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.