Mycorrhizal symbioses in lycopods

Lycopods represent a significant diversification point on the land plant phylogenetic tree, being the earliest divergent extant tracheophyte lineage (Kenrick, 1994) and marking the transition from nonvascular to vascular plants. Several lycophytes (Huperzia, Lycopodium, Lycopodiella and Phylloglossum; Supporting Information Fig. S1a) possess an ‘alternation of generations’ life cycle (Kenrick, 1994) that features fully independent gametophyte (haploid) and dominant sporophyte (diploid) generations (Haufler et al., 2016; Fig. S1b). In nature, all members of the Lycopodiaceae require mycorrhizal symbionts for growth and for the production of gametes (Winther & Friedman, 2008). These fungal symbionts are of particular interest as they are reported to be present across both free‐living generations of the plants: from the gametophyte to the young sporophyte (protocorm), while still attached to the gametophyte, through to the mature sporophyte (Bierhorst, 1971; Winther & Friedman, 2008).

Initially, it was thought that the fungal symbionts of the Lycopodiaceae were arbuscular mycorrhizal (AM)‐like with unique ‘lycopodioid’ features (Schmid & Oberwinkler, 1993). However, a recent global analysis of over 20 lycopod species determined that many form symbioses with both AM‐forming Glomeromycotina fungi and Mucoromycotina ‘fine root endophyte’ (MFRE) fungi, with MFRE partners being the only detectable fungal symbiont in the lycopod species Lycopodiella inundata (Rimington et al., 2015). MFREs, previously classified as the AM species Glomus tenue, have recently been reclassified as belonging within the Mucoromycotina (Orchard et al., 2017a,b) and renamed as Planticonsortium tenue (Walker et al., 2018). Emerging evidence suggests that, in contrast to the majority of studies on MFREs, which have so far focused primarily on the role of the fungal partners in phosphorus (P) transfer to host plants (Orchard et al., 2017a), MFRE partners also play a significant role in plant nitrogen (N) assimilation (Field et al., 2019; Hoysted et al., 2019), complementary to the role of AM fungi (AMFs) in P (Smith & Read, 2008) and potential N uptake (Hodge et al., 2001; Hodge & Storer, 2015). Such complementation with AMFs could help to explain the persistence of MFREs across nearly all modern plant lineages.

Mycorrhizal functioning in plants with alternating generations, such as L. inundata, is complex and poorly understood, with the only published research to date focusing on instantaneous measurements on a single life history stage; for example, photosynthetic sporophytes of Ophioglossum associating with AMFs (Field et al., 2015; Suetsugu et al., 2020). To date, only one study has dissected the symbiotic function of MFREs in L. inundata, or indeed in any vascular plant (Hoysted et al., 2019); however, like other studies investigating mycorrhizal function, experiments were limited to actively growing, photosynthetic adult sporophytes with erect fertile stems and thus provide only a snapshot in time of symbiotic function in a perennial plant. Given that MFREs have been reported to be present at each life stage of L. inundata – from the subterranean gametophyte to the retreating adult sporophyte (Hoysted et al., 2019) – these plants provide a unique opportunity to understand symbiotic function and enhance our knowledge of MFREs, not only in a vascular plant, but one with a complex life cycle.

We used a combination of isotope tracers and cytological analyses to investigate how MFRE fungal morphology and function may change across the transition from newly emerging, juvenile sporophytes to retreating adult sporophytes of L. inundata, how MFRE function changes as plants become photosynthetic, and how the loss of photosynthetic capacity of L. inundata may affect MFRE‐acquired nutrient assimilation in retreating sporophytes. We collected L. inundata (L.) gametophytes and sporophytes at three different life stages (Figs 1a–c, S1b) from Thursley National Nature Reserve, Surrey, UK (SU 90081 39754) in spring and late summer, 2017. Using the methods of Hoysted et al. (2019), we quantified carbon (C)‐for‐nutrient exchange between L. inundata and MFRE symbionts (Fig. 2). ³³P‐labelled orthophosphate and ¹⁵N‐labelled ammonium chloride were used to trace nutrient flow from MFRE to plant for each of the L. inundata life stages collected. We simultaneously traced the movement of C from plant to MFRE by generating a pulse of ¹⁴CO₂ and quantifying the activity of extraradical MFRE hyphae in the surrounding soil using sample oxidation (307 Packard Sample Oxidiser; Isotech, Chesterfield, UK) and liquid scintillation (see Methods S1 for details). Fungal symbionts from root samples of experimental plants were identified using molecular fungal identification methods as per Hoysted et al. (2019; see Methods S1 for details), with MFREs being detected in each life stage (GenBank/EMBL accession nos. MK673773–MK673803).

Our data show that MFRE fungi play distinct functional roles at each life stage of L. inundata, with evidence of bidirectional exchange of plant C for fungal‐acquired nutrients (N and P) between mature adult and retreating adult sporophytes and fungi, but no transfer of plant C to fungi and little fungal‐acquired nutrient gain in juvenile sporophytes. Furthermore, we show that these functional stages correspond with different cytologies of colonization across the L. inundata life cycle. Considered alongside the results of studies in other plants with complex life cycles (Roy et al., 2013; Gonneau et al., 2014; Suetsugu et al., 2018), our results emphasize the importance of investigating symbiotic fungal function across plant life histories.