Evidence for Genetic Drift in the Diversification of a Geographically

Isolated Population of the Hyperthermophilic Archaeon Pyrococcus

Patricia Escobar-Paramo,* Sulagna Ghosh, and Jocelyne DiRuggiero*
*Department of Cell Biology and Molecular Genetics, University of Maryland; and Center of Marine Biotechnology,
University of Maryland Biotechnology Institute

Genetic drift is a mechanism of population divergence that is important in the evolution of plants and animals but is thought
to be rare in free-living microorganisms because of their typically large population sizes and unrestricted means of dis-
persal. We used both phylogenetic and insertion sequence (IS) element analyses in hyperthermophilic archaea of the genus
Pyrococcus to test the hypothesis that genetic drift played an important role in the diversification of these microorganisms.
Multilocus sequence typing of a collection of 36 isolates of Pyrococcus, from different hydrothermal systems in the Pacific
Ocean and the Mediterranean Sea, revealed that Pyrococcus populations from different geographic locations are genet-
ically differentiated. Analysis of IS elements in these isolates exposed their presence in all individuals of only one geo-
graphically isolated lineage, that of Vulcano Island in the Mediterranean Sea. Detailed sequence analysis of six selected IS
elements in the Vulcano population showed that these elements cause deleterious genomic alterations, including inacti-
vation of gene function. The high frequency of IS elements in the sampled population together with their observed harmful
effects in the genome of Pyrococcus provide molecular evidence that the Vulcano Island population of Pyrococcus is geo-
graphically isolated and that those genetic mobile elements have been brought up to high frequency by genetic drift. Thus,
genetic drift resulting from physical isolation should be considered as a factor influencing differentiation in prokaryotes.

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Introduction
In sexually reproducing species, geographic isolation
can generate a barrier to gene flow that can result in genetic
drift and/or local adaptation and differentiation (Futuyma
1998). In contrast, microorganisms are generally believed
to have unrestricted dispersal capabilities due to their small
size, extremely large populations, and highly plastic
genomes, providing for worldwide distribution (Finlay
2002). Recent studies have challenged this view and
demonstrated that prokaryotes colonizing thermophilic
environments undergo genetic differentiation as a result
of geographic barriers to dispersal (Papke et al. 2003;
Whitaker, Grogan, and Taylor 2003; Papke and Ward
2004). Because nucleotide substitution is a recognized evo-
lutionary mechanism in all organisms, these studies used
high-resolution multilocus sequence typing (MLST) anal-
ysis of strains isolated from different geographic locations
and, in the case of Whitaker, Grogan, and Taylor (2003),
calculated the variance of genetic diversity (Cockerhams
FST parameter) between groups of strains to show popula-
tion differentiation between regions. However, an alterna-
tive explanation would be that the observed biogeographic
patterns are the result of sampling biases toward dominant
clones at a particular time, in different locations (Fenchel
2003). Therefore, the ability to detect geographical isolation
of microbial population necessarily relies on the use of
high-resolution genetic markers that undoubtedly demon-
strate the effects of genetic drift on genetic differentiation.
Insertion sequence (IS) elements constitute such mo-
lecular markers. IS elements are short segments of DNA,
typically 12 kb in length, with the ability to move within
and between genomes, without a need for DNA homology
(Galas and Chandler 1989). Although some elements can
persist in the genome because they bring a selective advan-
Key words: genetic drift, Pyrococcus, transposons, archaea,
phylogeny, evolution.
E-mail: diruggie@umd.edu.
Mol. Biol. Evol. 22(11):22972303. 2005
doi:10.1093/molbev/msi227
Advance Access publication August 3, 2005
Published by Oxford University Press 2005.
tage to their host, i. e., antibiotic resistance (Lupski 1987),
most IS elements seem to behave as selfish genes or para-
sites, persisting without major consequences or being del-
eterious to the host genome. The reduced host fitness is then
the result of gene inactivation, adjacent gene deletions, and
other chromosomal rearrangements (Doolittle and Sapienza
1980; Orgel and Crick 1980; Brookfield 2005). It is known
that the frequency of IS elements per site (any locus where
an IS is present in at least one of the genomes of the host
population) depends on the rates of transposition, excision,
and recombination (Langley, Brookfield, and Kaplan
1983). Natural selection limits the spread of IS by transpo-
sition due to the deleterious fitness effects associated with
gene disruption and inactivation, and an equilibrium is
reached when the effects of transposition and selection
are balanced (B. Charlesworth and D. Charlesworth
1983). In large populations the number of sites for transpo-
sitions is high enough that finding elements linked to a
particular site is an unlikely event. In other words, the
probability of finding two individuals with the same IS
at a particular site is much higher in a small population than
in a large population (Slatkin 1985). Therefore, stochastic
processes rather than selection better explain the presence
and maintenance at high frequencies of IS elements in nat-
ural populations.
Hyperthermophilic archaea of the genus Pyrococcus
are found in deep sea hydrothermal vents and shallow ma-
rine hot springs and can only grow above 70C and under
anaerobic conditions (Fiala and Stetter 1986; Lepage et al.
2004). Their environment displays island-like character-
istics with high-temperature niches (hot springs) separated
by large areas of inhospitable conditions (cold oceans). The
genus comprises three species Pyrococcus furiosus, Pyro-
coccus abyssi, and Pyrococcus horikoshii for which com-
plete genome sequences were obtained (Kawarabayasi et al.
1998; Robb et al. 2001; Cohen et al. 2003). Full-length IS
elements are found in the genome P. furiosus but are absent
from that of P. abyssi and P. horikoshii (DiRuggiero et al.
2000; Lecompte et al. 2001). P. furiosus IS elements belong
2298 Escobar-Paramo et al.
to a novel family of transposable elements and can be clas-
sified into three major groups based on nucleotide identity
(83%84%): IS-pfu1 (11 copies), IS-pfu2 (8 copies), and
IS-pfu3 (4 copies) (Kanoksilapatham et al. 2004). Each
IS element contains a transposase gene (702 bp), a short
spacer (5457 bp), and two flanking 16-bp inverted repeats
(fig. 3).
We used both, MLST and IS element analyses, in
hyperthermophilic archaea of the genus Pyrococcus to test
the hypothesis that genetic drift played an important role in
the diversification of these microorganisms.
Materials and Methods
Strain Isolation and Growth Conditions
Water and sediment samples were obtained from mud
pools and underwater hot springs in Vulcano Island, Italy,
in fall 2002 and 2003. Samples were stored in reduced me-
dia and anaerobic conditions until further processing.
Enrichment cultures were grown in 5060 ml liquid P.
furiosus medium inoculated with 12 ml, as described pre-
viously (Lepage et al. 2004). Following incubation at 90C,
cell densities were determined using acridine orange direct
counts (Darzynkiewics 1990). Cell-containing enrichment
cultures were plated on solid gel media as described in
Lepage et al. (2004) and incubated at 90C in anaerobic jars
for 34 days. Eight individual colonies from the 2002 sam-
pling (VB81, VB82, VB83, VB85, VB96, VB93, VB112,
VB113) and 11 from the 2003 sampling (V61, V62,
V63, V72, V73, V211, V212, V221, V222, V231,
V232) were selected and subjected to another round of
liquid and solid media growth before final culture in liquid
medium and storage at 4C. Isolates from the Juan de Fuca
Ridge (MZ4 from 1995; MV4 and MV7 from 1996; AV5
from 1999; EX2 from 2002; and JT1 from 1982) and the
East Pacific Ridge (12/1, 30/3, 30/4, 31/2, 32/1, 32/2,
32/3, 32/4 from sampling in 1999) in the Pacific Ocean
were kindly supplied by J. Baross (University of Washing-
ton, personal communication), T. Tuttle (Tuttle and DiRug-
giero, personal communication), and P. Forterre (Lepage
et al. 2004), respectively.
Strain Characterization and Phylogenetic Analysis
Genomic DNA was extracted from 60 ml of liquid
cultures using the BIO 101 Genome Kit (Q-BIOgene,
Irvine, Calif.) as described by the manufacturer. Specific
16S rDNA primers, Arch0333aF15 (5#-TCCAGGCCC-
TACGGG-3#) and Arch0958R19 (5#-YCCGGCGTT-
GAMTCCAATT-3#) were used to obtain polymerase
chain reaction (PCR) products from all the Vulcano iso-
lates. All loci were amplified by PCR in 12.5 ll reactions
containing 10 pmoles of primers, 3.0 mM MgCl2, 50 mM
KCl, 10 mM Tris, 0.2 mM deoxynucleoside triphosphates,
and 1 U of AmpliTaq DNA polymerase (Fermentas,
Hanover, Md.). PCR conditions were as follows: 94C for
2 min and 30 cycles of 94C for 30 s, 55C or 60C for
30 s, and 72C for 30 s. Sequences were obtained by direct
sequencing of PCR products purified using exoSAP (USB,
Cleveland, Ohio) and sequenced using Big Dye terminator
reactions (Perkin Elmer, Boston, Mass.) and an ABI3100
automated sequencer (Applied BioSystems, Foster City,
Calif.). Sequences for 16S rDNA, approximately 700 bp
in length, were aligned with those of closely related species
of Thermococcales (as listed in the GenBank database
[http://www.ncbi.nlm.nih.gov]) with the ClustalV program
(Higgins, Bleasby, and Fuchs 1992) from the Sequence
Navigator package and used to generate the tree shown
in Figure S1 (Supplementary Material online) with the
Neighbor-Joining algorithm from PAUP* 4.0b10 (Swofford
1998). Sequences from the other loci were edited and
aligned using the same software. Phylogenetic reconstruc-
tion of the evolutionary history of 36 Pyrococcus strains
and one Thermococcus strain was performed by simulta-
neous analysis of the DNA sequences of four loci homol-
ogous to P. furiosus valyl-tRNA synthetase (PF0290),
putative DNA helicase (PF0572), hypothetical protein
PF1459, and a-glucan phosphorylase (PF1536) 1 hypo-
thetical protein PF1537 (IIV; table 1). Three additional
loci prephenate dehydratase (PF0291) 1 deoxyhypusine
synthase (PF0292), 3-hydroxyisobutyrate dehydrogenase
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(PF0716), and a putative protease PF1905 (IVI; table 1)
were used to expand on the phylogenetic relationship
among the 19 Vulcano isolates and P. furiosus (IVII; table
1). These loci were selected to represent regions of high-
nucleotide variation among the three reference strains
and include protein-encoding genes and intergenic regions.
Primers used to amplify these loci are reported in Table S1
(Supplementary Material online). Phylogenetic analyses
were performed using the maximum likelihood analysis
from PAUP* 4.0b10 (Swofford 1998). In the likelihood
analysis, the general time reversible model with invariant
site and gamma correction was used (GTR 1 I 1 C) with
four rate categories. Likelihood and parsimony bootstrap
proportions were calculated from 1,000 iterations.
IS Analysis
Six primer pairs were designed to amplify specific IS
elements homologous to P. furious PF0069, PF0242,
PF0946, PF0536, PF1736, and PF0898, in all Vulcano
strains (Table S1, Supplementary Material online). The
PCR and sequencing conditions used were similar to
those described above. Sequences were aligned using the
ClustalV program in the Sequence Navigator package
(Applied BioSystems).
Southern Blot Analysis
Genomic DNA was isolated as described above. Re-
striction digest with HindIII and Southern hybridizations
were performed according to the procedure of Sambrook,
Fritsch, and Maniatis (1989). The probe, 210 bp in length
containing P. furiosus transposase sequence, was generated
by PCR and labeled using the DIG probe synthesis kit
(Roche Diagnostics Inc., Indianapolis, Ind.) as described
by the manufacturer.
Results and Discussion
Phylogenetic Analysis
We evaluated the existence of geographic genetic dif-
ferentiation by MLST of 36 Pyrococcus strains from three
geographical locations (fig. 1) comprising 3 reference
 Genetic Drift in the Evolution of Pyrococcus 2299

Table 1
Loci Considered in the Phylogenetic (IVII) and IS Elements (VIIIXIII) Analyses
Variable Sites (nucleotide
substitution rateb)
Segment
Loci ORF Numbers Genome Locationsa Description Analyzed (bp) All Strainsc Vulcano Strainsd
I PF0290 304315306990 Valyl-tRNA synthetase 654 252 (38.5%) 77 (12%)
II PF0572 592552594519 Putative DNA helicase 543 310 (57%) 119 (21%)
III PF1459 13642501365701 Hypothetical protein 773 394 (50%) 40 (5%)
IV PF1535 1 14329101435938 a-Glucan phosphorilase 1 718 383 (53%) 40 (5%)
PF1536 hypothetical protein
V PF0291 1 307149308978 Prephenate dehydratase 1 778 N/A 116 (14%)
PF0292 deoxyhypusine synthase
VI PF0716 715679716515 3-Hydroxyisobutyrate 699 N/A 13 (1.8%)
dehydrogenase
VII PF1905 17559141757236 Putative protease 1,182 N/A 141 (12%)
VIII PF0069 7299373694 Transposase 651 N/A 82 (13%)
IX PF0242 253396253584 Transposase 583 N/A 68 (12%)
X PF0946 909695910639 Transposase 730 N/A 30 (4%)
XI PF0536 552553553254 Transposase 702 N/A 2 (0.3%)e
XII PF1736 16123831613132 Transposase 750 N/A 24 (3%)f
XIII PF0898 870647871348 Transposase 702 N/A N/A

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NOTE.N/A: not applicable.
a
Genome locations in P. furiosus.
b
Number of variable nucleotides per analyzed segment.
c
Thirty-six strains considered.
d
Twenty strains considered unless indicated.
e
Eight strains considered.
f
Six strains considered.

strains fully sequenced, P. furiosus DSM3638 (Fiala and
Stetter 1986), P. horikoshii OT3 (Gonzalez et al. 1998),
and P. abyssi GE5 (Erauso et al. 1993), 19 strains isolated
from shallow hot spring samples collected in Vulcano Is-
land (Italy) by our group in 2002 and 2003 and identified
as Pyrococcus sp. based on 16S rDNA sequence analysis
(Fig. S1, Supplementary Material online), and 14 Pyrococcus
strains previously isolated in the Pacific Ocean from the
Juan de Fuca Ridge and the East Pacific Ridge (Lepage
et al. 2004) (samples collected in 1982, 1995 to 2002).
As demonstrated in previous studies with Sulfolobus and
two groups of thermophilic cyanobacteria (Papke et al.
2003; Whitaker, Grogan, and Taylor 2003), the patchy geo-
graphic distribution of their environment was reflected in
the genetic composition of the Pyrococcus strains. Six ma-
jor lineages of Pyrococcus, corresponding to the main geo-
graphical locations considered in the study, were resolved
in the maximum likelihood phylogenetic tree derived from
the simultaneous analysis of nucleotide sequence data for
four loci (IIV; table 1) from 36 strains (fig. 1). Isolates from
the East Pacific Ridge formed the most ancestral group fol-
lowed by the group of isolates from the Juan de Fuca Ridge
(fig. 1). The reference strains P. abyssi and P. horikoshii
(isolated from the North Fiji Basin and the Okinawa Trough
in the Pacific Ocean, respectively) stand alone each as sep-
arate lineages, whereas P. furiosus and the isolates collected
from the shallow hot springs of Vulcano Island (Italy) in the
Mediterranean Sea constitute a more recent group. How-
ever, within this latter group, P. furiosus, also isolated in
Vulcano Island in 1986 (Fiala and Stetter 1986), is highly
divergent from the more recently isolates from Vulcano.
The data represented in figure 1 suggest that physical bar-
riers to dispersal, and therefore geographical isolation, is in
part responsible for driving the observed genetic divergen-
ces in those hyperthermophilic microorganisms (Papke
et al. 2003; Papke and Ward 2004), although alternative ex-
planations such as the presence of dominant clones cannot
be ruled out.
IS Analysis
The relationship between geographical isolation and
genetic divergence in the Pyrococcus populations was fur-
ther supported by our observation that P. furiosuslike IS
elements were present in all the isolates from Vulcano Is-
land (Mediterranean Sea) and completely absent from that
of the Pacific Ocean (Fig. S2, Supplementary Material on-
line). We used standard Southern blot hybridization and
a 210-bp probe of highly conserved sequences for P.
furiosus ISs (DiRuggiero et al. 2000). The number of bands
visualized by this method in the Vulcano isolates ranged
from 15 to 23, and the band patterns differed among isolates
and with that of P. furiosus illustrating diversity in the
number and chromosomal positions of ISs among isolates.
Sequence analysis of the complete genome sequence of
P. abyssi and P. horikoshii revealed the presence of a
211-bp DNA fragment at position 761435 and a 244-bp
fragment at position 368535 in each genome, respectively.
Both fragments have high level of nucleotide identity (89%
and 91%) to P. furiosus transposase sequences (DiRuggiero
et al. 2000; Lecompte et al. 2001), suggesting that IS ele-
ments might have been present in the Pyrococcus popula-
tions of the Pacific Ocean at some point in time and were
subsequently lost. IS element diversity in the Vulcano
isolates was further analyzed by direct sequencing of six
selected ISs and their flanking regions (table 1). We found
three categories of IS elements evolutionary dynamics based
on our sequence analysis (fig. 2). In the first category, ISs and
2300 Escobar-Paramo et al.

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FIG. 1.Maximum likelihood tree inferred from the analysis of a concatenated nucleotide alignment of four loci: valyl-tRNA synthetase (PF0290),
putative DNA helicase (PF0572), hypothetical protein PF1459, and a-glucan phosphorylase (PF1536) 1 hypothetical protein PF1537 (IIV; table 1) in 36
Pyrococcus strains and 1 Thermococcus strain used as the out-group. (a) World map indicating the locations where the samples were collected. (b) The
tree was constructed using PAUP* 4.0b10 (Swofford 1998) with a GTR 1 I 1 C4 model of sequence evolution. Numbers above branches indicate
maximum likelihood (.60%)/maximum parsimony bootstrap proportions (.60%) when the same branches were recovered. Location names to the right
of the strains indicate their origin.

their flanking open reading frames (ORFs) were found in the
same order in all the Vulcano isolates and in P. furiosus
(PF0069, PF0242, PF0946), and ISs accumulated neutral
point mutations at the same level as protein-encoding genes
(table 1) indicative of coevolution with their host. In the sec-
ond category, ISs were present in some isolates but absent in
others and/or showed modifications of the flanking ORFs
(PF0536, PF1736), suggesting potential selection against
the retention of those mobile elements (fig. 2). For example,
in the genome of P. furiosus, IS-pfu2 PF1736 flanks one side
of a composite transposon, with IS-pfu2 PF1752 flanking the
other side. This composite transposon contains a 16-kb frag-
ment acquired by horizontal gene transfer and carrying an ac-
tively transcribed ABC transport system for maltose and
trehalose (DiRuggiero et al. 2000). This 16-kb DNA frag-
ment together with PF1752 were absent from all the Vulcano
isolates (from 2002 to 2003) and PF1736 was absent from 13
isolates (fig. 2), suggesting a short-term selective advantage
for these newly acquired genes. In the third category, ISs of
one type were replaced by ISs, or fragments of ISs, of another
type with significant alterations of the flanking regions
(PF0898; fig. 3, D, E, and F). We also found extensive rear-
rangement within the sequence of IS-pfu3 PF0898 by re-
placement of part or of the whole transposase gene,
including in some cases the spacer and left inverted repeat
(fig. 3, A, C, E, and F). The observed rearrangements asso-
ciated with IS elements were the result of both transposition
and intragenic recombination events, and the high number of
insertions/deletions among closely related isolates suggests
that these regions are hot spots for genome shuffling. It was
indeed suggested that IS elements were the cause of major
genomic rearrangements in the genome of P. furiosus when
compared to that of P. abyssi and P. horikoshii (Lecompte
et al. 2001; Zivanovic et al. 2002). The deleterious effects
of IS elements observed in the genomes of Pyrococcus
are related to their movements and include gene deletions
(fig. 3), chromosomal rearrangements (DiRuggiero et al.
2000; Lecompte et al. 2001), and gene inactivation such as
the napA gene in Pyrococcus woesei (Kanoksilapatham
et al. 2004). In this Pyrococcus strain, also isolated from
 Genetic Drift in the Evolution of Pyrococcus 2301

a) b)
PF0069 PF0242 PF0946 PF0536 PF1736 PF0898
V62
I D II III II Pfu
V232 I D II - - B
V72 I D II III - Pfu
V212 I D II III - D
V61 I D II III II Pfu
2002
V63 I D II III II Pfu
V211 I D II - - A
V221 I D II - - D
V222 I D II - - C
V231 I D II - - C
VB83 I D II ND II C
VB112 I D II - II Pfu
VB93 I D II - - E
VB85 I D II - - B
2003
VB86 I D II - - F
V73 I D II III II A
VB81 I D II - - F

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VB113 I D II III - Pfu
VB82 I D II - - F
Pyrococcus I D II III II Pfu
furiosus
0.005 substitutions/site

FIG. 2.Maximum likelihood tree inferred from the analysis of a concatenated nucleotide alignment of seven loci (prephenate dehydratase [PF0291] 1
deoxyhypusine synthase [PF0292], 3-hydroxyisobutyrate dehydrogenase [PF0716], and a putative protease PF1905 in addition to the four genes
from fig. 1) (IVI; table 1) in the 19 Vulcano isolates and Pyrococcus furiosus. (a) The tree was constructed as described in figure 1. Numbers to
the right indicate collection years. (b) Summary of the analysis of six P. furiosus-like IS elements in all isolates. Numerals I, II, III indicate the
type of IS element: IS-pfu1, IS-pfu3, and IS-pfu3, respectively; D indicates truncated IS elements of approximately 300700 bp, found in the
genome of P. furiosus (four copies) and classified as IS-D (Kanoksilapatham et al. 2004); - indicates absence of IS; and ND 5 nondetermined.
Letters in PF0898 are according to figure 3.

Vulcano and considered to be a subspecies of P. furiosus (Zil-
lig et al. 1987), the putative Na1/H1 antiporter (napA) gene
was disrupted by a IS-pfu3, whereas the napA gene from P.
furiosus remained intact. Inactivation of gene function by
transposition events in the domain Archaea is not unique
to Pyrococcus and has been shown to occur in Sulfolobus
solfataricus (Schleper et al. 1994) and Halobacteria
(DasSarma, RajBhandary, and Khorana 1983).
The frequency of IS elements per site in a population is
the result of their rate of proliferation through transposition
and their removal by natural selection, given the deleterious
fitness effects caused by their presence (B. Charlesworth
and D. Charlesworth 1983). In large host populations the
number of sites available for transposition is high, produc-
ing low-allele frequencies per site (Slatkin 1985). There-
fore, high-allele frequencies are only possible in a small
host population due to stochastic drift (B. Charlesworth
and D. Charlesworth 1983; Langley, Brookfield, and
Kaplan 1983; Slatkin 1985; Brookfield 1986), which is
what we observed in Vulcano Island (fig. 2). This brings
the question as to whether the presence of IS elements only
in the Vulcano Island population is the result of local ad-
aptation through selective advantage from harboring IS ele-
ments or the result of random genetic drift. Given the high
frequency per site of the IS elements analyzed here and their
potentially deleterious fitness effects in the genome of
Pyrococcus, we suggest that in the case of the population
of Vulcano Island these genetic mobile elements were pres-
ent in the colonizing population and brought up to high fre-
quency by genetic drift, whereas they disappeared from
the Pacific Ocean populations. Advantages associated with
the potential acquisition of novel genes through horizontal
gene transfer, possibly to cope with the highly dynamic en-
vironment of the shallow waters of Vulcano Island (J. P.
Amend, A. C. Amend, and Valenza 1998; Aiuppa et al.
2000) and the constant input of xenobiotic compounds in
the ecosystem, are only transient and are not sufficient to
explain the invasion of these genetic elements in the pop-
ulation (Top and Springael 2003). In contrast, the arms race
against the invasion of the Pyrococcus genome by these
hostile IS elements may explain the rapid genetic evolu-
tion of specific regions of the genome (Lecompte et al.
2001) as a mechanism to disrupt their optimal transposition
site sequences. This strategy has been shown in bacteria
where fast-evolving DNA regions were shown to be asso-
ciated with genes involved in defense against bacteriophage
invasion (Zheng, Roberts, and Kasif 2004). The observed
temporal clustering between Pyrococcus isolates from 2002
(8 isolates) and 2003 (11 isolates) (fig. 2), P. furious iso-
lated in 1986, might be the result of a similar process. In-
deed, selective sweep cannot explain the observed temporal
variation as the diversity of the strains is high from one year
to the next.
Conclusion
The presence of IS elements in only one population of
Pyrococcus, together with MLST, revealed genetic diver-
gence between populations occupying different geographic
locations. Physical barriers to their dispersal might be the
2302 Escobar-Paramo et al.
FIG. 3.Schematic representation of IS variations observed in the region coding for ORFs PF0894PF0899. A detailed description of IS PF0898 is
shown at the top. RIR, right inverted repeat; LIR, left inverted repeat; letters on the left indicate type of rearrangement. Strains representing each type are
listed in figure 2. Similar colors/patterns indicate similar ORFs. ORF sizes are not to scale.
result of severe constraints on their growth conditions
together with the discontinuous nature of their environ-
ment. The observed high frequency of IS elements and ev-
idence of their deleterious effects strongly suggest that
genetic drift occurred in the Vulcano Island population
and that it is an important mechanism of genetic divergence
in Pyrococcus. Thus, these observations and the island-like
nature of their environment make hyperthermophiles good
candidates to be the microbial counterpart of the giant
tortoise of the Galapagos (Fenchel 2003).
Supplementary Material
Table S1 and Figures S1 and S2 are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
We thank E. Marguet, P. Forterre, J. Tuttle, and J.
Baross for generously providing Pyrococcus strains from
the Pacific Ocean and I. Zivanovic for providing sequences
from Thermococcus radiotolerans. Nucleotide sequences
have been deposited in GenBank (accession numbers
AY904068AY904329). This work was supported by a
Department of Energy grant DE-FG02-01ER63133, an
Oak Ridge Associated Universities Ralph E. Powe Junior
Faculty Enhancement Award, and a Howard Hughes
Medical Institute undergraduate student fellowship to S.G.
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Martin Embley, Associate Editor
Accepted July 25, 2005