INTRODUCTION
Root-nodule symbioses (RNS) between plants and nitrogen (N) fixing
bacteria are ecologically and economically important interactions
(Graham & Vance, 2003; Vitousek et al. 2002; Wheeler & Miller,
1990), and provide model systems for studying the evolutionary ecology
of mutualism (Heath & Grillo, 2016). RNS are phylogenetically
widespread, occurring across a diverse set of organisms that include the
legume plants (Fabaceae) and Parasponia spp. that form
‘rhizobial’ associations with bacterial symbionts from three
proteobacterial phyla, a different set of plants from 8 families and 25
genera that form ‘actinorhizal’ associations with the single
actinomycete genus Frankia , and some species of cycads that
associate with cyanobacteria (Dawson, 2008; Piex et al . 2015;
Vessey et al . 2005). In all RNS, host plants acquire bacterial
symbionts ‘horizontally’; i.e., plants form nodules de novo with
bacteria found in the soil environment, rather than receiving symbionts
‘vertically’ from their parents, as in some endosymbiotic interactions
(Denison & Kiers 2011; Frank, 1996; Wall & Berry, 2008). The
independent existence of plant and bacterial partners in RNS affects the
evolution of the symbiotic interaction in several ways.
One result of this independence is the potential for evolutionary
conflict between host plants and symbiotic bacteria. Because individual
plants associate simultaneously with bacteria from multiple lineages
that can vary widely in the benefits they provide to the host (Denison,
2000; Kiers and Denison, 2008; Markham, 2008; Parker, 1995; Sachset al ., 2010), a Tragedy-of-the-Commons can occur in bacterial
communities (Denison 2000), favoring potentially ‘selfish’ behavior such
as decreased allocation of host-derived carbon to N-fixation (Oono &
Denison, 2010), or bacterial reproduction within host nodules that
evades host regulation (Cotin-Galvan et al ., 2016). Over
evolutionary time, selection for such ‘cheating’ behavior should
destabilize the mutualism, in the absence of other factors (Sachs and
Simms, 2006). However, bacterial cheating is thought to be held in check
by several countermeasures evolved by host plants. A multi-level system
of pre-nodulation communication mechanisms (Clúa et al ., 2018)
between roots and bacteria allows the plant to recognize bacterial
genotypes likely to provide effective N-fixation (‘partner choice’ Kiers
& Denison, 2008). Post-nodulation detection of bacterial N fixation
allows plants to ‘punish’ cheating genotypes via decreased reproduction
within nodules (termed ‘host sanctions’ (Denison 2000)) and/or
selectively allocate resources to especially beneficial bacterial
genotypes (‘differential rewards’ Simms & Taylor, 2002).
Horizontal transmission of RNS symbionts also means that the bacterial
partner occupies two distinct niches: one in which they live actively or
dormantly in soil, often forgoing N-fixation entirely, and one in which
they essentially act as plant organelles, fixing N using host-derived
carbon sources that, in the most effective symbiotic genotypes, are
allocated preferentially to N-fixation over bacterial reproduction
(Benson & Silvester, 1993; Denison & Kiers 2004). These two niches are
likely to exert different selection pressures on N-fixing bacteria,
which may result in tradeoffs between symbiotic and free-living
lifestyles (West et al ., 2002) and/or drive repeated gains and
losses of symbiotic capability in bacterial lineages (Sachs et
al . 2010; Sachs & Simms, 2006). Among the symbiotic fraction of the
bacterial community, passage between soil-dwelling and nodule-dwelling
phases has the potential to exert strong but complex influences on the
mixture of genotypes available to host plants. In order to be available
for nodule-formation in the first place, bacterial genotypes must be
able to maintain a viable existence in soil. Once chosen by a host
plant, a bacterial cell can be cloned to much greater densities within a
nodule before re-entering the soil during nodule senescence (Denison
2000), increasing the proportional representation of the chosen genotype
in the soil fraction, as well as its availability for future rounds of
nodule formation.
Little is known about the relationship between nodule and soil
assemblages of N-fixing bacteria in natural environments. Historically,
investigation of diversity patterns in soil-dwelling bacterial symbionts
have been based on the use of N-fixing ‘trap plants’ to sample bacterial
communities via nodule formation or, less frequently, on direct
isolation of bacteria from soils (Chaia et al ., 2010; McInneset al ., 2004). Both approaches are subject to significant and
well-known biases (Chaia et al ., 2010; Kirk et al . 2004).
In recent years, molecular methods such as DNA cloning, qPCR, and
next-generation sequencing (NGS) methods have been increasingly used to
characterize soil communities of N-fixing bacteria (e.g., Ben Tekayaet al . 2017, 2018; Miranda-Sanchez et al . 2016, Rodriguezet al ., 2016). While these methods are not without their own
biases (Acinas et al ., 2005; Chandler et al ., 1997;
Gołębiewski & Tretyn, 2020; Sáenz et al ., 2019; Sanchez-Cidet al ., 2022; van Elsas & Boersma, 2011), they allow
investigation of a much broader swath of the bacterial community not
limited to genotypes able to induce host nodules and/or grow under
laboratory conditions. In the present study, we use PCR and DNA cloning
to examine patterns of genetic diversity in naturally-occurring
soil-dwelling populations of the actinorhizal symbiont Frankiaspp. associated with the host species Alnus incana ssp.tenuifolia (hereafter ‘A. tenuifolia ’) across a primary
successional sere in the boreal forest of Alaska. We utilize a subset of
research sites in which we have previously observed stable
nodule-dwelling assemblages over a range of 3 to 5 years, with large
differences in composition among successional stages that are consistent
among replicate sites (Figure S1) (Anderson et al ., 2009, 2013;
Ruess et al . 2013). In the present study, we investigated three
questions: 1) how do soil and nodule assemblages compare in phylogenetic
diversity of Frankia , 2) do soil and nodule assemblages change in
parallel in response to environmental variation: both across succession,
and in response to long-term fertilization with nitrogen (N) and
phosphorus (P), and 3) do soil assemblages differ in the presence and
absence of A. tenuifolia individuals?