Phenology and function in lycopod–Mucoromycotina symbiosis

Abstract

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. Lycopodiella inundata forms associations with MFRE fungi in each stage of its life cycle (Rimington et al., 2015; Hoysted et al., 2019), and previous research in mature sporophytes has demonstrated that these associations represent nutritional mutualisms, akin to AM fungal associations in other vascular plants (Hoysted et al., 2019). However, despite there being copious MFRE colonization within juvenile sporophytes (Fig. 1d–f), we found no transfer of plant C to MFREs (Fig. 2a,b; Tables S1, S2) even though green leaves were present with potential photosynthetic capabilities. By contrast, transfer of C from plants to MFREs in both the mature and retreating adult sporophyte growth stages was evident (Fig. 2a,b; Tables S1, S2), with c. 2.4 times the amount of C being transferred from the plant to MFREs in mature adult sporophytes compared with retreating adult sporophytes, although this difference was not significant (Mann–Whitney U = 142.000, P = 0.144; Table S3). Winther & Friedman (2008) suggested a form of parental nurture may occur in lycopods with achlorophyllous subterranean gametophytes, such as L. inundata, where fidelity of fungal partners and shared mycelial networks between generations allow autotrophic sporophytes to supply the small but critical amounts of carbohydrates required to support heterotrophic gametophytes (Leake et al., 2008). Our findings may corroborate this idea of intergenerational support, with adult and retreating sporophytes transferring C to MFRE partners and C transfer by juveniles being undetectable. However, the absence of C transfer by juveniles in our experiments does not necessarily equate to a total lack of C transfer by juveniles; further research is needed to determine this. Movement of 33P from MFRE associates was detected in all L. inundata plants tested, although the amounts transferred varied among growth stages (Fig. 2c,d; Table S4), with juvenile L. inundata sporophytes receiving approximately 10-fold less 33P from their fungal partner compared with mature adult L. inundata sporophytes (Mann–Whitney U = 13.000, P = 0.012; Fig. 2c; Table S3). However, there was no significant difference in the amounts of 33P received from MFREs between mature adult sporophytes and juvenile sporophytes when aboveground plant-tissue 33P content was normalized to plant biomass (Mann–Whitney U = 45.000, P = 0.813; Fig. 2d; Table S3). In addition to 33P, significant amounts of 15N were transferred from MFREs to the shoots of mature and retreating adult L. inundata sporophytes (Fig. 2e,f; Table S4). Mature adult sporophytes received around nine times more 15N from MFREs than retreating ones did. However, there was no 15N transferred from MFREs to any of the juvenile sporophytes tested (Fig. 2e,f; Table S4). Although there was little to no exchange of plant-fixed C for fungal-acquired nutrients in juvenile sporophytes, we observed abundant bidirectional exchange of C for 33P and 15N between the mature adult sporophyte of L. inundata and MFRE fungi (Fig. 2a–f; Tables S1, S2, S4). These results are similar to those of a previous investigation into the function of AMF symbionts of green sporophytes of the fern Ophioglossum vulgatum, also defined by a characteristic alternation of generations (Field et al, 2015), which showed mutualistic exchange of plant-fixed C for nutrients between symbionts. Scanning electron microscopy results confirm distinct differences in fungal colonization between gametophytes, juvenile sporophytes and roots of adult plants. Colonization of the protocorm of newly developing sporophytes, which remain attached to the gametophyte (Fig. S2a–c), occurs de novo, with no evidence of the fungal symbiont crossing the gametophyte–sporophyte junction (placenta; Fig. S2d). Fungal colonization in newly developing sporophytes is both intra and intercellular (Fig. S2e) and, like in the gametophytes, consists of thin (> 2 µm diameter), branching hyphae with small intercalary and terminal vesicles (Fig. S2d), typical of MFRE colonization. The intercellular hyphae enlarge as the young sporophytes develop, reaching diameters well in excess of 3 µm, whereas the vesicles disappear (see Hoysted et al., 2019). By the time young sporophytes have reached the developmental stage used in our isotope tracer experiments (up to seven leaves, remnants of protocorm, rhizoids, and no or rarely one newly developing rootlet; Fig. 1a), the system of large, mucilage-filled intercellular spaces almost completely fills the remnants of the protocorm (Fig. 1d) and is packed with pseudoparenchymatous hyphal masses (Fig. 1e), which are mostly collapsed (Fig. 1f). Roots of actively growing (Fig. 1g) and retreating (Figs 1, 2, S2f,g) adult plants both display the same cytology of colonization, consisting of intracellular thin hyphae and vesicles (Fig. S2f,g; Hoysted et al., 2019); however, in the latter, the fungus is largely confined to the epidermal and outermost cortical layers (Fig. 1h). MFRE fungi have a distinct zonation in the gametophytes and protocorms of newly developed L. inundata sporophytes consisting of intracellular and intercellular phases. The intracellular phase of colonization is characterized by fine hyphae with small swelling/vesicles (and, in the gametophyte only, also hyphal coils with larger vesicles – see Hoysted et al., 2019). In the intercellular phase, the fungus proliferates in the system of mucilage-filled intercellular spaces, forming masses of large pseudoparenchymatous hyphae that eventually collapse and degenerate (Hoysted et al., 2019). This colonization is the same as that reported in other lycopod gametophytes and protocorms (Duckett & Ligrone, 1992; Schmid & Oberwinkler, 1993) and is strikingly similar to that described in the earliest diverging Haplomitriopsida liverworts Treubia and Haplomitrium (Carafa et al., 2003; Duckett et al., 2006), the only two liverwort genera known to date to be colonized exclusively by MFRE fungi (Bidartondo et al., 2011; Field et al., 2015; Rimington et al., 2020). In Treubia and Haplomitrium, the intracellular fungal swellings or ‘lumps’ are relatively short-lived; it has been suggested that these structures are involved in active metabolic interactions with the host cells (Carafa et al., 2003) and that their eventual collapse and lysis may also provide nutrients, such as N, to the host plant (Duckett et al., 2006). The MFRE fungal colonization in the roots of adult sporophytes is only intracellular and consists of fine aseptate hyphae with intercalary and terminal swellings/vesicles but without arbuscules (Hoysted et al., 2019). It is possible that the small swellings/vesicles may play an important role in host–fungus physiological relationships, as has been suggested for Haplomitriopsida liverworts (Carafa et al., 2003; Duckett et al., 2006). Further studies are urgently needed to determine the functional role of the diverse structures produced by MFREs in the different stages of Lycopodiella's life cycle, and indeed other plants. In retreating sporophytes, fungal colonization appears much reduced compared with fully photosynthesizing sporophytes, being mostly restricted to the outermost cortical layers (Fig. 1h). This may explain why retreating sporophytes receive smaller amounts of N and P from their fungal symbionts (Fig. 2c–f). Previous descriptions of Lycopodiella have highlighted the crucial role played by symbiotic fungi in the continued growth of the gametophyte; growing green portions of older gametophytes of Lycopodiella alopecuroides were often observed to be embedded in older, yellow portions with abundant fungal hyphae (Koster, 1941). Coupled with the absence of C and N transfer between L. inundata and MFRE fungi in the juvenile sporophyte in our experiments (Fig. 2a,b,e,f; Tables S1, S2, S4), this may suggest the presence of intergenerational support between alternating life stages whereby later life stages need to be present to transfer essential nutrients and nurture younger plants. In our experiments, the juvenile sporophytes were sustained throughout the experimental period despite the apparent lack of photosynthetic C being transferred from plant to fungus and without hyphal connections to mature sporophytes. It is possible that residual C reserves within the sporophyte tissues were mobilized and used for plant growth and allocation to fungi and recent photosynthates restricted for use only in plant tissues, suggestive of there being intricate temporal dynamics in allocation of C resources to fungal partners in this key transitional stage. Alternatively, the presence of collapsed and degenerating pseudoparenchymatous hyphal masses filling the extensive system of intercellular spaces in the remnants of protocorms may suggest a different scenario. This juvenile sporophytic stage just precedes root development, and therefore formation of a mycorrhizal association sensu stricto between Lycopodiella sporophytes and MFRE symbionts. It is likely that very early stages of sporophyte development are, like the gametophytes, completely, or largely, mycoheterotrophic (i.e. where plant C and nutrients are acquired entirely via mycorrhizal fungi), as fungal colonization is ubiquitous and extensive in their subterranean protocorms with only the apical parts of newly developing green leaves emerging above the ground. It is possible, therefore, that juvenile sporophytes just before root development maintain a partially mycoheterotrophic lifestyle, the masses of collapsed and degenerating intercellular hyphae releasing nutrients that support early sporophyte development. Further investigations are now required that include structural and functional assessment of subterranean gametophytes associating with MFRE fungi. This investigation represents the first functional assessment of fungal symbiosis across the changing phenology of the marsh clubmoss, L. inundata. We show that MFRE fungi play critical and distinct functional roles across different developmental stages and that these correspond with different cytologies of colonization. Our results show that MFREs have considerable plasticity in their interactions with plants that appears to relate to the developmental stage of the host and is suggestive of intergenerational support between sporophytes and gametophytes via shared MFRE symbionts. We gratefully acknowledge support from the NERC to KJF, SP (NE/N00941X/1; NE/S009663/1) and MIB (NE/N009665/1). KJF is funded by a BBSRC Translational Fellowship (BB/M026825/1) and a Philip Leverhulme Prize (PLP-2017-079). We thank James Giles (Natural England) for field support, Julia Masson and the RSPB for access to the Norfolk site, and The Species Recovery Trust for access to the Dorset site. KJF, SP, MIB and JGD conceived and designed the investigation. SP and JGD collected plant material. GAH undertook physiological analysis. SP undertook the cytological analysis. GAH led the writing; all authors discussed results and comments on the article. GAH agrees to serve as the author responsible for contact and ensure communication. Fig. S1 Land plant phylogeny of the Lycopodiales and life cycle of Lycopodiella inundata. Fig. S2 Patterns of fungal colonization in Lycopodiella inundata. Methods S1 Summary of materials and methods used during the study. Table S1 Summary of % carbon detected in experimental microcosms. Table S2 Summary of total amount of carbon detected in experimental microcosms. Table S3 Summary of statistical differences in MFRE functionality. Table S4 Summary of amounts of 15N and 33P detected in experimental microcosms. Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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