

28 1C was reached at 5 mm depth by c. 14:00 hours and

minimum nighttime temperature of 10–12 1C by

04:00 hours AM (Jon Wraith, pers. comm.). Winter tem-

peratures are normally below 0 1C and often plunge to

�30 1C or lower. Only one of the sites was possibly influ-

enced by water from a neighboring active spring. Narrow

Gauge Lower Terrace (NGL) had experienced water flow 3–4

years prior to collecting, and this more porous and delicate

deposit retained 8–20% water, probably for several days.

Samples were collected from various compass faces of the

travertine deposits. Although not quantified, northerly or

more shaded exposures appeared to harbor an endolithic

green band more frequently and more prominently than in

other exposures.

Analysis of community diversity using DGGE

DGGE community profiles of cyanobacterial 16S rRNA gene

sequences for each site were generated (data not shown).

The profiles for each community were characterized by

a few (two to seven) distinct bands over a background of

many more poorly resolved, indistinct bands. By visual

inspection, it appears that the communities may share some

common phylotypes, as evidenced by the equidistant migra-

tion of bands from different profiles. Each profile also

contained some uniquely migrating bands as well. BLAST

search results for the sequences that were retrieved from

FEMS Microbiol Ecol 57 (2006) 470–483 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

473Endolithic photosynthetic microorganisms

DGGE bands are included in Table 1. In most cases the

nearest relative was an uncultured (and usually unidenti-

fied) cyanobacterium or occasionally a chloroplast of a

green alga or moss from another study.

Clone library analysis

A total of 75 cyanobacterial 16S rRNA gene sequences were

determined from the clone libraries, which included 12–15

clones from each site. The clones were divided into

phylotype groups (Table 1). We have defined members of a

phylotype group as sequences with greater than 97.5%

sequence identity to each other (Stackebrandt & Goebel,

1994), although this does not guarantee species or

genus identity. The combined sequence data for clones and

DGGE bands from all sites can be divided into 28 phylotype

‘groups’, of which 15 have two or more sequences.

An additional 13 phylotypes are each represented by a

single sequence. Within the phylotype groups there were

some sequences that were 100% identical (over �380 bp)

to GenBank database sequences. From the consoli-

dated clone library there were eight sequences that were

retrieved more than once, each of these appearing two or

three times. A BLAST search analysis revealed that 16 of

the 28 phylotype groups have a nearest relative in the

GenBank database with sequence identity of 97% or greater.

In almost all cases, the nearest relative was an un-

cultivated cyanobacterial sequence. The sources of these

nearest relatives were wide ranging environments that

included hot and cold deserts as well as other cryptoendo-

lithic sites.

The two largest phylotype groups dominating the clone

library were phylotypes C (12 clones) and E (13 clones11

DGGE band). These two phylotype groups were 98–99%

identical to uncultivated cyanobacterial clone sequences

originating from soil crusts of the Colorado Plateau (Red-

field et al., 2002; Yeager et al., 2004). Group C included

sequences from Gardiner Lower Terraces (GLT), Narrow

Gauge Upper Terrace and Painted Pool Terrace (PAP).

Group E included sequences from the same sites in addition

to Narrow Gauge Ancient Terrace (NGA). Another major

phylotype was group I (6 clones, 1 DGGE band, 1 culture

isolate), which was distantly related to a Plectonema sp. F3

strain (94% identity) and therefore may represent a novel

cyanobacterial lineage. Interestingly, group I is a cluster

composed entirely of sequences from Narrow Gauge Lower

Terrace (NGL).

In addition to cyanobacterial sequences, our clone library

recovered sequences of representatives of moss and green

algal lineages. Phylotype group G (4 clones, 1 DGGE band)

is 95% identical to the plastid sequence of the green alga

Koliella sempervirens. All of the sequences in this group were

recovered from the Hoodoos (HDO). Mosses (epilithic)

are also represented in the clone library by phylotype

group H (5 clones, 1 DGGE band), which shares 99%

identity to a plastid sequence of the moss Physcomitrella

patens. Sequences in this phylotype group originated from

both the Hoodoos (HDO) and the Gardiner Lower Terrace

(GLT).

Isolation of clonal cultures

In addition to molecular methods, culture isolation was

used to identify members of the phototrophic endolithic

communities. A straightforward approach of dilution to

extinction and direct enrichment in BG11 medium (or ND

medium) was used to isolate cyanobacterial strains from

each of the sites. The most apparent result of cultivation

is that the majority of isolates are filamentous cyanobacteria.

Subsequent to clonal isolation, the 16S rRNA gene sequence

was determined for 36 isolates of cyanobacteria and

three green algal isolates (Table 2). Of the 36 cyanobacterial

culture sequences, 27 are unique; the rest represent identical

isolates that were retrieved more than once. Duplicate

isolates are included in Table 2 to indicate the frequency

of retrieval. The Table also includes four culture isolates

from three additional endolithic sites in Yellowstone Na-

tional Park for which clone library analyses were not

performed.

In general, the cyanobacterial and green algal isolates have

high similarity to other cultivated strains. For the 27 unique

cyanobacterial isolates, 14 have nearest BLAST search rela-

tives of 97–100% identity to cultivated cyanobacterial

strains. The sequence of one isolate from Narrow Gauge

Lower Terrace (NGL) was 100% identical to the sequence of

Cyanobium gracile, a unicellular cyanobacterium with a

global distribution in freshwater lakes and brackish seas

(Crosbie et al., 2003). Of the other 13 strains with close

cultivated relatives, 10 are related to species of the genus

Leptolyngbya and three to Nostoc species. Only two of the

cultures, CCMEE 6048 and CCMEE 6034, had nearest

relatives that were environmental clones. Seven of the

unique culture sequences have 94% or less identity to

database sequences and may represent novel lineages. These

cultures encompassed the less common morphotypes culti-

vated in this study, including the genera Synechococcus,

Chroococcidiopsis, Gloeocapsa, and Schizothrix. In addition

to cyanobacteria, we also retrieved two green algal strains:

CCMEE 6027, with 99% identity to Bracteacoccus aerius,

and CCMEE 6038/6039, with 99% identity to Paradoxia

multiseta.

The majority of the culture sequences could not be

assigned to phylotype groups. The 16S rRNA gene sequences

from the culture isolates that could be assigned to phylotype

groups from environmental clones or DGGE bands are

included in Table 1 and are also listed in bold in Table 2.

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

474 T.B. Norris & R.W. Castenholz

Table 1. Summary of rRNA gene sequence data combined from clone libraries, DGGE and cultures

Phylotype

group� Sequenced clones, DGGE bands and culturesw Nearest GenBank relative % Identity

A (3) NGA2, NGA10, NGH2 Microcoleus steenstrupii clone 177-7A (AF355395),

Uncultured bacterium, quartz pebbles in the

Southern Mojave Desert (AF493857)

93

B (2) NGL2, NGL11 Cyanobacterium sp. MBIC10216 (AB058249) 98

C (12) GLT9, GLT1, GLT5, GLT7, GLT12, GLT15, NGH14,

GLT8, PAP4, GLT13, GLT6, PAP6

Uncultured soil crust cyanobacterium clone lichen23,

Colorado Plateau (AF428529)

98

D (2) PAP7, PAP3 Uncultured soil crust cyanobacterium clone lichen37,

Colorado Plateau (AF428542)

97

E (14) (PAP10, PAP12, PAP1band), GLT10, (NGH5, NGH6),

NGH11, PAP5, (PAP9, PAP11), NGA3, NGA11, PAP8,

PAP2

Uncultured bacterium clone CD14, Colorado Plateau

or Chihuahuan Desert (AY360997)

99

F (5) (NGH4, HDO4, HDO2band), HDO2, NGA6 Uncultured cyanobacterium DGGE gel band B15,

Swiss dolomite (AY153458)

99

G (5) HDO5, (HDO3, HDO11, HDO15), HDO1band Koliella sempervirens plastid (AF278747) [green alga] 95

H (6) (HDO8, HDO14, HDO3band), GLT11, GLT14, HDO7 Physcomitrella patens plastid (AP005672) [moss] 99

I (8) (NGL9, NGL10, NGL5band, CCMEE6011), NGL1,

NGL3, NGL4, NGL12

Plectonema sp. F3 (AF091110) 94

J (9) (NGL5, CCMEE6004, CCMEE6007), NGL13,

(CCMEE6024, CCMEE6021), (CCMEE6035,

CCMEE6036, CCMEE6037)

Leptolyngbya sp. CNP1-Z1-C2 (AY239601) 99

K (3) (NGL7, NGL8), NGL4band Uncultured cyanobacterium isolate DGGE band Lb,

petroleum polluted microbial mat (AF423349)

99

L (2) NGH1, NGH9 Uncultured cyanobacterium DGGE gel band B15,

Swiss dolomite (AY153458)

95

M (2) GLT4, NGA7 Uncultured soil crust cyanobacterium clone lichen36,

Colorado Plateau (AF428541)

94

N (2) NGH15, NGA9 Uncultured soil crust cyanobacterium clone lichen36,

Colorado Plateau (AF428541)

97

O (3) NGH7, NGA4, NGH10 Uncultured soil crust cyanobacterium clone lichen5,

Colorado Plateau (AF428511)

99

P NGL2band Nostoc sp. pcA (AJ437559) 99

Q NGL3band Uncultured Antarctic cyanobacterium Fr132

(AY151730)

99

R NGH3 Uncultured soil crust cyanobacterium clone lichen5,

Colorado Plateau (AF428511)

97

S NGH8 Uncultured bacterium clone CD14, Colorado Plateau

or Chihuahuan Desert (AY360997)

94

T NGH12 Uncultured bacterium clone 1790-2, Hawaiian

volcanic deposit (AY425770)

96

U PAP1 Uncultured soil crust cyanobacterium clone lichen36,

Colorado Plateau (AF428541)

97

V NGA1 Uncultured soil crust cyanobacterium clone lichen37,

Colorado Plateau (AF428542)

94

W NGA5 Uncultured soil crust cyanobacterium clone lichen21,

Colorado Plateau (AF428527)

96

X NGA8 Uncultured cyanobacterium isolate DGGE gel band

B3-14, Swiss dolomite (AY153452)

97

Y NGA12 Uncultured cyanobacterium DGGE gel band B15,

Swiss dolomite (AY153458)

97

Z HDO1 Uncultured soil crust cyanobacterium clone lichen21,

Colorado Plateau (AF428527)

94

AA HDO10 Koliella sempervirens plastid (AF278747) [green alga] 94

BB HDO12 Acaryochloris sp. Awaji-1 (AB112435) 93

�Number of sequences of each phylotype indicated in parentheses.wIdentical sequences are enclosed in parentheses.

DGGE, denaturing gradient gel electrophoresis.

FEMS Microbiol Ecol 57 (2006) 470–483 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

475Endolithic photosynthetic microorganisms

Table 2. Culture isolates retrieved from cryptoendolithic travertine communities

Culture isolate

CCMEE number Source Accession number Nearest GenBank relative%Identity Morphotype

6004, 6007� Narrow Gauge Lower Terrace(NGL)

AY790836, AY790837 Leptolyngbya sp. CNP1-Z1-C2(AY239601)

99 Leptolyngbya

6011 Narrow Gauge Lower Terrace(NGL)

AY790838 Plectonema sp. F3 (AF091110) 94 Leptolyngbya

6012 Narrow Gauge Lower Terrace(NGL)

AY790839 Cyanobium gracile PCC6307(AF001477)

100 Cyanobium

6105 Narrow Gauge Lower Terrace(NGL)

AY790854 Nostoc sp. strain NIVA-CYA 124(Z82776)

99 Nostoc

6109 Narrow Gauge Lower Terrace(NGL)

AY790856 Lyngbya aestuarii kopara-LY(AJ621838)

92 Synechococcus

6132 Narrow Gauge Lower Terrace(NGL)

AY790866 Oscillatoriales cyanobacteriumIL-9.1 (AF401743)

94 Leptolyngbya

6021, 6024 Narrow Gauge Upper Terrace(NGH)

AY790840, AY790841 Leptolyngbya PCC7104(AB039012)

97 Leptolyngbya

6026 Narrow Gauge Upper Terrace(NGH)

AY790842 LPP-group MBIC10597 (AB058267) 97 Leptolyngbya

6108, 6117 Narrow Gauge Upper Terrace(NGH)

AY790855, AY790871 Nostocales cyanobacteriumIL-15.1 (AF401744)

100 Nostoc

6122 Narrow Gauge Upper Terrace(NGH)

AY790863 Cyanothece sp. PCC 7424(AF132932)

93 Synechocystis

6133 Narrow Gauge Upper Terrace(NGH)

AY790867 Leptolyngbya sp. CNP1-Z1-C2(AY239601)

98 Leptolyngbya

6048 Painted Pool (PAP) AY790848 Uncultured bacterium clone CD14(AY360997), Colorado Plateau orChihuahuan Desert

95 Chroococcidiopsis

6104 Painted Pool (PAP) AY790853 Nostoc commune (AB094352) 99 Nostoc6111 Painted Pool (PAP) AY790858 Oscillatoriales cyanobacterium

IL-9.1 (AF401743)92 Leptolyngbya

6119 Painted Pool (PAP) AY790861 Leptolyngbya sp. SV1-MK-52(AY239604)

99 Leptolyngbya

6124 Painted Pool (PAP) AY790864 Leptolyngbya sp. SV1-MK-49(AY239605)

97 Leptolyngbya

6028 Narrow Gauge Ancient Terrace(NGA)

AY790843 Phormidium autumnale(AF218371)

97 Leptolyngbya

6034 Narrow Gauge Ancient Terrace(NGA)

AY790844 Uncultured bacterium Tui1-3 16S(AF353281), acid mine drainagesystem in New Zealand

97 Leptolyngbya

6027w Narrow Gauge Ancient Terrace(NGA)

AY790872 Bracteacoccus aerius (U63101)[green alga]

99 Bracteacoccus

6125 Narrow Gauge Ancient Terrace(NGA)

AY790865 Uncultured bacterium clone CD31(AY361002)

99 Leptolyngbya

6058 Gardiner Lower Terrace (GLT) AY790852 Gloeocapsa sp. PCC 73106(AF132784)

96 Gloeocapsa

6130, 6131/6120

Gardiner Lower Terrace (GLT)/ AY790868, AY790869, Leptolyngbya sp. SEV5-5-C6(AY239591)

92 Schizothrix

Painted Pool (PAP) AY7908626035, 6036,6037

Hoodoos (HDO) AY790845, AY790846,AY790847

Leptolyngbya PCC7104(AB039012)

98 Leptolyngbya

6038, 6039w Hoodoos (HDO) AY790873, AY790874 Paradoxia multiseta (AY422078)[green alga]

99 Chlorella

6110, 6126 Hoodoos (HDO) AY790857, AY790870 Nostocales cyanobacteriumIL-15.1 (AF401744)

96 Nostoc

6116 Hoodoos (HDO) AY790860 Oscillatoriales cyanobacteriumIL-9.1 (AF401743)

93 Leptolyngbya

6112, 6056 Rabbit Creek Terracez AY790851, AY790859 Phormidium autumnale(AF218371)

97 Leptolyngbya

6052 New Mound AY790849 Uncultured cyanobacteriumisolate DGGE gel band R2A(AY354465)

99 Leptolyngbya

6054 Reave’s Mound AY790850 Leptolyngbya sp. CNP1-B3-C9 16S(AY239600)

98 Leptolyngbya

�Sequences in bold belong to phylotype clusters that include clones and/or denaturing gradient gel electrophoresis bands.w18S rRNA gene sequences.zSilica sinter terrace.

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

476 T.B. Norris & R.W. Castenholz

Phylotype group J includes the most culture isolates, with

seven isolates retrieved from the Narrow Gauge Lower

Terrace, Hoodoos, and Narrow Gauge Upper Terrace. Phy-

lotype group J also includes two environmental clones from

the Narrow Gauge Lower Terrace; one with 100% identity to

isolates CCMEE 6004/6007. These sequences are 99% iden-

tical to Leptolyngbya sp. CNP1-Z1-C2, and microscope

observations confirm a Leptolyngbya morphotype for these

strains (Fig. 2). Phylotype I is the only other group with

overlap between environmental clones and cultures. Within

this group the culture CCMEE 6011 has 100% identity to

two environmental clone sequences and a DGGE band. This

phylotype group is a distant relative (94% identity) of the

filamentous cyanobacterium Plectonema sp. F3. There were

23 other culture isolate sequences that were unique and did

not fall into phylotype groups composed of multiple

sequences.

Morphotypic diversity

Figure 2 displays photomicrographs of selected culture

isolates that are included in Table 2 and in the phylogenetic

tree (Fig. 3). The nearest GenBank relative is indicated in

Table 2. The cyanobacteria shown in panels a, b, c, and f have

close morphological similarities to the genera to which they

are most closely related by sequence identity [i.e. Cyanobium

(CCMEE 6012), Nostoc (CCMEE 6104, CCMEE 6110) and

Gloeocapsa (CCMEE 6058)]. The filamentous cyanobacteria

shown in panels g, i, and j all resemble their closest

phylotypes with 97–99% identities, the artificial ‘form-

genus’ Leptolyngbya (Fig. 2). The Leptolyngbya-type in panel

h (CCMEE 6132), however, has only a 94% identity to a

loosely named member of the order Oscillatoriales (which

includes Leptolyngbya) (Table 2, Fig. 3). Panel e displays a

unicellular (or aggregated unicellular) type with some

10 µm

CCMEE 6012 CCMEE 6104 CCMEE 6110

CCMEE 6048

CCMEE 6122 CCMEE 6058

(a) (b) (c)

(d) (e) (f)

(g) (h) (i)

(j) (k)

CCMEE 6119 CCMEE 6132

CCMEE 6034

CCMEE 6109

CCMEE 6036

Fig. 2. Diversity of selected cyanobacterial

morphotypes in clonal culture isolated from

travertine samples. All photomicrographs are

shown at the same scale (bars = 10mm). Panel a

(CCMEE 6012 Cyanobium gracile); Panel b

(CCMEE 6104) and c (CCMEE 6110) Nostoc-

types); Panel d (CCMEE 6048 Chroococcidiop-

sis-like), Panel e (CCMEE 6122 Synechocystis-

like); Panel f (CCMEE 6058 Gloeocapsa-type);

Panels g, h, i, and j (CCMEE 6119, CCMEE 6132,

CCMEE 6034, and CCMEE 6036 Leptolyngbya-

types); Panel k (CCMEE 6109 Synechococcus-

type).

FEMS Microbiol Ecol 57 (2006) 470–483 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

477Endolithic photosynthetic microorganisms

NGH15NGA9PAP1

GLT4NGA7Phylotype C (12)Colorado Plateau soil crust clone lichen 23Phylotype D (2)Mojave desert clone18 (quartz pebbles)

Phylotype E (14)Colorado Plateau soil crust clone CD14

CCMEE6048HDO1

NGA1Colorado Plateau soil crust clone lichen 5

NGH7NGA4

NGH10NGH3

NGA5NGA8

Nostc commune UTEX 584CCMEE6104

NGL2bandNostoc sp. ATCC 53789Nostoc sp. PCC 9709CCMEE6105

CCMEE6108CCMEE6110

Nostoc sp. PCC7120Phylotype B (2)

Cyanobacterium sp. MBIC1021Phylotype A (3)NGH8

Microcoleus steenstrupii clone 177-7AMicrocoleus vaginatus PCC9802

Microcoleus sociatus MPI 96MS.KIDNGH12

Chroococcidiopsis sp. 029 (Negev)Chroococcidiopsis sp. 171 (Antarctica)

Chroococcidiopsis sp. SAG2026Chroococcidiopsis thermalis PCC 7203

NGH1NGH9

HDO12Phylotype F (5)

DGGE gel band B15 (Swiss Dolomite)NGA12

Phylotype G (5)HDO10

Koliella sempervirens plastidPhylotype H (6)

Colorado Plateau soil crust plastid moss35Physcomitrella patens plastid

Phylotype K (3)CCMEE6109

CCMEE6028CCMEE6125CCMEE6034

CCMEE6112Phormidium autumnale

CCMEE6052CCMEE6124

CCMEE6119CCMEE6120

CCMEE6132CCMEE6116CCMEE6111

NGL3bandAntarctic clone Fr132

CCMEE6054Synechocystis sp. PCC 6803

CCMEE6122CCMEE6058

Gloeocapsa sp. PCC73106CCMEE6026

Antarctic clone FBP290Phylotype I (8)

Plectonema sp. F3Phylotype J (9)CCMEE6133Leptolyngbya sp. PCC 7104

Leptolyngbya sp. CNP1-Z1-C2CCMEE6012

Cyanobium gracile PCC 6307Gloeobacter violaceus PCC 7421

Chloroflexus aurantiacus DSM 637

0.01 substitutions/site

I

II

III

IV

V

VI

VII

VIII

IX

X

XI

XII

XIII

XIV

X V

83

97

100

91

87

99

92

100

6692

100

10093

100

99

58

98

100

72

100

87

100

100

Fig. 3. Distance tree based on cyanobacterial 16S rRNA gene sequences from Yellowstone travertine samples. The tree includes 75 environmental

clone sequences (blue), 8 DGGE bands (blue with ‘‘band’’ suffix) and 24 culture isolate sequences (green). Lettered phylotypes (red) represent a

phylotype of sequences with 97.5% or greater sequence identity and correspond to information presented in Table 1 and may include cultures, clones

or DGGE bands or all three. Roman numerals identify sequence clusters discussed in the text. Bootstrap values from 1000 re-samplings are given for

some relevant nodes.

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

478 T.B. Norris & R.W. Castenholz

similarity to the morphotype Synechocystis but with only a

93% identity to the nearest relative, the unicellular genus

Cyanothece (Table 2). The cultures shown in panels d and k

are anomalous with respect to nearest relatives. Culture

CCMEE 6048 (panel d) resembles a Chroococcidiopsis mor-

phologically but does not have a close identity to the

previously sequenced members of this genus (Table 2,

Fig. 3). The unicellular rods shown in panel k resemble the

artificial ‘form-genus’ Synechococcus, but the closest relative

(92% identity) is a well-known marine, filamentous cyano-

bacterium (Table 2). However, identities below 97–98%

have little meaning even at the genus level, and reflect the

large gaps in the database at the present time.

Phylogenetic analysis of molecular and culturesequences

A phylogenetic tree constructed by the neighbor-joining

method is shown, using the sequence data from the envir-

onmental clones, DGGE bands and culture isolates (Fig. 3).

Although the basal branching order of the tree is mostly

unsupported, the sequences from this study generally fell

into well-supported sequence clusters at the tips of these

branches and bootstrap values are given where relevant. The

sequences are distributed in 15 distinct sequence clusters or

cyanobacterial lineages. Phylogenetic trees were also con-

structed by Parsimony methods, and although the basal

branching order differed considerably, no differences were

observed in the major sequence clusters (data not shown).

Novel clusters

Clusters I and II are novel cyanobacterial lineages that

comprise the majority of the environmental clone se-

quences. Cluster I is the largest, with 45% of the environ-

mental clone sequences, including the two largest phylotype

groups. There is only one culture isolate sequence (CCMEE

6048) that belongs to this group. Morphologically this

culture resembles a Chroococcidiopsis, but it clearly does not

fall into a clade with other known Chroococcidiopsis isolates.

Cluster I includes environmental clone sequences from

every sample location except Narrow Gauge Lower Terrace

(NGL). An additional five environmental clone sequences

from this study belong to Cluster II. Clusters I and II are

comprised entirely of sequences from this study or GenBank

sequences of environmental clones from desert soil crusts;

they have no close relatives that are cultivated cyanobacterial

species. Cluster VII is another group without close culti-

vated relatives. This cluster is composed of eight environ-

mental clone sequences and one DGGE band. The major

phylotype group in this cluster is 99% identical to a DGGE

band retrieved from a cryptoendolithic environment of

dolomite in Switzerland. Clusters V and VI are small groups

of unique environmental clone sequences that are distantly

related to Microcoleus-like cyanobacteria.

Leptolyngbya clusters

There are four clusters of Leptolyngbya-like sequences, based

on morphology, sequence similarity or both. These four

clusters include a majority of the culture isolates from this

study. Cluster IX is composed entirely of culture isolate

sequences related to Leptolyngbya-like cultures and Phormi-

dium autumnale. Cluster X is a novel lineage that includes

one DGGE band and four culture isolates for which the

closest relative is an uncultivated Antarctic microbial mat

clone. Clusters XIII and XIV are two more distinct Lepto-

lyngbya-like clusters that each include a phylotype group

composed of sequences retrieved by both culture and

molecular methods. Cluster III is a well-supported clade of

heterocystous type sequences that include four culture

isolates of Nostoc-like morphology and one DGGE band.

Other sequences

A few of the 16S rRNA gene sequences do not fall into

distinct clusters. Members of Phylotype group K are closely

related (99% identity) to an environmental clone sequence

from a petroleum-polluted microbial mat, but have no close

cultivated relatives. Two culture isolates from this study,

CCMEE 6109 and CCMEE 6120, also lack close relatives and

represent completely novel culture isolates. Culture CCMEE

6109 is morphologically similar to Synechococcus (Fig. 2,

panel K), and culture CCMEE 6120 has a Schizothrix-like

morphology.

Discussion

Advantages of a multifaceted approach

This study has provided the first multifaceted (polyphasic)

survey of the photosynthetic, travertine-inhabiting endo-

liths in a temperate climate and laid the groundwork for

future environmental and physiological work on these

extreme phototrophs and their habitats. This study includes

whole community and culture molecular analyses, as well as

morphological information on culture isolates. Molecular

methods, such as the cloning of environmental DNA

sequences, are now generally regarded as providing a more

accurate description of microbial community diversity than

culture-based methods. But although molecular methods

are extremely useful tools for genetic inventories, they

provide limited information about the physiology or eco-

logy of community members and so a more ideal tactic is to

use a combination of approaches. The methods employed in

this study retrieved different but complementary informa-

tion about the variety of phototrophs in the travertine

FEMS Microbiol Ecol 57 (2006) 470–483 c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

479Endolithic photosynthetic microorganisms

habitat. Molecular methods allowed us to propose that

unicellular cyanobacteria may be the most abundant

type of photosynthetic endoliths in this community, infor-

mation that will aid the development of methods used in

future culture isolation attempts. It is important to note that

there is no close similarity at the 16S rRNA gene level

between the photosynthetic microorganisms of these com-

munities and those that occur in active travertine-depositing

hot springs of the Mammoth area of Yellowstone National

Park (Fouke et al., 2003; Pentecost, 2003). None of the

sequences that we obtained from the cultures or travertine

came close to matching the sequences retrieved from the

active springs.

Comments on the methods used

DGGE was found to be of limited usefulness in this

particular study because, rather than being dominated by a

few sequence types, the DGGE profiles were composed of

many unresolved bands, making excision and sequencing

almost impossible. This is not an uncommon issue

and recent literature documents the limitations of DGGE

(Liu & Stahl, 2002). Therefore, we relied more on

environmental clone library construction for our molecular

survey. Almost all of the sequences retrieved in our environ-

mental clone library analysis were closely related to

other environmental clones from analogous environments,

rather than to cultivated strains. There are a few reasons

for the lack of database sequences of cultured strains of

cyanobacteria from environments where desiccation and

freezing tolerance are important for survival. Relatively few

habitats of this type have been surveyed outside of polar or

hot desert regions, and most of the recent studies have been

limited to molecular analyses of bulk DNA from environ-

mental samples [e.g. (Redfield et al., 2002; de la Torre et al.,

2003)]. Another reason is the apparent difficulty of cultiva-

tion of some types. In this study we were only able to isolate

a single culture belonging to sequence cluster I, which

contained 45% of the environmental cyanobacterial

sequences. Morphologically this isolate appears to be similar

to Chroococcidiopsis, but its phylogenetic placement is

clearly outside the clade of other Chroococcidiopsis strains

(Fig. 3, Panel d). Thus, morphological information alone

may be misleading, but the combination of genetic and

morphological data may be of great help in clarifying

taxonomic units.

Culture isolates from this study also complemented

information retrieved by molecular methods. Using stan-

dard cyanobacterial culturing techniques (Castenholz,

1988), we isolated over 100 strains of cyanobacteria. Of the

36 isolates that have been sequenced, 27 are unique strains of

cyanobacteria, four of which belong to phylotype groups

defined by our clone library sequences. Although there was

some overlap between cultures and environmental se-

quences, many of the cultures fall into separate sequence

clusters apart from the environmental sequences. A majority

of these cultures are filamentous cyanobacteria, which are

typically easier to isolate, and, therefore, more frequently

obtained than unicellular types. Culture isolates CCMEE

6004, 6007, 6133, 6119, 6125 and 6054 had 98–99% 16S

rRNA gene identity to cyanobacterial strains or sequences

originating from North American desert environments, and

the 16S rRNA gene sequence of culture CCMEE 6052 is 99%

identical to a DGGE band sequence originating from a hot

spring source elsewhere in Yellowstone National Park (Black

Sand Pool). Two of the Leptolyngbya sequence clusters, X

and XIII, include relatives that are environmental clone

sequences from Antarctica. Many of the culture sequences

from this study are most closely related (but not identical)

to sequences obtained from other extreme environments,

including deserts. Little is known about dissemination of

cyanobacteria, particularly from one continent to another.

However, a convincing case of endemism has been demon-

strated in thermophilic cyanobacteria from disjunct transo-

ceanic hot springs (Papke et al., 2003). The method of

dispersal of the endolithic microorganisms of our study is

unknown, but it is possible that exfoliation of rock surfaces

that exposes the microbial layer with subsequent dissemina-

tion by wind or insects may result in inoculation of other

surfaces (e.g. Friedmann & Weed, 1987; Sun & Friedmann,

1999), but a gradual lateral spread within the rock may also

occur (Van Thielen & Garbary, 1999).

Problems using 16S rRNA gene sequencesimilarities for species identification

It is also important to note that, although 16S rRNA gene

sequences may show close relationships (i.e. the sharing of a

common ancestor), similarities do not discriminate taxa at

the species level and extended periods of evolution may still

separate cyanobacteria that have 98–100% identities of 16S

rRNA gene sequences. In the present study, an excellent

example is that of culture CCMEE 6012 for which a BLAST

search indicates 100% 16S rRNA gene identity with a

few sequenced strains in the database that have been

identified as Cyanobium gracile (Table 2). The fact that the

other strains were isolated from lakes (Germany, Hungary)

and the Baltic Sea would indicate that the genomes of

these strains must differ greatly, at least in the genes related

to the obvious ecological and physiological disparities.

Two strains of the marine pico-cyanobacterium, Prochloro-

coccus marinus have 16S rRNA gene sequence identities of

over 97% but the complete genomes of both show enor-

mous differences, some of which equate with the different

light regime preferences for these two strains (Rocap et al.,

2003).

FEMS Microbiol Ecol 57 (2006) 470–483c� 2006 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved

480 T.B. Norris & R.W. Castenholz

Comparisons with other endolithic microbialcommunities

Endolithic communities in hot deserts tend to be dominated

by unicellular cyanobacteria (Whitton & Potts, 2000). How-

ever, cold deserts of Antarctica have endolithic habitats

dominated in some cases by eukaryotes (e.g. green algae

and protolichens) and in others by cyanobacteria, many of

which are unicellular (Van Thielen & Garbary, 1999; de la

Torre et al., 2003). de la Torre et al. (2003) found that an

environmental clone library derived from one of these

Antarctic cryptoendolithic communities (one of the

McMurdo Dry Valleys) was, in fact, dominated by a single

cyanobacterial phylotype. Cultivation of a cyanobacterial

strain with 100% 16S rRNA gene identity to this phylotype

revealed that it had a coccoid morphology. The limited

information that exists at present would suggest that tempe-

rate endolithic habitats tend to have a greater diversity of

taxa than those of hot and cold deserts. Surveys of endolithic

communities in dolomitic rocks of Switzerland (Sigler et al.,

2003) and limestones of the Niagara Escarpment (Gerrath

et al., 1995, 2000) in Canada recovered a wide range of

cyanobacterial genera, including both filamentous and uni-

cellular types. In both of these studies the green algae

Chlorella and Stichococcus made a major contribution to

the community structure, and yellow-green algae also oc-

curred in the Niagara limestones. Endolithic communities

have also been found in moist acidic geothermal environ-

ments (Gross et al., 1998; Walker et al., 2005), in which case,

the only phototrophs of the community were members of

the acidophilic red algal order Cyanidiales. In contrast, the

travertine cryptoendolithic community of Yellowstone was

dominated by cyanobacteria, and few green algae were

retrieved in cultures or clone libraries. Although our clone

library pool was small, it was evident that a similar distribu-

tion of phylotypes was retrieved from all of the sampling

sites except Narrow Gauge Lower Terrace (NGL), which did

not share any phylotypes with other sites. This area is the

most recently formed travertine (3–4 years) and is also

much softer, and more porous than in the other locations,

properties that may allow longer retention of moisture and

therefore may be inhabited by less desiccation-tolerant

strains. In the travertine system overall, a broad range of

cyanobacterial genera are present. This suggests that adapta-

tions to desiccation and freezing tolerance are not limited to

a few cyanobacterial genera, but rather arose convergently

or, alternatively, early in the cyanobacterial radiation.

Future prospects

A future goal of this research is to describe in some detail the

physical and chemical properties of selected ancient traver-

tines of Yellowstone National Park, measurements of rock

porosity, water retention, nutrient composition, and light

attenuation. In addition, the physiology of desiccation- and

freezing-tolerance of cultured community members repre-

sents an ultimate objective. However, the tolerances of many

culture isolates of this study to desiccation stress at low

relative humidity (equivalent to summer values in Yellow-

stone National Park), to high temperature, to freezing, and

to various salinities have already been assayed in a prelimin-

ary study. Results indicate enhanced tolerances of travertine

isolates to these stresses compared to tolerance levels in most

cyanobacteria from aquatic or moister habitats (Norris and

Castenholz, unpublished data). The importance of continu-

ing to study endolithic environments and their microbial

communities is underscored by their relevance as analogues

for life on early terrestrial Earth and for possible past or

present life on Mars.

Acknowledgements

This work was performed while T.B.N. held a National

Research Council Research Associateship Award at the

University of Oregon in affiliation with NASA-Ames Re-

search Center. Special thanks go to undergraduate Pamela

N. Johnston who contributed greatly to the culture isolation

work and to undergraduate Julie Fox who assayed tolerances

to desiccation and other stresses. This work was supported

by a research grant from the Thermal Biology Institute at

Montana State University to T.B.N. and by a NASA/ARC

Cooperative grant NCC 2-5524 to R.W.C. We would like to

thank the Yellowstone Center for Resources, Yellowstone

National Park, for permission to collect within the park

(Permit No. 0185). R.W.C. is a member of the NASA

Astrobiology institute.

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483Endolithic photosynthetic microorganisms