Nicole A. Hynson

The Physiological Ecology of Mycoheterotrophy

The purpose of this chapter is to provide a practical and theoretical framework for the study of the ecophysiology of mycoheterotrophic plants. We accomplish this by providing a comparative overview of our current knowledge on carbon and nitrogen isotope natural abundance in partially and fully mycoheterotrophic plants associated with ectomycorrhizal, wood- and litter-decomposer saprotrophic, and arbuscular mycorrhizal fungi, and discuss their ecophysiological implications. We present a meta-analysis of all stable carbon and nitrogen isotope values from the majority of species of partially and fully mycoheterotrophic plants investigated thus far. We summarize our current understanding of the ecophysiology of fully mycoheterotrophic plants in the families Orchidaceae and Ericaceae as well as nonvascular plants, and species from the tropics that associate with arbuscular mycorrhizal fungi. We also review the occurrence of initial mycoheterotrophy among orchids and ericaceous plants that are autotrophic upon reaching adulthood. We highlight current studies of cryptic or partial mycoheterotrophy in green plants that appear to be fully autotrophic, but meet some portion of their C demands via fungi in a mixotrophic nutrition. Furthermore, we explore the utility of ecophysiological methods such as radioactive and stable isotope probing, measuring plant assimilatory and respiratory responses to environmental gradients such as light availability, and natural abundance stable isotope analysis for future studies of mycoheterotrophic food webs. Finally, methodological limitations and considerations for the study of physiological ecology of mycoheterotrophy are also outlined in this chapter.

Measuring carbon gains from fungal networks in understory plants from the tribe Pyroleae (Ericaceae): a field manipulation and stable isotope approach.

Partial mycoheterotrophy, a newly discovered form of mixotrophy in plants, has been described in at least two major lineages of angiosperms, the orchids and ericaceous plants in the tribe Pyroleae. Partial mycoheterotrophy entails carbon gains both directly from photosynthesis and via symbiotic mycorrhizal fungi, but determining the degree of plant dependence on fungal carbon is challenging. The purpose of this study was to determine if two chlorophyllous species of Pyroleae, Chimaphila umbellata and Pyrola picta, were receiving carbon via mycorrhizal networks and, if so, if their proportional dependency on fungal carbon gains increased under reduced light conditions. This was accomplished by a field experiment that manipulated light and plants’ access to mycorrhizal networks, and by using the stable carbon isotope composition (δ13C) of leaf soluble sugars as a marker for the level of mycoheterotrophy. Based on leaf soluble sugars δ13C values, we calculated a site-independent isotope enrichment factor as a measure of fungal contributions to plant C. We found that, under each treatment and over time, the two test species demonstrated different isotopic responses caused by their different intrinsic physiologies. Our data, along with previously published studies, suggest that Chimaphila umbellata is primarily an autotrophic understory plant, while Pyrola picta may be capable of partial mycoheterotrophy. However, in this study, a 50% decrease in light availability did not significantly change the relative dependency of P. picta on carbon gains via mycoheterotrophy.

Fungal host specificity is not a bottleneck for the germination of Pyroleae species (Ericaceae) in a Bavarian forest

Plants that produce dust seeds can recruit fungi to meet their earliest requirements for carbon and other nutrients. This germination strategy, termed initial mycoheterotrophy, has been well investigated among the orchid family, but there are numerous other plant lineages that have independently evolved mycoheterotrophic germination strategies. One of these lineages is the tribe Pyroleae (Ericaceae). While the fungi associated with mature plants in Pyroleae have been fairly well documented, their mycobionts at the germination and seedling stages are largely unknown. Here, we use an in situ seed baiting experiment along with molecular fingerprinting techniques and phylogenetic tests to identify the fungi associated with seedlings of two Pyroleae species, Pyrola chlorantha and Orthilia secunda. Our results indicate that similar to adult plants, Pyroleae seedlings can associate with a suite of ectomycorrhizal fungi. Some seedlings harboured single mycobionts, while others may have been inhabited by multiple fungi. The dominant seedling mycobiont of both Pyroleae species was a fungus of unknown trophic status in the order Sebacinales. This taxon was also the only one shared among seedlings of both investigated Pyroleae species. We discuss these results juxtaposed to orchids and one additional Pyrola species in the context of ontogenetic shifts in fungal host specificity for mycoheterotrophic nutrition.

Are there geographic mosaics of mycorrhizal specificity and partial mycoheterotrophy? A case study in Moneses uniflora (Ericaceae)

The majority of plants require symbiotic interactions with other organisms to complete at least a portion of their life cycles. However, the reliance of plants on these interactions varies, and the net benefit to plant individuals is dependent on the environmental context in which they occur (Thompson, 2005). One particularly interesting group of obligate symbiotic plants are mycoheterotrophs. Rather than deriving carbon from photosynthesis, mycoheterotrophic plants meet all or a portion of their carbon demands via symbiotic interactions with fungi that are often simultaneously engaged in mutualisms with surrounding autotrophic trees (Merckx, 2013). Via these tripartite networks, autotrophic trees are the ultimate carbon source for many mycoheterotrophic species. Mycoheterotrophic plants can be either fully mycoheterotrophic – where they have lost the ability to photosynthesize and are completely dependent on fungi to meet their carbon demands, or partially mycoheterotrophic – a kind of mixotrophy where some autotrophy is retained (Merckx et al., 2009). Similar to many host–parasite interactions, a hallmark of many fully mycoheterotrophic plants is extreme specificity to species (Bidartondo & Bruns, 2005), or even genotypes of fungal hosts (Barrett et al., 2010). Although some exceptions exist (Hynson & Bruns, 2009; Roy et al., 2009), the reason(s) for this frequent extreme specificity are not fully understood. However, two non-exclusive explanations prevail: (1) as exploiters of the mycorrhizal mutualism, mycoheterotrophs fine-tune their physiology tomaximize their interactions with specific fungal hosts, thus preventing broad host switching (Hynson & Bruns, 2010); (2) neighborhood interactions such as partner filtering, prevent mycoheterotrophs from exploiting certain fungi (Egger&Hibbett, 2004). Because they retain some autotrophy (and thus the ability to reciprocate on the mycorrhizal mutualism), under each of these scenarios partial mycoheterotrophs would not be expected to form specificmycorrhizal associations unless the fitness benefits of fungal exploitation outweigh those of cooperation. Accordingly, among partial mycoheterotrophs studied thus far evidence of fungal partner specificity is somewhat limited; Pyrola japonicaKlenze ex Alef. (Ericaceae) and Limodorum abortivum (L.) Sw. (Orchidaceae) associate with a range of ectomycorrhizal taxa, but predominantly partner with Russula Pers. spp., and Corallorhiza trifida Châtel (Orchidaceae) partners solely with ectomycorrhizal Tomentella Pers. ex Pat. spp., but appears to gain little carbon from photosynthesis (Girlanda et al., 2006; Zimmer et al., 2008; Cameron et al., 2009; Matsuda et al., 2012). Furthermore, themajority of studies on partial mycoheterotrophs examine the degree of partial mycoheterotrophy and fungal partner specificity within single or among a few plant populations (Bidartondo et al., 2004; Julou et al., 2005; Abadie et al., 2006; Tedersoo et al., 2007;Matsuda et al., 2012; Johansson et al., 2015). While these efforts provide goodbaseline data, amore complete test of mycorrhizal specificity among partial mycoheterotrophs would include wider geographic samplings of these species and their fungal symbionts. Assuming coevolution, across a species range its symbiotic partnerships will be shaped by geographic selection mosaics that depend upon population-specific biotic and abiotic conditions (sensu Thompson, 2005). Here we set out to examine the fungal partnerships of a putative partial mycoheterotroph, Moneses uniflora (L.) A. Gray (Ericaceae), across a large portion of its natural range. Analyzing the naturally abundant carbon and nitrogen stable isotope ratios of understory plants has been a useful technique to infer partialmycoheterotrophy in nature (Gebauer&Meyer, 2003; Julou et al., 2005; Hynson et al., 2013). The carbon stable isotope composition of partial mycoheterotrophs tends to be enriched in the heavy isotope of carbon (C) compared to surrounding autotrophic species, but depleted in C relative to full mycoheterotrophs (Hynson et al., 2013). Within an ecological foodweb as substrates are processed and consumed there is a corresponding isotopic enrichment that can be detected in the consumer (Fry, 2006). Therefore, the enrichment in C found in mycoheterotrophs is owed to all or a portion of their carbon demands being met through the uptake of compounds that have been previously processed by fungi, rather than direct uptake of atmospheric CO2 through photosynthesis (Gebauer & Meyer, 2003; Hynson et al., 2013). Many partially and fully mycoheterotrophic taxa are also enriched in the heavy isotope of nitrogen (N) relative to surrounding autotrophic species, and some leafy green ericaceous understory species are enriched only in N without any detectable differences in their C composition from surrounding autotrophs (Hynson et al., 2013). This latter group has been referred to as ‘cryptic mycoheterotrophs’ where their N enrichment may be indicative of the uptake of fungal-assimilated organic compounds that would inherently include carbon. However, whether this carbon is used by plants for their own growth is unclear (Hynson et al., 2013). Therefore, green plants that associate with ectomycorrhizal fungi and are enriched in both C and N relative to surrounding autotrophs are the clearest examples of

Uncovering unseen fungal diversity from plant DNA banks

Throughout the world DNA banks are used as storage repositories for genetic diversity of organisms ranging from plants to insects to mammals. Designed to preserve the genetic information for organisms of interest, these banks also indirectly preserve organisms’ associated microbiomes, including fungi associated with plant tissues. Studies of fungal biodiversity lag far behind those of macroorganisms, such as plants, and estimates of global fungal richness are still widely debated. Utilizing previously collected specimens to study patterns of fungal diversity could significantly increase our understanding of overall patterns of biodiversity from snapshots in time. Here, we investigated the fungi inhabiting the phylloplane among species of the endemic Hawaiian plant genus, Clermontia (Campanulaceae). Utilizing next generation DNA amplicon sequencing, we uncovered approximately 1,780 fungal operational taxonomic units from just 20 DNA bank samples collected throughout the main Hawaiian Islands. Using these historical samples, we tested the macroecological pattern of decreasing community similarity with decreasing geographic proximity. We found a significant distance decay pattern among Clermontia associated fungal communities. This study provides the first insights into elucidating patterns of microbial diversity through the use of DNA bank repository samples.

Biological invasions increase the richness of arbuscular mycorrhizal fungi from a Hawaiian subtropical ecosystem

Biological invasions can have various impacts on the diversity of important microbial mutualists such as mycorrhizal fungi, but few studies have tested whether the effects of invasions on mycorrhizal diversity are consistent across spatial gradients. Furthermore, few of these studies have taken place in tropical ecosystems that experience an inordinate rate of invasions into native habitats. Here, we examined the effects of plant invasions dominated by non-native tree species on the diversity of arbuscular mycorrhizal (AM) fungi in Hawaii. To test the hypothesis that invasions result in consistent changes in AM fungal diversity across spatial gradients relative to native forest habitats, we sampled soil in paired native and invaded sites from three watersheds and used amplicon sequencing to characterize AM fungal communities. Whether our analyses considered phylogenetic relatedness or not, we found that invasions consistently increased the richness of AM fungi. However, AM fungal species composition was not related to invasion status of the vegetation nor local environment, but stratified by watershed. Our results suggest that while invasions can lead to an overall increase in the diversity of microbial mutualists, the effects of plant host identity or geographic structuring potentially outweigh those of invasive species in determining the community membership of AM fungi. Thus, host specificity and spatial factors such as dispersal need to be taken into consideration when examining the effects of biological invasions on symbiotic microbes.