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Key words: community assembly, convergence, divergence, ecosystem
impact, functional traits, habitat filtering, invasion.
Letters
Effect of segregation and
genetic exchange on
arbuscular mycorrhizal fungi
in colonization of roots
Introduction
Arbuscular mycorrhizal fungi (AMF) are abundant soil
organisms and form symbioses with roots of the majority of
terrestrial plants (Smith & Read, 2008). The symbiosis with
AMF can promote plant productivity and diversity, and
tolerance to pathogens and to herbivores (Newsham et al.,
1995; van der Heijden et al., 1998; Bennett et al., 2006;
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Bennett & Bever, 2007). The hyphae produced by spores
are coenocytic, harbouring many nuclei in a common cytoplasm. Moreover, genetic differences among co-occurring
nuclei have been observed and this explains the high intraindividual genetic diversity found in AMF (Pringle et al.,
2000; Clapp et al., 2001; Kuhn et al., 2001; Rodriguez
et al., 2004; Hijri & Sanders, 2005). Two processes related
to the within-individual genetic variation in AMF have
been recently demonstrated in the species Glomus
intraradices and can affect the nucleotype content of an
AMF in a very short time span (Croll et al., 2009; Angelard
et al., 2010). First, genetic exchange between two genetically different AMF lines can lead to new spores having a
mixture of parental nucleotypes (Croll et al., 2009).
Second, one mother spore can produce new spores with
different nucleotype contents as a result of the segregation
of nucleotypes at spore formation (Angelard et al., 2010).
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It has recently been shown that progeny obtained
through genetic exchange and segregation (called crossed
AMF and segregated AMF, respectively) can differentially
alter plant growth and plant gene transcription compared
with their parents or other progeny (Angelard et al., 2010).
The plant genes studied were previously shown to be specifically expressed in the symbiosis at both early and late stages
of fungal development (Gutjahr et al., 2008). Additionally,
the alterations in plant growth caused by different AMF
lines were not fixed in that they differed among different
plant species (Angelard et al., 2010). So far, the effects
of genetic exchange on fungi have been conducted using
an in vitro system, and crossed lines can have different
phenotypes compared with their parents or other progeny
(Croll et al., 2009). Croll et al. (2009) only measured extraradical hyphae and spores produced outside the roots in
in vitro cultured fungal lines. However, nothing is currently
known about the effect of segregation on the fungal phenotypes, or about how genetic exchange and segregation affect
the colonization and development of the fungus inside plant
roots. G. intraradices form three structures inside plant
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roots, namely hyphae, arbuscules (where the plant and the
fungus exchange nutrients) and vesicles (propagules and
storage organs) (Smith & Read, 2008).
In the present study, we determined the effect of genetic
exchange and segregation in G. intraradices on the development and growth of the fungus in plant roots. Our analysis
was based on the glasshouse experiments described by
Angelard et al. (2010), where the author investigated the
effects of these two mechanisms on plant growth and plant
gene transcription. Angelard et al. (2010) inoculated two
plant species, Plantago lanceolata and Oryza sativa, with
AMF lines of G. intraradices in two independent experiments. In the genetic-exchange experiment, plants were
inoculated with parental and crossed lines. The segregated
lines were obtained by cultivating separately single spores
from crossed lines, and both the crossed and segregated lines
were used in the segregation experiment of Angelard et al.
(2010). Ten replicates were made in individual pots for each
treatment (a combination of one AMF line with one plant
species) in individual pots. Here, we determined the fungal
growth of all of these AMF lines by measuring the proportion
Fig. 1 Fungal colonization in the genetic exchange experiment. Mean hyphal, arbuscular and vesicular colonization of Oryza sativa and
Plantago lanceolata by parental lines (closed columns) and crossed lines (open columns). Arrows indicate the phenotypic (white arrows) and
genetic (black arrows) similarity between crossed and parental lines (only shown when fungal colonization was different among arbuscular
mycorrhizal fungi (AMF) lines). For example, vesicular colonization in P. lanceolata by crossed lines S1, S3 and S5 was most similar to
colonization by parental line C2. Conversely, those crossed lines were genetically more similar to parental line C3 than to parental line C2 (as
determined by amplified fragment length polymorphism analysis (AFLP); Angelard et al., 2010). Error bars represent the SD, and different
letters above bars indicate a significant difference (P < 0.05) according to the Tukey–Kramer Honestly Significant Difference (HSD) test.
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of hyphae, arbuscules and vesicles formed inside plant roots
using the method of McGonigle et al. (1990). The methods
are fully described in the Supporting Information Notes S1.
Based on previous results, we hypothesized that crossed and
segregated AMF lines would have different fungal traits
inside plant roots compared with their parents, even at an
early stage of colonization. Moreover, we hypothesized that
the phenotypic changes among AMF lines would differ
depending on the plant species.
Genetic exchange and segregation alter fungal
growth traits
Our results show that both genetic exchange and segregation can result in fungi that colonize plants differently
compared with their parents or other offspring (Figs 1, 2).
For example, the hyphal colonization of the crossed line
Sc2 on P. lanceolata was significantly different compared
with the hyphal colonization of both parents (Fig. 1).
Also, the crossed line, Sb, exhibited vesicular colonization
on P. lanceolata that was significantly different from that in
either of the parents (Fig. 1). Several segregated lines also
had different phenotypes compared with other segregated
lines and compared with their respective crossed lines (see,
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for example, the colonization of the segregated line S4c
inside roots of P. lanceolata, Fig. 2). Therefore, the main
conclusion of our study was that the two processes by which
AMF can genetically change, namely genetic exchange and
segregation, affect how the fungus develops inside the host.
Host effects on the growth of crossed and
segregated lines
In addition to the effect of the AMF line reported here, we
found a host species effect on the growth of AMF (Tables 1
and 2). Moreover, this host effect was not the same for the
different growth traits measured. Indeed, for both genetic
exchange and segregation experiments, significant results
show that AMF made overall more arbuscules and hyphae
inside roots of O. sativa than inside roots of P. lanceolata,
while we found the opposite for vesicles. Additionally, the
changes in the different fungal growth traits among AMF
lines were affected by the plant species, but these responses
were not the same in each AMF line (shown by a significant
AMF line by plant species interaction; Tables 1, 2 and
Figs 1, 2). For example, in the segregation experiment,
arbuscular colonization of the segregated line S4b was higher
inside roots of O. sativa than inside roots of P. lanceolata,
Fig. 2 Fungal colonization in the segregation experiment. Mean hyphal, arbuscular and vesicular colonization of Oryza sativa and Plantago
lanceolata by crossed lines (closed columns) and segregated lines (open columns). Error bars represent the SD, and different letters above bars
indicate a significant difference (P < 0.05) according to the Tukey–Kramer Honestly Significant Difference (HSD) test.
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whereas the difference in arbuscular colonization between
the two hosts was not as large for the crossed line S4 (Fig. 2).
Intriguingly, in the genetic-exchange experiment, these
AMF line by plant species interactions can result in crossed
lines growing similarly to one parent in one host, but growing
similarly to the other parent in the other host. This is
represented in Fig. 1 by the different direction of the white
arrows showing the phenotypic similarity between parental
and crossed lines. For example, most crossed lines between
C3 and D1 made similar amounts of arbuscular colonization as the parental line D1 in O. sativa, but arbuscular
colonization in P. lanceolata was similar to that in the other
parent C3 (Fig. 1). This emphasizes that the crossed lines
indeed grew in symbiosis in a different way to the parents.
Overall, these results stress the importance of the genetics of
the fungus on the potential interactions that can occur in
symbiosis with different host species.
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Relationship between fungal colonization and
genotype
From the studies of Croll et al. (2009) and Angelard et al.
(2010), we know that all the crossed AMF lines used here
were genetically more related to the parental line C3 (represented by the black arrows in Fig. 1). However, depending
on both the trait measured and the host species, the crossed
lines did not necessarily grow like the parental line C3
(shown by the different direction of the white and black
arrows). The relationship between the genetic relatedness
and the phenotypes of the segregated lines compared with
their parent are more complex and difficult to analyse.
Indeed, the variations in phenotypes among segregated lines
originating from the same parent were mostly larger and
occurred more frequently than variation among crossed
lines. A potential explanation could be that genetic exchange
Table 1 Results of the two-way crossed mixed-model ANOVA for the different traits measured (arbuscular, vesicular and hyphal colonization)
in the genetic exchange experiment
Trait
Pairing between parental lines C2 · C3
Hyphal colonization
Arbuscular colonization
Vesicular colonization
Pairing between parental lines C3 · D1
Hyphal colonization
Arbuscular colonization
Vesicular colonization
AMF line
Plant species
AMF · Plant
interaction
F4,85 = 2.92*
F4,85 = 0.67ns
F4,85 = 5.52***
F1,4 = 3.54ns
F1,4 = 15.46*
F1,4 = 12.14*
F4,85 = 1.21ns
F4,85 = 1.33ns
F4,85 = 5.11***
F4,82 = 15.12***
F4,82 = 8.70***
F4,82 = 16.19***
F1,4 = 0.45ns
F1,4 = 6.63ns
F1,4 = 4.71ns
F4,82 = 5.09***
F4,82 = 3.08*
F4,82 = 13.00***
AMF, arbuscular mycorrhizal fungi.
Significance levels: ***, P < 0.001; *, P < 0.05; ns, P > 0.05. Results of the Shapiro-Wilks tests for normal distribution of the data are shown
in Supporting Information Table S1.
Table 2 Results of the two-way crossed mixed-model ANOVA for the different traits measured (arbuscular, vesicular and hyphal colonization)
in the segregation experiment
Trait
Crossed line Sc2 and segregated lines
Hyphal colonization
Arbuscular colonization
Vesicular colonization
Crossed line S3 and segregated lines
Hyphal colonization
Arbuscular colonization
Vesicular colonization
Crossed line S4 and segregated lines
Hyphal colonization
Arbuscular colonization
Vesicular colonization
AMF line
Plant species
AMF · Plant
interaction
F6,120 = 6.02***
F6,120 = 5.86***
F6,120 = 2.62*
F1,6 = 0.14ns
F1,6 = 9.76*
F1,6 = 7.04*
F6,120 = 1.72ns
F6,120 = 0.81ns
F6,120 = 1.63ns
F6,121 = 7.38***
F6,121 = 10.03***
F6,121 = 2.89*
F1,6 = 6.83*
F1,6 = 13.55*
F1,6 = 11.08*
F6,121 = 2.81*
F6,121 = 5.82***
F6,121 = 1.07ns
F3,67 = 18.13***
F3,67 = 15.72***
F3,67 = 6.32***
F1,6 = 9.87ns
F1,6 = 16.51*
F1,6 = 1.21ns
F3,67 = 6.94***
F3,67 = 8.15***
F3,67 = 9.16***
AMF, arbuscular mycorrhizal fungi.
Significance levels: ***, P < 0.001; *, P < 0.05; ns, P > 0.05. Results of the Shapiro-Wilks tests for normal distribution of the data are shown
in Supporting Information Table S1.
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is a less random phenomenon than segregation because of
the constraints that can emerge from mixing genetically different nuclei. Consequently, the panel of new progeny
obtained with different symbiotic characteristics would be
larger through segregation than through genetic exchange.
Correlation between fungal colonization and
plant growth
Combined with the results of Angelard et al. (2010) on the
dry weight of the plants, we found significant, positive
correlations between rice dry weight and arbuscular colonization, for both the genetic exchange and the segregation
experiments (Pearson correlation coefficient r = 0.46,
P < 0.001 and r = 0.17, P = 0.027, respectively). However,
we found significant negative correlations between the dry
weight of P. lanceolata and arbuscular colonization, for both
the genetic exchange and the segregation experiments
(r = )0.22, P = 0.047 and r = )0.42, P < 0.001, respectively). The correlations were similar with the other fungal
colonization traits (data not shown). These results stress again
the importance of the interaction between host species and
AMF. However, the measures of the fungal colonization and
the plant dry weight were made only once, at the end of the
experiment. A time course of fungal colonization would be
more accurate in order to make conclusions about the potential effect of fungal colonization on plant growth (or vice versa)
as, indeed, such interactions could change through time.
Angelard et al. (2010) conducted two independent experiments (genetic exchange and segregation experiments).
Several controlled (the amount of soil and the amount of
watering) and uncontrolled (such as temperature and
humidity) parameters were not the same between the two
experiments. This can explain the quantitative differences in
colonization between the experiments found here. However,
the parameters were standardized within each experiment
with considerable replication, and statistical analyses have
been performed, allowing us to compare the treatments
within each experiment and to state accurately which treatments had a significant effect. Nevertheless, it is interesting
to note that similar patterns (concerning fungal growth and
the correlation between plant and fungal growth) have been
found for both experiments.
Conclusion
A previous study has shown that genetic exchange in AMF
can lead to progeny having different phenotypes (spore and
hyphal density) in in vitro culture systems compared with
their parents and compared with each other (Croll et al.,
2009). Here, we investigated the effect of both genetic
exchange and segregation in AMF on the development and
growth of the fungus in roots of non-transformed plants in
glasshouse conditions. Our results show that the two
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processes can alter the pattern of development of fungus in
the roots. Moreover, the fungal development was also
affected by different plant species. We previously knew that
genetic exchange and segregation can lead to progeny that
differentially alter plant growth (Angelard et al., 2010).
Combined with those results, our findings suggest that specific interactions could occur between different plant species
and different AMF genotypes, and that the specificities
could appear in the initial weeks of the establishment of the
symbiosis. Genetic exchange and segregation could be two
mechanisms, owing to the particular genetic structure of
AMF, that can create new progeny with different symbiotic
effects in a very short time span and that can adapt rapidly
to different environmental conditions, such as different
plant species.
Acknowledgements
This work was supported by grants from the Swiss National
Science Foundation (grant numbers 31000AO-105790 ⁄ 1
and 31003A-127371).
Caroline Angelard and Ian R. Sanders*
Department of Ecology and Evolution, University of
Lausanne, Biophore, Lausanne, Switzerland
(*Author for correspondence: tel +41(0)21 692 42 61;
email [email protected])
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Supporting Information
Additional supporting information may be found in the
online version of this article.
Notes S1 Supporting methods provide a full description of
the materials and methods used in the study.
Table S1 Results of the Shapiro–Wilk tests (W) performed
to test the null hypothesis that the data were normally distributed
Please note: Wiley-Blackwell are not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the New Phytologist Central
Office.
Key words: arbuscular mycorrhizal fungi, fungal colonization, genetic
exchange, Glomus intraradices, segregation.
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