bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.316760; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Phenology and function in lycopod-Mucoromycotina symbiosis. 2 3 Grace A. Hoysted1*, Martin I. Bidartondo2,3, Jeffrey G. Duckett4, Silvia Pressel4 and 4 Katie J. Field1 5 6 1Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 7 2TN, UK 8 2Comparative Plant and Fungal Biology, Royal botanic Gardens, Kew, Richmond, 9 TW9 3DS, UK 10 3Department of Life Sciences, Imperial College London, London, SW7 2AZ, UK 11 4Department of Life Sciences, Natural History Museum, London, SW7 5BD, UK 12 13 *Corresponding author: 14 Grace A. Hoysted ([email protected]) 15 16 Words: 2338 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.316760; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 35 Abstract 36 Lycopodiella inundata is a lycophyte with a complex life cycle. The gametophytes 37 and the juvenile, mature and retreating sporophytes form associations with 38 Mucoromycotina fine root endophyte (MFRE) fungi, being mycoheterotrophic as 39 gametophytes and mutualistic as mature sporophytes. However, the function of the 40 symbiosis across juvenile and retreating sporophyte life stages remains unknown. 41 We measured carbon-for-nutrient exchanges between L. inundata and MFRE across 42 the transition from newly emerging sporophytes to mature sporophytes and in 43 retreating adult sporophytes. We show MFRE fungi play distinct functional roles at 44 each plant life stage, with evidence of bidirectional exchange of plant C for fungal 45 acquired nutrients (N and P) between mature adult and retreating adult sporophytes 46 and fungi, but no transfer of plant C to fungi and little fungal-acquired nutrient gain in 47 juvenile sporophytes. Furthermore, we show that these functional stages correspond 48 with different cytologies of colonisation. Our results show that MFRE have 49 considerable plasticity in their interactions with the host plant which is related to the 50 developmental stage of the host. This highlights the need for further research into 51 symbiotic fungal function across plant life histories. 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.316760; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 69 Introduction 70 Lycopods represent a significant node on the land plant phylogenetic tree, 71 being the earliest divergent extant tracheophyte lineage (Kenrick, 1994) and marking 72 the transition from non-vascular to vascular plants. Several lycophytes (Huperzia, 73 Lycopodium, Lycopodiella and Phylloglossum; Supplementary Fig. 1a) possess an 74 “alternation of generations” lifecycle (Kenrick, 1994) which features fully independent 75 gametophyte (haploid) and dominant sporophyte (diploid) generations (Haufler et al, 76 2016; Supplementary Fig. 1b). In nature, all members of the Lycopodiaceae require 77 mycorrhizal symbionts for growth and for the production of gametes (Winther and 78 Friedman, 2008). These fungal symbionts are of particular interest as they are 79 reported to be present across both free-living generations of the plants: from the 80 gametophyte to the young sporophyte (protocorm), while still attached to the 81 gametophyte, through to the mature sporophyte (Bierhorst, 1971; Winther and 82 Friedman, 2008). 83 Initially, it was thought that the fungal symbionts of the Lycopodiaceae were 84 arbuscular mycorrhizal (AM)-like with unique “lycopodioid” features (Schmid and 85 Oberwinkler, 1993). However, a recent global analysis of over 20 lycopod species 86 determined that many form symbioses with both AM-forming Glomeromycotina fungi 87 and Mucoromycotina “fine root endophyte” (MFRE) fungi, with Lycopodiella inundata 88 being the only lycopod species found to engage in exclusive associations with MFRE 89 partners (Rimington et al. 2015). MFRE, previously classified as the AM species 90 Glomus tenue, have recently been reclassified as belonging within the 91 Mucoromycotina (Orchard et al, 2017a, b) and renamed as Planticonsortium tenue 92 (Walker et al. 2018). Emerging evidence suggests that, in contrast to the majority of 93 studies on MFRE which have so far focussed primarily on the role of the fungal 94 partners in phosphorus (P) transfer to host plants (Orchard et al, 2017a), and in 95 contrast to AM fungi that are predominantly involved in P uptake (Smith & Read, 96 2008), MFRE partners play a significant role in plant nitrogen (N) assimilation 97 (Hoysted et al, 2019; Field et al, 2019). 98 Mycorrhizal functioning in plants with alternating generations, such as L. 99 inundata, is complex and poorly understood with the only published research to date 100 focussing on instantaneous measurements on a single life history stage, e.g. 101 photosynthetic sporophytes of Ophioglossum associating with AMF (Field et al, 102 2015; Suetsugu et al, 2020). To date, only one study has dissected the symbiotic bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.316760; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 103 function of MFRE in L. inundata, or indeed in any vascular plant (Hoysted et al, 104 2019); however, like other studies investigating mycorrhizal function, experiments 105 were limited to actively growing, photosynthetic adult sporophytes with erect fertile 106 stems and thus provide only a snapshot in time of symbiotic function in a perennial 107 plant. Given that MFRE have been reported to be present at each life stage of L. 108 inundata – from the subterranean gametophyte to the retreating adult sporophyte 109 (Hoysted et al, 2019), these plants provide a unique opportunity to understand 110 symbiotic function and enhance our knowledge of MFRE, not only in a vascular 111 plant, but one with a complex lifecycle. 112 We used a combination of isotope tracers and cytological analyses to 113 investigate how MFRE fungal morphology and function may change across the 114 transition from newly emerging, juvenile sporophytes to retreating adult sporophytes 115 of L. inundata, how MFRE function changes as plants become photosynthetic and 116 how the loss of photosynthetic capacity of L. inundata may affect MFRE-acquired 117 nutrient assimilation in retreating sporophytes. 118 119 Materials and Methods 120 We collected Lycopodiella inundata (L.) gametophytes and sporophytes at 121 three different life stages (Figure 2a-c, Figure S1b) from Thursley National Nature 122 Reserve, Surrey, UK (SU 90081 39754) in spring and late summer, 2017. Using the 123 methods of Hoysted et al, (2019), we quantified carbon-for-nutrient exchange 124 between L. inundata and MFRE symbionts. 33P-labelled orthophosphate and 15N- 125 labelled ammonium chloride were used to trace nutrient flow from MFRE-to-plant for 126 each of the L. inundata life stages collected. We simultaneously traced the 14 127 movement of carbon from plant-to-MFRE by generating a pulse of CO2 and 128 quantifying the activity of extraradical MFRE hyphae in the surrounding soil using 129 sample oxidation (307 Packard Sample Oxidiser, Isotech, Chesterfield, UK) and 130 liquid scintillation (see Supplementary Information for details). 131 L. inundata gametophytes, juvenile sporophytes (up to 7 leaves, remnants of 132 protocorm, rhizoids) and roots of mature and retreating adult plants (both wild and 133 experimental), were either stained with trypan blue (Brundrett et al, 1996), fixed and 134 embedded in Spur’s resin following Hoysted et al (2019), or processed for scanning 135 electron microscopy (SEM) (Hoysted et al. 2019), within 48 hrs of collection (Orchard 136 et al, 2017c). bioRxiv preprint doi: https://doi.org/10.1101/2020.09.28.316760; this version posted September 29, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 137 Fungal symbionts from root samples of experimental plants were identified 138 using molecular fungal identification methods as per Hoysted et al. (2019; see 139 Supplementary Information for details) with MFRE being detected in each life stage 140 (GenBank/EMBL accession numbers: MK673773-MK673803). 141 142 Results and Discussion 143 Our data show MFRE fungi play distinct functional roles at each life stage of 144 L. inundata, with evidence of bidirectional exchange of plant C for fungal acquired 145 nutrients (N and P) between mature adult and retreating adult sporophytes and fungi, 146 but no transfer of plant C to fungi and little fungal-acquired nutrient gain in juvenile 147 sporophytes. Furthermore, we show that these functional stages correspond with 148 different cytologies of colonisation across the L. inundata life cycle. Considered 149 alongside the results of studies in other plants with complex life cycles (Roy et al., 150 2013; Gonneau et al., 2014; Suetsugu et al., 2018), our results emphasise the 151 importance of investigating symbiotic fungal function across plant life histories. 152 153 C-for-nutrient exchange between L. inundata and MFRE across life stages 154 L. inundata forms associations with MFRE fungi in each stage of its life cycle 155 (Rimington et al, 2015; Hoysted et al, 2019) and previous research in mature 156 sporophytes has demonstrated that these associations represent nutritional 157 mutualisms, akin to AM fungal associations in other vascular plants (Hoysted et al, 158 2019).