The plant kingdom exhibits a diversity of nutritional strategies, extending beyond complete autotrophy. In addition to full mycoheterotrophs and holoparasites, it is now recognized that a greater number of green plants than previously assumed use partly of fungal carbon. These are termed partial mycoheterotrophs or mixotrophs. Notably, some species exhibit a dependency on fungi exclusively during early ontogenetic stages, referred to as initial mycoheterotrophy. Japonolirion osense, a rare plant thriving in serpentinite soils, emerges as a potential candidate for initial mycoheterotrophy or mixotrophy. Several factors support this hypothesis, including its diminutive sizes of shoot and and seeds, the establishment of Paris-type arbuscular mycorrhizal associations, its placement within the Petrosaviales-largely composed of fully mycoheterotrophic species-and its ability to face the challenging conditions of its environment. To explore these possibilities, our study adopts a multidisciplinary approach, encompassing stable isotope abundance analyses, in vitro experiments, anatomical analyses, and comparative plastome analyses. Our study aims to (1) determine whether J. osense relies on fungal carbon during germination, indicating initial mycoheterotrophy, (2) determine if it employs a dual carbon acquisition strategy as an adult, and (3) investigate potential genomic reductions in photosynthetic capabilities. Contrary to expectations, our comprehensive findings strongly indicate that J. osense maintains complete autotrophy throughout its life cycle. This underscores the contrasting nutritional strategies evolved by species within the Petrosaviales.

Department of Biology Graduate School of Science Kobe University 1 1 Rokkodai Nada ku Kobe 657 8501 Japan

Department of Botany University of British Columbia Vancouver BC V6T 1Z4 Canada

Department of Evolutionary and Population Biology Institute for Biodiversity and Ecosystem Dynamics University of Amsterdam Amsterdam 1098 XH The Netherlands

Department of Experimental Plant Biology Faculty of Science Charles University Viničná 5 Prague 12844 Czech Republic

Department of Mycorrhizal Symbioses Institute of Botany Czech Academy of Sciences Lesní 322 Průhonice 25243 Czech Republic

Department of Plant Taxonomy and Nature Conservation University of Gdansk Wita Stwosza 59 Gdansk 80 308 Poland

Field Science Center for Northern Biosphere Hokkaido University Forests Hokkaido University Sapporo Hokkaido 060 0811 Japan

Institut Systématique Evolution Biodiversité Muséum National d'Histoire Naturelle CNRS Sorbonne Université EPHE 57 Rue Cuvier CP39 Paris 75005 France

Institut Universitaire de France Paris France

Institute of Organic Chemistry and Biochemistry Czech Academy of Sciences Flemingovo náměstí 2 Praha Dejvice 160 00 Czechia

Naturalis Biodiversity Center Darwinweg 2 Leiden 2333 CR The Netherlands

Prague Botanical Garden Trojská 800 196 Prague 17100 Czech Republic

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BMC Plant Biol. 2024 Nov 23;24(1):1113. doi: 10.1186/s12870-024-05838-3. PubMed

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Merckx VSFT. Mycoheterotrophy: The biology of plants living on fungi. In: Mycoheterotrophy. 2013. pp. 1–356.

Wicaksono A, Mursidawati S, Molina J. A plant within a plant: insights on the development of the Rafflesia Endophyte within its host. Bot Rev. 2021;87:233–42. DOI

Fahmy GM, Hassan AH. Haustorial structure of the holoparasitic angiosperm Cynomorium Coccineum L. invading host roots. Flora: Morphology Distribution Funct Ecol Plants. 2021;274(June 2020):151731.

Yoshida S, Cui S, Ichihashi Y, Shirasu K. The Haustorium, a Specialized Invasive Organ in parasitic plants. Annu Rev Plant Biol. 2016;67:643–67. PubMed DOI

Selosse MA, Charpin M, Not F. Mixotrophy everywhere on land and in water: the grand écart hypothesis. Ecol Lett. 2017;20:246–63. PubMed DOI

Selosse MA, Petrolli R, Mujica MI, Laurent L, Perez-Lamarque B, Figura T, et al. The Waiting Room Hypothesis revisited by orchids: were orchid mycorrhizal fungi recruited among root endophytes? Ann Bot. 2022;129:259–70. PubMed DOI PMC

Selosse MA, Richard F, He X, Simard SW. Mycorrhizal networks: des liaisons dangereuses? Trends Ecol Evol. 2006;21:621–8. PubMed DOI

Rasmussen HN, Rasmussen FN. Orchid mycorrhiza: implications of a mycophagous life style. Oikos. 2009;118:334–45. DOI

Simard SW, Beiler KJ, Bingham MA, Deslippe JR, Philip LJ, Teste FP. Mycorrhizal networks: mechanisms, ecology and modelling. Fungal Biol Rev. 2012;26:39–60. DOI

Giesemann P, Rasmussen HN, Gebauer G. Partial mycoheterotrophy is common among chlorophyllous plants with Paris-type arbuscular mycorrhiza. Ann Bot. 2021;127:645–53. PubMed DOI PMC

Giesemann P, Rasmussen HN, Liebel HT, Gebauer G. Discreet heterotrophs: green plants that receive fungal carbon through Paris-type arbuscular mycorrhiza. New Phytol. 2020;226:960–6. PubMed DOI

Giesemann P, Eichenberg D, Stöckel M, Seifert LF, Gomes SIF, Merckx VSFT, et al. Dark septate endophytes and arbuscular mycorrhizal fungi (Paris-morphotype) affect the stable isotope composition of ‘classically’ non-mycorrhizal plants. Funct Ecol. 2020;34:2453–66. DOI

Gomes SIF, Merckx VSFT, Kehl J, Gebauer G. Mycoheterotrophic plants living on arbuscular mycorrhizal fungi are generally enriched in 13 C, 15 N and 2H isotopes. J Ecol. 2020;108:1250–61. DOI

Suetsugu K, Taketomi S, Tanabe AS, Haraguchi TF, Tayasu I, Toju H. Isotopic and molecular data support mixotrophy in Ophioglossum at the sporophytic stage. New Phytol. 2020;228:415–9. PubMed DOI

Suetsugu K, Matsubayashi J, Ogawa NO, Murata S, Sato R, Tomimatsu H. Isotopic evidence of arbuscular mycorrhizal cheating in a grassland gentian species. Oecologia. 2020;192:929–37. PubMed DOI

Selosse M-A, Roy M. Green plants that feed on fungi: facts and questions about mixotrophy. Trends Plant Sci. 2009;14:64–70. PubMed DOI

Jacquemyn H, Merckx VSFT. Mycorrhizal symbioses and the evolution of trophic modes in plants. J Ecol. 2019;107:1567–81. DOI

APG IV. An update of the Angiosperm Phylogeny Group classification for the orders and families of flowering plants: APG IV. Bot J Linn Soc. 2016;181:1–20. DOI

Funamoto D. Pollination biology of a rare serpentine plant, Japonolirion osense (Petrosaviaceae). Nord J Bot. 2023. 10.1111/njb.04121. DOI

Yamato M, Ogura-Tsujita Y, Takahashi H, Yukawa T. Significant difference in mycorrhizal specificity between an autotrophic and its sister mycoheterotrophic plant species of Petrosaviaceae. J Plant Res. 2014;127:685–93. PubMed DOI

Cameron KM, Chase MW, Rudall Cameron PJ. Recircumscription of the monocotyledonous family Petrosaviaceae to include Japonolirion. Brittonia. 2003;55:214–25. DOI

Nuraliev MS, Sokoloff DD, Averyanov LV, Remizowa MV. How many species are there in the monocot order Petrosaviales? Synonymization of Petrosavia amamiensis with P. Sakuraii. Phytotaxa. 2022;548:277–87. DOI

Tobe H. Embryology of Japonolirion (Petrosaviaceae, Petrosaviales): a comparison with other monocots. J Plant Res. 2008;121:407–16. PubMed DOI

Lallemand F, Martin-Magniette ML, Gilard F, Gakière B, Launay-Avon A, Delannoy É, et al. PubMed

Lallemand F, Logacheva M, Le Clainche I, Bérard A, Zheleznaia E, May M, et al. Thirteen New Plastid genomes from Mixotrophic and Autotrophic species provide insights into Heterotrophy Evolution in Neottieae Orchids. Genome Biol Evol. 2019;11:2457–67. PubMed DOI PMC

Van Waes JM, Debergh PC, Waes V. In vitro germination of some western European orchids. Physiol Plant. 1986;67:253–61. DOI

Figura T, Tylová E, Šoch J, Selosse M-A, Ponert J. In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development. Ann Bot. 2019;123:625–39. PubMed DOI PMC

Ponert J, Figura T, Vosolsobě S, Lipavská H, Vohník M, Jersáková J. Asymbiotic germination of mature seeds and protocorm development of DOI

Shapiro SS, Wilk MB. An analysis of variance test for normality (complete samples). Biometrika. 1965;52(3/4):591–611. DOI

Bartlett M. Properties of sufficiency and statistical tests. Proc R Soc Lond Math Phys Sci. 1937;160:268–82.

Soukup A, Tylová E. Essential Methods of Plant Sample Preparation for Light Microscopy. In: Clifton NJ, editor. Methods im molecular biology. Totowa, NJ: Humana; 2014. pp. 1–23. PubMed

Gutmann M, von Aderkas P, Label P, Lelu M-A. Effects of abscisic acid on somatic embryo maturation of hybrid larch. J Exp Bot. 1996;47:1905–17. DOI

Soukup A. Selected simple methods of plant cell wall histochemistry and staining for light microscopy. Plant Cell Morphogenesis: Methods Protocols. 2019:27–42. PubMed

Tillich M, Lehwark P, Pellizzer T, Ulbricht-Jones ES, Fischer A, Bock R, et al. GeSeq - Versatile and accurate annotation of organelle genomes. Nucleic Acids Res. 2017;45:W6–11. PubMed DOI PMC

Graham SW, Lam VKY, Merckx VSFT. Plastomes on the edge: the evolutionary breakdown of mycoheterotroph plastid genomes. New Phytol. 2017;214:48–55. PubMed DOI

Wicke S, Schneeweiss GM, dePamphilis CW, Müller KF, Quandt D. The evolution of the plastid chromosome in land plants: gene content, gene order, gene function. Plant Mol Biol. 2011;76:273–97. PubMed DOI PMC

Bateman A, Martin MJ, Orchard S, Magrane M, Ahmad S, Alpi E, et al. UniProt: the Universal protein knowledgebase in 2023. Nucleic Acids Res. 2023;51:D523–31. PubMed DOI PMC

Buell MF. Seed and Seedling of Acorus calamus. 1935.

Brundrett MC, Kendrick B, Peterson CA. Efficient lipid staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in polyethylene glycol-glycerol. Biotech Histochem. 1991;66:111–6. PubMed DOI

Tobe H, Takahashi H. Embryology of Petrosavia (Petrosaviaceae, Petrosaviales): evidence for the distinctness of the family from other monocots. J Plant Res. 2009;122:597–610. PubMed DOI

Yeung EC. A perspective on orchid seed and protocorm development. Bot Stud. 2017;58:1–14. PubMed DOI PMC

Eriksson O, Kainulainen K. The evolutionary ecology of dust seeds. Perspect Plant Ecol Evol Syst. 2011;13:73–87. DOI

Martin JN. Botany for agricultural students. Wiley; 1919.

Leake J. The biology of myco-heterotrophic (‘saprophytic’) plants. New Phytol. 1994;127:171–216. PubMed DOI

Penzig OAJ. Beiträge Zur Kenntniss Der Gattung Epirrhizanthes Bl. Ann Du Jardin Botanique De Buitenzorg. 1901;17:142–70.

Verkerke W. Ovules and seeds of the Polygalaceae. J Arnold Arboretum. 1985;66:353–94. DOI

Gebauer G, Preiss K, Gebauer AC. Partial mycoheterotrophy is more widespread among orchids than previously assumed. New Phytol. 2016;211:11–5. PubMed DOI

Gebauer G, Meyer M. 15 N and 13 C natural abundance of autotrophic and myco-heterotrophic orchids provides insight into nitrogen and carbon gain from fungal association. New Phytol. 2003;160:209–23. PubMed DOI

Courty PE, Walder F, Boller T, Ineichen K, Wiemken A, Rousteau A, et al. Carbon and nitrogen metabolism in mycorrhizal networks and mycoheterotrophic plants of tropical forests: a stable isotope analysis. Plant Physiol. 2011;156:952–61. PubMed DOI PMC

Murata-Kato S, Sato R, Abe S, Hashimoto Y, Yamagishi H, Yokoyama J, et al. Partial mycoheterotrophy in green plants forming Paris-type arbuscular mycorrhiza requires a thorough investigation. New Phytol. 2022;234:1112–8. PubMed DOI

Merckx V, Stöckel M, Fleischmann A, Bruns TD, Gebauer G. 15 N and 13 C natural abundance of two mycoheterotrophic and a putative partially mycoheterotrophic species associated with arbuscular mycorrhizal fungi. New Phytol. 2010;188:590–6. PubMed DOI

Matsuda Y, Yamaguchi Y, Matsuo N, Uesugi T, Ito J, Yagame T, et al. Communities of mycorrhizal fungi in different trophic types of Asiatic Pyrola Japonica Sensu lato (Ericaceae). J Plant Res. 2020;133:841–53. PubMed DOI

May M, Jąkalski M, Novotná A, Dietel J, Ayasse M, Lallemand F, et al. Three-year pot culture of Epipactis helleborine reveals autotrophic survival, without mycorrhizal networks, in a mixotrophic species. Mycorrhiza. 2020;30:51–61. PubMed DOI

Minasiewicz J, Zwolicki A, Figura T, Novotná A, Bocayuva MF, Jersáková J et al. Stoichiometry of carbon, nitrogen and phosphorus is closely linked to trophic modes in orchids. BMC Plant Biol. 2023;23. PubMed PMC

Mennes CB, Smets EF, Moses SN, Merckx VSFT. New insights in the long-debated evolutionary history of Triuridaceae (Pandanales). Mol Phylogenet Evol. 2013;69:994–1004. PubMed DOI

Ramírez-Barahona S, Sauquet H, Magallón S. The delayed and geographically heterogeneous diversification of flowering plant families. Nat Ecol Evol. 2020;4:1232–8. PubMed DOI

Correction: Japonolirion osense, a close relative of the mycoheterotrophic genus Petrosavia, exhibits complete autotrophic capabilities