ARTICLE Received 7 Oct 2011 | Accepted 10 Apr 2012 | Published 15 May 2012 DOI: 10.1038/ncomms1831 Contrasting arbuscular mycorrhizal responses of vascular and non-vascular plants to a simulated Palaeozoic CO2 decline Katie J. Field1, Duncan D. Cameron1, Jonathan R. Leake1, Stefanie Tille1, Martin I. Bidartondo2,3 & David J. Beerling1 The arbuscular mycorrhizal (AM) fungal symbiosis is widely hypothesized to have promoted the evolution of land plants from rootless gametophytes to rooted sporophytes during the mid-Palaeozoic (480–360 Myr, ago), at a time coincident with a 90% fall in the atmospheric 14 CO2 concentration ([CO2]a). Here we show using standardized dual isotopic tracers ( C and 33P) that AM symbiosis efficiency (defined as plant P gain per unit of C invested into fungi) of liverwort gametophytes declines, but increases in the sporophytes of vascular plants (ferns and angiosperms), at 440 p.p.m. compared with 1,500 p.p.m. [CO2]a. These contrasting responses are associated with larger AM hyphal networks, and structural advances in vascular plant water- conducting systems, promoting P transport that enhances AM efficiency at 440 p.p.m. [CO2]a. Our results suggest that non-vascular land plants not only faced intense competition for light, as vascular land floras grew taller in the Palaeozoic, but also markedly reduced efficiency and total capture of P as [CO2]a fell. 1 Department of Animal and Plant Sciences, University of Sheffield, S10 2TN, UK. 2 Department of Biology, Imperial College London, SW7 2AZ, UK. 3 Jodrell Laboratory, Royal Botanic Gardens, Kew TW9 3DS, UK. Correspondence and requests for materials should be addressed to K.J.F. (e-mail: [email protected]). NatURE COMMUNicatiONS | 3:835 | DOI: 10.1038/ncomms1831 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. ARTICLE NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1831 rbuscular mycorrhizal (AM) fungi are among the most Devonian Rhynie chert flora, together with exceptionally well-pre- ancient and widespread obligate symbionts in contempo- served spores representative of several of the major glomeralean Arary floras, and are dependent on host plants for carbon, as fungal groups8,9,10,11,12. These associations with AM fungi formed 1 they lack saprotrophic capabilities . AM fungi are highly adapted to under a CO2-rich atmosphere, even before true roots evolved mobilize and translocate phosphorus (P) from beyond root deple- around 400 Myr, ago (Fig. 1). tion zones and from pores too small for entry by root hairs, in return The subsequent diversification of embryophytes in the mid- for receiving photosynthate carbon1. Physiological adaptations Palaeozoic (480–360 Myr, ago) is marked by the transition from a include phosphate transporters within hyphal membranes with gametophyte-dominant generation lifecycle to one with an increas- very high affinities for soil P, and phosphate transporters located in ingly elaborate sporophyte-dominant generation, and the evolution- arbuscules that transfer P from the fungus to the plant cortex cells2. ary appearance of roots, leaves and other innovations13. As terrestrial Phylogenetic relationships of mycorrhiza-specific genes coding for plants and ecosystems evolved, they progressively grew in stature, AM-inducible plant P transporters indicate their involvement in biomass, nutrient demand and rooting depth to extensively colonize symbiotic P transport for hundreds of millions of years, with highly uplifted continental land masses. The resulting increase in below- conserved regulation of gene expression2. The basis of the AM mutu- ground export of photosynthate carbon energy drove enhanced alism involves a stable cooperation between partners. Host plants silicate rock weathering14, the development of soil profiles, pedog- preferentially reward fungal partners that provide greater access to enic clay minerals15,16,17 and organic carbon burial18,19. Enhanced P with photosynthate, and the fungi allocate P preferentially to plant biological weathering from 410–340 Myr, ago is linked to a 90% 3 partners providing a greater carbohydrate supply . reduction in the atmospheric CO2 concentration ([CO2]a) through Phosphorus is an essential element required by plants for core the release of Ca and Mg from silicate minerals, and their export to metabolism (ATP) and growth (phospholipid membranes, RNA the oceans where they were precipitated into carbonate sediments and DNA), and its low availability in soils is often a primary (Fig. 1)18,20. During the mid Palaeozoic, co-evolution of the AM constraint on the productivity of terrestrial ecosystems4. This fungal symbiosis and rooting systems likely had a pivotal role in requirement has driven the establishment of symbiotic partnerships provisioning the expanding biomass, stature and biogeography of between soil-dwelling fungi and the earliest land plants (embryo- forested ecosystems with their rising demand for essential soil nutri- phytes) to increase the uptake of P ever since plants’ initial con- ents. This would be especially important as P became increasingly quest of the land5,6,7. Intercellular hyphae and arbuscules have been limited owing to depletion of the primary mineral apatite (calcium documented in gametophytes and sporophytes from the Early phosphate) by chemical and biological weathering4,21 (Fig. 1). a Angiosperms*†Vascular plants Gymnosperms* Plantago lanceolata Roots Ferns*† and 385–400 Myr, ago horsetails Lycopods* Gametophyte Hornworts*† Bryophytes Osmunda regalis (1n) AM fungi Sporophyte 445–450 Myr, ago Mosses (2n) Liverworts*† 445–450 Myr, ago Preissia quadrata Charophytic algae 400 300 200 100 0 Time (Myr, ago) b 4,000 Marchantia paleacea 3,000 ] (p.p.m.) 2 2,000 1,000 Atmospheric [CO 0 400 300 200 100 0 Time (Myr, ago) Figure 1 | Co-evolution of land plant lifecycles and atmospheric CO2 concentrations. (a) depicts land plant phylogeny (adapted from (refs. 47,48) and (b) the modelled global [CO2]a history (adapted from (ref. 24)) for the past 500 Myr, ago. Mapped onto the phylogeny is the occurrence of AM fungal associations across plant taxa*, and fossil evidence of AM fungi†. Species investigated in this work are highlighted in bold and pictured right. Dominant stages of plant life cycles are shown as 1 n (gametophyte) and 2 n (sporophyte). NatUre cOMMUNicatiONS | 3:835 | DOI: 10.1038/ncomms1831 | www.nature.com/naturecommunications © 2012 Macmillan Publishers Limited. All rights reserved. NatUre cOMMUNicatiONS | DOI: 10.1038/ncomms1831 ARTICLE Here we investigate the hypothesis that the evolutionary transi- Geosiphon pyriformis X86656 Archaeospora leptoticha AJ006466 tion from non-vascular to vascular embryophytes with sporophyte- 70 Archaeospora trappei AJ006800 dominant lifecycles as Palaeozoic [CO2]a declined, drove changes in Paraglomus brasilianum AJ301862 the plant–fungal cost benefits of the AM symbiosis. Our approach is 100 Paraglomus occultum AJ006899 33 14 Glomus claroideum AJ276075 based on the development and application of dual P and C isotope Glomus claroideum AJ276080 tracers to measure the reciprocal fluxes of carbon from plant-to-fun- Osmunda regalis gus, and phosphorus from fungus-to-plant. These fluxes are quan- Glomus etunicatum AJ852598 98 tified using mesh-walled cores filled with soil set around the plants Preissia quadrata that can be colonized by AM hyphae but not plant roots or rhizoids. Glomus mosseae Z14007 Glomus coronatum AJ276086 Static cores preserve intact hyphal networks running into the core, 99 Glomus verruculosum AJ301858 whereas rotated cores severe the network. AM symbiosis efficiency Glomus geosporum AJ245637 Glomus caledonium Y17635 is defined as P gained by the plant through its fungal partners per Glomerales 89 Glomus caledonium Y17653 unit of photosynthate carbon invested into the P absorbing hyphal Glomus sinuosum AJ133706 network in the undisturbed mesh cores. This novel metric allows Glomus manihotis Y17648 Glomus clarum AJ852597 85 AM symbiosis efficiency to be determined for whole plants growing 90 Glomus clarum AJ276084 in natural soil, and facilitates comparisons across taxa and environ- Glomus proliferum AF213462 Glomus intraradices AY635831 ments. Experiments were conducted with plant species represent- 70 ing three key nodes on the land plant evolutionary tree spanning several hundred million years of terrestrial ecosystem evolution 83 (Fig. 1). These nodes encompass the critical transition from non- Preissia quadrata vascular plant gametophyte-dominant to vascular plant sporo- 13 phyte-dominant lifecycles and appearance of true roots . We 76 investigated two species of basal non-vascular land plants (complex 87 thalloid liverworts) with rhizoid systems (Preissia quadrata and Glomus fasciculatum Y17640 Glomus sp. AJ301857 Marchantia paleacea), grown either as prostrate mats of gameto- 94 Preissia quadrata phytes (P. quadrata), or as individual gametophytes (M. paleacea). Plantago lanceolata Both species of liverworts belong to the phylogenetic sub-group of 22 Glomus versiforme X86687 Marchantiopsida . Liverwort gametophyte [CO2]a responses were compared with those of the sporophytes of the basal vascular land plant Osmunda regalis, a fern with a well-developed rooting sys- Osmunda regalis tem, and the advanced angiosperm Plantago lanceolata, which has 93 Diversispora spurca Y17650 a coarse rooting system and high mycorrhizal dependency. DNA Osmunda regalis sequencing of the fungal partners within host root/thallus samples Acaulospora longula AJ306439 of