Introduction
A key prediction in evolutionary biology is that relatives will
cooperate more and compete less
(Griffin & West 2002; West
2002; West et al. 2007). Kin selection emerges because relatives
share high proportions of their genes, and by cooperating, more of these
genes are passed to the next generation. Kin selection has been applied
to understand a diversity of cooperative phenomena, from cooperating RNA
molecules (Levin & West
2017a, b) to cooperation among human beings
(dos Santos & West 2018).
However, kin selection can also be vulnerable to competition among
relatives, especially in cases where there is high spatial structuring
in the population. Under these cases, increased competition among
relatives can totally negate benefits of cooperation
(Queller 1992; West 2002).
While there is an increasing understanding of when and why relatives
cooperate, it is unknown how these dynamics affect organisms interacting
with a group of relatives. In symbiotic partnerships, for example, a
host interacts with communities of microbes, which can vary in levels of
relatedness (Fosteret al. 2017). The host may benefit from interacting with highly
related strains because of reduced conflict and competition within the
community (Frank 1996a, 2003; West
2002). However, interacting
with less related strains may not always entail a cost for the host, and
could even be beneficial. Specifically, if there is a greater relative
difference among the symbiont species in their ability to acquire
different, or complementary resources, the host could benefit from
interacting with non-relatives
(Jansa et al. 2008;
Wagg et al. 2011). Likewise, if competition drives an
underbidding scenario, which results in symbionts providing more
benefits, for less in return, the host could benefit from interacting
simultaneously with competing symbiotic strains
(Wyatt et al. 2014;
Noë & Kiers 2018).
Manipulating relative relatedness in symbiotic communities has
historically been challenging, making direct tests of these ideas
difficult. Here, we use the arbuscular mycorrhizal symbiosis to study
the effects of symbiont relatedness on host plants interacting via
competing or cooperating mycorrhizal fungal networks. The vast majority
of land plants are colonized by arbuscular mycorrhizal fungi. The fungi
trade soil bound nutrients such as phosphorus and nitrogen for
photosynthetic carbon from the host plant
(Jiang et al. 2017;
Keymer et al. 2017; Luginbuehl et al. 2017). The fungi
form underground networks that can connect roots of different plant
individuals. Hyphal fusion, otherwise known as anastomosis, can occur
among closely-related arbuscular mycorrhizal fungi
(Giovannetti et al.2004; Jakobsen 2004). This has the potential to increase resource
sharing across the fungal network
(Johansen & Jensen 1996;
Walder et al. 2012), which could increase the fitness of the
fungi (Giovannetti et
al. 2015) and potentially their hosts
(Roger et al. 2013).
However, when fungi are genetically less related, the hyphae can be
vegetative incompatible and fusion will not occur
(Giovannetti et al.2003; Croll et al. 2009). Direct antagonism among competing
arbuscular mycorrhizal strains has been shown to lead to negative
outcomes for fungal abundance and plant growth (Engelmoer et al.2014), and can also
influence fungal co-existence within host roots
(Roger et al. 2013).
For example, past work has shown how competition between distantly
related arbuscular mycorrhizal fungal isolates resulted in almost
complete exclusion of one isolate by the other, whereas more related
isolates shared the roots space in an almost 50:50 proportion
(Roger et al. 2013).
While this suggests that level of relatedness can affect fungal
competitive dynamics within a root, it is unknown how relatedness
affects the functioning of the hyphal network, especially when the
hyphae connect multiple plants.
Our aim was to understand how fungal relatedness affects the physical
formation and nutrient transfer in a fungal network formed between host
plants. To study phosphorus distribution and transfer, we employed a
recently developed technique in which we tag phosphorus rock (apatite)
with fluorescent quantum-dot nanoparticles
(van ’t Padje et al.in press; Whiteside et al. 2019; van’t Padje et al. 2020).
Quantum-dots fluoresce in bright and pure colors when excited with
UV-light. We used a class of quantum-dots that were highly fluorescent,
stable and well characterized in terms of toxicity, uptake and transfer
by fungal hyphae, with accumulation patterns in root (and leaf) tissue
as expected (Whitesideet al. 2009; Gustafsson et al. 2015). This allowed us to
determine how much phosphorus was transferred across the fungal network
per unit of fungal biomass.
We grew a host root colonized by a single focal strain. The arbuscular
mycorrhizal hyphae of this focal plant were allowed to interact with a
fungal network of a second host plant that was either the same fungal
strain (“selfing”) or two genetically less-related fungal strains
(both “non-selfing”). In order of highest to lowest relatedness, these
treatments included: (i) the same fungal strain (selfing), (ii) a
different fungal strain within the same species (non-selfing), or (iii)
a fungal strain of a different species in the same genus (non-selfing).
We grew these plant and fungal treatments as both whole plants in soil
and as in-vitro root organ cultures in petri plates. The latter
allowed us to determine where phosphorus was distributed across the
network using our quantum-dot tagging technique, as well as to quantify
the physical fungal network structure using imaging techniques
(Boddy 1999; Heatonet al. 2012a, b). We determined how varying relatedness in fungal
networks between the two host plants influenced: (i) host growth, (ii)
fungal colonization inside root tissue (intraradical colonization),
(iii) network formation outside the root tissue (extraradical
colonization), and (iv) transfer of nutrients across the network to the
host root.