ES42CH22-Reed
ARI
ANNUAL
REVIEWS
27 September 2011
14:24
Further
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
Click here for quick links to
Annual Reviews content online,
including:
• Other articles in this volume
• Top cited articles
• Top downloaded articles
• Our comprehensive search
Functional Ecology of
Free-Living Nitrogen Fixation:
A Contemporary Perspective
Sasha C. Reed,1 Cory C. Cleveland,2
and Alan R. Townsend3
1
U.S. Geological Survey, Canyonlands Research Station, Moab, Utah 84532;
email: [email protected]
2
Department of Ecosystem and Conservation Sciences, University of Montana, Missoula,
Montana 59812
3
Department of Ecology and Evolutionary Biology and the Institute of Arctic and Alpine
Research (INSTAAR), University of Colorado, Boulder, Colorado 80309
Annu. Rev. Ecol. Evol. Syst. 2011. 42:489–512
Keywords
First published online as a Review in Advance on
September 2, 2011
asymbiotic, biogeochemistry, nonsymbiotic, nutrients, nifH gene
The Annual Review of Ecology, Evolution, and
Systematics is online at ecolsys.annualreviews.org
Abstract
This article’s doi:
10.1146/annurev-ecolsys-102710-145034
c 2011 by Annual Reviews.
Copyright All rights reserved
1543-592X/11/1201-0489$20.00
Nitrogen (N) availability is thought to frequently limit terrestrial ecosystem processes, and explicit consideration of N biogeochemistry, including
biological N2 fixation, is central to understanding ecosystem responses to
environmental change. Yet, the importance of free-living N2 fixation—a
process that occurs on a wide variety of substrates, is nearly ubiquitous in
terrestrial ecosystems, and may often represent the dominant pathway for
acquiring newly available N—is often underappreciated. Here, we draw from
studies that investigate free-living N2 fixation from functional, physiological, genetic, and ecological perspectives. We show that recent research and
analytical advances have generated a wealth of new information that provides
novel insight into the ecology of N2 fixation as well as raises new questions
and priorities for future work. These priorities include a need to better integrate free-living N2 fixation into conceptual and analytical evaluations of
the N cycle’s role in a variety of global change scenarios.
489
ES42CH22-Reed
ARI
27 September 2011
14:24
1. INTRODUCTION
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
Nitrogen (N) is essential to all life, and a single process—biological N2 fixation—is thought to
account for more than 97% of “new” N inputs into unmanaged terrestrial ecosystems (Vitousek
et al. 2002, Galloway et al. 2004). In light of its importance within the global N cycle, as well as
data suggesting that N often limits terrestrial net primary production (NPP) (Vitousek & Howarth
1991, Elser et al. 2007, LeBauer & Treseder 2008), N2 fixation is central to our understanding of
ecosystem function and to predicting future ecosystem responses to global environmental change
(Hungate et al. 2003, Reich et al. 2006, Wang & Houlton 2009). However, key uncertainties
remain in our understanding of the rates of and controls over this process (Vitousek et al. 2002,
Houlton et al. 2008, Hedin et al. 2009, Cleveland et al. 2010).
In this review, we focus on free-living N2 fixation, defined here as any form of biological
N2 fixation that does not consist of a demarcated symbiotic relationship between plants and
microorganisms (see below). Free-living N2 fixation—also commonly called asymbiotic N2 fixation
or nonsymbiotic N2 fixation—is nearly ubiquitous in terrestrial ecosystems, occurring on the
surface of plants and in their leaves, on leaf litter and decaying wood, and in soil (e.g., Roskoski
1980, DeLuca et al. 2002, Matzek & Vitousek 2003, Benner et al. 2007, Barron et al. 2008).
However, although free-living N2 fixation is relatively easy to measure in small samples of a given
substrate (e.g., soils, leaves), profound spatial and temporal variability in the process limit our ability
to assess ecosystem-scale N inputs via the free-living N2 fixation pathway. Nevertheless, current
evidence suggests that free-living N2 fixation represents a critical N input to most terrestrial
ecosystems, particularly those lacking large numbers of symbiotic N2 -fixing plants.
Here, we synthesize existing information in an attempt to provide a contemporary evaluation of
the extent and magnitude of free-living N2 fixation in terrestrial ecosystems, as well as the controls
on the process. To that end, we draw from a wide range of studies that offer insight into freeliving N2 fixation from functional, organismal, and ecological perspectives. In addition, we address
remaining uncertainties in free-living N2 fixation research and offer a set of recommendations
aimed at providing a clearer understanding of this fundamental ecosystem process.
2. TWO N2 FIXATION PATHWAYS: SYMBIOTIC AND FREE-LIVING
N availability is frequently thought to limit NPP, as well as other processes, in a wide range of
ecosystems (e.g., Vitousek & Howarth 1991, Elser et al. 2007, LeBauer & Treseder 2008), and
although most organisms rely on fixed forms of N to meet their N demand (i.e., NH4 + , NO3 − ,
or organic N), N2 -fixers are unique in their ability to exploit an essentially limitless supply of N2
in the atmosphere. Biological N2 fixation occurs via two primary pathways: symbiotic and freeliving. However, the line separating these two pathways is not as clearly defined (nor as mutually
exclusive) as it may seem, as relationships between N2 -fixing microorganisms and plants occur in a
diversity of forms (Sprent & Sprent 1990). Nonetheless, for the purposes of this review we define
symbiotic N2 fixation as N2 fixation that occurs via relationships between plants (e.g., legumes)
and the N2 -fixing microbial symbionts occupying plant root nodules (e.g., Rhizobia or Frankia).
In contrast, we classify all other forms of N2 fixation (including N2 fixation by epiphytes on plant
leaf surfaces and the symbiotic N2 fixation that occurs in lichens) as free-living N2 fixation.
Rates of symbiotic N2 fixation have been assessed in numerous terrestrial ecosystems, but in effect, all large-scale estimates of N2 fixation in natural systems represent substantial extrapolations
of a few point measurements to the biome scale (e.g., Cleveland et al. 1999). Such extrapolations
suggest that symbiotic fixers can achieve very high rates of fixation, reaching >150 kg N/ha/year in
some unmanaged ecosystems. As a reference, N fertilizer added to modern agricultural systems is
490
Reed
·
Cleveland
·
Townsend
ES42CH22-Reed
ARI
Table 1
27 September 2011
Biome-scale estimates of terrestrial free-living and symbiotic N2 fixation rates based on the literaturea
Free-living N2
Range in
free-living N2
fixation rates
(kg N/ha/year)
fixation rates
(kg N/ha/year)
n
N2 fixation rates
(kg N/ha/year)
Moist tundra and alpine tundra
1.5
0.4–3.0
7
1.0–4.9
3
Boreal forest and woodland
1.2
0.3–3.8
14
0.3–6.6
2
Temperate forests
1.7
0.01–12
38
1–160
17
Temperate grasslands
4.7
0.1–21
13
0.1–10
8
3–30
3
3–90
6
Biome
Tropical savanna
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
14:24
15.0
Range in symbiotic
n
Tropical evergreen forest
7.8
0.1–60
19
5.5–16
4
Tropical floodplain
7.5
4.1–12
3
14–28.5
2
Tropical deciduous forest
3.3
3.3
1
7.5–30
3
Mediterranean shrubland
1.0
1.0
1
0.1–10
4
Desert
4.0
0.01–13
15
0.7–29.5
3
a
Values are N2 fixation rate means or ranges and represent data from n sites per biome. When more than one component of N2 fixation was measured
from a pathway within a single site (e.g., soil and leaf litter N2 fixation), we summed the values. When there was more than one value from a single
substrate for a single site, we took the average. References that added data to this table but are not included in the Literature Cited are provided in
Supplemental Text 1 (follow the Supplemental Material link from the Annual Reviews home page at http://www.annualreviews.org). Rates from the
two N2 fixation pathways (symbiotic and free-living) are not from simultaneous assessments and are typically not from the same individual sites. We draw
attention to the data to illustrate the potential for free-living N2 fixation to provide important inputs of N to many ecosystems.
Supplemental Material
often ∼100 kg N/ha/year, but in some parts of the world can reach >500 kg N/ha/year (Vitousek
et al. 2009). By contrast, published estimates of free-living N2 fixation are usually much lower, suggesting inputs between 1 and 20 kg N/ha/year when extrapolated to the ecosystem scale (Table 1)
(Boring et al. 1988, Cleveland et al. 1999, Son 2001). The differences between published estimates
of symbiotic and free-living N2 fixation rates contribute to a common perception that N inputs
via free-living fixation are small relative to those derived from symbiotic pathways. However,
many estimates of N2 fixation rates are inherently biased because they frequently represent maximum potential rates rather than more realistic, spatially explicit estimates of ecosystem-scale N
inputs.
Four main lines of evidence challenge the assumption that symbiotic inputs may in most
ecosystems far outweigh free-living inputs. First, many ecosystems lack large numbers of symbiotic
N2 -fixers (Woodmansee et al. 1981, Crews 1999, Menge et al. 2010), and free-living fixation likely
represents the dominant biological source of new N to those ecosystems. Second, the presence of
potentially symbiotic N2 -fixing plants may not be a reliable indicator of actual symbiotic N inputs.
Actual N2 fixation rates have been measured only in relatively few putative symbiotic N2 -fixers
(e.g., Sprent 2009, Barron et al. 2011), and some evidence suggests significant intraspecific
variation in N2 fixation rates of nodulated species (Galiana et al. 2002, Sprent 2005). Moreover,
symbiotic N2 -fixers with the potential to nodulate do not always fix N2 in natural environments,
and thus the presence or absence of symbiotic N2 -fixers may not aid in our prediction of N2 fixation
rates (e.g., Barron et al. 2011). Third, large-scale estimates of symbiotic and free-living N2 fixation
suggest that in many locations and in some biomes, free-living fixation could contribute a greater
proportion of N inputs than does symbiotic N2 fixation, and when scaled by biome area, free-living
fixation could account for a substantial proportion of the biologically fixed N2 globally (Table 1)
(Cleveland et al. 1999). Finally, free-living N2 fixation rates vary significantly within sites, and N2
www.annualreviews.org • Free-Living Nitrogen Fixation
491
ARI
27 September 2011
14:24
fixation may be most active in areas of the ecosystem where N demand or N losses are relatively
high (e.g., in decaying litter, where high carbon (C):N ratios increase decomposer demand for N)
(Hedin et al. 2009). Thus, free-living N inputs could be critical to particular aspects of function
(e.g., N supplied to litter during decomposition) or to subsidizing N losses (Vitousek et al. 2002,
Hedin et al. 2009), regardless of whether or not they are the dominant N input at the ecosystem
scale.
Tropical rain forests provide a good example of how widely accepted views of free-living N2
fixation belie the importance of the process relative to symbiotic N2 fixation. N2 fixation rates
in tropical forests are commonly thought to be among the highest of any natural ecosystem
(Soderland & Roswall 1982, Cleveland et al. 1999), and indeed relatively high symbiotic rates
have been suggested for a few tropical sites (e.g., Roggy et al. 1999). However, estimates from
multiple rain forests suggest that free-living pathways also fix significant amounts of N2 (>10 kg
N/ha/year) ( Jordan et al. 1983, Reed et al. 2008, Cusack et al. 2009). Moreover, although legumes
are abundant in many tropical forests (Crews 1999), data suggest that, at least in mature forests, they
may not be actively fixing much N2 (Barron et al. 2011). Cleveland and others (2010) recently used
mass balance and modeling approaches to corroborate these observations. They showed that after
accounting for free-living N2 fixation and atmospheric N deposition, only modest inputs of N via
symbiotic fixation were necessary to balance the N budget of a mature tropical forest in Amazonia.
Furthermore, additional new evidence synthesized by Hedin and others (2009) suggested that freeliving N2 fixation and atmospheric deposition often provide enough N to balance high N losses
and to maintain tropical forest N richness. Indeed, even beyond tropical regions, biomes suggested
to have high free-living N2 fixation rates are also thought to have high rates of soil denitrification
(Cleveland et al. 1999, Seitzinger et al. 2006). Although such comparisons are based on relatively
few data and reflect significant variability among those that do exist, taken together the patterns
suggest a coupling between terrestrial N inputs and losses that has been observed at a variety of
scales. For example, a similar spatial link between N2 fixation and denitrification has been seen
in oceans, highlighting a potential global nature of spatial correlations between N2 fixation and
denitrification rates (Deutsch et al. 2001).
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
3. VARIATION WITHIN AND BETWEEN ECOSYSTEMS
Cleveland and others (1999) assessed rates of both free-living and symbiotic N2 fixation in
biomes globally, and their analysis showed large spatial variation in the relative contributions of
each pathway. More data—in particular simultaneous measurements of free-living and symbiotic
fixation within individual sites—are clearly needed to define the rates of each N2 -fixing pathway
within and across biomes. However, the data we do have suggest that the relative contribution of
free-living N2 fixation varies considerably at the biome scale (Table 1). For example, although
incomplete, existing data suggest that, relative to symbiotic N2 fixation, free-living N2 fixation
may be most important in tropical rain forests, temperate grasslands, arctic tundra, and boreal
ecosystems (Table 1). In addition, rough calculations of N demand from NPP [assuming
conservative C:N ratios of 30:1 and using NPP estimates from Cleveland and others (1999)]
suggest that cumulative new N inputs via free-living pathways could generate N pools similar in
magnitude to the annual N requirements of NPP in a surprisingly short period of time, ranging
from ∼2 years in deserts to ∼100 years in temperate forests.
Studies have also used ecological chronosequences to investigate temporal variation in freeliving N2 fixation rates over long timescales, such as those occurring over the course of ecosystem
development and succession (Vitousek 1994; Crews et al. 2000, 2001; Perez et al. 2004; Schmidt
et al. 2008; Menge & Hedin 2009). In particular, data suggest that free-living N2 fixation could
492
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
represent a critical N input at early stages of primary succession and ecosystem development
(Vitousek 1984, Schmidt et al. 2008), and in some cases free-living N2 -fixers arrive long before N2 fixing plants (e.g., Schmidt et al. 2008). Theory suggests that N2 fixation should downregulate at
later stages of development—after ecosystem N capital increases. However, successional patterns
in free-living N2 fixation vary among sites, as do the types of variation. For example, consistent
with theoretical predictions, a number of studies have demonstrated declines in free-living N2
fixation rates over the course of succession, with the lowest rates in late successional-stage forests
(e.g., Dawson 1983, Skujins et al. 1987, Hope & Li 1997). However, other studies observe the
opposite trend (Zackrisson et al. 2004, DeLuca et al. 2008, Jackson et al. 2011). For example, Perez
and coauthors (2004) showed that litter-layer N2 fixation provided a steady N input to unpolluted
southern hemisphere temperate forests even in late successional stages. In contrast, on a set of
Hawai’ian lava flows on Mauna Loa, there was no real difference among free-living N2 fixation
rates across the chronosequence, but the dominant source of fixed N changed from a cyanolichen
(at 10 years) to a cyanoalga (at 52 years) to heterotrophic N2 -fixers in leaf litter and detritus
(at 142 years) (Crews et al. 2001). Different patterns among chronosequences likely result from
variation in a host of ecosystem properties, such as symbiotic N2 fixation inputs, hydrology, and
the extent of N losses (Vitousek 1984, Hedin et al. 1995, Perez et al. 2004, Menge & Hedin 2009).
For free-living pathways, substantial spatial and temporal variations in N2 fixation rates within
any given ecosystem are also common. A number of studies have assessed temporal (e.g., seasonal)
variation in free-living N2 fixation within ecosystems, and most show notable variation among
seasons (> ± 50%) (e.g., Chapin et al. 1991; Reed et al. 2007a; Perez et al. 2004, 2010; Menge
& Hedin 2009; Stewart et al. 2011). In general, seasonal differences are likely driven by variation
in light, temperature, and precipitation (Bentley 1987), but chemical changes in the substrate
on which N2 fixation occurs may also be important (Thompson & Vitousek 1997, Vitousek &
Hobbie 2000, Reed et al. 2007a, Barron et al. 2008). Rates of free-living N2 fixation on leaf litter
are often high, and chemical changes in leaf litter during decomposition (e.g., Cleveland et al.
2006, Parton et al. 2007) appear to drive significant changes in N2 fixation rates as decomposition
proceeds. For example, leaf litter phosphorous (P) immobilization and declining N:P ratios over
the course of decomposition correlated with increasing N2 fixation rates in a tropical forest (Reed
et al. 2007a). However, regardless of the factors that drive temporal variation in N2 fixation, the
fact that rates vary over relatively short timescales casts doubt on the accuracy of annual rates
calculated by scaling measurements made at a single point in time.
There is also notable spatial variation in free-living N2 fixation rates within ecosystems. Freeliving N2 fixation occurs on many substrates (e.g., in leaves, wood, soil) that occupy different
zones of the ecosystem (e.g., horizontal gradients from soil to the top of the forest canopy), with
associated N2 fixation rates varying by orders of magnitude (Figure 1a). Although studies that
simultaneously assess free-living N2 fixation across multiple habitats within a single ecosystem are
rare, those that do exist often show higher mass-based rates in bulk leaf litter than in soil (e.g.,
Heath et al. 1988, Perez et al. 2004, Hofmockel & Schlesinger 2007, Reed et al. 2008), and the
lowest mass-based rates on canopy leaves (Figure 1a). However, this pattern does not hold true for
all ecosystems. For example, in some Hawai’ian forests, data suggest that the rate of N2 fixation can
be higher in the canopy than in the forest floor owing to an abundance of N2 -fixing lichens found
at the sites (Crews et al. 2000, Benner et al. 2007). This spatial pattern highlights the importance
of N2 -fixer community composition in helping to determine N2 fixation rates within ecosystems.
For instance, data from a tropical rain forest showed that increased light caused increased N2
fixation rates in the epiphyll community of canopy leaves but not among free-living fixers in leaf
litter or soil; this pattern further suggests that fixation is tied to autotrophy in the canopy but
heterotrophy in the forest floor (Figure 1a) (Reed et al. 2008).
www.annualreviews.org • Free-Living Nitrogen Fixation
493
ES42CH22-Reed
ARI
27 September 2011
14:24
a
Canopy leaves
PAR-incubated
canopy leaves
Leaf litter
Topsoil
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
1
10
102
103
104
105
Log scale of N2 fixation rates
(pg N/g/h)
0.06
10
Canopy
Leaf litter
8
0.05
Soil
0.04
6
0.03
4
0.02
2
Canopy N2 fixation rates
(kg N/ha/year)
Leaf litter and soil N2 fixation rates
(kg N/ha/year)
b
0.01
0
0
Symphonia
Manilkara
Caryocar
Qualea
Brosimum
Schizolobium
Genus
Figure 1
Spatial patterns of free-living N2 fixation rates along a canopy-to-soil profile and among different canopy
tree species in a tropical rain forest ecosystem. (a) Along the canopy-to-soil profile, rates of free-living N2
fixation are shown for sunlit canopy leaves incubated at the forest floor, sunlit canopy leaves incubated under
elevated photosynthetically active radiation (PAR), and mixed-species leaf litter and topsoil (0–2 cm)
incubated at the forest floor. N2 fixation rates (pg N/g/h) showed significant variation along the vertical
profile (P < 0.001), and means ( ± 1 SE) are presented on a logarithmic scale (x-axis; n ≈ 48 trees per
component). (b) Species-specific variation in N2 fixation rates (kg N/ha/year) occurring on and beneath six
canopy tree species in a tropical rain forest (n ≈ 8 trees per species per substrate). N2 fixation rates in canopy
leaves, bulk leaf litter, and topsoil (0–2 cm depth) varied significantly between species (P < 0.02). All data are
from Reed et al. (2008), and the figure is reproduced with the permission of the Ecological Society of
America.
There is also spatial variation in N2 fixation rates within a single component of an ecosystem
(e.g., leaf litter), for example, across leaves and litter derived from different plant species (Figure 1b)
(Reed et al. 2008, Cusack et al. 2009, Perez et al. 2010). These data suggest that species traits, such
as foliar P concentrations and C:N ratios, are important in influencing free-living N2 fixation rates
across canopy leaves, bulk leaf litter, and even in soils beneath individual trees (Figure 1b). Thus,
494
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
although there may not be a defined symbiotic relationship between plants and free-living N2 fixers, plants can still indirectly affect free-living N2 fixation rates through a number of mechanisms.
Finally, another common source of spatial variation in free-living N2 fixation rates comes from
“hotspots” of high fixation activity (Alexander & Schell 1973, Perez et al. 2008, Reed et al. 2010).
Hotspots are spatially explicit zones of activity (a hotspot can be a few centimeters away from a
non-hotspot; Reed et al. 2010) where rates are much higher than average, and hotspots are a common occurrence for free-living N2 fixation and other microbially mediated processes, including
denitrification and methanogenesis (Davidson et al. 2004, Groffman et al. 2009). The frequency
and magnitude of hotspots can strongly influence scaled-up estimates of N2 fixation rates, and
the factors that lead to fixation hotspots remain a significant unknown in our understanding of
free-living N2 fixation. Some recent genetic research suggests that hotspots in a lowland rain forest
contained distinct N2 -fixing communities (Reed et al. 2010), and these data highlight the potential
for N2 -fixer community composition to play a role in mediating small-scale variation in free-living
N2 fixation rates (Figures 2 and 3). Thus, even over very small spatial scales, free-living N2 -fixer
community composition can differ in profound ways, and associated N2 fixation rates may vary by
more than an order of magnitude across those same scales (Figure 3).
4. PHYSIOLOGY AND FUNCTIONAL BIOLOGY
N2 fixation is an exclusively prokaryotic metabolic process and is performed by a phylogentically diverse group of organisms that occupy both the Bacterial and Archaeal domains (Eady 1991, Young
1992, Raymond et al. 2004). N2 -fixers can be autotrophic, heterotrophic, chemolithotrophic, photoheterotrophic, and methanogenic. Field and laboratory evidence suggests that when fixed N is
available in the environment, free-living N2 -fixers can preferentially use it rather than fixing N2
(Drozd et al. 1972, Buhler et al. 1987, Ludden 1994, Barron et al. 2008, Cusack et al. 2009).
However, this does not mean that fixers always do so in natural environments, and free-living N2
fixation can persist even when fixed N availability is relatively high (e.g., Menge & Hedin 2009).
Although a diverse group, the physiology and functional biology of all free-living N2 -fixers are
markedly affected by the properties and requirements of the nitrogenase enzyme. In particular,
the enzyme’s activity is characterized by (a) O2 sensitivity, (b) response to metal content due to
the enzyme’s component proteins [iron (Fe), vanadium (V), and/or molybdenum (Mo)], (c) a need
for adequate supplies of reducing power and adenosine triphosphate (ATP), and (d ) common
suppression by N availability.
4.1. O2
Free-living N2 -fixers can be obligate anaerobes, facultative anaerobes, or obligate aerobes and
thus exist in a range of environments that span gradients of O2 availability. Yet, O2 has the potential
to inhibit nitrogenase and thereby suppress N2 fixation. N2 -fixers avoid the potentially toxic
effects of O2 by isolating N2 fixation in space using cellular components where O2 concentrations
are kept low (e.g., heterocysts), by separating N2 fixation in time from O2 -evolving processes
such as photosynthesis, or by increasing respiration to draw down O2 levels (Robson & Postgate
1980). For obligate aerobes, optimal conditions are met when O2 concentrations are balanced
by respiratory demand: Under low O2 concentrations, nitrogenase is limited by energy, but at
higher concentrations nitrogenase can be inhibited directly by O2 (Robson & Postgate 1980).
O2 inhibition can occur at two levels, both by reducing nitrogenase activity and by reducing
nitrogenase production (Hill 1988).
www.annualreviews.org • Free-Living Nitrogen Fixation
495
ARI
27 September 2011
R. r
rum
R. sph
aeroid
is
XY
es XY
atu
N
Group I
Mo-dependent
nitrogenase
95 64
100
97
100
100
Y
XY
R. capsulatus XY
100
100 95
sL
0.2 substitutions/site
100
99
M. bar
94 100
86
100
keri N
fl
D. hafniense 1208+1210
H2
83
100
100
100
87
100
100
100
100
99
94
100
ri N
flH
100
rke
448
ba
639+1
M.
M. the
rmoau
totrop
hicum
1451
M. jann
a
schii 90
1+
381
2+1
M. k
and
leri 1
41
100
100
29
35
8+
52
17
Frankia NifHD
D. hafniens
e 2827
C. acetobutylicum 247+250
C. tepidu
m 1511+
1514
100
M
49 M . mazei 719
+722
M . ac
. b etiv
ar
ora
ke
ns
379
ri N
2+
M.
ifH
379
the
A.
D
5
vin
rm
ela
oa
u
Group II
nd
tot
ii 6
rop
9+
hic
Mo-dependent
71
um
15 nitrogenase
25
+1
52
8
81
83
11
12
8+
7+
17
35
s1
s3
an
tri
or
HD
175
tiv
lus
ce
Vnf
0+1
pa
eri
.a
117
R.
M bark
ans
vor
M.
ceti
M. a. barkeri AnfHD
M
R. capsulatus 1460+1461
R.
A palustris 3 +33
3
.
3
0
v
31
R. inelandii
rub
593+
594
rum
s3
99 86
100
9
vo
ra
n
88
77
M
.a
ce
ti
99
8+
Group IV
Uncharacterized
18
+2828
90
F. nucleatum 889+890
77
R. rubrum 1555+1556
100
1
8
9
1
+
0
is 198
tr
s
lu
8
a
4
R. p
+29
944
ris 2
t
s
alu
290
R. p
9+1
128
9
e
s
96
5
ien
+1
24
afn
67
h
9
.
1
D
+2
s
i
4
r
5
st
22
8
alu
us
58
R. p
at
l
+1
u
9
s
p
58
ca
5 100
s1
51
R.
an
r
4+
1
5
it vo
ei
ce
az
. a M. m
M
+
30
0
18
05
20
+7
71
49
0
C
C
e7
c.P orm
77
sto nctif
o
6+3
100
N . pu
100
ii 37
d
N
n
71
ela
HD
97
. vin
iae Nif
100 82 100 A . pneumon
K
100
77
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ub
str
N.
pu
n
c
tifo
rm
Nost
e
o
c
1
.P
74
CC7
120
5451 8 LN
Synechoc
LN
ys
tis
.P
CC
6803 LN
tus LN
sX
lu
sul
a
T. elong
cu
pa
a
ph
tia
pa
ra
n
R.
s
R.
au
R. c
ap
XY
R.
m
idu
tep
C.
C.
C. tepidum LN
s LN
iacu
rant
C. au
N
rum L
R. rub
N
sL N
t ri
sL
lus
de
oi
er
Group V
Pigment biosynthesis
14:24
3
459
92+
5
4
loti
+450
M. meliloti 449
S.
R. rubrum 407+408
R
R. sp . capsulatus N
ifHD
R. B. f haero
pa ung
ides
lu
315
str orum
9+3
160
is
946
40
1+
91
946
+4
2
09
2
ES42CH22-Reed
Group III
Mo-independent
nitrogenase
Figure 2
Phylogenetic tree constructed using nifH and nifD homologs from complete genomes. Groups I–III represent functional nitrogenases,
including Mo-dependent groups I and II and the V- and Fe-dependent group III. Group IV consists of uncharacterized nitrogenase
paralogs of nifH and nifD proteins, and group V includes the subunits of enzymes involved in the late steps of photosynthetic pigment
biosynthesis. The tree was constructed using the Neighbor-Joining method with 500 bootstrap replicates: >60% bootstrap support
percentages are shown at each node. From Raymond et al. (2004), and reproduced with the permission of The Society for Molecular
Biology and Evolution.
496
Reed
·
Cleveland
·
Townsend
ES42CH22-Reed
ARI
27 September 2011
a
14:24
b
500
ln N2 fixation rate (ng N/g/h)
300
200
100
0
0.5
0.6
0.7
0.8
0.9
1
c
1
2, 3
2
7
6
5
4
–8
–6
–4
–2
0
2
ln nifH/16S abundance
Diversity estimate (H'/H'max)
3
100
Relative abundance (%)
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
N2 fixation rate (μg N/kg/day)
8
400
Firmicutes
Methylobacterium
Rhizobium
Sinorhizobium
Gluconacetobacter
Deltaproteobacteria
Azospirillum
Zymomonas
Gamma proteobacterium BAL286
Rhodopseudomonas
Uncultured
50
0
Average
Hotspot
unamended unamended
Average
+P
Hotspot
+P
Figure 3
Relationships between N2 -fixer community structure and N2 fixation rates. (a) Saturating relationship
between N2 -fixer diversity and N2 fixation rate in a New York Raynam silt loam soil that received a range of
agricultural treatments. Diversity was estimated from nifH clone libraries and defined as the evenness
component of the Shannon index (H /H max ). Consistent with the observation of a unimodal response, a
second-order polynomial function was used to fit the data. Values represent means ± 1 SD. Data are from
Hsu & Buckley (2009), and the figure was reproduced with the permission of The International Society for
Microbial Ecology. (b) Relationship between N2 fixation rates and nifH gene relative abundance in tropical
rain forest leaf litter (n = 14; P = 0.025; r2 = 0.35). Data are from unamended and P-fertilized (+P) plots
and were taken from Reed et al. (2010). (c) Community composition in litter samples from unamended and
+P plots, and in samples within treatments that had different N2 fixation rates (samples with average N2
fixation rates and hotspot samples with very high rates). Significant differences among free-living N2 -fixer
communities (denoted by numbers above columns) were determined at P < 0.05 using Unifrac analysis. Data
are from Reed et al. (2010).
4.2. Metals
All known forms of the nitrogenase enzyme require Fe and most also contain Mo or V. Research
on species such as Rhodobacter capsulatus and Azotobacter chroococcum has shown that a single species
can synthesize Mo or alternative, non-Mo nitrogenases (using V or Fe instead of Mo) (Bishop et al.
1980, Eady 1996). Genetic and environmental factors (e.g., Mo availability, O2 concentrations,
and light) determine which nitrogenase is synthesized (Robson et al. 1986, Masepohl et al. 2002,
www.annualreviews.org • Free-Living Nitrogen Fixation
497
ES42CH22-Reed
ARI
27 September 2011
14:24
Bellenger et al. 2011). Alternative nitrogenases are widely distributed among N2 -fixers (Figure 2),
but it is commonly accepted that Mo-nitrogenase is most efficient at N2 fixation, and that N2 fixers use alternative nitrogenases when Mo is in relatively low concentrations (Masepohl et al.
2002, Bellenger et al. 2011). However, the vast majority of this research has focused on specific,
culturable N2 -fixing species in the laboratory, and thus the commonality and activity of nonMo nitrogenases in the natural environment remains unknown. This is an important issue to
resolve. For example, if Mo-free nitrogenases are more common and active than suggested by
culture-based studies, conceptual models relating N2 fixation to trace element controls could need
amendment.
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
4.3. ATP and Reducing Power
N2 fixation is one of the most metabolically costly processes on Earth (Simpson & Burris 1984). For
example, in highly controlled chemostat studies under variable conditions, Azotobacter vinelandii
can use >16 mol of ATP per mol of N2 fixed and >100 g of glucose to fix one gram of N2 (Gutschick
1981, Hill 1992). These high-energy demands translate into relatively high demands for ATP and
reducing power, and N2 -fixers acquire this reducing power in varied forms: Photosynthetic N2 fixers use the sun’s energy, and heterotrophic fixers rely on catabolic pathways to derive energy
from organic matter. The efficiency of N2 fixation depends on the particular energy source used
and is also regulated by environmental conditions (Hill 1992). For example, the efficiency of N2
fixation for heterotrophic N2 -fixers is different depending on both the substrate used (e.g., malate
versus sucrose) and on environmental O2 concentrations (Hill 1992). Laboratory studies also
show that active N2 fixation rapidly decreases ATP pools and lowers ATP/ADP ratios (Hill 1988),
and that suboptimal environmental conditions can reduce the efficiency of ATP use during N2
fixation (Hill 1992). High demand for ATP may also help explain the high P demands of N2 -fixing
organisms (Vitousek et al. 2002).
4.4. Nitrogen
N2 -fixers may meet their N demands by fixing N2 , by acquiring mineral N from the external
environment, or by enzymatic breakdown and reallocation of internal cellular N. Studies conducted in chemostats have shown that when mineral forms of N (i.e., NH4 + or NO3 − ) are readily
available in the environment, many N2 -fixing organisms will switch off N2 fixation; however, in
multiple organisms the efficacy of this switch can depend on dissolved organic C concentrations,
pH, and/or the C:N ratio of the cell (Cejudo & Paneque 1988). In particular, under laboratory
conditions nitrogenase activity is inversely related to the supply of NH4 + (Drozd et al. 1972) and
directly related to the C:N ratio of the growth medium (Buhler et al. 1987). In most cases, when
external supplies of mineral N are depleted, low N availability favors N2 fixation. Yet, in some
instances N supplies can drop to levels that are insufficient to meet the N demands of producing
nitrogenase, and some N2 -fixers use proteases to break down internal organic N, which, in turn,
can be reallocated to nitrogenase production (Hill 1992). Under severely low N conditions, organisms may actually lack sufficient N to synthesize proteases, limiting nitrogenase synthesis and N2
fixation. Such effects have been observed only in the laboratory under conditions of extremely low
N availability (Hill 1992), but potential N constraints on fixation rates in natural environments
remain wholly unknown. Nonetheless, this observation informs an intriguing hypothesis: In some
N limited environments, N limitation may persist because N constrains nitrogenase production
(and hence N2 fixation).
498
Reed
·
Cleveland
·
Townsend
ES42CH22-Reed
ARI
27 September 2011
14:24
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
5. GENETIC INSIGHTS
Genetic techniques have provided notable recent advances in terrestrial free-living N2 fixation
research, and assessments of N2 -fixer community composition and structure are setting the stage
for a deeper understanding of terrestrial N2 fixation. N2 fixation lends itself especially well to
genetic research and, in particular, the nifH functional gene—which encodes for dinitrogenase
reductase—is well-suited to studies of natural N2 -fixer communities (Raymond et al. 2004). The
nifH gene is an ideal marker because (a) it is highly conserved; (b) it parallels 16S rRNA gene
distributions; and (c) it can be used as an indicator of N2 -fixer community assemblage, abundance,
and activity (Young 1992, Widmer et al. 1999, Poly et al. 2001, Zehr et al. 2003, Raymond et al.
2004, Yeager et al. 2004). Investigations of nifH genes have greatly enhanced our understanding
of the phylogenetic history and biology of N2 -fixing organisms (Figure 2) (e.g., Normand &
Bousquet 1989, Fani et al. 2000). Moreover, not only has this research allowed us to identify novel
organisms, but nifH gene studies in terrestrial ecosystems have also begun to reveal how N2 -fixing
communities are affected by their environment, and, in turn, how community shifts may influence
N2 fixation rates.
A number of themes have emerged from N2 -fixer genetic research and are beginning to reveal
mechanistic links between free-living N2 -fixer community composition and function. N2 -fixing
communities mediate N2 fixation, and thus changes to the abundance and structure of free-living
N2 -fixer communities could directly translate into differences in rates and timing of N2 fixation.
Indeed, multiple studies have shown that within-site variations in abiotic characteristics (e.g., C
chemistry, P availability, pH) correlate with variations in N2 -fixer community composition and
with differences in N2 fixation rates (Nelson & Mele 2006, Hsu & Buckley 2009, Teng et al. 2009,
Lindsay et al. 2010). In particular, metrics of both C quantity and quality relate to variations in N2 fixer community structure, and research suggests that differences in C stocks, chemistry, and/or
input rates help regulate N2 -fixer abundance, diversity, community composition, and activity (Poly
et al. 2001, Forbes et al. 2009, Wakelin et al. 2010).
Next, changes in nutrient availability may alter N2 fixation rates by driving changes in N2 fixer community composition as well as by inducing higher rates for individual organisms (e.g.,
Benner & Vitousek 2007, Hsu & Buckley 2009, Wakelin et al. 2007, Lindsay et al. 2010, Reed
et al. 2010). Multiple studies have concluded that nutrient availability helps regulate free-living N2
fixation, but the role of N2 -fixer community composition in mediating this control remains largely
unknown. Reed et al. (2010) showed that P fertilization in a tropical rain forest increased N2 -fixer
abundance and diversity, altered N2 -fixer community composition, and stimulated N2 fixation
rates (Figure 3b,c). In addition, Orchard et al. (2009) demonstrated that nifH gene expression in
ocean N2 -fixers was downregulated under low-P conditions, further highlighting molecular-level
controls over N2 -fixer responses to P. Another study showed that higher soil N concentrations
were negatively correlated with nifH gene abundance (Lindsay et al. 2010) in a managed ecosystem,
suggesting a community-level control for the declines in N2 fixation rates commonly observed
after N additions (Crews et al. 2000, Barron et al. 2008, Cusack et al. 2009). However, nifH
gene abundance is not always a reliable predictor of N2 fixation rates (Wallenstein & Vilgalys
2005), and in fact some observations suggest that N2 -fixer abundance may more strongly relate
to N2 fixation rates when N availability is low or when P availability is high (Lindsay et al. 2010,
Reed et al. 2010). More work is needed to elucidate the relationship between nutrient availability
and N2 -fixer community in regulating N2 fixation rates, but these studies indicate that community
composition may play an important role in regulating N2 fixation rates and, more broadly, provide
additional evidence that changes in microbial communities have relevance for ecosystem-scale
function.
www.annualreviews.org • Free-Living Nitrogen Fixation
499
ARI
27 September 2011
14:24
Observations from nutrient fertilization experiments and across natural fertility gradients
suggest positive relationships between the diversity and fixation rates of free-living N2 -fixers
(Figure 3a) (Hsu & Buckley 2009, Moseman et al. 2009, Reed et al. 2010, Wakelin et al. 2010).
For example, in a tillage experiment, Hsu & Buckley (2009) showed that differences in N2 -fixer
community structure had a greater effect on fixation rates than measured soil characteristics,
and, in particular, N2 fixation rates displayed a positive but saturating response to increased
N2 -fixer diversity (Figure 3a). This response offers a microbial perspective to our understanding
of diversity-ecosystem function relationships (Huston 1997, Tilman et al. 2001, Hooper et al.
2005, Balvanera et al. 2006, Ackerly & Cornwell 2007). More broadly, these studies suggest
that, analogous to observations in plant ecology, aspects of microbial ecosystem function may be
predictable from microbial community composition.
Moreover, variations in the community composition of N2 -fixers in response to changes in
nutrient availability (e.g., Tan et al. 2003, Coelho et al. 2009, Lindsay et al. 2010, Reed et al. 2010)
may influence our interpretation of some process-based fertilization experiments. For example,
nutrient manipulations often use large amounts of fertilizer to ensure the alleviation of nutrient
limitation (inputs are in some cases orders of magnitude higher than natural input rates). However,
if large nutrient pulses drive shifts in microbial community composition that create communities
unlikely to occur under realistic conditions, process-only-based interpretations of responses to
fertilizer may lead to an erroneous understanding of the underlying mechanisms.
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
6. CONTROLS OVER FREE-LIVING N2 FIXATION
Perhaps the most active area of research on terrestrial free-living N2 fixation focuses on the
environmental factors that control N2 fixation rates. A number of possible controls have been
proposed; the effects of climate and of C, N, P, and Mo availability have been most commonly
explored (Figure 4). Predatory controls over free-living terrestrial N2 -fixers (e.g., preferential
grazing on N2 -fixers by protozoa, nematodes, etc.) have received less attention, likely due to the
difficulty of assessing population-to-community level interactions in a diverse terrestrial microbial
environment.
Like all organisms, N2 -fixers may be affected by a wide variety of biotic and abiotic factors: there
can be a limiting supply of resources; it can be too warm or too cold, too wet or too dry (Figure 5).
Controls that singularly or more strongly affect N2 -fixers (relative to non-fixers) are of particular
interest because these controls help determine ecosystem N availability and limitation to other
processes (Vitousek et al. 2002, 2010). Although a variety of investigations using chemostats or in
other highly controlled laboratory environments have revealed potential controls over particular
N2 -fixing organisms, here we focus on results from field settings to most directly speak to the
active controls over terrestrial N2 fixation in situ.
N2 fixation is an enzymatic process; hence, in the absence of other constraints, potential rates
of N2 fixation should increase with temperature to some maximum and then decline (e.g., Houlton
et al. 2008). Whether different free-living N2 -fixing organisms or those from different environments converge at a similar temperature maximum is not well known, but temperature has been
shown to constrain N2 fixation rates (Hicks et al. 2003, Houlton et al. 2008) and could help explain
the elevated free-living N2 fixation often observed in warmer biomes (Table 1) (Cleveland et al.
1999). Moisture can also regulate free-living N2 fixation; increasing water availability results in
increased N2 fixation rates (Roskoski 1980, Chapin et al. 1991, Hofmockel & Schlesinger 2007,
Reed et al. 2007a). Small changes in substrate moisture can have large effects on overall rates, particularly in drier climates; thus, the altered precipitation patterns predicted with climate change
500
Reed
·
Cleveland
·
Townsend
ES42CH22-Reed
ARI
27 September 2011
14:24
a
b
Fertilized N2 fixation rates relative
to unfertilized rates
0.30
Nitrogenase activity
(μmol C2H4/sample/h)
0.25
0.20
0.15
0.10
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
0.05
0
1:6
1:3
1:2
1:1
aN:P
2:1
3:1
6:1
7
n=6
6
5
4
n = 14
n=6
3
2
1
0
n = 10
+N
+P
+N+P
+Micronutrients
Treatment
Figure 4
Relationships between free-living N2 fixation rates and nutrient availability. (a) Relationship between prairie
soil cyanobacterial nitrogenase activity and the available N:available P ratio (aN:P) of fertilization treatments
(measured 33 days after treatments began). Data suggest the aN:P ratio of the N2 -fixer environment helps
regulate N2 fixation rates (shown here as ethylene production per sample per hour). Data are from Eisele
et al. (1989), and the figure was reproduced with the permission of Springer Science and Business Media.
(b) Data from the literature showing field-assessed, free-living N2 fixation responses to fertilization with N,
P, N + P, and micronutrients (all fertilization experiments represented by the micronutrient-treatmentadded Mo; some studies added other micronutrients as well). Values represent means ± 1 SE of the
free-living N2 fixation rate response (fertilized rates divided by unamended rates for each site, n sites per
average). Values <1 indicate a net decrease in N2 fixation in response to the treatment, and values >1
indicate that the treatment elicited an increase in N2 fixation rates. Although the data suggest that N
suppresses N2 fixation rates and P/micronutrient additions stimulate rates, new research suggests that some
commercial P fertilizer can be contaminated with enough Mo to confound interpretation of N2 fixation
responses to P additions (Barron et al. 2008). Responses to nutrient fertilization varied significantly both
within and between ecosystems.
have the potential to significantly affect free-living N2 fixation rates (Gundale et al. 2009, Jackson
et al. 2011).
Free-living heterotrophs in litter and soil environments often use organic matter as a resource
both to fix N2 and to maintain high respiration rates to avoid O2 deactivation of nitrogenase (Hill
1992). In light of the high demand for reduced C, it is not surprising that a variety of data suggest
that low C availability can constrain free-living N2 fixation rates. For example, Vitousek & Hobbie
(2000) showed that leaf litter lignin concentrations were negatively correlated with leaf litter N2
fixation rates, which suggests the potential for rates to be positively related to the supply of relatively
labile litter C. Furthermore, data from these fertilization experiments suggested an interaction
between litter C and nutrient controls; that is, data suggested that P fertilization exerted stronger
controls over leaf litter N2 fixation rates in the low-lignin litter (Vitousek & Hobbie 2000). Other
studies support the notion that C availability and quality can constrain free-living N2 fixation rates,
and multiple studies suggest that rates are positively correlated with C:N ratios (e.g., Maheswaran
& Gunatilleke 1990, Cusack et al. 2009, Perez et al. 2010). Taken together, these data suggest
that N2 fixation is enhanced when C is relatively plentiful and N relatively scarce and that, beyond
assays of total C, C chemistry may help determine C availability to heterotrophic N2 fixation.
P is typically identified as the nutrient that most frequently limits terrestrial free-living N2
fixation (Figure 4b). To a certain extent, the origin of this paradigm can be traced to aquatic
www.annualreviews.org • Free-Living Nitrogen Fixation
501
ES42CH22-Reed
ARI
27 September 2011
14:24
P, Mo
N
C
H2O
T
2
1
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
N2-fixer
community
6
NPP
3
Free-living
N2 fixation
rates
4
Soil P
availability
5
Denitrification
1
2
Benner & Vitousek (2007)
Coelho et al. (2009)
Lindsay et al. (2010)
Moseman et al. (2009)
Nelson & Mele (2006)
Reed et al. (2010)
Stewart et al. (2011)
Tan et al. (2003)
Teng et al. (2009)
Wakelin et al. (2007)
2
Alexander & Schell (1973)
Barron et al. (2008)
Benner et al. (2007)
Chapin et al. (1991)
continued
Crews et al. (2000)
Cusack et al. (2009)
Dawson (1983)
Deluca et al. (2008)
Eisele et al. (1989)
Gundale et al. (2009)
Heath et al. (1988)
Hicks et al. (2003)
Hofmockel & Schlesinger (2007)
Houlton et al. (2008)
Hsu & Buckley (2009)
Jackson et al. (2011)
Reed et al. (2007a)
Reed et al. (2007b)
Reed et al. (2008)
Roskoski (1980)
Silvester (1989)
2
continued
Thompson & Vitousek (1997)
Vitousek & Hobbie (2000)
Zackrisson et al. (2004)
4
Houlton et al. (2008)
Wang et al. (2007)
5
3
Cleveland et al. (1999)
Elser et al. (2007)
Hungate et al. (2003)
LeBauer & Treseder (2008)
Reich et al. (2006)
Thornton et al. (2009)
Townsend et al. (2011)
Wang et al. (2007)
Deutsch et al. (2001)
6
Benner et al. (2007)
Hsu & Buckley (2009)
Lindsay et al. (2010)
Menge & Hedin (2009)
Moseman et al. (2009)
Reed et al. (2010)
Stewart et al. (2011)
Figure 5
Schematic representation showing potential biotic and abiotic controls on free-living N2 fixation rates and a number of other terrestrial
ecosystem processes that free-living N2 fixation could potentially affect. Numbers in circles relate the controls to associated references
listed below. P, Mo, N, C (quality and availability), temperature (T), and moisture (H2 O) have all been shown to regulate free-living
N2 fixation rates in terrestrial ecosystems, but the relative importance of any given control—and the importance of interactions among
controls—are ecosystem specific. Contemporary genetic data suggest that many abiotic controls are mediated through different
N2 -fixing communities and that N2 -fixer physiology, abundance, diversity, and community composition all play a role in regulating N2
fixation rates. Data also suggest that free-living N2 fixation represents a significant input of N to many terrestrial ecosystems with the
potential to help regulate denitrification losses, soil P availability (via altered phosphatase activity), net primary productivity, and
ecosystem responses to global environmental change.
ecological literature, which often describes P as the master element owing in large part to its
ability to regulate N availability via N2 fixation (Redfield 1958). There are also physiological reasons to expect P limitation. In particular, high ATP requirements may elevate N2 -fixers’ demand
for P, and the organic matter that many free-living N2 -fixers metabolize (e.g., leaf litter) is often
supplied at N:P ratios above their biological demand (Cleveland & Liptzin 2007). In fact, many
fertilization studies have observed a stimulatory response to added P (Figure 4b) (Eisele et al. 1989;
502
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
Vitousek & Hobbie 2000; Benner et al. 2007; Reed et al. 2007a,b), and an investigation along a
natural P gradient in a Costa Rican forest supported the idea that P availability constrains freeliving N2 fixation rates (Reed et al. 2008). In contrast, fertilization with N commonly suppresses
free-living N2 fixation (Figure 4b) (e.g., Crews et al. 2000, Zackrisson et al. 2004, Barron et al.
2008, Cusack et al. 2009), a response that makes sense from cellular, physiological, and biogeochemical perspectives. However, estimates of the extent to which N or P fertilization affects N2
fixation rates—if at all—vary widely in the literature (Figure 4b).
Fertilization with either N or P does not always elicit the same (or any) response, but due to the
overall suppressing effects of N and the often stimulatory effects of P (Figure 4b), N:P ratios may
predict N2 fixation rates better than either N or P alone. Work by Eisele et al. (1989) suggested
that N2 fixation rates may be tied to the ratio of available N to available P (Figure 4a), paralleling
the response of cyanobacteria in many freshwater and managed terrestrial ecosystems (Schindler
1977, Smith 1992). Thus, variations in substrate stoichiometry may help explain N2 fixation rate
differences that metrics of single-nutrient availability alone do not reveal. However, relationships
between soil N:P ratios and soil N2 fixation were not seen in a restored prairie characterized by
much lower concentrations of both N and P (Reed et al. 2007b). In this relatively infertile system,
the absolute abundance of available P was a better predictor of N2 fixation rates than were N:P
ratios.
Moreover, when considering P fertilization results, it is important to take into consideration
that Barron et al. (2008) showed that Mo was a hidden contaminant in some commercial P fertilizers and that, although leaf litter free-living N2 fixation rates in a Panamanian rain forest responded
significantly to fertilization with commercial P, fixation did not respond to P sources that were
not contaminated with Mo. Furthermore, free-living N2 fixation rates responded significantly to
additions of Mo. These results showed conclusively that Mo alone limited leaf litter N2 fixation
rates in this tropical forest. Accordingly, this result calls into question the results of N2 fixation
fertilization experiments in which Mo could represent an incidental nutrient addition, thus confounding the interpretation of N2 fixation responses (Figure 4b). Furthermore, when designing
future fertilization experiments, care must be taken in purchasing and testing fertilizer for unwanted trace Mo contaminants. However, as Barron et al. (2008) note, these results do not imply
that P does not limit N2 fixation in other ecosystems. For example, Vitousek & Hobbie (2000)
used a full-factorial P × micronutrient fertilization experiment to show that in a site in Hawai’i,
leaf litter placed in P-fertilized plots maintained significantly higher rates of N2 fixation relative
to controls, whereas leaf litter in plots fertilized with a suite of trace elements (including Mo) did
not fix more N2 .
These data and others stress the probable importance of a multielement perspective when assessing nutrient controls over terrestrial free-living N2 fixation. Yet, we currently lack the ability
to predict free-living N2 fixation rates across multiple systems as a function of multiple resource
controls (e.g., C, N, P, Mo). In part, this inability results from insufficient data. For example,
few fertilization studies have been published in which free-living N2 fixation rates and P and Mo
concentrations were simultaneously assessed (Vitousek & Hobbie 2000, Barron et al. 2008). Thus,
identifying critical ratios or analogous stoichiometric controls (e.g., Sterner & Elser 2002) remains
difficult. This challenge is exacerbated by a lack of information on N2 -fixer cellular stoichiometry
in terrestrial ecosystems; stoichiometric insights typically involve estimates of both substrate and
cellular ratios (Redfield 1958, Reiners 1986, Sterner & Elser 2002, Cleveland & Liptzin 2007).
A thorough picture of cellular requirements and their potential variation across N2 -fixer phylogenies would require separating free-living N2 -fixers from the non-fixing community in natural
environments; methodological difficulties make this task problematic. However, isotopic labeling
techniques could offer some breakthroughs. For example, 15 N labeling and ultracentrifugation are
www.annualreviews.org • Free-Living Nitrogen Fixation
503
ES42CH22-Reed
ARI
27 September 2011
14:24
now allowing researchers to separate N2 -fixer from nonfixer DNA (after incubating samples with
15
N2 and separating out the heavier DNA of organisms that fixed N2 and built genetic material)
(Buckley et al. 2007). Perhaps similar approaches would be possible at the cellular level.
7. RESPONSIVENESS AND ECOSYSTEM FUNCTION
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
N-containing molecules are highly mobile, in the sense that they occur in multiple gaseous and
soluble forms that move within and between terrestrial ecosystems. Free-living N2 fixation is
similarly dynamic, and variable rates—driven by cellular regulation or community composition
shifts, for example—may help explain responsive relationships among N2 fixation, N loss, and N
demand (e.g., Deutsch et al. 2001). The environment of free-living N2 -fixers is small relative to
plants, which integrate across space and time with canopies, root structures, and relatively large
storage capacities. Accordingly, even in ecosystems with relatively rich N economies at coarse
spatial and temporal scales, free-living N2 fixation may persist in microsites or components of
the ecosystem where N is relatively low (Hedin et al. 2009), or at times when N is low. If so,
persistent niches of N2 fixation could act to replenish N that is lost or immobilized (sensu Hedin
et al. 2009). Thus, although a biogeochemical perspective on free-living N2 fixation has long
held that higher rates should occur where and when N is relatively less available and non-N
nutrients more available, a contemporary conceptual framework for free-living N2 fixation should
also explicitly consider scale and community dynamics. For example, even during successional or
developmental stages when overall soil N availability is relatively high, zones within the ecosystem
may be favorable for high rates of free-living N2 fixation (e.g., litter layers with high C:N ratios)
(Hedin et al. 2009). In such sites, N2 -fixing organisms may maintain higher N2 fixation rates than
would be predicted on the basis of soil N alone, and thus N2 fixation could sustain both a N-rich
economy and substantial N loss (Hedin et al. 2009).
The high biological diversity of free-living N2 -fixers, combined with the fact that microbial
communities can undergo profound compositional shifts within very short timescales, suggests
that free-living N2 fixation may be more responsive to environmental change [e.g., climate change,
elevated atmospheric carbon dioxide (CO2 ) concentrations] than are symbiotic forms. For example, the general increase in litter C:N ratios observed in elevated CO2 experiments (Luo et al.
2006) might be predicted to elicit increases in litter N2 fixation rates. However, data from a pine
forest and a pine plantation showed that elevated atmospheric CO2 did not increase forest floor
free-living N2 fixation, at least over a five-year span (Hofmockel & Schlesinger 2007, Zheng et al.
2008). Investigations of symbiotic fixation responses to CO2 experiments suggest that N2 fixation
increases only in response to elevated CO2 when other nutrients (e.g., P, Mo) are added, and nutrient availability could also constrain free-living N2 fixation responses (Figure 4b) (van Groenigen
et al. 2006). Other data suggest that free-living N2 -fixing communities and rates could rapidly
respond to subtle changes in temperature and precipitation (Deslippe et al. 2005, Hofmockel &
Schlesinger 2007, Houlton et al. 2008, Gundale et al. 2009). More frequent incorporation of freeliving N2 fixation measurements into global change experiments would significantly enhance our
understanding of how this process might respond to environmental change.
Next, we know that N responses to global change could dramatically affect future C-climate
feedbacks (e.g., Hungate et al. 2003, van Groenigen et al. 2006, Thornton et al. 2009, Wang &
Houlton 2009). However, most current terrestrial ecosystem models incorporate phenomenological relationships only between N2 fixation and other variables, if N2 fixation is represented at
all. Free-living rates are almost never mechanistically modeled, and, although multiple models
attempt to simulate symbiotic N2 fixation, namely, its controls and effects on global C and climate
504
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
predictions (Vitousek & Field 1999, Rastetter et al. 2001, Vitousek et al. 2002, Wang et al. 2007,
Menge et al. 2009), the same cannot be said for free-living fixation.
Finally, potential variation in N2 fixation rates is expected in ecosystems that are not at steady
state (e.g., those recovering from disturbance such as fire or deforestation). Following disturbance,
many ecosystems have been shown to undergo dramatic changes to N cycling (Ojima et al. 1994,
Reich et al. 2001, Davidson et al. 2007), and free-living N2 fixation offers a likely mechanism
for replacing disturbance-induced N losses. For example, we know that fire can stimulate freeliving N2 fixation in some grassland ecosystems (e.g., Eisele et al. 1989), but the generality of
this response across multiple ecosystem types remains unresolved. Fire, deforestation, and/or
conversion of forests to low-diversity managed plantations are all likely to have substantial effects
on free-living N inputs, yet our understanding of how N2 fixation helps determine the trajectory of
response after disturbance remains in its infancy. Nevertheless, the few studies that have explored
free-living N2 fixation rates and communities following disturbance suggest they do indeed change
(Widmer et al. 1999, Zackrisson et al. 2004, DeLuca et al. 2008), and determining the magnitude
and extent of such changes is a contemporary challenge.
8. CRITICAL UNKNOWNS AND NEXT STEPS
The role of N2 fixation in the past, current, and future N cycle remains a major focus in ecology
and is critical to our understanding of both current and future ecosystem function (Vitousek et al.
1997, Hungate et al. 2004, Reich et al. 2006, Galloway et al. 2008, Thornton et al. 2009, Townsend
& Howarth 2010). In particular, N2 fixation is essential for understanding how biogeochemical
cycles interact and are coupled in space and time, as well as for clarifying proximate versus ultimate
nutrient constraints on ecosystem function (Vitousek et al. 2010, Finzi et al. 2011, Townsend et al.
2011). For example, although data suggest that changes in N availability can alter both the structure
and function of ecosystems, N may represent a proximate limitation to factors such as NPP if N
inputs via N2 fixation are regulated by non-N resources (Figure 5) (Vitousek et al. 2010).
Determining proximate versus ultimate controls is increasingly important in a world that is
experiencing major changes to multiple biogeochemical cycles. Furthermore, an incomplete understanding of free-living N2 fixation rates and controls represents a significant gap in our ability
to assess the degree to which the N cycle has been changed by human activity (Hedin et al. 1995)
and to accurately predict ecosystem responses to global change (e.g., Reich et al. 2006, Thornton
et al. 2009, Wang & Houlton 2009).
We contend that a systematic effort to better quantify and model free-living N2 fixation in a
range of terrestrial ecosystems, and across a variety of environmental gradients that include human
disturbance, should be viewed as a priority in both the ecosystem ecology and global change scientific communities. As reviewed here, our increasing ability to couple process-based measurements
with community-to-cellular-scale data is an opportunity to improve our understanding not only
of free-living N2 fixation itself but of a number of broader questions in ecology.
SUMMARY POINTS
1. N2 fixation is a fundamental ecosystem process, and free-living N2 fixation represents a
ubiquitous and perhaps dominant source of new N to many terrestrial ecosystems.
2. Free-living N2 fixation rates vary significantly in both space (from centimeters to kilometers) and time (from hours to years), and this variation is likely driven by both N2 -fixer
community and environmental controls.
www.annualreviews.org • Free-Living Nitrogen Fixation
505
ES42CH22-Reed
ARI
27 September 2011
14:24
3. Recent advances in free-living N2 fixation research offer novel insight into the role of
N2 -fixing community structure in regulating N2 fixation rates and provide important
new physiological, functional, and phylogenetic perspectives on N2 fixation.
4. Studies suggest a suite of possible controls over N2 fixation (e.g., N, Mo, P, and C
availability), highlighting the need for simultaneous measurements of multiple ecosystem
characteristics. Recent work suggests that conceptual models that integrate multiple
controls—e.g., a stoichiometric perspective—can offer improved insight.
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
5. Beyond acquiring more data, we should use our current understanding of mechanistic
controls over free-living N2 fixation to more deeply integrate this fundamental process
into conceptual and numerical models.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
This synthesis represents a convergence of ideas that has benefitted enormously from numerous
discussions with Diana Nemergut and Steve Schmidt. We are indebted to Lars Hedin for valuable comments on earlier versions of the manuscript. We are also grateful to Tim Seastedt, Bill
Bowman, Jason Neff, Alex Barron, Ben Houlton, and Peter Vitousek for insight and conversations
that helped shape the perspectives offered here. Finally, we offer special thanks to those scientists who willingly offered new data and insight to our understanding of N2 fixation. Neither this
review nor much of the work that informed it would have been possible without support from
the National Science Foundation, the Andrew W. Mellon Foundation, and the U.S. Geological
Survey.
LITERATURE CITED
Ackerly DD, Cornwell WK. 2007. A trait-based approach to community assembly: partitioning of species trait
values into within- and among-community components. Ecol. Lett. 110:135–45
Alexander V, Schell DM. 1973. Seasonal and spatial variation of nitrogen fixation in the Barrow, Alaska,
tundra. Arc. Alp. Res. 5:77–88
Balvanera P, Pfisterer B, Buchman N, He J-S, Nakashizuka T, et al. 2006. Quantifying the evidence for
biodiversity effects on ecosystem functioning and services. Ecol. Lett. 9:1146–56
Barron AR, Purves DW, Hedin LO. 2011. Facultative nitrogen fixation by canopy legumes in a lowland
tropical forest. Oecologia 165:511–20
Barron AR, Wurzburger N, Bellenger JP, Wright SJ, Kraepiel AML, Hedin LO. 2008. Molybdenum limitation
of asymbiotic nitrogen fixation in tropical forest soils. Nat. Geosci. 2:42–45
Bellenger J-P, Wichard T, Xu Y, Kraepiel AML. 2011. Essential metals for nitrogen fixation in a free-living
N2 -fixing bacterium: chelation, homeostasis and high use efficiency. Environ. Microbiol. 13:1395–411
Benner JW, Conroy S, Lunch CK, Toyoda N, Vitousek PM. 2007. Phosphorus fertilization increases the
abundance and nitrogenase activity of the cyanolichen Pseudocyphellaria crocata in Hawaiian montane
forests. Biotropica 39:400–5
506
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
Benner JW, Vitousek PM. 2007. Development of a diverse epiphyte community in response to phosphorus
fertilization. Ecol. Lett. 10:628–36
Bentley BL. 1987. Nitrogen fixation by epiphylls in a tropical rain forest. Ann. Mo. Bot. Gard. 74:234–41
Bishop PE, Jarlenski DML, Heatherington DR. 1980. Evidence for an alternative nitrogen fixation system in
Asotobacter vinelandii. Proc. Natl. Acad. Sci. USA 77(12):7342
Boring LR, Swank WT, Waide JB, Henderson GS. 1988. Sources, fates, and impacts of nitrogen inputs to
terrestrial ecosystems: review and synthesis. Biogeochemistry 6:119–59
Buckley DH, Huangyutitham V, Hsu S-F, Nelson TA. 2007. Stable isotope probing with 15 N2 reveals novel
noncultivated diazotrophs in soil. App. Environ. Microbiol. 73:3196–204
Buhler T, Sann R, Monter U, Dingler C, Kuhla J, Oelze J. 1987. Control of dinitrogen fixation in ammoniumassimilating culture of Azotobacter vinelandii. Arch. Microbiol. 148:247–51
Cejudo FJ, Paneque A. 1988. Effect of nitrogen starvation on ammonium-inhibition of nitrogenase in Azotobacter chroococcum. Arch. Microbiol. 149:481–84
Chapin FS, Bliss LC, Bledsoe LJ. 1991. Environmental regulation of nitrogen fixation in a high arctic lowland
ecosystem. Can. J. Bot. 69:2744–55
Cleveland CC, Houlton BZ, Neill C, Reed SC, Townsend AR, Wang Y. 2010. Using indirect methods to
constrain symbiotic nitrogen fixation rates: a case study from an Amazonian rain forest. Biogeochemistry
99:1–13
Cleveland CC, Liptzin D. 2007. C:N:P stoichiometry in soil: Is there a “Redfield ratio” for the microbial
biomass? Biogeochemistry 85:235–52
Cleveland CC, Reed SC, Townsend AT. 2006. Nutrient regulation of organic matter decomposition in a
tropical rain forest. Ecology 87:492–503
Cleveland CC, Townsend AR, Schimel DS, Fisher H, Howarth RW, et al. 1999. Global patterns of terrestrial
biological nitrogen (N2 ) fixation in natural ecosystems. Glob. Biogeochem. Cy. 13:623–45
Coelho MRR, Marriel I, Jenkins SN, Lanyon CV, Seldin L, O’Donnell AGO. 2009. Molecular detection and
quantification of nifH gene sequences in the rhizosphere of sorghum (Sorghum bicolor) sown with two
levels of N fertilizer. Appl. Soil Ecol. 42:48–53
Crews TE. 1999. The presence of nitrogen fixing legumes in terrestrial communities: evolutionary versus
ecological considerations. Biogeochemistry 46:233–46
Crews TE, Farrington H, Vitousek PM. 2000. Changes in asymbiotic, heterotrophic nitrogen fixation on
leaf litter of Metrosideros polymorpha with long-term ecosystem development in Hawaii. Ecosystems 3:
386–95
Crews TE, Kurina LM, Vitousek PM. 2001. Organic matter and nitrogen accumulation and nitrogen fixation
during early ecosystem development. Biogeochemistry 52:259–279
Cusack DF, Silver W, McDowell WH. 2009. Biological nitrogen fixation in two tropical forests: ecosystemlevel patterns and effects of nitrogen fertilization. Ecosystems 12:1299–315
Davidson EA, de Carvalho CJR, Figueira AM, Ishida FY, Ometto JPHB, et al. 2007. Recuperation of nitrogen
cycling in Amazonian forests following agricultural abandonment. Nature 447:995–98
Davidson EA, Ishida FY, Nepstad DC. 2004. Effects of an experimental drought on soil emissions of carbon dioxide, methane, nitrous oxide, and nitric oxide in a moist tropical forests. Glob. Change Biol. 10:
718–30
Dawson JO. 1983. Dinitrogen fixation in forest ecosystems. Can. J. Microbiol. 29:979–92
DeLuca TH, Zackrisson O, Gundale MJ, Nilsson N-C. 2008. Ecosystem feedbacks and nitrogen fixation in
boreal forests. Science 320:1181
DeLuca TH, Zackrisson O, Nilsson M-C, Sellstedt A. 2002. Quantifying nitrogen-fixation in feather moss
carpets of boreal forests. Nature 419:917–20
Deslippe JR, Egger KN, Henry GHR. 2005. Impacts of warming and fertilization on nitrogen-fixing microbial
communities in the Canadian High Arctic. FEMS Microb. Ecol. 53:41–50
Deutsch C, Sarmiento JL, Sigman DL, Gruber N, Dunne JP. 2001. Spatial coupling of nitrogen inputs and
losses in the ocean. Nature 445:163–67
Drozd JW, Tubb RS, Postgate JR. 1972. A chemostat study of the effect of fixed nitrogen sources on nitrogen
fixation membranes and free amino acids in Azotobacter chroococcum. J. Gen. Microbiol. 73:221–32
www.annualreviews.org • Free-Living Nitrogen Fixation
507
ARI
27 September 2011
14:24
Eady RR. 1991. The dinitrogen-fixing bacteria. In The Prokaryotes, ed. A Balows, HG Truper, M Dworkin,
W Harder, K-H Schleifer, pp. 534–53. New York: Springer-Verlag
Eady RR. 1996. Structure-function relationships of alternative nitrogenases. Chem. Rev. 96:3013–30
Eisele KA, Schimel DS, Kapustka LA, Parton WJ. 1989. Effects of available P and N:P ratios on non-symbiotic
dinitrogen fixation in prairie soils. Oecologia 79:471–74
Elser JJ, Bracken MES, Gruner DS, Harpole WS, Hillebrand H, et al. 2007. Global analysis of nitrogen and
phosphorus limitation of primary producers in freshwater, marine and terrestrial ecosystems. Ecol. Lett.
10:1135–42
Fani R, Gallo R, Lio P. 2000. Molecular evolution of nitrogen fixation: the evolutionary history of the nifD,
nifK, nifEE, and nifN genes. J. Mol. Evol. 51:1–11
Finzi AC, Austin AT, Cleland EE, Frey SD, Houlton BZ, Wallenstein BZ. 2011. Responses and feedbacks
of coupled biogeochemical cycles to climate change: examples from terrestrial ecosystems. Front. Ecol.
Environ. 9:61–67
Forbes MS, Broos K, Baldock JA, Gregg AL, Wakelin SA. 2009. Environmental and edaphic factors drive
bacterial communities involved in soil N-cycling. Aus. J. Soil Res. 47:380–88
Galiana A, Balle P, Kanga AN, Domenach AM. 2002. Nitrogen fixation estimated by the 15 N natural abundance
method in Acacia mangium Willd inoculated with Bradyrhizobium sp. and grown in silvicultural conditions.
Soil Biol. Biochem. 34:251–62
Galloway JN, Dentener FJ, Capone DG, Boyer EW, Howarth RW, et al. 2004. Nitrogen cycles: past, present
and future. Biogeochemistry 70:153–226
Galloway JN, Townsend AR, Erisman JW, Bekunda M, Cai ZC, et al. 2008. Transformation of the nitrogen
cycle: recent trends, questions, and potential solutions. Science 320:889–92
Groffman PM, Butterbach-Bahl K, Fulweiler RW, Gold AJ, Morse JL, et al. 2009. Challenges to incorporating
spatially and temporally explicit phenomena (hotspots and hot moments) into denitrification models.
Biogeochemistry 93:49–77
Gundale MJ, Gustafsson H, Nilsson M-C. 2009. The sensitivity of nitrogen fixation by a feathermosscyanobacteria association to litter and moisture variability in young and old boreal forests. Can. J. Forest
Res. 39:2542–49
Gutschick VP. 1981. Evolved strategies in nitrogen acquisition by plants. Am. Nat. 118:607–37
Heath B, Sollins P, Perry DA, Cromack K Jr. 1988. Asymbiotic nitrogen fixation in litter from Pacific Northwest forests. Can. J. Forest Res. 18:68–74
Hedin LO, Armesto JJ, Johnson AH. 1995. Patterns of nutrient loss from unpolluted, old-growth temperate
forests: evaluation of biogeochemical theory. Ecology 76:493–509
Hedin LO, Brookshire ENJ, Menge DNL, Barron AR. 2009. The nitrogen paradox in tropical forest ecosystems. Annu. Rev. Ecol. Syst. 40:613–35
Hicks WT, Harmon ME, Griffiths RP. 2003. Abiotic controls on nitrogen fixation and respiration in selected
woody debris from the Pacific Northwest, USA. Ecoscience 10:66–73
Hill S. 1988. How is nitrogenase regulated by oxygen? FEMS Microbiol. Rev. 54:111–30
Hill S. 1992. Physiology of nitrogen fixation in free-living heterotrophs. In Biological Nitrogen Fixation, ed. G
Stacey, RH Burris, J Evans, pp. 87–129. New York: Chapman and Hall
Hofmockel KS, Schlesinger WH. 2007. Carbon dioxide effects on heterotrophic dinitrogen fixation in a
temperate pine forest. Soil Sci. Soc. Am. J. 71:140–44
Hooper DU, Chapin FS, Ewel JJ, Hector A, Inchausti P, et al. 2005. Effects of biodiversity on ecosystem
functioning: a consensus of current knowledge. Ecol. Monogr. 75:3–35
Hope SM, Li C-Y. 1997. Respiration, nitrogen fixation, and mineralizable nitrogen spatial and temporal
patterns within two Oregon Douglas-fir stands. Can. J. For. Res. 27:501–9
Houlton BZ, Wang Y-P, Vitousek PM, Field CB. 2008. A unifying framework for dinitrogen fixation in the
terrestrial biosphere. Nature 454:327–30
Hsu S-F, Buckley DH. 2009. Evidence for the functional significance of diazotroph community structure in
soil. ISME J. 3:124–36
Hungate BA, Dukes JS, Shaw MR, Luo Y, Field CB. 2003. Nitrogen and climate change. Science 302:1512–
13
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
508
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
Hungate BA, Stiling PD, Dijkstra P, Johnson DW, Ketterer ME, et al. 2004. CO2 elicits long-term decline
in nitrogen fixation. Science 304:1291
Huston MA. 1997. Hidden treatments in ecological experiments: re-evaluating the ecosystem function of
biodiversity. Oecologia 110:449–60
Jackson BG, Martin P, Nilsson M-C, Wardle DA. 2011. Response of feather moss associated N2 fixation and
litter decomposition to variations in simulated rainfall intensity and frequency. Oikos 120:570–81
Jordan C, Caskey W, Escalante G, Herrera R, Montagnini F, et al. 1983. Nitrogen dynamics during conversion
of primary Amazonian rain forest to slash and burn agriculture. Oikos 40:131–39
LeBauer DS, Treseder KK. 2008. Nitrogen limitation of net primary productivity in terrestrial ecosystems is
globally distributed. Ecology 89:371–79
Lindsay EA, Colloff MJ, Gibb NL, Wakelin SA. 2010. The abundance of microbial functional genes in grassy
woodlands is influenced more by soil nutrient enrichment than by recent weed invasion or livestock
exclusion. Appl. Environ. Microbiol. 76:5547–55
Ludden PW. 1994. Reversible ADP-ribosylation as a mechanism of enzyme regulation in prokaryotes. Mol.
Cell. Biochem. 138:123–29
Luo Y, Hui D, Zhang D. 2006. Elevated CO2 stimulates net accumulations of carbon and nitrogen in land
ecosystems: a meta-analysis. Ecology 87:53–63
Maheswaran J, Gunatilleke IAUN. 1990. Nitrogenase activity in soil and litter of a tropical lowland rain forests
and an adjacent fernland in Sri Lanka. J. Trop. Ecol. 6:281–89
Masepohl B, Drepper T, Paschen A, Gross S, Pawlowski A, et al. 2002. Regulation of nitrogen fixation in the
phototrophic purple bacterium Rodobacter capsulatus. J. Mol. Microbiol. Biotechnol. 4:243–48
Matzek V, Vitousek P. 2003. Nitrogen fixation in bryophytes, lichens, and decaying wood along a soil-age
gradient in Hawaiian montane rain forest. Biotropica 35:12–19
Menge DNL, DeNoyer JL, Lichstein JW. 2010. Phylogenetic constraints do not explain the rarity of nitrogenfixing trees in late-successional temperate forests. PLoS ONE 5:e12056
Menge DNL, Hedin LO. 2009. Nitrogen fixation in different biogeochemical niches along a 120,000-year
chronosequence in New Zealand. Ecology 90:2190–201
Menge DNL, Levin SA, Hedin LO. 2009. Facultative versus obligate nitrogen fixation strategies and their
ecosystem consequences. Am. Nat. 174:465–77
Moseman SM, Zhang R, Qian PY, Levin LA. 2009. Diversity and functional responses of nitrogen-fixing
microbes to three wetland invasion. Biol. Invasions 11:225–39
Nelson DR, Mele PM. 2006. The impact of crop residue amendments and lime on microbial community
structure and nitrogen-fixing bacteria in the wheat rhizosphere. Soil Res. 44:319–29
Normand P, Bousquet J. 1989. Phylogeny of nitrogenase sequences in Frankia and in other nitrogen-fixing
organisms. Mol. Gen. Genet. 213:238–46
Ojima DS, Schimel DS, Parton WJ, Owensby CE. 1994. Long- and short-term effects of fire on nitrogen
cycling in tallgrass prairie. Biogeochemistry 24:67–84
Orchard ED, Webb EA, Dyhrman ST. 2009. Molecular analysis of the phosphorus starvation response in
Trichodesmium spp. Environ. Microbiol. 11:2400–11
Parton W, Silver WL, Burke IC, Grassens L, Harmon ME, et al. 2007. Global-scale similarities in nitrogen
release patterns during long-term decomposition. Science 315:361–64
Perez CA, Carmona MR, Aravena JC, Armesto JJ. 2004. Successional changes in soil nitrogen availability, nonsymbiotic nitrogen fixation and carbon/nitrogen ratios in southern Chilean forest ecosystems. Oecologia
140:617–25
Perez CA, Carmona MR, Armesto JJ. 2010. Non-symbiotic nitrogen fixation during leaf litter decomposition
in an old-growth temperate rain forest of Chiloe Island, southern Chile: effects of single versus mixed
species litter. Austral Ecol. 35:148–56
Pérez S, Pérez CA, Carmona MR, Farina JM, Armesto JJ. 2008. Effects of labile phosphorus and carbon on
non-symbiotic N2 fixation in logged and unlogged evergreen forests in Chiloé Island, Chile. Rev. Chil.
Hist. Nat. 81:267–78
Poly F, Ranjard L, Nazaret S, Gourbiere F, Monrozier LJ. 2001. Comparison of nifH in soils and microenvironments with contrasting properties. Appl. Environ. Microb. 67:2255–62
www.annualreviews.org • Free-Living Nitrogen Fixation
509
ARI
27 September 2011
14:24
Rastetter EB, Vitousek PM, Field C, Shaver GR, Herbert D, Agren GI. 2001. Resource optimization and
symbiotic nitrogen fixation. Ecosystems 4:369–88
Raymond J, Siefert JL, Staples CR, Blankenship RE. 2004. The natural history of nitrogen fixation. Mol. Biol.
Evol. 21:541–54
Redfield AC. 1958. The biological control of chemical factors in the environment. Am. Sci. 64:205–21
Reed SC, Cleveland CC, Townsend AR. 2007a. Controls over leaf litter and soil nitrogen fixation in two
lowland tropical rain forests. Biotropica 39:585–92
Reed SC, Cleveland CC, Townsend AR. 2008. Tree species control rates of free-living nitrogen fixation in a
tropical rain forest. Ecology 89:2924–34
Reed SC, Seastedt TR, Mann CM, Suding KN, Townsend AR, Cherwin KL. 2007b. Phosphorus fertilization
stimulations nitrogen fixation and increases inorganic nitrogen concentrations in a restored prairie. Appl.
Soil Ecol. 36:238–42
Reed SC, Townsend AR, Cleveland CC, Nemergut DR. 2010. Microbial community shifts influence patterns
in tropical forest nitrogen fixation. Oecologia 264:521–31
Reich PB, Hungate BA, Luo Y. 2006. Carbon-nitrogen interactions in terrestrial ecosystems in response to
rising atmospheric carbon dioxide. Annu. Rev. Ecol. Evol. Syst. 37:611–36
Reich PB, Peterson DW, Wedin DA, Wrage K. 2001. Fire and vegetation effects on productivity and nitrogen
cycling across a forest-grassland continuum. Ecology 82:1703–19
Reiners WA. 1986. Complementary models for ecosystems. Am. Nat. 127:59–73
Robson RL, Eady RR, Richardson TH, Miller RW, Hawkins M, Postgate JR. 1986. The alternative nitrogenase
of Azotobacter chroococcum is a vanadium enzyme. Nature 322:388–90
Robson RL, Postgate JR. 1980. Oxygen and hydrogen in biological nitrogen fixation. Annu. Rev. Microbiol.
34:183–207
Roggy JC, Prevost MF, Garbaye J, Domenach AM. 1999. Nitrogen cycling in the tropical rain forest of French
Guiana: comparison of two sites with contrasting soil types using delta N-15. J. Trop. Ecol. 15:1–22
Roskoski JP. 1980. Nitrogen fixation in hardwood forests of the northeastern United States. Plant Soil 54:33–44
Schindler DW. 1977. Evolution of phosphorus limitation in lakes. Science 195:260–62
Schmidt SK, Reed SC, Nemergut DR, Grandy AS, Cleveland CC, et al. 2008. The earliest stages of ecosystem
succession in high-elevation (5000 metres above sea level), recently deglaciated soils. Proc. R. Soc. B.
275:2793–802
Seitzinger S, Harrison JA, Bohlke JK, Bouwman AF, Lowrance R, et al. 2006. Denitrification across landscapes
and waterscapes: a synthesis. Ecol. Appl. 16:2064–90
Silvester WB. 1989. Molybdenum limitation of asymbiotic nitrogen fixation in forests of Pacific Northwest
America. Soil Biol. Biochem. 21:283–89
Simpson FB, Burris RH. 1984. A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen
by nitrogenase. Science 224:1095–97
Skujins J, Tann CC, Borjesson I. 1987. Dinitrogen fixation in a montane forest sere dominated by 15N2
assimilation and in situ acetylene-reduction methods. Soil Biol. Biochem. 19:465–71
Smith VH. 1992. Effects of nitrogen:phosphorus supply ratios on nitrogen fixation in agricultural and pastoral
ecosystems. Biogeochemistry 18:19–35
Soderland R, Rosswall T. 1982. The nitrogen cycle. In The Natural Environment and Biogoechemical Cycles, ed.
O Hutzinger, pp. 61–81. New York: Springer
Son Y. 2001. Non-symbiotic nitrogen fixation in forest ecosystems. Ecol. Res. 16:183–96
Sprent JI. 2005. Nodulated legume trees. In Nitrogen Fixation in Agriculture, Forestry and the Environment, ed.
D Werner, WE Newton. pp. 113–141. Amsterdam: Springer
Sprent JI. 2009. Legume Nodulation. Chichester, UK: Wiley-Blackwell
Sprent JI, Sprent P. 1990. Nitrogen-Fixing Organisms. Pure and Applied Aspects. New York: Chapman Hall
Sterner RW, Elser JJ. 2002. Ecological Stoichiometry: The Biology of Elements from Molecules to the Biosphere.
Princeton, NJ: Princeton Univ. Press
Stewart KJ, Coxson D, Siciliano SD. 2011. Small-scale spatial patterns in N2 -fixation and nutrient availability
in an arctic hummock-hollow ecosystem. Soil Biol. Biochem. 43:133–40
Tan XY, Hurek T, Reinhold-Hurek B. 2003. Effect of N fertilization, plant genotype and environmental
conditions on nifH gene pools in roots of rice. Environ. Microbiol. 5:1009–15
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
510
Reed
·
Cleveland
·
Townsend
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
ES42CH22-Reed
ARI
27 September 2011
14:24
Teng QH, Sun B, Fu XR, Li SP, Cui ZL, Cao H. 2009. Analysis of nifH gene diversity in red soil amended
with manure in Jiangxi, south China. J. Microbiol. 47:165–41
ter Steege H, Pitman NCA, Phillips OL, Chave J, Sabatier D, et al. 2006. Continental-scale patterns of canopy
tree composition and function across Amazonia. Nature 443:444–47
Thompson MV, Vitousek PM. 1997. Asymbiotic nitrogen fixation and litter decomposition on a long soil-age
gradient in Hawaiian montane rain forest. Biotropica 29:134–44
Thornton PE, Doney SC, Lindsay K, Moore JK, Mahowald NM, et al. 2009. Carbon-nitrogen interactions
regulate climate-carbon cycle feedbacks: results from an atmosphere-ocean general circulation model.
Biogeosciences 6:2099–120
Tilman D, Reich PB, Knops J, Wedin D, Mielke T, Lehman C. 2001. Diversity and productivity in a long-term
grassland experiment. Science 294:843–45
Townsend AR, Cleveland CC, Houlton BZ, Alden CB, White JWC. 2011. Multi-element regulation of the
tropical forest carbon cycle. Front. Ecol. Environ. 9:9–17
Townsend AR, Howarth RW. 2010. Fixing the global nitrogen problem. Sci. Am. 2:64–71
van Groenigen K-J, Six J, Hungate BA, de Graaff M-A, van Breemen N, van Kessel C. 2006. Element
interactions limit soil carbon storage. Proc. Natl. Acad. Sci. USA 103:6571–74
Vitousek PM. 1984. Litterfall, nutrient cycling, and nutrient limitation in tropical forests. Ecology 65:285–
98
Vitousek PM. 1994. Potential nitrogen fixation during primary succession in Hawai’i Volcanoes National
Park. Biotropica 26:234–40
Vitousek PM, Aber JD, Howarth RW, Likens GE, Matson PA, et al. 1997. Human alteration of the global
nitrogen cycle: sources and consequences. Ecol. App. 7:737–50
Vitousek PM, Cassman K, Cleveland C, Crews T, Field CB, et al. 2002. Towards an ecological understanding
of biological nitrogen fixation. Biogeochemistry 58:1–45
Vitousek PM, Field CB. 1999. Ecosystem constraints to symbiotic nitrogen fixers: a simple model and its
implications. Biogeochemistry 46:179–202
Vitousek PM, Hobbie S. 2000. Heterotrophic nitrogen fixation in decomposing litter: patterns and regulation.
Ecology 81:2366–76
Vitousek PM, Howarth RW. 1991. Nitrogen limitation on land and in the sea: How can it occur? Biogeochemistry
13:87–115
Vitousek PM, Naylor R, Crews T, David MB, Drinkwater LE, et al. 2009. Nutrient imbalances in agricultural
development. Science 324:1519–20
Vitousek PM, Porder S, Houlton BZ, Chadwick OA. 2010. Terrestrial phosphorus limitation: mechanisms,
implications and nitrogen-phosphorus interactions. Ecol. Appl. 20:5–15
Wakelin SA, Colloff MJ, Harvey PR, Marschner P, Gregg AL, Rogers SL. 2007. The effects of stubble
retention and nitrogen application on soil microbial community structure and functional gene abundance
under irrigated maize. FEMS Microbiol. Ecol. 59:661–70
Wakelin SA, Guptac VVSR, Forrester ST. 2010. Regional and local factors affecting diversity, abundance and
activity of free-living, N2 -fixing bacteria in Australian agricultural soils. Pedobiologia 53:391–99
Wallenstein MD, Vilgalys RJ. 2005. Quantitative analyses of nitrogen cycling genes in soils. Pedobiologia
49:665–72
Wang Y-P, Houlton BZ. 2009. Nitrogen constraints on terrestrial carbon uptake: implications for the global
carbon-climate feedback. Geophys. Res. Lett. 36:L24403
Wang Y-P, Houlton BZ, Field CB. 2007. A model of biogeochemical cycles of carbon, nitrogen and phosphorus
including symbiotic nitrogen fixation and phosphatase production. Glob. Biogeochem. Cycles 21:1–15
Widmer F, Shaffer BT, Porteous LA, Seidler RJ. 1999. Analysis of nifH gene pool complexity in soil and litter
at a Douglas Fir forest site in the Oregon Cascade Mountain range. Appl. Environ. Microb. 65:374–80
Woodmansee RG, Vallis I, Mott JJ. 1981. Grassland nitrogen. In Terrestrial Nitrogen Cycles, ed. FR Clark, T
Rosswall. Ecol. Bull. (Stockh.) 33:443–62
Yeager CM, Kornosky JL, Housman DC, Grote EE, Belnap J, Kuske CR. 2004. Diazotrophic community
structure and function in two successional stages of biological soil crusts from the Colorado Plateau and
Chihuahuan Desert. Appl. Environ. Microb. 70:973–83
www.annualreviews.org • Free-Living Nitrogen Fixation
511
ES42CH22-Reed
ARI
27 September 2011
14:24
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
Young JPW. 1992. Phylogenetic classification of N-fixing organisms. In Biological Nitrogen Fixation, ed. G
Stacy, RH Burris, HK Evans, pp. 43–86. New York: Chapman Hall
Zackrisson O, DeLuca TH, Nilsson M-C, Sellstedt A, Berglund LM. 2004. Nitrogen fixation increases with
successional age in boreal forests. Ecology 85:3327–34
Zehr JP, Jenkins BD, Short SM, Steward GF. 2003. Nitrogenase gene diversity and microbial community
structure: a cross-system comparison. Environ. Micro. 5:539–55
Zheng J-Q, Han S-J, Ren F-R, Zhou Y-M, Zhang Y. 2008. Effects of long-term elevated CO2 on N2 fixing, denitrifying and nitrifying enzyme activities in forest soils under Pinus sylvestriformis in Changbai
Mountain. J. Forest. Res. 19:283–87
512
Reed
·
Cleveland
·
Townsend
ES42-FrontMatter
ARI
11 October 2011
16:5
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
Contents
Annual Review of
Ecology, Evolution,
and Systematics
Volume 42, 2011
Native Pollinators in Anthropogenic Habitats
Rachael Winfree, Ignasi Bartomeus, and Daniel P. Cariveau p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Microbially Mediated Plant Functional Traits
Maren L. Friesen, Stephanie S. Porter, Scott C. Stark, Eric J. von Wettberg,
Joel L. Sachs, and Esperanza Martinez-Romero p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p23
Evolution in the Genus Homo
Bernard Wood and Jennifer Baker p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p47
Ehrlich and Raven Revisited: Mechanisms Underlying Codiversification
of Plants and Enemies
Niklas Janz p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p71
An Evolutionary Perspective on Self-Organized Division of Labor
in Social Insects
Ana Duarte, Franz J. Weissing, Ido Pen, and Laurent Keller p p p p p p p p p p p p p p p p p p p p p p p p p p p p91
Evolution of Anopheles gambiae in Relation to Humans and Malaria
Bradley J. White, Frank H. Collins, and Nora J. Besansky p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 111
Mechanisms of Plant Invasions of North America and European Grasslands
T.R. Seastedt and Petr Pyšek p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 133
Physiological Correlates of Geographic Range in Animals
Francisco Bozinovic, Piero Calosi, and John I. Spicer p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 155
Ecological Lessons from Free-Air CO2 Enrichment (FACE) Experiments
Richard J. Norby and Donald R. Zak p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 181
Biogeography of the Indo-Australian Archipelago
David J. Lohman, Mark de Bruyn, Timothy Page, Kristina von Rintelen,
Robert Hall, Peter K.L. Ng, Hsi-Te Shih, Gary R. Carvalho,
and Thomas von Rintelen p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 205
Phylogenetic Insights on Evolutionary Novelties in Lizards
and Snakes: Sex, Birth, Bodies, Niches, and Venom
Jack W. Sites Jr, Tod W. Reeder, and John J. Wiens p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 227
v
ES42-FrontMatter
ARI
11 October 2011
16:5
The Patterns and Causes of Variation in Plant Nucleotide Substitution Rates
Brandon Gaut, Liang Yang, Shohei Takuno, and Luis E. Eguiarte p p p p p p p p p p p p p p p p p p p p p 245
Long-Term Ecological Records and Their Relevance to Climate Change
Predictions for a Warmer World
K.J. Willis and G.M. MacDonald p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 267
The Behavioral Ecology of Nutrient Foraging by Plants
James F. Cahill Jr and Gordon G. McNickle p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 289
Climate Relicts: Past, Present, Future
Arndt Hampe and Alistair S. Jump p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 313
Annu. Rev. Ecol. Evol. Syst. 2011.42:489-512. Downloaded from www.annualreviews.org
by Dr. Diego Rodriguez on 02/24/12. For personal use only.
Rapid Evolutionary Change and the Coexistence of Species
Richard A. Lankau p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 335
Developmental Patterns in Mesozoic Evolution of Mammal Ears
Zhe-Xi Luo p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 355
Integrated Land-Sea Conservation Planning: The Missing Links
Jorge G. Álvarez-Romero, Robert L. Pressey, Natalie C. Ban, Ken Vance-Borland,
Chuck Willer, Carissa Joy Klein, and Steven D. Gaines p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 381
On the Use of Stable Isotopes in Trophic Ecology
William J. Boecklen, Christopher T. Yarnes, Bethany A. Cook, and Avis C. James p p p p 411
Phylogenetic Methods in Biogeography
Fredrik Ronquist and Isabel Sanmartı́n p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 441
Toward an Era of Restoration in Ecology: Successes, Failures,
and Opportunities Ahead
Katharine N. Suding p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 465
Functional Ecology of Free-Living Nitrogen Fixation:
A Contemporary Perspective
Sasha C. Reed, Cory C. Cleveland, and Alan R. Townsend p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 489
Indexes
Cumulative Index of Contributing Authors, Volumes 38–42 p p p p p p p p p p p p p p p p p p p p p p p p p p p 513
Cumulative Index of Chapter Titles, Volumes 38–42 p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 517
Errata
An online log of corrections to Annual Review of Ecology, Evolution, and Systematics
articles may be found at http://ecolsys.annualreviews.org/errata.shtml
vi
Contents