Functional analysis of liverworts in dual symbiosis with Glomeromycota and Mucoromycotina fungi under a simulated Palaeozoic CO2 decline

Most land plants form mutualistic associations with arbuscular mycorrhizal fungi of the Glomeromycota, but recent studies have found that ancient plant lineages form mutualisms with Mucoromycotina fungi. Simultaneous associations with both fungal lineages have now been found in some plants, necessitating studies to understand the functional and evolutionary significance of these tripartite associations for the first time. We investigate the physiology and cytology of dual fungal symbioses in the early-diverging liverworts Allisonia and Neohodgsonia at modern and Palaeozoic-like elevated atmospheric CO2 concentrations under which they are thought to have evolved. We found enhanced carbon cost to liverworts with simultaneous Mucoromycotina and Glomeromycota associations, greater nutrient gain compared with those symbiotic with only one fungal group in previous experiments and contrasting responses to atmospheric CO2 among liverwort–fungal symbioses. In liverwort–Mucoromycotina symbioses, there is increased P-for-C and N-for-C exchange efficiency at 440 p.p.m. compared with 1500 p.p.m. CO2. In liverwort–Glomeromycota symbioses, P-for-C exchange is lower at ambient CO2 compared with elevated CO2. No characteristic cytologies of dual symbiosis were identified. We provide evidence of a distinct physiological niche for plant symbioses with Mucoromycotina fungi, giving novel insight into why dual symbioses with Mucoromycotina and Glomeromycota fungi persist to the present day.

Rainwater solution was applied to plants within experimental systems as a fine mist every two days. Plants were misted until the surface of the plant was visibly wet.

Construction of mesh-covered cores
Based on the methods of Johnson et al. (2001), each core had two windows cut into the below-ground portion (20 mm x 50 mm, see Fig. S1). The windows and base were covered by 10 µm pore size nylon mesh and sealed with a fast-setting cement (Polypipe Building Products, UK). This mesh size is fine enough to exclude liverwort rhizoids but allows the ingrowth of fungal hyphae. We perforated a fine-bore capillary tube using a needle, and installed it to run the full length of each of the cores (100 mm in length, 1.02 mm internal diameter; Portex, UK). The tubing ensured isotope was introduced and distributed evenly throughout the core volume. We sealed the capillary tube using fast-setting cement 5 mm from the bottom of the core in order to prevent excess isotope leaching from the bottom of the core.

Molecular identification of fungal associates
Wild Neohodgsonia and Allisonia were prepared for molecular analysis within one day of collection. We dissected both plant species in the same way to leave the central part of the thallus and rhizoidal ridge (2-3 mm 2 ) where fungal colonisation is highest. We performed two independent DNA extractions per plant using the method of Gardes & Bruns (1993) in combination with the QBioGene Gene-Clean kit. Genomic DNA was amplified using the 18S universal fungal primer set NS1 (White et al., 1990) and EF3 (Smit et al., 1999) (White et al., 1990) which were edited and assembled into contigs (ca. 1,600 bp) using Geneious v5.6.4 (Biomatters, NZ). An identical method was used for the sequencing of the plants used in the isotope labelling experiments.

Plant harvest and sample analyses
Upon detection of maximal belowground 14 C flux following release of 14 CO 2 , 2ml of 2 M KOH was introduced to each chamber to trap residual 14 CO 2(g) in the chamber headspace.
Soil cores were removed from pots and incubated in gas-tight containers with a further 2 ml 2 M KOH to trap 14 CO 2 emitted through respiration. Soil cores were transferred to fresh containers and KOH every two hours for a further four hours. One ml of each 'used' KOH trap was transferred to vials containing 10 ml of the scintillation cocktail Ultima Gold (Perkin Elmer, Beaconsfield, UK) and the radioactivity of each sample determined through liquid scintillation. These data were used to calculate carbon budgets for each experimental pot (see SI for equations).
Plant and soil materials were separated, freeze-dried, weighed and homogenised using a Yellowline A10 Analytical Grinder (IKA, Germany).

P transfer from fungus to plant
The 33 P transferred from fungus to plant was determined using published equations (Cameron et al., 2007): (1) Where M 33 P = Mass of 33 P (mg), cDPM = counts as disintegrations per minute, SAct = specific activity of the source (Bq mmol -1 ), Df = dilution factor and Mwt = molecular mass of P.

Carbon transfer from plant to fungus
The difference in carbon between the static and rotated cores is equivalent to the total C transferred from plant to symbiotic fungus within the soil core. Total carbon assimilated by the plant was calculated using equation 2 (Cameron et al., 2006): (2) Where T pf = Transfer of carbon from plant to fungus, A = radioactivity of the tissue sample (Bq); A sp = specific activity of the source (Bq Mol −1 ), m a = atomic mass of 14 C, P r = proportion of the total 14 C label supplied present in the tissue; m c = mass of C in the CO 2 present in the labelling chamber (g) (from the ideal gas law; Eqn. 3): ( 3) where m cd = mass of CO 2 (g), M cd = molecular mass of CO 2 (44.01 g mol -1 ) P = total pressure (kPa); V cd = volume of CO 2 in the chamber (0.003 m 3 ); R = universal gas constant (J K -1 mol -1 ); T, absolute temperature (K); m c , mass of C in the CO 2 present in the labelling chamber (g), where 0.27292 is the proportion of C in CO 2 on a mass fraction basis (Cameron et al., 2008). Figure S1. Schematic diagram of mesh-covered core (not drawn to scale).

Supplementary figures
] A B Figure S2. Illustration of experimental procedure for labelling with (a) 33 P-orthophosphate and (b) 14 CO 2. Figure S3. Phylogenetic tree showing the placement of mutualistic fungi colonising Allisonia cockaynii and Neohodgsonia mirabilis. The 18S ribosomal gene was sequenced using molecular cloning. Control plants were prepared for sequencing within one day of collection. Experimental plants (ambient or elevated CO2) were prepared for sequencing when plants were harvested at the end of the isotope labelling experiments. New sequences are highlighted in black while reference sequences from GenBank are in grey. Bayesian analysis was performed using an HKY85 model and invgamma rates with four simultaneously run heated chains (chain length: 1.1x10 6 ). The sequence of Mucoromycotina fungi from Neohodgsonia grown under elevated CO2 is not included in this tree as only a 450bp segment was produced.