WEWeb EcologyWEWeb Ecol.1399-1183Copernicus PublicationsGöttingen, Germany10.5194/we-18-105-2018Do mycorrhizal fungi create below-ground links between native plants and Acacia longifolia? A case study in a coastal maritime pine forest in PortugalDo mycorrhizal fungi create below-ground links between native plants and Acacia longifolia?CarvalhoPedropedro.carvalho@itqb.unl.ptMartinsRuiPortugalAntóniohttps://orcid.org/0000-0003-1748-6345GonçalvesM. TeresaCFE – Centre for Functional Ecology, Department of Life Sciences,
University of Coimbra, Calçada Martim de Freitas, 3000-456 Coimbra,
PortugalPedro Carvalho (pedro.carvalho@itqb.unl.pt)19June20181811051148January20184June20186June2018This work is licensed under the Creative Commons Attribution 4.0 International License. To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/This article is available from https://we.copernicus.org/articles/18/105/2018/we-18-105-2018.htmlThe full text article is available as a PDF file from https://we.copernicus.org/articles/18/105/2018/we-18-105-2018.pdf
Maritime pine forests are a major ecosystem throughout the
Portuguese coast and are severely affected by the invasion of Acacia longifolia.
The presented study investigated the diversity of ectomycorrhizal
fungi (ECM) of major plant species in these ecosystems to find possible links
between Pinus pinaster, three native Cistaceae shrubs and the
Acacia invasive species. We successfully identified 13 ECM fungal
taxa. Within those, two species from the order Helotiales were found in all
plant species, and over half of the fungal ECM species found in Pinus pinaster were also common to the Cistaceae shrubs. Network analysis points to
the Cistaceae shrubs having a central role in these below-ground communities,
therefore enforcing the idea that they are key to these communities
and should not be underestimated. Our results also point to the evolving role
of invasive plant species in the ecosystem dynamics in the rhizosphere, which
host fungal species that are common to native plants, although it is not
yet clear whether these fungal taxa are native or a consequence of the presence of
Acacia longifolia.
Introduction
Plants can obtain many benefits from establishing mycorrhizal associations,
for instance, better access to nutrient and water supplies and higher
resilience to biotic and abiotic stresses (Smith and Read, 2006). Although there are
some exceptions, the majority of ectomycorrhizal
(ECM) fungal species are not truly host-specific and can colonize the roots
of several individuals from different plant species, from trees to shrubs
and even herbaceous plants (Bruns et al., 2002; Buscardo et al., 2012; Dickie et al., 2004; Ishida et al.,
2007; Kennedy et al., 2003). The fundamental role of ECM fungi in ecosystem
dynamics, nutrient cycling and plant performance is well established (Itoo and Reshi, 2014; Smith and
Read, 2006) and approximately 25 000 species of fungi have been described as
being able to establish ectomycorrhizal associations (Rincón
et al., 2015; Tedersoo et al., 2010; Tedersoo and Smith, 2013),
Pinus pinaster, the “maritime pine”, is distributed along the western Mediterranean basin (Campelo et al., 2015), inhabiting mainly acid
and silicon coastal soils (Berthier, 2001). It is one
of the most important forest species in Portugal (Campelo et al., 2015;
Monteiro-Henriques et al., 2016), covering 27.3 % of the forested area and
being the most extensively distributed forest species (Godinho-Ferreira et al., 2005). All Pinaceae species
are reported as establishing mainly ectomycorrhizas, although some studies
describe the occurrence of arbuscular mycorrhizas (AM) in Pinus spp. and in other
Pinaceae (e.g. Horton et al., 1998). In
recent years the ectomycorrhizal fungal communities of Pinus spp. have been
studied through molecular approaches using individual ectomycorrhizal root
tips, soil and whole roots (Buscardo et al., 2010, 2011, 2012; Cox et al., 2010; Jarvis et al., 2013; Pestaña
Nieto and Santolamazza Carbone, 2009; Rincón et al., 2015; Walbert et
al., 2010). Maritime pine forests are a major ecosystem throughout the
Portuguese coast and are severely affected by the invasion of Acacia longifolia.
The south-western Australian species Acacia longifolia is one of the most prolific invasive
species in Portugal (Marchante et al., 2003). Acacia species are
known to form AM and ECM associations, with AM being predominantly found in
both native and invaded areas (Aswathappa et
al., 1987; Rodríguez-Echeverría et al., 2009). To the extent of
our knowledge, there are no reports of ECM associations with this species in
Portugal.
Cistaceae shrubs are common understory species in the Mediterranean basin.
They can establish both ECM and AM (Smith and Read, 2006) and have been
reported to share ECM fungi with co-occurring Pinus pinaster
(Buscardo et al., 2012). From the 12 species of the Mediterranean genus
Cistus occurring in the Iberian Peninsula (Águeda et al., 2006;
Alonso Ponce et al., 2011), nine are native in Portugal and compose 7 %
of the forested area (Godinho-Ferreira et al., 2005). Being woody, evergreen
and pyrophytic shrubs (Arrington and Kubitzki, 2003; Comandini et al., 2006)
all species of Cistus are among the first colonizers after a
disturbance event (e.g. fire or grazing) and thus are pioneer species of
ecological successions (Águeda et al., 2006; Alonso Ponce et al., 2011;
Comandini et al., 2006). About 230 species of Ascomycota and Basidiomycota
fungi have been described as ECM symbionts of Cistus sp, with 35
being Cistus-specific mycobionts nearly all belonging to Russulaceae
and Cortinariaceae families (Comandini et al., 2006). Halimium halimifolium is another Mediterranean Cistaceae shrub that occurs in sandy
soil, becoming the dominant species in sand ridges where the water table
depth ranges from 2 to 4 m (Zunzunegui et al., 2002). Based on a fruiting
body survey, 12 ECM fungal species have been
identified associated with H. halimifolium in Corsica (Taudiere et
al., 2015), of which four species were also associated with Pinus pinaster.
We hypothesized that there is a high degree of similarity between the ECM fungal
communities associated with the Cistaceae species and the ones associated
with the maritime pine. Furthermore, we looked for putative ECM symbionts in
the root system of nearby Acacia longifolia individuals with the aim of evaluating whether it shares
putative symbionts with Cistaceae shrubs and with the dominant tree, Pinus pinaster.
In this study we investigated the ECM fungal communities of the most frequent
plant hosts in a coastal maritime pine forest, namely Pinus pinaster, three Cistaceae shrubs (Cistus salviifolius, Cistus psilosepalus and Halimium halimifolium), and also the invasive
species (Acacia longifolia). We achieve this by sorting
ectomycorrhizal root tips into “ad hoc morphological groups” and carrying out further
molecular identification by DNA barcoding using ITS sequencing (Schoch
et al., 2012; Seifert, 2009). We observed a high degree of similarity between
the ECM communities found in Pinus pinaster and in the Cistaceae
shrubs. Although Acacia longifolia had a less diverse ECM fungal
community, the few detected common ECM fungal species imply that
it is already part of the below-ground dynamics of the fungal communities.
Materials and methodsStudy site
The study area was an even-aged managed forest of Pinus pinaster located in the central coast of Portugal (40.35834∘ N,
8.81903∘ W). The area has a typical Mediterranean climate with oceanic
influence and a marked summer drought. The average annual temperature and
precipitation are 16.2 ∘C and 953 mm and the soil is
acidic with a sandy texture and low water-holding capacity from the
order Inceptisol (Campelo et al., 2015). The area is dominated by
Pinus pinaster trees with an average age of 45 years and a high
occurrence of the exotic invasive Acacia longifolia. Cistus psilosepalus, Cistus salviifolius, Halimium halimifolium and Corema album are the most common understory shrubs.
Results from the BLAST analysis based on ITS sequences obtained
from field-collected ECM root tips. The most similar sequences from NCBI are
represented by their accession numbers. Species identification was done at
sequence similarity higher than 97.
The obtained sequences were edited using Geneious® software,
sequences quality (HQ) was assessed and low-quality sequences were discarded.
Basic Local Alignment Search Tool (BLAST) was performed with the National Center
for Biotechnology Information (NCBI) database to confirm species taxonomic
identification (Altschul et al., 1997). Species identification was achieved
using 97 % sequence similarity. Identical sequences, i.e. with
sequence similarity higher than 99 %, were grouped and only one
representative of each group was considered in subsequent analyses. Sequences
obtained in this study were submitted to the European Nucleotide Archive
(ENA) (Table 2). Sequences were aligned using the MUSCLE algorithm available
in MEGA7® software. Phylogenetic analysis of the obtained
sequences, together with sequences retrieved from online databases (GenBank),
was performed using maximum likelihood methods with the GTR model of
evolution, which was selected as the best-fitting model using the Akaike
information criterion (AIC) in jModelTest (Guindon and Gascuel, 2003; Posada,
2008). The phylogenetic analysis was performed using PhyML (Guindon and
Gascuel, 2003) in Phylemon 2.0 (Sánchez et al., 2011). Branch support was
assessed using the bootstrap likelihood ratio test with 1000 repetitions. The
resulting tree was represented using the R software (R Development Core Team,
2011) package ggtree from BioConductor (Yu et al., 2017) (Fig. 1).
Data analysis
Maximum likelihood tree based on ITS
sequences obtained from field-collected ECM root tips and reference sequences
obtained from Genbank (included with their accession numbers). Sequences
obtained in this study are named in Table 1. Sequences with similarity
> 99 % are grouped and listed together. Numbers at the
nodes are values for branch support estimated using the bootstrap likelihood
ratio test (1000 resamplings, values 0–1, values above 0.98 are not
displayed for easier viewing). The scale bar indicates the evolutionary
distances.
To characterize and compare the fungal communities associated with each plant
species we calculated the Shannon index (H′) of diversity, using abundance as
the number of ECM tips in each identified morphotype group, according to
H′=-∑pi.log2(pi), where pi is the
proportion of each fungal species in the community (Shannon and Weaver,
1949). Based on the results, the Pielou index (E) of evenness was
calculated according to P=H′/log2(S), where S is defined
as the total number of species in the community (Species richness). For this,
values closer to the unit indicate a community where the ECM tips are equally
distributed among the species identified. The Margalef index (Dm) for species
richness was calculated as follows: D=(S-1)/ln(N), where N is
defined as the total number of ECM tips identified (Gamito, 2010) (Table 3).
All indexes were obtained using R software (R Development Core Team, 2011)
with the package vegan (Dixon, 2003).
To analyse community composition and similarity, we resorted to the
Bray–Curtis index (BC) for all pairwise combinations of plant species (Chao et al., 2005) (Table 4). This index
estimates the similarity between communities. With these results we obtained
a cladogram (Fig. 2), which allows the results to be clearly viewed using the R
package vegan. Community composition comparisons can be seen in the
ordination analysis, performed using a non-metric multidimensional scaling
(NMDS). The two dimensions of separation of the communities are represented
in graphic form (Fig. 3) with stress values of 0.033, using the R software
package vegan.
Ecological indexes for each plant species considering the
identified ECM symbionts. S is the total number of species, N is the total number of ECM
tips, H′ is the Shannon–Wienner index, E is the Pielou index and D is the Margalef index.
To further understand the relationship between the communities associated
with each species, we computed the Euclidean distance between each fungal
plant community, obtaining a metric distance matrix that was used to create
a weighted and undirected network, using the R package qgraph (Epskamp et al., 2012) (Fig. 4). This algorithm
allows the Euclidean distances to be visualized by resizing and shading the
edges (Euclidean distances) between each node (plant-associated community)
according to the values of each edge that translate the number of
commonly identified species as well as the relationships between identified
species in each community.
In order to further understand the network, we obtained values for
centrality, specifically the value for betweenness centrality (node
strength). This gives us a measure for centrality, not only a topological one but
also one that influences the network of each node. The
shortest path of the edges through the node are taken into account, which is found by the
minimal sum of the edges that link any pairs of nodes in the network, as
well as the weight of the edges, given by the Euclidean distances.
If a high number of shortest paths that pass through a node it
is considered a central node in the network. We also obtained the value for
closeness centrality, which is calculated based on the sum of the lengths of
the shortest paths between the node and all the other nodes in the graph, so
that a central node is closer to all the other nodes. This is computed by the R
package qgraph (Table 5). Moreover, two clustering coefficients were
determined for each community: the Zhang and Horvath's weighted clustering
coefficient (Zhang and Horvath, 2005)
and Onnela's clustering coefficient (Onnela et al.,
2005).
Topological indexes based on the network obtained with the Euclidian
distances between each plant fungal community. Betweenness centrality,
represented here by the node strength, closeness centrality and clustering
coefficients (Zhang and Horvath; Onnela) indicate the importance and
influence of each community in the network.
From 433 observed root tips, the morphological sorting resulted in 27
morphotypes for all plant species. After PCR sequencing and BLAST a total of
13 fungal taxa were identified (Table 2): two Ascomycota in association with
Acacia longifolia, four Basidiomycota and three Ascomycota in association with Pinus pinaster, three
Basidiomycota and one Ascomycota in association with Cistus psilosepalus, six Basidiomycota and
three Ascomycota in association with Cistus salviifolius, and three Basidiomycota and one
Ascomycota in association with Halimium halimifolium (Table 2).
Cluster dendrogram using average linkage between groups, given by
the Bray–Curtis dissimilarity index matrix. The y axis shows rescaled distance
cluster combinations.
Identification up to species level was not possible for 19 morphotypes that
corresponded to four taxa (Table 2). Morphotypes identified as “Uncultured
ECM 1” and “Uncultured ECM 2” belonged to the Helotiales order (Fig. 2).
Two unidentified Russula species were named Russula sp. 1 and Russula sp. 2 and were closely
related to Russula sardonia and Russula cascadensis, respectively (Fig. 1).
The most frequent genus was Russula, with three different identified taxa, and the
most frequent taxon was the Uncultured ECM 1, found in all plant-associated communities.
Species richness and diversity, calculated according to the Margalef index
and Shannon index, were the highest for Cistus salviifolius and
Pinus pinaster while the lowest values were found in Acacia longifolia, Halimium halimifolium and Cistus psilosepalus
communities show to be the most even.
The highest values of similarity were found between Halimium halimifolium and Cistus psilosepalus, which was an expected result since they
host the same fungal species. The lowest levels were found between
Pinus pinaster and Halimium halimifolium. When plotted,
similar clusters were shown between Halimium halimifolium and
Cistus psilosepalus, and between Cistus salviifolius and Pinus pinaster, with Acacia longifolia hosting the most dissimilar community (Table 3; Fig. 2). NMDS analysis
showed that the plant-related communities were separated in two dimensions. The
observed separations support the Bray–Curtis cladogram, showing the cluster
of Halimium halimifolium with Cistus psilosepalus and
Pinus pinaster with Cistus salviifolius as well as the
separation of Acacia longifolia (Figs. 2 and 3).
Pinus pinaster shared four species with Cistus salviifolius
(Uncultured ECM1, Uncultured ECM2, Archaeozhizomyces borealis and
Russula sp. 1), two with Cistus psilosepalus (Uncultured ECM1
and Russula sp. 1) and two with Halimium halimifolium
(Uncultured ECM 1 and Russula sp. 1). Over half of the fungal species
found in Pinus pinaster were also present in the Cistaceae shrubs.
The fungal species found in Acacia longifolia, Uncultured ECM 1 and
Uncultured ECM 2, were also found in Cistaceae shrubs and in Pinus pinaster.
Non-metric multidimensional scaling (NMDS) of the plant-associated
fungal communities, showing the clustering of similar communities in the two
dimensions represented here. Plant communities are represented by the name
of the plant species. Each red circle represents one identified fungal taxa
except for Pinus pinaster, where the red circle represents three different fungal taxa, and
Cistus salviifolius, where the red circle represents four fungal taxa.
The network analysis suggests that Cistus psilosepalus is the most central and important node
in the network, as shown by the topological indexes. On the other hand,
Acacia longifolia is the least central and weakest node, but it showed the highest value in
one of the clustering indexes.
Discussion
We identified 13 different fungal taxa associated with the most common ECM
host plants in the studied habitat. ECM fungal species associated with
Halimium halimifolium are described for the first time and the
results are in line with those obtained with above-ground fruiting
body surveys (Taudiere et al., 2015).
By taking into account the small scale of this study and comparing our
results with those obtained in a similar habitat (Pestaña Nieto and
Santolamazza Carbone, 2009), we may conclude that this ecosystem has an
appreciable diversity of ECM fungi and that most of the identified fungal
species were common to two or more plant partners. These results are in
agreement with previous studies showing the existence of common ECM fungi
partners of Pinuspinaster and Cistaceae shrubs,
suggesting the existence of a common mycorrhizal network (CMN) in this
ecosystem in which putative partners have been identified.
Our study is also among the first to report ECM fungi associated with
Acacia longifolia outside its native range. The above-ground negative impact of this invasive
species in Portuguese forests is well documented (e.g. Marchante et al., 2015).
Some authors have already investigated the below-ground symbiotic relations
of A. longifolia (Rodríguez-Echeverría,
2010; Rodríguez-Echeverría et al., 2009). They were able to
correlate its invasive success to the introduction of exotic rhizobia and to
the ability to profusely nodulate with native or invasive bacteria, giving a
glimpse into the effects and the ecology of invasive species in the new
range.
In the present study, in contrast with the data available
in terms of bacteria and AM fungi, we investigated another symbiotic association of A. longifolia, and
our results pointed out another facet of the invasiveness of this species.
We hypothesized that Acacia longifolia could already be a member of the community as the host
of a fungal species that could colonize neighbouring native plant species. Our
results support this hypothesis by showing that two fungal taxa were found
both in Acacia longifolia and in the native species. This observation places Acacia longifolia within the
network formed by the fungal species, although it is an outlink in the
broader studied plant community as shown in the cladogram (Fig. 2). The
ordination test also showed a clear separation from the other communities
in both dimensions (Fig. 3).
Network diagram created using the R package qgraph. Circles
indicate plant-related communities (nodes) and grey lines (edges) link
plant-related communities based on the Euclidean distances between them.
Weight of each line represents the weight of each edge in terms of the
Euclidean distance results between each node. Css is Cistus salviifolius, Hlh is Halimium halimifolium, Pnp is Pinus pinaster, Csp is Cistus psilosepalus and Acl is Acacia longiolia.
The lack of information regarding the identity of some fungal taxa,
particularly the Uncultured ECM 1 and Uncultured ECM 2, undermined further
conclusions on the putative role of the invasion in these communities
(Vrålstad et al., 2002), Moreover, the lack of information about the
fungal associations of A. longifolia in its native range and in
other older places of introduction, leave open the hypothesis that
Acacia longifolia may be acting as a vector of dispersion and
possibility of an invasion of fungal taxa to and from the invaded locations,
as has been observed for bacterial symbionts (Rodríguez-Echeverría,
2010). The hosts of native fungal species may be expanded by establishing
themselves as new hosts, deeply influencing the dynamics of these species
(Pringle et al., 2009).
More studies should be conducted to further characterize the fungal
communities of Acacia longifolia, both in their native location and in the places where
they have been introduced, with varying levels of invasiveness.
In this study we described a novel host of the recently identified fungus
Archaeorhizomyces borealis (Menkis et al., 2014), Cistus salviifolius, which is also its first
non-gymnosperm host. It is possible that the structural similarities of the
cortical Hartig net described for both the genus Cistus and
gymnosperms hosts (Smith and Read, 2006) may explain this novel combination.
The high number of ECM fungal species shared between the studied plant
species suggests that the role of the Cistaceae shrubs in these ecosystems is important.
Moreover, the high similarity between the ECM fungal species found in the
three surveyed species may indicate a certain specificity at the family
level, since they harbour fungal species that also colonize pines. These
shrubs can be of crucial importance as a reservoir for ECM fungal species
after disturbances that affect mainly tree species, such as logging or
recurrent wildfires (Buscardo et al., 2012).
Contrary to what we expected, the dominant tree species, Pinus pinaster, is neither the
most central nor the strongest node of the network, which is mainly driven
by the high number of species shared between the Cistaceae shrubs and their
weight on the network. Cistus psilosepalus is the most central node, mainly because all the
associated species are the same for Halimium halimifolium, and there is a high number of species
shared with all the plants studied. Overall, these results point to the
central role of these understory species in the network that formed below-ground
and highlight the need for awareness of their importance to the
ecosystems.
Concerning Acacia longifolia, it shared symbionts with all the studied shrub species. It is
possible that the shrubs play an important role by allowing the putative new
fungal symbiont species to enter the network and establish themselves in the
ecosystem.
These shrubs may be working as “donors” of symbionts or rather
creating bridges between the already established community and the
native communities by being general hosts or using other uncharacterized
mechanisms.
Conclusions
This small-scale study revealed an appreciable diversity of ECM fungal taxa
and associations in an understudied ecosystem. We found that closely related
plant species showed high similarity in terms of associated fungal
communities and that an invasive species has common fungal symbionts with
native plants, possibly indicating that it is already part of the below-ground
mutualistic network. Furthermore, a complex ECM fungal network was
identified between five plant species, even in the small-scale studied area.
This opens further research questions about the ecology of ECM in this
coastal ecosystem. More thorough and extensive studies are needed to unravel
the identity and diversity of ECM fungal species, their specificity and the
ecological role of the putative CMN that connects the native species with the
invasive species. A better description of how similar the communities of
invasive species are in their invasive range and a comparison with their
native systems will certainly provide valuable information that is needed to understand
the below-ground dynamics.
A data matrix of the identified fungal taxa for each plant
species can be found in Supplement 2.
The supplement related to this article is available online at: https://doi.org/10.5194/we-18-105-2018-supplement.
The authors declare that they have no conflict of
interest.
Acknowledgements
We would like to thank Susana Rodriguez-Echeverria for the valuable
input in the experimental design and on data interpretation crucial for this
paper. We would like to thank Susana Gonçalves for the valuable
input through the work and expertise in the field. We would like to thank Mika Tarkka and anonymous reviewers for their valuable comments and
suggestions on an earlier version of the manuscript. This work was financed
by FCT/MEC through national funds and the co-funding by the FEDER, within
the PT2020 Partnership Agreement, and COMPETE 2020, within the Project
UID/BIA/04004/2013.
Edited by: Jutta Stadler
Reviewed by: Mika Tarkka and one anonymous referee
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