Introduction
Mutualistic interactions with microsymbionts are ubiquitous in the roots
of almost all terrestrial plants. Patterns and processes of community
assembly of diverse mutualistic symbionts in host plants have begun to
receive great attention along with the recent advances in
high-throughput molecular barcoding techniques (Vályi et al., 2016;
Bitterbiere et al., 2020). It has been increasingly reported how local
symbiont communities assemble through abiotic and biotic filters from a
regional pool, especially for mycorrhizal fungi (de Souza & Freitas,
2017; Lekberg et al., 2011; López-García et al., 2017; Bitterbiere et
al., 2020). On the other hand, for decades, researchers have also
demonstrated significant effects of genetic variation within a species
on shaping ecological communities (Bailey et al., 2009; Crutsinger et
al., 2014; Kagiya et al., 2018; Whitham et al., 2006). In
plant-microsymbiont interactions, many previous studies have
demonstrated that mutualistic interactions with microbial symbionts
increase or decrease the performance of host plants, depending on
genetic variation in infected symbionts, including the growth, survival,
reproduction, and defense (Ballhorn et al. 2017; Barrett et al, 2012;
2015; Broughton et al., 2003; Clawson et al., 1998; 1999; Heath &
Stinchcombe, 2014; Mensah et al., 2015). Nevertheless, it remains
unclear how local interaction networks between a host and symbionts are
shaped from the pool of genetically diverse microorganisms in the
natural rhizosphere.
Genetic variation in a host plant population may also provide great
insights for ecological and evolutionary perspectives in microsymbiont
assembly in nature. This is because genetic variation within a plant
species is generally known for one of primary factors that shape the
assemblages of soil microbiomes in terms of species richness and
abundance (Evans et al., 2016; Genung et al., 2013; Lamit et al., 2015;
Lankau, 2011; Schweitzer et al., 2008). However, the influences of
genetic variation in host plants on the assembly of interacting
microsymbionts remain unclear. Because inefficient variants necessarily
prosper and break down the reciprocal mutualism, there are filtering
mechanisms for the reciprocal mutualism between hosts and symbionts
(Archetti et al., 2011; Heath & Stinchcombe, 2014). Several mechanisms
are involved in the stabilization of mutualistic partnerships in
environments with lower quality partners; for example, partner choice,
in which host plants can choose the optimal partner based on
preinfection signals (Kiers & Denison, 2008), and sanctions, in which
host plants monitor the mutualistic benefits from bacteria and punish
less nitrogen-fixing nodules to favor cooperation nodules (Denison,
2000; Kiers & Denison, 2008; Kiers et al., 2003; 2007; Materon &
Zibilske, 2001; West et al., 2002b). Given that plants are ubiquitously
exposed to genetically diverse microsymbionts in nature, including both
efficient and inefficient variants, the maintenance of plant-microbe
mutualistic symbiosis in the field should be achieved through these
filtering mechanisms. The filtering processes from the microbial
community pool into host roots are important for the understanding of
network structure, community dynamics, and dispersal of microbial
symbionts in natural ecosystems (Valyi et al., 2016). However, because
theoretical and experimental approaches have been primarily applied, to
our knowledge, no studies have successfully demonstrated the filtering
activities and/or their outcomes in genetic associations between plants
and microsymbiont mutualists in natural ecosystems.
To address host plants’ filtering specific symbiont strains into their
roots from surrounding microsymbiont communities in nature, we need to
distinguish between infecting and non-infecting microsymbiont
communities. The mutualism between the nitrogen-fixing bacteriaFrankia sp. (Frankiaceae) and the alder tree (Alnus ),
known as “Frankia–Alnus mutualism,” provides a good
opportunity to study the communities of both co-infecting symbionts and
non-infecting relatives in nature. Frankia form nodules on
actinorhizal host plants, such as Alnus , Myrica andCasuarina . Because Frankia bacteria are also free-living
in soil (Benson & Dawson, 2007), this root nodule symbiosis is likely
to be effective to successfully distinguish communities of symbionts
infecting hosts and communities of close relatives that are present in
the soil around hosts but are non-infecting.
The goal of this study is to elucidate the filtering effects of a host
plant on associated microsymbiont communities in a natural condition. We
aim to (1) comprehensively investigate local and regionalAlnus–Frankia symbiotic communities in host root nodules and
rhizosphere soils in the field, using next-generation sequencing (NGS);
(2) compare the genotypic composition of Frankia bacteria between
nodules and soils to examine the consequences of filtering forces; (3)
examine how abiotic environmental factors and genetic variation in hosts
affect Frankia communities, and (4) examine how host-assembled
symbionts are associated with host genetic variation.