RESEARCH ARTICLE
Are Archaea inherently less diverse than Bacteria in the same
environments?
Josephine Y. Aller1 & Paul F. Kemp2
1
School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, USA; and 2Center for Microbial Oceanography, Research and
Education, School of Ocean and Earth Science, University of Hawaii, Honolulu, HI, USA
Correspondence: Josephine Y. Aller, School
of Marine and Atmospheric Sciences, Stony
Brook University, Stony Brook, NY 117945000, USA. Tel.: 11 631 632 8655;
fax: 11 631 632 3066;
e-mail: [email protected]
Received 25 September 2007; revised 12 March
2008; accepted 20 March 2008.
First published online 8 May 2008.
DOI:10.1111/j.1574-6941.2008.00498.x
Editor: Gary King
Keywords
Archaea; phylogenetic diversity; Bacteria.
Abstract
Like Bacteria, Archaea occur in a wide variety of environments, only some of which
can be considered ‘extreme’. We compare archaeal diversity, as represented by 173
16S rRNA gene libraries described in published reports, to bacterial diversity in 79
libraries from the same source environments. An objective assessment indicated
that 114 archaeal libraries and 45 bacterial libraries were large enough to yield
stable estimates of total phylotype richness. Archaeal libraries were seldom as large
or diverse as bacterial libraries from the same environments. However, a relatively
larger proportion of libraries were large enough to effectively capture rare as well as
dominant phylotypes in archaeal communities. In contrast to bacterial libraries,
the number of phylotypes did not correlate with library size; thus, ‘larger’ may not
necessarily be ‘better’ for determining diversity in archaeal libraries. Differences in
diversity suggest possible differences in ecological roles of Archaea and Bacteria;
however, information is lacking on relative abundances and metabolic activities
within the sampled communities, as well as the possible existence of microhabitats.
The significance of phylogenetic diversity as opposed to functional diversity
remains unclear, and should be a high priority for continuing research.
Introduction
Once thought to only inhabit a limited number of extreme
environments or specialized ecological niches, prokaryotes
of the domain Archaea are now thought to be globally
widespread (Delong, 1992; McInerney et al., 1995; Preston
et al., 1996; Bintrim et al., 1997; Munson et al., 1997; Marc
et al., 1998; Murray et al., 1998; Massana et al., 2000; Church
et al., 2003; Zhang et al., 2005; Teske, 2006) and a significant
component of microbial assemblages in a broad range of
habitats (Fuhrman et al., 1992; DeLong et al., 1994; Karner
et al., 2001; Herndl et al., 2005), including man-made
environments (Godon et al., 1997; Sekiguchi et al., 1998;
Zhu et al., 2003; Leclerc et al., 2004). Archaea are now
recognized to be phylogenetically (Fuhrman et al., 1993;
Ovreas et al., 1997; Bowman & McCuaig, 2003; Kormas
et al., 2003; Mills et al., 2003; Elshahed et al., 2004) and
physiologically (Ouverney & Fuhrman, 2000; Wuchter et al.,
2003; Könneke et al., 2005; Robertson et al., 2005) diverse.
In a previous paper, we reviewed what is known of the
phylogenetic diversity of bacterial assemblages in aquatic
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and other environments (Kemp & Aller, 2004a), and in a
companion paper (Kemp & Aller, 2004b) asked how well
bacterial diversity in the source environment is represented
by 16S rRNA gene libraries, by far the most commonly used
method to assess phylogenetic diversity. In this paper, we
turn to the Archaea and assess what is known of archaeal
phylogenetic diversity in different environments, again as
represented by 16S rRNA gene libraries. Very few 16S rRNA
gene libraries sample microbial diversity exhaustively and
most published studies do not include an evaluation of how
well the library represents diversity in the source environment (Kemp & Aller, 2004a). Therefore, we apply an
assessment procedure described in Kemp & Aller (2004b)
and available online (http://www.aslo.org/lomethods/free/
2004/0114a.html) to determine whether the library is large
enough to yield a stable estimate of total phylotype richness
using the abundance-based richness estimators SChao1
(Chao, 1984, 1987) and SACE (Chao et al., 1993). We
compare archaeal to bacterial diversity, and then consider
the implications of the observed differences and similarities
to address two very basic questions: (1) is it important to
FEMS Microbiol Ecol 65 (2008) 74–87
75
Archaeal vs. bacterial diversity
measure and understand phylogenetic diversity and (2) are
there unique characteristics of both Archaea and Bacteria
which can explain the observed diversity patterns?
We note that studies of phylogenetic diversity have fallen
somewhat out of favor, as techniques to explore functional
diversity have been developed. While functional diversity
cannot be mapped directly onto rRNA gene-based phylogenetic diversity, a phylogenetically diverse community
certainly is more likely to be functionally diverse than a
community with few phylotypes. A phylogenetically diverse
community also allows the possibility of functional redundancy, in which more than one phylotype can perform a given
transformation. Arguably, an understanding of functional
diversity requires an understanding of phylogenetic diversity.
Materials and methods
Terminology
Consistent with our earlier treatment of the diversity of
Bacteria (Kemp & Aller, 2004a), we use the term ‘clones’ to
denote the identified products of PCR amplification; ‘library’
to denote a collection of such identified PCR products derived
from a given environmental source; ‘library size’ to denote the
number of PCR products that were actually characterized; and
‘phylotype’ to denote a group of PCR products judged by the
original authors to be essentially identical, regardless of the
method or criteria they used to assess phylogenetic similarity.
As with our previous examination of bacterial libraries, most
studies used sequence similarity as the criterion for distinguishing among phylotypes, and in most published studies,
phylotypes are considered identical if their 16S rRNA gene
sequences are Z97% identical. We make no assumptions
regarding the relationship of ‘phylotype’ to traditional concepts of genera and species.
Source data
Most of what is known of the phylogenetic diversity of
Archaea in aquatic and other environments is based on
distinguishing among different organisms using PCR amplification of 16S rRNA gene, without actually culturing
them or having any direct knowledge of their morphology,
physiology, or ecology. While 16S rRNA gene libraries are
ideally representative of the diversity present in the source
environment, technically they are only samples of the PCRamplified material from which clones are made. Another
limitation in phylogenetic studies is that likely none of the
primers commonly used to amplify 16S rRNA gene are truly
‘universal’ and therefore no single set will successfully target
all Archaea in a given environment. Many others have
commented on potential PCR biases and their effect on any
PCR-based representation of diversity. While this concern is
appropriate, rRNA gene libraries are still the primary means
FEMS Microbiol Ecol 65 (2008) 74–87
by which phylogenetic diversity is assessed, and we note that
most researchers recognize and take precautions to minimize such biases.
We have located 173 libraries from at least 12 different
types of environments (Moyer et al., 1994, 1995, 1998;
Bintrim et al., 1997; Bowman et al., 1997, 2000a, b; Fuhrman
& Davis, 1997; Godon et al., 1997; Munson et al., 1997;
Chandler et al., 1998; Dojka et al., 1998; Sekiguchi et al.,
1998; Vetriani et al., 1998, 1999; Crump et al., 1999; Tajima
et al., 1999, 2001; Takai & Sako, 1999; Bond et al., 2000;
Cifuentes et al., 2000; Crump & Baross, 2000; Cytryn et al.,
2000; Jurgens et al., 2000; Lueders & Friedrich, 2000;
Massana et al., 2000; Orphan et al., 2000; Reysenbach et al.,
2000; Skirnisdottir et al., 2000; Watanabe et al., 2000, 2002;
Brambilla et al., 2001; Casamayor et al., 2001, 2002; Hjorleifsdottir et al., 2001; Jackson et al., 2001; Karner et al.,
2001; Lanoil et al., 2001, 2005; Madrid et al., 2001a, b;
Marteinsson et al., 2001; Rudolph et al., 2001; Takai et al.,
2001; Bano & Hollibaugh, 2002; Huang et al., 2002, 2004;
Huber et al., 2002, 1986, 1998; Moissl et al., 2002; Oren,
2002; Pesaro & Widmer, 2002; Reed et al., 2002; Stein et al.,
2002; Teske et al., 2002; Bowman & McCuaig, 2003; Chen
et al., 2003; Church et al., 2003; Elshahed et al., 2003, 2004;
Gonzalez-Toril et al., 2003; Itoh, 2003; Kormas et al., 2003;
Mouné et al., 2003; Nercessian et al., 2003; O’Connell et al.,
2003; Schrenk et al., 2003, 2004; Zhu et al., 2003; Chachkhiani et al., 2004; Gallagher et al., 2004; Higaski et al., 2004;
Leclerc et al., 2004; Lysnes et al., 2004; Rutz & Kieft, 2004;
Shin et al., 2004; Grabowski et al., 2005; Heijs et al., 2005;
Høj et al., 2005; Knittel et al., 2005; Kvist et al., 2005; MeyerDombard et al., 2005; Mills et al., 2005; Pašić et al., 2005;
Snell-Castro et al., 2005; Zhang et al., 2005; Gihring et al.,
2006; Karr et al., 2006; Lin et al., 2006; Schwarz et al., 2006;
Siering et al., 2006; Clementino et al., 2007; Perreault et al.,
2007; Zeng et al., 2007) for which source data met the same
criteria used in our previous analyses of bacterial diversity
(Kemp & Aller, 2004a). In 79 cases both bacterial and archaeal
libraries were constructed from the same source environment.
We excluded studies that did not use archaeal-specific
primers (Casamayor et al., 2002); that intentionally selected
amplification products (e.g. based on intensity and frequency of bands: Heck et al., 1975; Demergasso et al.,
2004); or did not provide sufficient data regarding the
frequency distribution of phylotypes (e.g. McInerney et al.,
1995; Munson et al., 1997; Marc et al., 1998; Piñar et al.,
2001; Furlong et al., 2002; Lysnes et al., 2004; Zhang et al.,
2005; Galand et al., 2006; Li et al., 2007). We excluded
pooled data derived from several libraries.
Large enough library assessment
The approach described in Kemp & Aller (2004b) was used
to determine whether libraries were large enough to yield
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76
stable and unbiased richness estimates with application of
either SACE (Chao et al., 1993) or SChao1 (Chao, 1984, 1987),
or both. SChao1 and SACE estimators are highly correlated when
most phylotypes are present only once or twice, as is true of
many prokaryotic libraries. The assessment procedure is
similar to rarefaction analysis (Heck et al., 1975). For each
library, 1000 data subsets are derived by random sampling
with replacement. These derived data subsets range in size ni
from i = 1–100% of the total size N of the library, in
increments of 1% of N rounded to the nearest integer value.
For each subsample size ni, 10 replicate data subsets are drawn
with replacement from the model library; at size ni = N, all 10
data subsets are identical to the complete library. SChao1 and
SACE are calculated for each derived data subset, and plotted
against subsample size ni to determine whether an asymptote
has been reached. If the estimated phylotype richness reached
an asymptote (for our purposes usual examination was
sufficient), we infer that the library was large enough to yield
a stable estimate of phylotype richness. The stable estimates
can be compared between libraries. In this study, the numbers
of clones in each phylotype from a given library were entered
into the spreadsheet found at http://www.aslo.org/lomethods/
free/2004/0114a.html. The form processor subsampled the
library, calculated values of SChao1 and SACE, and plotted
richness estimates against subsample size to predict whether
a library adequately represents the diversity in the source
environment.
Nonparametric abundance-based estimators
of coverage
Two nonparametric abundance-based estimators are useful
when sampling moderately even communities (K.A. Keating
and J.F. Quinn, pers. commun.): Good’s coverage (Good,
1953) and Chao et al.’s CACE (Chao et al., 1993; Colwell &
Coddington, 1994).
Estimating the number of unseen phylotypes
The most basic estimate of phylotype richness is the
observed number of different phylotypes in a library, which
provides a minimum estimate of the total number of
phylotypes present in the source assemblage. The estimates
of SChao1 and SACE are usually higher than the number of
observed phylotypes, and the difference is the number of
unseen phylotypes; that are believed to occur in the source
environment, but were not actually collected or amplified.
Results
Large enough library determination
One hundred of the 151 archaeal libraries from aquatic
systems, and 14 of 22 libraries from nonaquatic systems
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J.Y. Aller & P.F. Kemp
were determined to be large enough to provide stable
phylotype richness estimates (Table 1). Among the 79
companion bacterial libraries, 31 of the 62 libraries from
aquatic systems and 14 of the 17 from nonaquatic libraries
were determined to be large enough.
Library size and sources
The average size of the 173 archaeal libraries was 52 42
(mean SD) and ranged from five clones in sediments from
Guanabara Bay between Rio de Janeiro and Niteroi, Brazil
(Clementino et al., 2007) to an Icelandic hot spring with 201
clones (Kvist et al., 2005) (Table 1 and Fig. 1). Eight libraries
had more than 150 clones, six from hyperthermal environments and two from cold briny springs. Bacterial libraries
from the same source environments varied widely in size
averaging 76 71 phylotypes (mean SD, n = 79) with
several libraries from anoxic sediments containing over 275
clones (Fig. 1).
Library composition
The average number of observed phylotypes for archaeal
libraries from aquatic systems was 9 7 (mean SD,
n = 151, range 2–40; Table 1). The most diverse libraries
were from a microbial mat in an acidic thermal spring (40
unique phylotypes in 84 clones) (Jackson et al., 2001), and
from sediments at the source of an artesian sulfide and
hydrocarbon-rich spring (39 unique phylotypes in 83
clones) (Elshahed et al., 2003). In nonaquatic systems, the
average number of unique phylotypes was 8 5
(mean SD, n = 22, range 2–19; Table 1). The number of
observed phylotypes in archaeal libraries was not correlated
with library size (r2 = 0.11, n = 173).
In contrast, bacterial libraries from the same source
environment in aquatic systems contained on average 29
phylotypes in both aquatic systems (29 37, n = 62, range
2–227), and nonaquatic systems (29 29, n = 17, range
7–106). The number of observed phylotypes in bacterial
libraries was correlated with library size (r2 = 0.63, n = 79).
Rare species are defined operationally by various authors
as those species occurring once, up to two times, or up to 10
times in samples of a community. We defined rare phylotypes as those appearing only once or twice in a library.
Figure 2 compares rare phylotypes among archaeal and
bacterial libraries across all environments. Rare phylotypes
contributed 66% of the phylotypes observed in all archaeal
libraries (Fig. 2a; linear regression slope = 0.66, r2 = 0.80,
n = 173) even though 26 had no phylotypes which occurred
only once, and in 17 of these, all phylotypes appeared three
or more times. When libraries without singletons and
doubles are excluded, rare phylotypes contributed a majority [67% (linear regression slope = 0.67, r2 = 0.79, n = 156)]
of phylotypes. Library size was unrelated to the percentage
FEMS Microbiol Ecol 65 (2008) 74–87
77
Archaeal vs. bacterial diversity
Table 1. Comparison of coverage and phylotype richness estimates for large enough archaeal and bacterial libraries from different environments
Archaeal Libraries
173 (114 large enough)
Aquatic systems
Plankton
Biofilms
Gas Hydrates
Groundwater
Hyperthermal
Sediment
Suspended Particles
Microbial Matsnonhyperthermal
All aquatic systems
observed/predicted (%)
Other Systems
Digestive
Soils
Bioreactors
Liquid Natural gas
All other systems
observed/predicted (%)
n (large
enough)
Library size
(range)
Observed
Phylotypes
CACE
Coverage
Good’s C
SACE
Schao1
37 (23)
1
11 (10)
23 (15)
51 (30)
21 (15)
3 (2)
4
40 (6–129)
10
72 (35–125)
58 (21–181)
59 (17–201)
41 (5–190)
34 (9–83)
37 (14–76)
10 8
4
87
97
97
11 6
11 4
75
0.831 0.153
0.900
0.885 0.149
0.799 0.161
0.866 .154
0.787 0.200
0.293 0.217
0.679 0.222
0.638 .310
0.900
0.973 0.026
0.928 0.068
0.946 0.100
0.808 0.197
0.293 0.100
0.837 0.167
16 16
5
12 13
15 13
12 17
22 18
22
14 17
14 15
4
10 10
15 19
11 17
18 15
18
11 11
151 (100)
52 42
9 7 (2–40)
0.827 0.183
0.895 0.163
15 16
13 16
5 (4)
9 (3)
6 (5)
2
22 (14)
30 (19–45)
54 (30–190)
28 (19–44)
26 (21–31)
39 35
64
12 10
63
18 (16,19)
8 5 (2–19)
0.894 0.151
0.878 0.129
0.805 0.178
0.561
0.784 0.326
0.894 0.151
0.942 0.074
0.935 0.044
0.561
0.832 0.268
13 12
71
93
32 (30–32)
13 10
14 19
7
83
43 (36–52)
15 17
Bacterial Libraries 79 (44 large enough)
n
Aquatic systems
Plankton
Biofilms
Gas Hydrates
Groundwater
Hyperthermal
Sediment
Suspended Particles
Microbial Matsnonhyperthermal
All aquatic systems
observed/predicted (%)
Other Systems
Digestive
Soils
Bioreactors
Liquid Natural gas
All other systems
13 (4)
1 (0)
7 (5)
4 (2)
16 (10)
11 (4)
6 (2)
4 (3)
Size
Phylotypes
CACE large enough
Coverage Good’s C
SACE
Schao1
33 (7–72)
29
60 (34–127)
123 (46,174)
73 (20–171)
130 (7–347)
46 (16–86)
112 (39–301)
16 14
14
16 6
40 (28,57)
19 17
60 68
19 13
36 21
0649 0.229
1.0
0.879 0.120
0.639 0.131
0873 0.140
0.653 0.204
0.588
0.705 0.092
0.733 0.183
1.0
0.897 0.124
0.760 0.128
0.735 0.240
0.717 0.246
0.378
0.753 0.125
37 56
4
25 17
109 39
39 49
157 193
87,49
89 36
28 44
4
23 14
113 64
33 43
136 170
64,34
70 23
62 (31)
76 71
29 37
0.714 0.198
0.781 0.198
63 85
56 76
2 (1)
3
10 (8)
2
17 (14)
44
71 (23–167)
78 (19–202)
20 (15–24)
64 60
34
42 55
29 29
12,14
29 29
0.478
0.693 0.185
0.816 0.109
0.471
0.711 0.172
0.478
0.718 0.148
0.843 0.125
0.471
0.731 0.182
78
89 125
57 73
44,34
63 75
72
82 117
62 100
29,21
62 88
Phylotype estimates are for libraries judged to be large enough to yield a stable prediction of richness where n = number in parentheses from n column.
of rare phylotypes in archaeal libraries (linear regression
slope = 0.013, r2 = 0.0071, n = 173).
Among the bacterial libraries from the same source
environments (Fig. 2b), rare phylotypes contributed 63%
of the observed phylotypes (Fig. 2b; linear regression
slope = 0.63, r2 = 0.72, n = 79). Only four libraries had no
phylotypes that occurred only once and of those, only two
lacked any rare phylotypes. In three source environments:
microbial mats, bioreactors, and lithotrophic biofilms, rare
FEMS Microbiol Ecol 65 (2008) 74–87
phylotypes were more common in archaeal than in bacterial
libraries (Fig. 2c). There was no significant relationship
between library size and the percentage of rare phylotypes
(linear regression slope = 1.44, r2 = 0.42, n = 79).
Coverage
The two coverage estimators summarized in Table 1, suggest
that on average archaeal libraries captured more than half of
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78
J.Y. Aller & P.F. Kemp
1000
Observed phylotypes
Archaea
Archaeoplankton
Sediment
Groundwater
Gas hydrates
Digestive systems
Soils
Bioreactors
Hyperthermal
Biofilm
Suspended particles
Microbial mat
Natural gas
100
10
1
1
10
(a)
50
30
1000
10 000
Archaea
(b) Bacteria
Sediment
Groundwater
Gas hydrates
Digestive systems
Archaeoplankton
Soils
40
Rare phylotypes
100
Library size
Fig. 1. Total reported diversity of Archaea at a
glance compared with bacterial diversity which is
illustrated by the ellipse (from Kemp & Aller,
2004a). We provide Table 1 as a service to readers who might like the details.
100
Bioreactors
Biofilm
Suspended particles
Microbial mat
Natural gas
Hyperthermal
Predominantly
rare
y = 0.7569x − 1.333
r = 0.8243
20
75
50
y = 0.63x − 1.31
r = 0.71
Equally
abundant
10
25
0
0
0
10
20
30
40
Observed phylotypes
% Incidence of rares
(c)
50
0
20
40
60
80
100
120
Observed phylotypes
1
0.75
0.5
0.25
0
s
ats ors gas nt tive al ton ter ils cle tes film
l m act ral ime es erm ank wa So par ti ydra c bio
h hi
bia iore natu Sed Dig er th kopl ound
d
s
o
r
e
a
B
r
p e
c
d G trop
Hyrcha G
Mi
uid
o
en
sp
Liq
A
lith
Su
Fig. 2. (a) Comparison of the number of phylotypes which occur only once or twice to the total number observed in an archaeal library. Libraries which
fall below the 1 : 1 line tend to have uniformly abundant phylotypes, while libraries above the line have a skewed distribution with many rare phylotypes.
Rare phylotypes constituted 76% of the total observed phylotypes although 17 of 173 libraries had no rare phylotypes. (b) In bacterial libraries 63% of
the observed phylotypes were rares with only two of the 79 libraries having no rare phylotypes. (c) Rare phylotypes contributed at least 50% of all
phylotypes observed in libraries from all environments except liquefied natural gas. Rare phylotypes were more abundant in archaeal than bacterial
libraries from soils, microbial mats, sediments, groundwater, and bioreactors. Data from three large bioreactor libraries (13 rare/166 observed, 202 rare/
275 observed and 93 rare/118 observed) were not plotted to allow for more clear comparisons.
the estimated total number of phylotypes in their source
environments. In aquatic environments, capture rates were
close to 75%, and for large-enough libraries, capture rates
were still greater (CACE = 83 18%; C = 89 16%). The
highest coverage was found for gas hydrates and the lowest
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for liquid natural gas libraries. Good’s (1953) varied widely
(column 5 in Table 1), with the extremes found for an
archaeoplankton library composed entirely of rare phylotypes (occurring once or twice) (Moissl et al., 2002), to 17
archaeal libraries that had no rare phylotypes. Both coverage
FEMS Microbiol Ecol 65 (2008) 74–87
79
Fig. 3. Archaeal libraries determined to be large
enough to estimate phylotype richness compared with corresponding bacterial libraries
from the same source environment. Libraries of
all sizes for both Archaea and Bacteria were
determined to be large enough. Library size was
unrelated to the number of observed phylotypes
(P o 0.05).
Observed phylotypes
Archaeal vs. bacterial diversity
100
Archaea
Bacteria
(a)
(b)
10
10
1
1
1
250
100
1
Library size
Greater bacterial
diversity
Estimated phylotypes
150
Similar bacterial
to archaeal diversity
100
50
0
t
r
s
n
s
al
rs
ils al
as
ve
en ate
tes
ilm
sti icle acto ankto erm ral g
So herm
dra Biof
dim ndw
h tu
ge part
l
e
t
t
i
y
e
r
r
r
P
h
D
u
S ro
pe
s
pe na
Bio
ed
G
hy
Hy uid
Ga
nd
on
iq
pe
n
L
s
ts
Su
Ma
estimators were highest in libraries from gas hydrates,
groundwater, hyperthermal and bioreactor environments,
and were lowest for natural gas and suspended particles
(Table 1). They were unrelated to library size across all
environments although the largest libraries also have the
highest coverage.
For the ‘large-enough’ libraries, Good’s C and Chao et al.’s
CACE estimators were generally lower for bacterial than for
archaeal libraries derived from the same environment (Table
1). They were not correlated with library size for either
index. As in the archaeal libraries, coverage estimates for
bacterial libraries varied highest in libraries from gas hydrates and hyperthermal environments and lowest among
libraries from digestive systems and liquid natural gas
(Table 1).
Phylotype richness
In 26 instances both bacterial and archaeal libraries from the
same source environment were considered large enough, so
FEMS Microbiol Ecol 65 (2008) 74–87
100
Sace-Archaea
SChao1-Archaea
Sace-bacteria
SChao1-bacteria
200
Fig. 4. Comparison estimates of the total number of phylotypes in a source environment predicted by the SChao1 and SACE richness estimators
for large enough archaeal and corresponding
bacterial libraries. Both estimators predict greater bacterial than archaeal diversity for most
source environments. Similar bacterial diversity is
only predicted for biofilms and liquefied natural
gas environments. See Table 1 for numbers of
libraries.
100
that a valid comparison of the estimated phylotype richness
values can be made. The observed diversity of Archaea was
greater than that of Bacteria in only five cases (Table 1), and
the estimated total phylotype richness estimated by SACE or
SChao1 was nearly always lower for Archaea than for Bacteria.
Libraries of all sizes were found to be large enough for
both Archaea and Bacteria and library size was unrelated to
the number of observed phylotypes (P o 0.05) (Fig. 3). For
the large enough archaeal libraries from aquatic systems, we
estimate the source community contains 29 22% additional unique phylotypes than were actually observed, based
on SACE, and 20 21% based on SChao1. For nonaquatic
libraries, the corresponding values were 23 19% (SACE)
and 16 20% (SChao1; Fig. 4). In contrast, for bacterial
libraries both estimators predicted that the source community actually contains 44 25% (SACE estimator) or
38 25% (SChao1 estimator) additional unique phylotypes
than were actually observed in the large enough libraries
from aquatic systems, and 49 19% (SACE) and 43 23%
(SChao1; Fig. 4) from nonaquatic systems.
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Discussion
Diversity of Archaea vs. Bacteria
rRNA gene libraries are miniscule samples of extremely
abundant organisms, and it is highly likely that all but the
most abundant phylotypes would be missed, or found only
rarely (see Kemp & Aller, 2004b). As Colwell & Coddington
(1994) note, even exhaustive sampling can leave many
species represented by only a few specimens or even a single
specimen. Although some archaeal libraries have few or no
rare phylotypes, approximately two-thirds of both archaeal
and bacterial libraries of all sizes from many different kinds
of environments are composed of predominantly rare
phylotypes. This would suggest that a priori, researchers
might expect to spend equal effort exploring archaeal vs.
bacterial diversity. In reality, archaeal libraries are rarely as
large as corresponding bacterial libraries from the same
source environments (Table 1 and Fig. 1). We found that
the number of phylotypes observed is not correlated with
library size for Archaea (Figs 1 and 2), suggesting that larger
is not necessarily better when sampling Archaea in a new
environment. In contrast, the number of observed phylotypes is correlated with library size for bacterial libraries,
suggesting that few bacterial libraries had exhaustively
sampled diversity in the source environment, and larger is
indeed better.
Among the 173 libraries examined, there were only five
libraries where the observed archaeal diversity was greater
than bacterial diversity at the same sampling location: two
sets of plankton assemblages (Fuhrman & Davis, 1997; Bano
& Hollibaugh, 2002), an arsenite-oxidizing acidic thermal
spring (Fig. 5d) (Jackson et al., 2001), subterranean hot
spring (Marteinsson et al., 2001), and methane-rich sediments associated with a hydrocarbon seep (Fig. 5c) (Mills
et al., 2005). When libraries were judged large enough and
richness estimators could be used for a valid comparison, we
find that the diversity of Archaea and Bacteria may be
similar in some environments including suspended particles, biofilms, and liquid natural gas. In all other environments predicted bacterial diversity appears to be much
greater than archaeal (Figs 3 and 4).
The limitations of rarefaction analyses and usefulness of
our ‘large-enough’ library assessment procedure (31) are
demonstrated by the study, by Mills et al. (2005), of
methane-rich sediments associated with a hydrocarbon seep
in the Gulf of Mexico. While their published rarefaction
analyses did not indicate that a sufficient number of clones
had been screened to estimate the potential diversity of
Archaea and Bacteria, our assessment procedure suggests
that their libraries actually are large enough to yield stable
phylotype richness estimates. We predict that archaeal
diversity is 30% greater than bacterial diversity in their
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c
J.Y. Aller & P.F. Kemp
source environment (Fig. 5c). In another example, in liquid
natural gas pipelines (Zhang et al., 2005) the size and
diversity of the archaeal and bacterial libraries were almost
identical (15 phylotypes/24 clones for Bacteria, 16/21 for
Archaea). Because both libraries were judged to be large
enough to estimate total diversity, we can apply richness
estimators to predict that bacterial diversity is actually
greater than archaeal diversity in their source environment
(Fig. 5e).
Why does the diversity of Archaea differ from
that of Bacteria?
Like Bacteria, Archaea occur in a wide variety of environments only some of which would be considered ‘extreme’ in
some manner. In the majority of environments, Archaea
tend to have lower diversity than bacteria in the same
environment. However, Archaea do contribute a large
proportion of the total prokaryotic phylotypes present in
most environments studied to date (Table 2). It is uncertain
how this view will change as additional data is acquired from
understudied environments such as the marine plankton,
previously not thought of as typical for Archaea.
Why are Archaea less diverse than Bacteria in most
environments where they co-occur? This is an obvious and
important question that bears upon any assessment of the
ecological roles of Archaea and Bacteria, but which may not
be answerable with conventional sampling approaches. We
speculate that Archaea may perceive and make use of the
environment in ways that are more restrictive than for
Bacteria. For example, Archaea might live in microniches
while Bacteria live more broadly or in different microniches
in the same macroenvironment. More generally, nearly any
sample collected for analysis of prokaryotic diversity will
contain a multitude of microenvironments; in a sense,
Archaea and Bacteria may not truly coexist even if they are
collected in the same sample. We would question whether
co-occurrence in a macroscale sample is a sufficient justification for comparisons of the diversity of prokaryotes found
in that sample. We note that the same comments could
be made regarding comparisons within the Archaea or
Bacteria, among groups occupying different microenvironments within a single microenvironment.
The energetic costs of metabolic processes carried out by
Archaea in at least some environments may be so great that
phylogenetic diversification is limited, in comparison to that
of Bacteria. For example, Oren (1999) suggested that the
diversity of halophilic Archaea was directly limited by the
energetic costs of maintaining themselves in extreme environments compared with the small free-energy change associated with dissimilatory reactions.
More broadly, we question whether Archaea possess less
physiological flexibility than Bacteria in environments that
FEMS Microbiol Ecol 65 (2008) 74–87
81
Archaeal vs. bacterial diversity
(a) 150
SACE 310
SChao1 237
Pig manure digester
Bacteria
Archaea
100
(b) 150
50
SACE 10
SChao1 8
SACE 47
SChao1 31
0
0
0
30
50
100
150
200
0
SACE 30
SChao1 28
Methane hydrate seep
sediments & gas
25
Phylotypes
SACE 435
SChao1 380
100
50
(c)
Antarctic
sediments
(d) 50
50
100
150
200
250
SACE 21
SChao1 20
10
SACE 93
SChao1 94
25
SACE 34
SChao1 34
5
0
0
0
(e)
30
350
Hyperthermal spring
20
15
300
50
100
150
(f) 75
Liquid natural gas
25
SACE 34
SChao1 25
20
0
50
100
150
SACE 116
SChao1 93
Microbial mat
50
15
SACE 45
SChao1 29
10
5
25
0
SACE 4
SChao1 4
0
0
10
20
30
40
50
0
50
100
150
200
250
Library size
Fig. 5. Examples of rarefaction curves for microbial diversity in different environments, and predicted total phylotype richness in the source community.
(a) Pig manure digester (Snell-Castro et al., 2005) where bacterial diversity is extensive and archaeal limited; (b) sediment at 20–21 cm depth on
Antarctic Continental Shelf with a diverse bacterial community and moderate archaeal diversity predicted (Bowman & McCuaig, 2003); (c) sediments
and gas from methane hydrate seep mounds in Gulf of Mexico where archaeal diversity is predicted to be somewhat higher than bacterial (Mills et al.,
2003); (d) arsenite-oxidizing acidic thermal spring where the predicted archaeal diversity is predicted to be three times higher than the bacterial (Jackson
et al., 2001); (e) liquified natural gas in pipelines where observed diversity would suggest similar or higher archaeal diversity, but richness estimators
predict higher bacterial diversity (Zhu et al., 2003); and (f) microbial mat from Lake Fryxell, McMurdo Dry Valleys, Antarctica with significantly higher
bacterial diversity (P o 0.05) and a 72 clone archaeal library had two phylotypes and richness estimators would predict four (Brambilla et al., 2001).
are not considered to be extreme. Although only limited
information is available on metabolic diversification among
Archaea beyond methane and hydrogen-based energy metabolisms, recent studies find Crenarchaeota that actively
assimilate amino acids, protein, and diatom extracellular
polymers in Arctic waters and at depths of 100–200 m in the
spring, accounting for up to 4 40% of dissolved organic
matter assimilation (Kirchman et al., 2007). Other studies
report isolation of a nitrifying Crenarchaeon (Könneke
et al., 2005) and Archaea that can function as chemorganotrophs (Wuchter et al., 2003) and assimilate free amino acids
(Ouverney & Fuhrman, 2000). Much more information is
FEMS Microbiol Ecol 65 (2008) 74–87
needed to assess whether Archaea are typically more, less or
equally physiologically diverse than Bacteria in the same
environment, but certainly these studies suggest that Archaea are physiologically diverse as a whole.
The relative abundance and metabolic activities of Bacteria and Archaea within a specific community are not
necessarily related to their diversity. Abundance estimates
of microbial cells based on FISH labeling with Archaea- and
Bacteria-specific probes, suggest that Archaea generally
account for only a minor fraction ( o 5%) of the total
prokaryotic community (Gonzalez-Toril et al., 2003; Wobus
et al., 2003; Koizumi et al., 2004; van der Wielen et al., 2005;
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c
82
J.Y. Aller & P.F. Kemp
Table 2. Relative proportion of archaeal and bacterial rRNA phylotypes
predicted by the SACE estimator in large enough companion libraries
from various environments
Phylotypes
Predicted
Environment
Number
studies
Archaea
Mean SD
Bacteria
Mean SD
Hyperthermal
Sediment
Liquified Natural Gas
Groundwater
Non Hyperthermal Mats
Suspended Particles
Gas Hydrates
Bioreactors
Rumen
Biofilm
Plankton
3
1
2
2
2
1
5
5
1
1
1
66 47
47
32 3
26 3
20 22
18
11 12
93
9
5
4
24 15
435
39 7
109 40
110 9
15
25 17
72 93
77
4
87
Kirchman et al., 2007) but can account for 10% (Watanabe
et al., 2002), 20% (Bowman & McCuaig, 2003) even 40%
(Koizumi et al., 2004; Kirchman et al., 2007) of some
communities. Even if they are not overwhelmingly abundant, the activities of Archaea particularly those forming
metabolic alliances with Bacteria, can be critical to the
formation of functional microbial communities (e.g.
Meyer-Dombard et al., 2005; Daffonchio et al., 2006;
Ariesyady et al., 2007; Yakimov et al., 2007) and even minor
changes in biotic and/or abiotic factors can alter the
diversity of both Archaea and Bacteria, the community
structure and ecosystem operation (e.g. Meyer-Dombard
et al., 2005; Fuhrman et al., 2006).
Finally, the diversity of Archaea may be linked to that of
Bacteria in quite another way. Studies of tundra wetlands
(Hoj et al., 2005); waste water sludge digesters (Ariesyady
et al., 2007), and methane hydrate outcrops (Orphan et al.,
2001; Teske et al., 2002; Kormas et al., 2003; Knittel et al.,
2005) suggest that factors influencing bacterial communities
and consequently the availability of substrates for methanogenic Archaea may indirectly limit archaeal diversity.
proposed that the performance stability of an ecosystem
dominated by microorganisms, even one facing frequent
disturbances, is the result not of phylogenetic diversity per
se, but of functional redundancy, which is ensured by the
presence of a reservoir of species able to perform the same
ecological function. In the mixed-methanogenic reactor
systems studied by Fernández et al. (1999) and Zumstein
et al. (2000) and considered by Briones & Raskin (2003),
fermenting Bacteria, syntrophic Bacteria, and methanogenic
Archaea coexist. The archaeal and synthrophic bacterial
phylotypes were less phylogenetically diverse and lacked
functional redundancy, and varied little in abundance over
time although they did exhibit patterns of dominance and
succession. The far more phylogenetically diverse fermenting bacteria varied temporally in response to substrate
changes, but system stability was maintained by functional
redundancy among the oscillating populations. Variations
in the methanogens and syntrophic bacteria actually controlled the overall operation of the system.
In frequently disturbed microbially dominated systems
such mobile muds (e.g. Madrid et al., 2001a, b; Aller & Aller,
2004) that are characterized by extremely diverse and
abundant Bacteria and few Archaea, system stability may
result from a high degree of functional redundancy. High
diversity with multiple organisms capable of the same
metabolic functions allows efficient remineralization of any
and all organic matter in spite of population oscillations.
If most microbial diversity represents functionally interchangeable taxa as suggested by models like that of Curtis
et al. (2002) then phylogenetic diversity data will provide
little information regarding microbial community function,
unless that functional redundancy is recognized and understood. However, in California coastal waters Fuhrman et al.
(1992) found repeating and predictable subsets of bacteria
within the microbial community that were not functionally
interchangeable and that occurred predictably under specific
combinations of biotic and abiotic variables. The significance of phylogenetic diversity as opposed to functional
diversity remains unclear, and should be a high priority for
continuing research.
Implications of abundance, diversity, and
activity measurements
Studies of engineered and natural ecosystems allow us to
consider whether there are fundamental differences in
diversity, dominance, and the extent of functional redundancy sensu Walker (1992) in archaeal and bacterial communities living in the same environments. Utilizing
bioreactors as model ecosystems, Briones & Raskin (2003)
drew upon theoretical concepts in higher ecological organization (Walker, 1992; Peterson et al., 1998) to examine
diversity, trophic interactions, and process efficiency. They
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c
Acknowledgements
This project was supported by National Science Foundation
OCE Grants 9818574 (to J.Y.A.) and 0083193 (to P.F.K. and
J.Y.A.) and CICEET Grant 35757 (to J.Y.A. and P.F.K.). P.F.K.
was supported by NSF grant EF-0424599. Contribution No.
1328 from the School of Marine and Atmospheric Sciences,
Stony Brook University. Contribution No. 45 of the Center
for Microbial Oceanography: Research and Education at the
University of Hawaii.
FEMS Microbiol Ecol 65 (2008) 74–87
83
Archaeal vs. bacterial diversity
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