A geothermal-linked biological oasis in Yellowstone Lake, Yellowstone National Park, Wyoming

A geothermal-linked biological oasis in Yellowstone Lake,Yellowstone National Park, WyomingD. LOVALVO,1 ,* S. R. CLINGENPEEL,2 ,* S. MCGINNIS,3 R. E. MACUR,2 J . D. VARLEY,3

W. P. INSKEEP,2 J . GLIME,4 K. NEALSON5 AND T. R. MCDERMOTT2

1Eastern Oceanics, West Redding, CT, USA2Thermal Biology Institute, Montana State University, Bozeman, MT, USA3Big Sky Institute, Montana State University, Bozeman, MT, USA4Department of Biological Sciences, Michigan Technological University, Houghton, MI, USA5Department of Earth Sciences, University of Southern California, Los Angeles, CA; and JC. Venter Institute, La Jolla, CA, USA

ABSTRACT

Hundreds of active and dormant geothermal vents have been located on the floor of Yellowstone Lake, although

characterization of the associated biology (macro or micro) has been extremely limited. Herein, we describe an

aquatic moss (Fontinalis) colony closely associated with vent emissions that considerably exceeded known

temperature maxima for this plant. Vent waters were supersaturated with CO2, likely accommodating a CO2

compensation point that would be expected to be quite elevated under these conditions. The moss was colo-

nized by metazoa, including the crustaceans Hyalella and Gammarus, a segmented worm in the Lumbriculidae

family, and a flatworm specimen tentatively identified as Polycelis. The presence of these invertebrates suggest

a highly localized food chain that derives from the presence of geothermal inputs and thus is analogous to the

deep marine vents that support significant biodiversity.

Received 17 December 2009; accepted 5 April 2010

Corresponding authors: Timothy R. McDermott. Tel.: 406-994-2190; fax: 406-994-3933; e-mail: timmcder@

montana.edu; John D. Varley. Tel.: 406-994-2320; fax: 406-994-5122; e-mail: [email protected]

INTRODUCTION

Yellowstone Lake contains hundreds of hydrothermal vents

(Morgan et al., 2007) that contribute roughly 10% to the

total geothermal venting activity in the Yellowstone geother-

mal complex (Balistrieri et al., 2007). The lake bottom has

been mapped three different times over the last 136 years,

with vent exploration being the focus of work during the last

quarter century. The majority of the Yellowstone Lake hydro-

thermal vents have been documented using bathymetric and

seismic approaches, and a submersible remote operating

vehicle (ROV) (see Morgan et al., 2007, for comprehensive

summary). The vents are primarily clustered in the northern

half of the lake, spanning from the West Thumb to the Mary

Bay regions, and all appear to be within the boundary of the

current Yellowstone caldera (Morgan et al., 2007).

Some vent activity has been located visually as gas bubbles

or turbulence on the lake surface. Evidence of high output

vents in the West Thumb area (Fig. S1) originally derived

from observations of open ice during the winter, which sug-

gested subsurface geothermal vent activity was maintaining

water column temperatures above freezing. Subsequent ROV

dives discovered a large vent cone and a rock outcrop shelf

structure that emits large volumes of warm water and gas.

Interestingly, this particular vent seemed unique relative to all

other active vents thus far observed in the lake in that it is

robustly colonized by plants. In all cases, the plants appeared

very closely associated with vent emissions (http://www.

tbi.montana.edu/media/Fontinalis_vent.html); i.e. only

where water and gas venting activity was visually obvious.

The occurrence of higher plants so closely associated with

venting activity was of interest not only because of plant–

temperature relationships, but also because of the relative

depth and very low light conditions at this site. Also, while

microbiological diversity and novelty associated with Yellow-

stone’s diverse terrestrial geothermal features has been well

documented (e.g., Brouns et al., 2005; Inskeep & McDer-

mott, 2005; Reysenbach et al., 2005; Sheehan et al., 2005;

Spear et al., 2005; Ward & Cohan, 2005; Young et al., 2005;

Madigan et al., 2005), except for cursory visual descriptions*Both authors contributed equally.

� 2010 Blackwell Publishing Ltd 327

Geobiology (2010), 8, 327–336 DOI: 10.1111/j.1472-4669.2010.00244.x

of microbial mats and a report of unidentified macrophytes in

the vicinity of vents at SCUBA depths (Remsen et al., 2002),

relatively little is known about the biology, micro or macro,

associated with the hydrothermal features on the Yellowstone

Lake floor. The study described herein provides the first

in-depth identification of the biology associated with any vent

in this lake, focusing in this case on the above-mentioned

plant colony and the mesofauna found to inhabit it.

MATERIALS AND METHODS

Sampling

The sampling site was in the West Thumb region of Yellow-

stone Lake, approximately 0.5 km offshore from the West

Thumb geyser basin (Fig. S1), with the coordinates

44�24.720¢ N, 110�33.616¢ W. The ROV used in this work

(Fig. 1) was first mentioned by Cuhel et al. (2002), but

additional description is provided here (see below) to more

thoroughly document the ROV’s instrumentation and

sampling capability important for the work conducted in this

study. The National Park Service boat ‘Cutthroat’ was used

for all sampling work.

Geochemical and light analyses

Geochemical analyses were either performed immediately or

samples were appropriately preserved and stored until further

analyses could be conducted at the field-based laboratory

located at Lake Village or at Montana State University in

Bozeman. Several redox sensitive species were immediately

analyzed onboard the ship, including FeII and FeIII using the

Ferrozine method with filtered samples (0.2 lm; To et al.,

1999) and total dissolved sulfide (DS) using the amine sulfuric

acid method (APHA, 1998) with unfiltered samples [to avoid

rapid degassing of H2S(aq) upon filtration]. Aqueous pH val-

ues were obtained using a Fisher Accumet AP-71 meter and

AP-55 probe equipped with temperature compensation.

Additional aqueous samples were filtered (0.2 lm) directly

into sterile 50 mL Falcon tubes and refrigerated at 4 �C. Two

tubes were preserved with trace metal grade HNO3 (1%) and

HCl (0.5%) for analysis using inductively coupled plasma

instrumentation [ICP-OES and ICP-MS (Aligent Model

7500)] for total dissolved elements including Ag, Al, As, Ba,

Be, Bi, B, Ca, Cd, Ce, Co, Cr, Cs, Cu, Dy, Er, Eu, Fe, Ga,

Gd, Ge, Hf, Ho, In, K, La, Li, Lu, Mg, Mn, Mo, Na, Nb, Nd,

Ni, P, Pb, Pr, Re, Rb, Sb, Sc, Se, Si, Sm, Sn, Sr, Ta, Te, Tb,

Th, Ti, Tl, Tm, U, V, W, Y, Yb, Zn, and Zr. One unacidified

tube was analyzed for predominant inorganic anions (F),

Cl), SO42), NO3

), CO32), S2O3

2), AsO43)) using anion

exchange chromatography (Dionex DX 500; AS16-4 mm

column), and aqueous NH4+ using the phenolate colorimetric

(A630nm) procedure on a flow injection analyzer (APHA,

1998). Dissolved inorganic C (DIC) and dissolved organic C

(DOC) were determined on separate samples taken in closed

headspace serum bottles (previously baked at 500 �C) using a

Shimatzu Model TOC-VCSH total C analyzer.

Concentrations of dissolved O2 in vent fluids were deter-

mined immediately after collection using the Winkler method

(APHA, 1998). Dissolved H2, CH4, and CO2 were deter-

mined using headspace gas chromatography as described

previously by Inskeep et al. (2005). Closed headspace

aqueous samples were collected by using either the ROV

syringe or a peristaltic pump to push vent fluids through

an inline 140 mm diameter filter (0.2 lm) and into sterile

(autoclaved) 160-mL serum bottles. After a three head space-

volume purge, the serum bottles were capped with zero

headspace using sterile (autoclaved) butyl stoppers. A known

Sampling arm

Sampling cup w/temp probe

Tether

Water/sample Water

A B

CBuoyancy

Sampling arm(deployed)

Tether

Water/sampleintake exit

Video

35 mm camera &

strobe Samplingsyringe

Tether

SonarSonarRunning light

Samplecamera basket

Fig. 1 Photographs of the ROV used for sampling

of the geothermal vent and moss. (A) Functional

description of ROV equipment. (B) Vacuum sampler

collection can. (C) Deployment of the ROV immedi-

ately after release from the boat tether.

328 D. LOVALVO et al .

� 2010 Blackwell Publishing Ltd

volume of liquid (�30 mL) was withdrawn and replaced with

an equivalent volume of filter-sterilized (0.2 lm) air and the

bottles were incubated with intermittent shaking for 60 min.

Samples of the gas headspace were injected into a portable

dual-channel Varian gas chromatograph (Model CP2900)

equipped with thermal conductivity detectors. One of the

channels utilized a 10-m 5-A molecular sieve column (60 �C)

and UHP Ar carrier gas (21 psi) to separate H2 and the other

channel utilized a 10 m PPQ (PoraPlotQ) column (70 �C)

and UHP He carrier gas (18 psi) to separate CH4 and CO2.

Gas concentrations measured in the headspace were then used

to calculate the gas concentrations in the original solution

using temperature-corrected Henry’s Law constants and a

mass balance equation:

Henry’s Law:

KH ¼ c(aq)=c(g)

Mass Balance:

½c(aq)sampleVL� ¼ ½c(g)equilVg;eq� þ ½c(aq)equilVL;eq�;

where the KH is Henry’s Law constant at the incubation tem-

perature (KH calculated using thermodynamic constants

reported in Amend & Shock, 2001), c is concentration in

either aqueous or gas phases (mol L)1), and V is volume of

liquid or gas.

Light intensity was measured using an International Light

Technologies Model ILT 1700 radiometer by lowering a light

sensor to various depths in the water column overlying the

vent. The light sensor was calibrated to read visible light at

400–700 nm, with primary sensitivity at 555 nm.

Scanning electron microscope energy-dispersive X-ray

analysis

Portions of the solid phase material collected were air-dried,

mounted and sputter-coated with carbon, and imaged with

the SEM (JEOL 6100). Elemental spectra were acquired from

representative spots using Energy Dispersive X-rays (NORAN

detector with ROENTEC software, Bruker AXS Microanalysis,

Ewing, NJ, USA). Quantitative analysis from each spectrum

was run to obtain an elemental profile.

Moss identification

Moss samples were stored frozen until identified. Initial iden-

tification was based on leaf morphology features followed by

phylogenetic assessments. For the latter, the chloroplast rbcL

gene, tRNA-Leu ⁄ Phe spacer, and nuclear ITS region DNA

were PCR cloned and sequenced. DNA was extracted from

washed leaves using methods previously described by Botero

et al. (2005), and then used as templates for 50 lL PCRs

containing 1.5 mM MgCl2, 20 lg BSA, 0.2 mM each dNTP,

1 lM each primer, and 1.25 u Taq polymerase. The PCR

programs were as follows:

1. The rbcL gene was amplified as two overlapping

fragments. The first fragment used the primers rbcL-73F (5¢-ATACCAAAGATGTTTTTTTATAAG-3¢) and rbcL804hR

(5¢-TGCAGTAAAACCACCTG-3¢) (Tsubota et al., 1999).

The PCR program was: 94 �C for 5 min, 30 cycles of 94 �Cfor 1 min, 40 �C for 1 min, and 72 �C for 1:30 min, 72 �Cfor 7 min, and 4 �C hold. The second fragment used primers

FontF (5¢-TYATGCGTTGGMGWGAYCGT-3¢) and FontR

(5¢-TCTAAGGCAACTCTATTAGCAAC-3¢). PCR condi-

tions were: 94 �C for 5 min, 30 cycles of 94 �C for 1 min,

50 �C for 1 min, and 72 �C for 1 min, 72 �C for 7 min, and

4 �C hold.

2. The tRNA-Leu ⁄ Phe region was amplified using primers

trnC (5¢-CGAAATCGGTAGACGCTACG-3¢) and trnF (5¢-ATTTGAACTGGTGACACGAG-3¢), using the program:

94 �C for 5 min, 30 cycles of 94 �C for 45 s, 52 �C for 45 s,

and 72 �C for 45 s, 72 �C for 7 min, and 4 �C hold.

3. The ITS region was amplified using primers BMBC-R

(5¢-GTACACACCGCCCGTCG-3¢) and LS4-R (5¢-TCAA

GCACTCTTTGACTCTC-3¢) (Shaw & Allen, 2000), using

the program: 94 �C for 5 min, 30 cycles of 94 �C for 1 min,

50 �C for 1:45 min, and 72 �C for 1 min, 72 �C for 7 min,

and 4 �C hold. In addition to these primers, sequencing

was accomplished using the internal primers ITS2

(5¢-GCTGCGTTCTTCATCGATGC-3¢), ITS3 (5¢-GCATC-

GATGAAGAACGCAGC-3¢), ITS4 (5¢-TCCTCCGCTTAT-

TGATATGC-3¢), and ITS5 (5¢-GGAAGTAAAAGTCGTAA-

CAAGG-3¢) (Baldwin, 1992).

Mesofauna identification

All samples were immediately preserved in 80% ethanol on

the boat. As with the plant, identification followed two

approaches: (i) traditional Linnean taxonomy and (ii)

molecular phylogeny. For the Linnean taxonomic approach,

the Pennak (1989) key was used for the crustacean and

flatworm specimens, whereas the segmented worm was

keyed from Kathman & Brinkhurst (1998). For phylo-

genetic analysis, DNA was obtained by using the FastDNA

Spin Kit for Soil (MP Biomedicals, Solon, OH, USA) on

tissue dissected from the organisms, and then PCRs ampli-

fied the 5¢ end of the 18S rRNA gene and a portion of

the cytochrome c oxidase subunit I (COI) gene using the

following protocols:

18S rRNA gene: Amplification used primers SSU F04

(5¢-GCTTGTCTCAAAGATTAAGCCC-3¢) and SSU R22

(5¢-GCCTGCTGCCTTCCTTGGA-3¢) (Meldal et al., 2007)

with a PCR program of 94 �C for 5 min, 30 cycles of 94 �Cfor 45 s, 50 �C for 45 s, and 72 �C for 45 s, 72 �C for 7 min,

and 4 �C hold.

Geothermal-linked biological oasis 329


COI gene: A portion of the COI gene was amplified using

primers pr-a2 (5¢-AGCTGCAGTTTTGGTTTTTTGGA-3¢)and pr-b2 (5¢-ATGAGCAACAACATAATAAGTATCATG-

3¢) for the planarian (Bessho et al., 1992) and primers

LCO1490 (5¢-GGTCAACAAATCATAAAGATATTGG-3¢)and HCO2198 (5¢-TAAACTTCAGGGTGACCAAAAAA-

TCA-3¢) for the other organisms (Hou et al., 2007). Both

used the PCR program given above for the 18S rRNA gene.

All amplicon sequences were compared against public data-

bases using BLAST 2.2.22 (Altschul et al., 1997). Phyloge-

netic analysis was performed on the amplicons. Sequences

were aligned using CLUSTALW (Thompson et al., 1997) and

then a Maximum Likelihood phylogenetic trees were con-

structed using PAUP 4.0b10 (Sinauer Associates Inc., Sunder-

land, MA, USA).

Accession numbers

GenBank accession numbers for all gene sequences generated

and described in this study are as follows: Gammarus 18S

rRNA gene, GU066807; Hyalella 18S rRNA gene,

GU066808; Lamprodrilus 18S rRNA gene, GU066809;

Polycelis 18S rRNA gene, GU066810; Gammarus COI (cyto-

chrome c), GU066811; Hyalella COI, GU066812; Lampro-

drilus COI, GU066813; Polycelis COI, GU066814;

Fontinalis rbcL, GU066815; Fontinalis ITS, GU066816;

Fontinalis tRNA, GU066817.

RESULTS

ROV Description

The ROV (Fig. 1) is fully operator controlled via thrusters that

allow for lake floor reconnaissance and precise ROV position-

ing for sampling, and is equipped with a mechanical arm that

allows for probing and sampling of vent openings. A sampling

cup at the end of the mechanical arm encloses suction tube

openings as well as two calibrated temperature sensors (deli-

berate redundancy) that allows for real-time temperature

determination. Live feed video allows for real-time viewing,

vent identification, and guides sampling efforts. Operator-

controlled syringes (1.0 L capacity each) are located on the

port and starboard sides of the ROV (Fig. 1A), and allow water

samples to be taken for gas and solute chemistry determina-

tions. In addition, a separate suction tube located in the

sampling cup is connected to a vacuum device (Fig 1B) that

passes water and sample into a 1.0-L container outfitted with a

screen covering (1 mm mesh) the exit port to allow water to

pass through while collecting solid phase material. This instru-

ment was used to obtain samples of plant branches and leaves,

and to glean vent emission sediment and metazoa attached to

the moss leaves and branches. Also, a continuous Norprene�

tubing connection from the sampling cup to the boat allows

for large volume (e.g. 300 L) water samples brought to the

surface via a peristaltic pump located on the boat deck. The

ROV is tethered to the boat via an electrical ⁄ optical umbilical,

which facilitates operator communication with the ROV and

also includes the peristaltic pump tubing. The ROV also

carries a solid sample collection basket, a color sector scan

sonar system, and a 35-mm camera and strobe.

Plant identification

This report focuses on the intriguing and robust colony of a

macrophyte that visually appeared to be of a single type and

that was closely associated with vent emissions (Fig. 2)

(http://www.tbi.montana.edu/media/Fontinalis_vent.html).

Initial inspection of leaf architecture identified the plant as the

moss Fontinalis. Moss leaf morphology included an absence

of keel, twist at upper part of leaf, and 30–40� spread of leaves.

These characteristics are consistent with Fontinalis novae-

angliae (Welch, 1960). Genetic characterization based first

on the PCR cloned rbcL (1251-bp amplicon) suggested it was

99.9% identical to GenBank accession AB050949, annotated

A

B

Fig. 2 Video excerpt and photograph of the Fontinalis moss. (A) Part of Fonti-

nalis colony adjacent to the high output vent and illuminated by the running

lights on the ROV. The ROV sampling arm is shown inserted into the vent hole

while sampling water from the vent. (B) Close up of moss leaf structure.



as Fontinalis antipyretica rbcL. Having placed the moss in the

genus Fontinalis, subsequent genetic analysis focused on the

ITS region spanning from the 3¢ portion of the 18S rRNA

gene and including ITS1, the 5.8S rRNA gene ITS2, and a 5¢portion of the 26S rRNA gene. In addition, the tRNA-

Leu ⁄ Phe spacer DNA was also PCR cloned and sequenced.

Both nuclear genomic DNA regions are well represented

among the Fontinalis entries. A maximum-likelihood analysis

of ITS DNA concatenated with tRNA spacer sequence (1807

nucleotides total) suggests that the Yellowstone Lake speci-

men is most closely related to a clade comprised of F. anti-

pyretica, Fontinalis gigantea, and Fontinalis chrysophylla

(Fig. 3), all of which differ from this moss by having keeled

leaves.

Mesofauna identification

The mesofauna found associated with the moss are shown in

Fig. 4. As with the moss, Linnean taxonomic approaches were

used initially to identify the organism, which then directed

DNA target selection and guided PCR primer design.

Sequence availability for the 18S rRNA and COI genes

differed for various species. A summary maximum-likelihood

tree for the 18S rRNA gene is shown in Fig. 5. Yellowstone

Lake specimen I4 (Fig. 4A) keyed out as Hyalella azteca,

which was in agreement with 18S rRNA gene phylogenetic

analysis that clearly placed it within the genus Hyalella

(Fig. 5), and an COI-based analysis which confirmed this

specimen as the species azteca (Fig. 6). Morphological fea-

tures of crustacean specimen I3 identified it as Gammarus

lacustris (Fig. 4B), which was also supported by phylogenetic

analyses of the 18S rRNA (Fig. 5) and COI (Fig. 7) genes.

The segmented worm specimen I6 (Fig. 4C) was sexually

immature so visual identification could only assign it to the

family Lumbriculidae; a genus designation was not possible.

Phylogenetic analysis (18S rRNA) confirmed it as a member

of the Lumbriculidae and potentially a member of the genus

Lamprodrilus (Fig. 5). The flatworm specimen I7 was appar-

ently damaged during suctioning from the moss leaves and

thus only a tentative identification was made as Polycelis coro-

nata. Phylogenetic analysis (18S rRNA) confirms that it is a

species of Polycelis (Fig. 5), but there are few sequences from

that genus available in GenBank, leaving us uncertain as to a

species level identification. Additional COI work with the

putative Lamprodrilus (Fig. S2) and Polycelis (Fig. S3) speci-

mens are in agreement with these genus-level designations.

Vent environment characterization

Vent water temperature was 34.6 �C, but slightly cooler at

32.3 �C within the moss cluster adjacent to the vent. Light

intensity was assessed throughout the water column above the

vent site. Incremental lowering of a light meter showed a

nearly two order of magnitude decrease in light irradiance in

the first 10 m (Fig. 8). Near vent depth (28 m), light irradi-

ance decreased further (total of >8000-fold decrease) and was

likely due in part to the constant emission of solid phase mate-

rial from the vent (http://www.tbi.montana.edu/media/

Fontinalis_vent.html) that resulted in the surrounding water

being somewhat clouded. SEM electron dispersive X-ray anal-

ysis of this beige-colored material found it to be comprised

primarily of an alumino-silicate material (% atom composition:

O, 62.3; Si, 17.7; Al, 5.1). The close association of the moss

with the vent resulted in the moss being covered with this

material.

Additional analysis of the vent water was directed towards

ascertaining whether it could be the source of important

nutrients for the moss. Total inorganic CO2(aq) concentra-

tion was 10.22 mM (Table 1) and 1.11 mM upon mixing with

96

65

81

59

94

83

67

67

51

55

95

51

0.005 substitutions per site

Fontinalis antipyretica (AF192108, AF191517)

Fontinalis neomexicana (AF192105, AF191514)

Fontinalis hypnoides (AF192101, AF191510)


Fontinalis duriaei (AF192126, AF191535)

Fontinalis squamosa (AF192111, AF191520)


Fontinalis chrysophylla (AF192103, AF191512)

Fontinalis gigantea (AF192127, AF191536)

Yellowstone Lake sample (GU066816, GU066817)

Fontinalis redfearnii (AF192098, AF191507)

Fontinalis dalecarlica (AF192125, AF191534)

Fontinalis flaccida (AF192104, AF191513)

Fontinalis novae-angliae (AF192131, AF191540)

Fontinalis sullivantii (AF192102, AF191511)

Fontinalis missourica (AF192106, AF191515)

Fontinalis welchiana (AF192133, AF191542)

Fig. 3 Maximum-likelihood tree illustrating the phylogenetic relationship of

the Yellowstone Fontinalis ITS-tRNA DNA concatenate. Bootstrap values

>50% are shown. Tree was rooted with Brachelyma subulatum (AF192094,

AF191503), Dichelyma falcatum (AF192096, AF191505), and Dichelyma

uncinatum (AF192095, AF191504).



lake water approximately 1 m above the vent. Presumably,

similar mixing might occur within the moss stand immediately

adjacent to the vent opening. Vent water was acidic (pH 5.5)

and significant levels of some important macronutrients were

present. Specifically, a constant flux of �24 lM NH4+ and

0.75 lM NO3) (Table 1) combine to provide significant levels

of available nitrogen. The same can be said for sulfur and

potassium (60 and 140 lM, respectively), although total phos-

phorus was below detection, which in this analysis was

<10 lM. Among the trace elements, B and Li were present at

significant concentrations, and the metalloids Sb and As were

also present, with the latter at significant levels (see below).

DISCUSSION

The literature contains numerous inventories and phy-

logenetic characterizations of the microbial communities

intimately linked with geothermal features, whereas such

relationships with eukaryotic organisms have been much less

common. The proliferation of complex higher organisms in

close association with a Yellowstone Lake geothermal vent

parallels that documented for deep marine vents (Rona et al.,

1986; Fustec et al., 1987; Galkin & Moskalev, 1990;

Cann et al., 1994; Desbruyeres et al. 1994; Chevaldonne

et al., 1997), although to our knowledge this is the first such

documentation for a freshwater habitat, and thus this report

extends and expands awareness of such ecological relation-

ships. Unidentified macrophytes have been reported in the

general vicinity of vents at SCUBA depths (Remsen et al.,

2002); however, the frequency of the moss-vent association

documented here is unknown at present. Given the lake’s vio-

lent volcanic past and relative youth (�11 000 years; Morgan

et al., 2007), fossil evidence of this or any other association

would likely be rare.

CBA

Fig. 4 Photos of mesofauna collected from the branches of the Fontinalis moss. (A) Hyalella azteca specimen, (B) Gammarus lacustris, and (C) the Lamprodrilus sp.

All samples are preserved in 70% ethanol (bar = 1 mm in each panel).

Lake Organism I7 Accession

Polycelis felina DQ665996

Polycelis tenuis Z99949

Phagocata vitta DQ6659998

Crenobia alpina M58345


Lamprodrilus achaetus DQ31323

Lamprodrilus stigmatias DQ313

Lumbriculus variegatus AF2094

Styloscolex baicalensis AJ308

Helobdella paranensis AF11598

Tubifex tubifex EU126846

Lake Organism I4 Accessions

Hyalella sp GreenHy AJ966716

Hyalella sp LGB1 AJ966715

Hyale nilssoni AY826958

Parhyale hawaiensis AY826957


Gammarus lacustris AY926786

Gammarus troglophilus AF20298

Dikerogammarus villosus EF582

Chaetogammarus stoerensis AY9

81 88

67

100

51

90

100

100

71

54

99

53

93

51 79

63

0.05 substitutions per site Chaetogammarus stoerensis (AY926762)

Dikerogammarus villosus (EF582898)

Gammarus troglophilus (AF202983) Gammarus lacustris (AY926786) Yellowstone Lake sample I3 (GU066807)

Parhyale hawaiensis (AY826957) Hyale nilssoni (AY826958)

Yellowstone Lake sample I7 (GU066810) Polycelis felina (DQ665996)

Polycelis tenuis (Z99949) Phagocata vitta (DQ665998)

Crenobia alpina (M58345) Yellowstone Lake sample I6 (GU066809)

Lamprodrilus achaetus (DQ313235) Lamprodrilus stigmatias (DQ313236) Lumbriculus variegatus (AF209457)

Styloscolex baicalensis (AJ308513) Helobdella paranensis (AF115987)

Tubifex tubifex (EU126846) Yellowstone Lake sample I4 (GU066808)Hyalella sp. GreenHy (AJ966716) Hyalella sp. LGB1 (AJ966715)

Fig. 5 Abbreviated maximum-likelihood tree illustrating the phylogenetic rela-

tionship of the 18S rRNA genes PCR cloned from the Yellowstone Lake mesofauna

associated with the moss. Phylogeny is based on alignments of nucleotides. Boot-

strap values >50% are shown. Tree was rooted with Dalatias licha (AY049827),

Pristiophorus cirratus (AY049849), and Squalus acanthias (M91179).

Yellowstone Lake sample I4 (GU066812)

Hyalella azteca (DQ464710)

Hyalella sp. isolate GreenHy (AJ968918)

Hyalella sp. isolate BeulCf (AJ968916)

Hyalella sp. isolate LGB1 (AJ968917)

Hyalella sp. isolate BeulHy (AJ968915)

Hyale nilssoni (AF520435)

Parhyale hawaiensis (EF989709)

Orchestia cavimana (EF989708)

98

100

100

100

96

53

0.05 substitutions per site

Fig. 6 A maximum-likelihood tree illustrating the phylogenetic relationship of

the COI gene PCR cloned from the Yellowstone Lake specimen I4. Phylogeny is

based on alignments of 626 nucleotides, representing the 65–690 bp region of

the gene. Bootstrap values >50% are shown. Tree was rooted with Gammarus

lacustris (AY926671).



Water temperature in the vent examined in this study

was significantly lower than that documented for marine vents

and is also much lower than most vents described for this lake

(Morgan et al., 2007). Nevertheless, the temperature

observed at this vent is considerably greater than the sustained

temperature range of the coldwater moss Fontinalis (Glime,

1987). Even tropical bryophytes do poorly above 25 �C(Frahm, 1990) and truly aquatic bryophytes are rare there.

However, we note the report of geothermal bryophytes:

Bryum japanense can grow at 40 �C, and Philonotis laxiretis

and Bryum cyclophyllum at 38 �C (Watanabe, 1957). Previous

physiology work with F. antipyretica demonstrated this moss

experiences a net carbon loss at 20 �C (Carballeira et al.,

1998). And while Fontinalis appears to have some adaptive

capacity to environmental temperature (Fornwall & Glime,

1982), the extremes noted for the Fontinalis colony described

herein exceeds the environmental plasticity documented for

this bryophyte. Consequently, at the temperatures observed

in this study CO2 lost due to respiration would be expected to

greatly exceed photosynthetically captured carbon (Sommer

& Winkler, 1982) under conditions where this moss is

normally documented to occur; i.e. not associated with a geo-

thermal vent.

In addition to high temperature stress, water turbidity lim-

ited light penetration at this location. As a group, bryophytes

are tolerant of shade (Martin, 1980; Martin & Churchill,

1982), and indeed Fontinalis has been documented to occur

at depths of up to 120 m in Crater Lake, where water clarity

Yellowstone Lake sample I3 (GU066811)

G. pseudolimnaeus (EF570333)

G. troglophilus (EF570350)

G. lacustris (AY926671)

G. bousfieldi (EF570299)

G. emeiensis (EF570306)

G. pulex (EF570334)

G. tigrinus (EF570348)

G. abstrusus (EF570304)

G. martensi (EF570325)

G. nipponensis (EF570312)

G. sinuolatus (EF570339)

G. duebeni (AY926669)

G. locusta (EF570324)

G. roeseli (EF570337)

G. glabratus (EF570307)

G. koreanus (EF570314)

G. lichuanensis (EF570356)

G. curvativus (EF570302)

G. gregoryi (EF570311)

G. comosus (EF570300)

G. nekkensis (EF570331)

56

81

100

100

0.05 substitutions/site

Fig. 7 A maximum-likelihood tree illustrating the phylogenetic relationship of

the COI gene PCR cloned from the Yellowstone Lake specimen I3 and to

Gammuras accessions available in GenBank. Phylogeny is based on alignments

of 593 nucleotides, representing the 95–687 bp region of the gene. Bootstrap

values >50% are shown. Tree was rooted with Sinogammarus chuanhui

(EF570355).

Fig. 8 Light penetration in the Yellowstone Lake water column above the

vent. Light irradiance is shown as percent relative to that recorded at the surface

(�800 W m)2) and as a function of depth.

Table 1 Geochemical analysis of the vent water

Major cations and neutrals

Na+ K+ Ca+2 Si NH4+ Mg+2

mM mM mM mM lM lM

1.23 0.06 0.12 0.27 27.4 70.5

Anions

Cl) SO4)2 F) NO3

) S2O3)2 PO4

)3

mM mM lM lM mM mM

0.55 0.14 95 0.75 bd bd

Dissolved gases

CO2 (aq) DIC S)2 (aq) O2 (aq) CH4 (aq) H2 (aq)

mM mM lM lM lM nM

10.22 10.2 bd 81.8 7.6 14

Trace elements

As Se Rb Sr Mo Sb

lM lM lM lM lM lM

1.85 bd 0.15 0.58 0.11 0.03

B Al Mn Fe Zn Ga

lM lM lM lM lM lM

28.62 3.23 bd bd 0.22 bd

Cs Ba W Pb V Li

lM lM lM lM lM lM

0.21 0.11 0.08 bd 0.25 32.20

bd, below detection.



allows light to penetrate to considerable depths (Hasler,

1938). This is in contrast to Yellowstone Lake, which is quite

turbid owing to lake sediments being carried by subsurface

currents generated by significant daily winds (Benson, 1961).

In addition to lake turbidity, mineral vent emissions no doubt

contributed to reduce light levels at vent depth by nearly four

orders of magnitude relative to the lake surface waters

(Fig. 8). Vent emissions also generated a constant showering

of mineral material that covered leaf surfaces of the moss

(http://www.tbi.montana.edu/media/Fontinalis_vent.html),

which presumably would further reduce access to light and

thus present additional physiological challenges to incorporat-

ing CO2 via photosynthesis.

All of the above apparent vent-associated growth con-

straints notwithstanding, occurrence of this moss appeared

restricted to the immediate areas surrounding vent emissions,

suggesting the latter provides a positive influence which

overcomes the presumably negative vent-associated effects of

high temperature, low light, and epiphytic mineral covering.

The high concentration of CO2(aq) in the vent water

(Table 1) and in the area immediately surrounding the vent

probably offers an explanation. Under these conditions,

the moss’ Rubisco enzyme is likely constantly saturated,

permitting it to efficiently capture and fix carbon at rates

that exceed respiratory costs, and thus allowing for biomass

accumulation and moss growth. Availability of substantial

fixed nitrogen (Table 1) would presumably also be a favorable

influence.

Seemingly, the vent environment would also be atypical for

the mesofauna found associated with the moss. The organisms

identified here are normally found in the littoral zone as

opposed to being found in the profundal zone. In this vent-

associated environment, these organisms presumably experi-

ence significantly elevated temperature and CO2 concentra-

tions, acidic pH, and depleted O2; conditions that depart

from optimum conditions known for these species. Further,

concentrations of aluminum and arsenic (Table 1) in the vent

waters are similar in magnitude to the LC50 reported for

members of Hyalella (Borgmann et al., 2005) and Gamma-

rus (Spehar et al., 1980). Although these animals are motile,

they are nevertheless subjected to lake currents that could

potentially carry them in and out of the vent water plume by

the swaying of the moss. Thus, at a minimum, they would be

exposed to these extreme conditions for brief periods. It is

currently unknown how these organisms are coping with this

environment.

Phylogenetic analysis of the mesofauna specimens resulted

in maximum-likelihood estimates that were in agreement

with Linnean-based approaches. Assessments of specimen I4

based on the 18S rRNA and the COI genes were consistent

with each other and with the classification based on

morphological features: all classified this organism as Hyalella

azteca (Figs 5 and 6). The same can be said for crustacean

specimen I3, which was identified as Gammarus lacustris

(Figs 4B, 5 and 7). The sexual immaturity of the segmented

worm specimen I6 (Fig. 4C) constrained visual identifica-

tion to a family-level (Lumbriculidae) designation. Phylo-

genetic analysis (18S rRNA) confirmed it as a member of

the Lumbriculidae and potentially within the genus Lampro-

drilus (Fig. 5), and was consistent with the results of the

COI-based analysis (Fig. S2). However, we note with

interest that the latter suggested significant phylogenetic

relatedness of segmented worms previously identified as

Agriodrilus and Teleuscolex (Fig. S2). This has been noted

previously with specimens collected from Lake Baikal

(Kaygorodova & Sherbakov, 2006), and is consistent with

the suggestion that Agriodrilus and Teleuscolex may actually

be subgenera of, or cogeneric with, Lamprodrilus based on

some key morphological features used in Linnean taxonomy

(Brinkhurst, 1989). To the best of our knowledge, the

aforementioned segmented worm genera have never been

reported in North America.

Identification of the moss using Linnean and molecular

genetic techniques yielded somewhat different results. Both

approaches clearly placed the moss in the genus Fontinalis,

however more precise taxonomic resolution was less certain.

Initial phylogenetic analysis of the plant based on rbcL sug-

gested high relatedness to F. antipyretica, although because

Fontinalis rbcL is not well represented in GenBank, subse-

quent analysis examined ITS and tRNA-Leu ⁄ Phe spacer DNA

sequences for which Fontinalis is much more robustly repre-

sented. A maximum likelihood-based assessment (Fig. 3)

illustrated F. antipyretica to be paraphyletic and thus similar

to that reported by Shaw & Allen (2000). The Yellowstone

specimen occupied the same clade with at least one F. antipy-

retica accession, but was separate from F. novae-angliae

(Fig. 2). Lack of congruency between the leaf morphology

and genetic characterization has been noted previously (Shaw

& Allen, 2000).

In summary, the vent water properties represent a paradox

with respect to bryophyte photosynthesis, but nevertheless

appears foundational to a food web comprised of Fontinalis

upon whose surfaces reside metazoa that in turn are known to

be consumed by fish. This linkage between a food web and

raw geochemical inputs from the Yellowstone caldera parallels

that documented for deep marine vents, where geochemical

energy is converted to biochemical energy that enables and

sustains complex communities in interesting, if not extraordi-

nary, environments. Ecologically, these deeply submerged

hydrothermal vents are oases that are essentially islands (sensu

MacArthur and Wilson) from which biological diversity

emerges.

ACKNOWLEDGMENTS

This research was supported primarily by a grant from the

Gordon and Betty Moore Foundation (Grant #1555), and

additional funding from the National Park Service Centennial



Challenge Match Program (PMIS #137808). Work was con-

ducted under NPS research permit No. 5700.

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SUPPORTING INFORMATION

Additional Supporting Information may be found in the

online version of this article:

Fig. S1 Maps relating location of study.

Fig. S2 A maximum-likelihood tree illustrating the phylogenetic relationship of

the COI gene PCR cloned from the Yellowstone Lake specimen I6.

Fig. S3 A maximum-likelihood tree illustrating the phylogenetic relationship of

the COI gene PCR cloned from the Yellowstone Lake specimen I7.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.



A geothermal-linked biological oasis in Yellowstone Lake, Yellowstone National Park, Wyoming

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