Rhizophagus irregularis, the model fungus in arbuscular mycorrhiza research, forms dimorphic spores Vasilis Kokkoris1,2,3 , Claudia Banchini2, Louis Par�e4, Lobna Abdellatif2 , Sylvie S�eguin2, Keith Hubbard2, Wendy Findlay2, Yolande Dalp�e2 , Jeremy Dettman2, Nicolas Corradi1 and Franck Stefani2 1Department of Biology, University of Ottawa, Ottawa, ON, K1N 6N5, Canada; 2Agriculture and Agri-Food Canada, Ottawa Research and Development Centre, Ottawa, ON, K1A 0C6, Canada; 3Amsterdam Institute for Life and Environment (A-LIFE), Faculty of Science, Section Systems Ecology, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV, Amsterdam, the Netherlands; 4Universit�e Laval, Centre d’�Etude de la Forêt (CEF) and Institut de Biologie Int�egrative et des Syst�emes (IBIS), 1030 Ave de la M�edecine, Qu�ebec, QC, G1V 0A6, Canada Author for correspondence: Franck Stefani Email: franck.stefani@agr.gc.ca Received: 4 May 2023 Accepted: 10 June 2023 New Phytologist (2023) doi: 10.1111/nph.19121 Key words: arbuscular mycorrhizal fungi, glomalin gene, morphology, Rhizophagus irregularis, spore dimorphism, spore wall layers, SSU-ITS-LSU rDNA region. Summary � Rhizophagus irregularis is the model species for arbuscular mycorrhizal fungi (AMF) research and the most widely propagated species for commercial plant biostimulants. � Using asymbiotic and symbiotic cultivation systems initiated from single spores, advanced microscopy, Sanger sequencing of the glomalin gene, and PacBio sequencing of the partial 45S rRNA gene, we show that four strains of R. irregularis produce spores of two distinct mor- photypes, one corresponding to the morphotype described in the R. irregularis protologue and the other having the phenotype of R. fasciculatus. � The two spore morphs are easily distinguished by spore colour, thickness of the subtending hypha, thickness of the second wall layer, lamination of the innermost layer, and the dextri- noid reaction of the two outer spore wall layers to Melzer’s reagent. The glomalin gene of the two spore morphs is identical and that of the PacBio sequences of the partial SSU-ITS-LSU region (2780 bp) obtained from single spores of the R. cf fasciculatus morphotype has a med- ian pairwise similarity of 99.8% (SD = 0.005%) to the rDNA ribotypes of R. irregularis DAOM 197198. � Based on these results, we conclude that the model AMF species R. irregularis is dimorphic, which has caused taxonomic confusion in culture collections and possibly in AMF research. Introduction Arbuscular mycorrhizal fungi (AMF, Glomeromycotina) are widespread soil fungi that form symbiotic associations with 72% of vascular plants (Brundrett & Tedersoo, 2018). Compared with Ascomycota or Basidiomycota, the species richness within Glomeromycotina is limited with c. 350 species described to date (amf-phylogeny.com). Despite this low species richness, AMF taxonomy is not a trivial endeavour, as several species have been described mainly on the basis of spore anatomical characters alone. Phylogenetic analyses have shown that these characters may be unreliable due to convergence in spore evolution and architecture, phenotypic plasticity, or dimorphism. Spore developmental and morphological plasticity, which can be defined as the natural variation in size, shape, and colour of genetically identical spores in response to environmental factors, are found throughout the AMF phylogeny. For example, the spore morphological characters used to discriminate R. intrara- dices from R. irregularis can vary with the propagation technique (in vivo or in vitro system), host species, and topology of spore production (intraradical vs extraradical; Walker et al., 2021). Since these characters are similarly variable for both species, DNA sequencing is required to separate them. Sequencing of the partial SSU-ITS-LSU regions of the nuclear ribosomal DNA (rDNA) allowed to correct the misidentification of the AMF strain ‘Glomus intraradices DAOM 197198’, which showed its conspecificity with Glomus irregulare (Stockinger et al., 2009). Spores of Rhizophagus clarus (syn. Glomus clarum Nicolson & Schenck) are another example of phenotypic plasticity, as they can vary considerably in size (100–240 lm) and colour (Benti- venga et al., 1997). Spore phenotypic variation has also been observed between single-spore cultures of Scutellospora pellucida with progeny spores exhibiting contrasting and heritable shapes (Bever & Morton, 1999). By contrast, spore dimorphism is less common in AMF. Beyond the morphological variation that falls under the defini- tion of spore plasticity, dimorphism usually involves different ontogenies leading to different spore types (i.e. acaulosporoid, entrosphosporoid, gigasporoid, glomoid, and scutellosporoid) or to sporocarps. For example, the spores of Glomus ambisporum and Glomus heterosporum were described as being dimorphic (two different glomoid morphs) depending on whether they are pro- duced in sporocarps or within roots (Smith & Schenck, 1985). Dimorphism has also been identified within AM fungal species producing acaulosporoid and glomoid spores: Within the genus Ambispora, individuals produce either glomoid spores (spheroidal � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) 1 www.newphytologist.com This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Research https://orcid.org/0000-0002-1667-0493 https://orcid.org/0000-0002-1667-0493 https://orcid.org/0000-0001-8221-0770 https://orcid.org/0000-0001-8221-0770 https://orcid.org/0000-0002-4807-5197 https://orcid.org/0000-0002-4807-5197 https://orcid.org/0000-0002-7932-7932 https://orcid.org/0000-0002-7932-7932 https://orcid.org/0000-0002-6025-2192 https://orcid.org/0000-0002-6025-2192 mailto:franck.stefani@agr.gc.ca http://amf-phylogeny.com http://creativecommons.org/licenses/by-nc-nd/4.0/ http://crossmark.crossref.org/dialog/?doi=10.1111%2Fnph.19121&domain=pdf&date_stamp=2023-07-11 or ovoid only) or both glomoid and acaulosporoid spores (spores produced either laterally or terminally within a saccule, see Walker et al., 2018 for more details). Each morphotype could be considered a distinct species based on morphological assessment alone, and in Ambispora gerdemannii, the glomoid morph was indeed first described as Glomus leptotichum (Shenck & Smith, 1982) while the acaulosporoid morph was described as Acaulospora gerdemannii (Nicolson & Schenck, 1979), before being merged into a new dimorphic species named Ambispora gerdemanii (Walker Demircik et al., 2007). Similarly, Acaulospora spinosa produces acaulosporoid spores along with small, delicate, and colourless glomoid spores (Taylor et al., 2014). Other types of dimorphism exist as both entrophosporoid and glomoid morphs have recently been observed in ‘single-species cultures’ [sic] of Entrophospora infrequens (Błaszkowski et al., 2022). Awareness of spore morphological plasticity and dimorphism has increased with advances in DNA barcoding, but ironically, molecular identification of AMF based on sequencing of the SSU-ITS-LSU region of the 45S rRNA gene may lead to taxo- nomic confusion. Specifically, the rRNA genes are not organized in tandem repeats with hundreds of identical or very similar sequences as in other fungi (Maeda et al., 2018). Rather, up to 10 rRNA genes with distinct sequences (pairwise similarity = 92.6–100% for the partial SSU-ITS-LSU regions) are present within the R. irregularis DAOM 197198 genome and are located on four chromosomes (Yildirir et al., 2022; Spersch- neider et al., 2023). This intragenomic rDNA heterogeneity can mislead molecular identification; a situation well-illustrated by the description of R. venetianum (Turrini et al., 2018), which was described as a new species based on only one of the multiple ribo- types of R. irregularis (Walker et al., 2021). Thus, phenotypic plasticity, dimorphism, and intragenomic rDNA heterogeneity make AMF taxonomy, species recognition, and inoculum purity a challenging endeavour. Here, we set out to investigate the possibility that the model AMF species R. irregularis is also dimorphic. We found that three strains of R. irregularis in in vivo cultures held at the Canadian Col- lection of Arbuscular Mycorrhizal Fungi (CCAMF) produce spores of two morphs, morph-1 corresponding to the morphotype described in the protologue of R. irregularis and morph-2 having the appearance of R. fasciculatus. The species identity of both morphs was investigated using in vivo and in vitro cultures with light microscopy, scanning, and transmission electron microscopy, as well as with Sanger sequencing of the glomalin gene, and PacBio sequencing of the partial 45S rRNA gene. We also demonstrated that single-spore cultures inoculated with spores of R. cf fasciculatus and R. irregularis (DAOM 197198) in modified M medium con- taining myristate and on a superabsorbent polymer in an auto- trophic cultivation system also produce dimorphic spores. Materials and Methods All methodological steps taken to investigate the possibility of dimorphism in R. irregularis (Błaszk., Wubet, Renker & Buscot) C. Walker & A. Sch€ußler are summarized in Supporting Infor- mation Fig. S1 and detailed below. In vivo and in vitro cultures Spores with the appearance of R. fasciculatus (morph-2) were recorded in three in vivo cultures (pot culture ID 562E, 864D, and 1184D) initially initiated with environmental soil samples collected in Qu�ebec (Les Îles-de-la-Madeleine, Outaouais) and Prince Edward Island (Table 1). The environmental soil was combined with an autoclaved (two cycles of 1 h at 121°C over 2 d) mixture of montmorillonite clay (turface) and vermiculite (1 : 1 ratio) in a 75 mm diameter plastic pot. Plantago maritima (infrutescences collected from Anse St-Denis, Rivi�ere-Ouelle, QC, Canada) or Allium porrum (Giant Musselburgh variety, Vesey’s Seeds Ltd, York, PEI, Canada) were used as hosts. Each pot was fertilized monthly with 20 ml of Long Ashton solution (Hewitt & Commonwealth Bureau of Horticulture and Planta- tion Crops, 1966) and watered as needed. In vivo cultures were individually enclosed in a sun bag (Sigma-Aldrich) to avoid cross-contamination (Walker & Vestberg, 1994). The in vivo cultures were grown in the glasshouse (22°C, 15 h light, photo- synthetic photon flux density was 100 lMol m�2 s�1 on top of the sun bags) and subcultured every 20 months: roots and c. 60 g of soil from the previous in vivo culture were inoculated into a new in vivo culture containing sterile 1 : 1 mixture of montmoril- lonite clay and vermiculite and a new host plant seedling. Differ- ent taxa were observed in the first subcultures, including spores morphologically related to R. irregularis and R. cf fasciculatus. Spores of R. cf fasciculatus were isolated by wet sieving c. 15 g of soil (stacked sieves, mesh sizes of 500, 300, 150, and 38 lm). Clusters of 10–20 spores were separated under an Olympus SZX10 stereomicroscope (Olympus, Toronto, ON, Canada). The morphological and molecular data obtained from the spores isolated in the in vivo cultures were compared with the morphological and molecular data obtained from the spores of R. irregularis (DAOM 197198). The fungus was cultivated under monoxenic conditions in association with Ri T-DNA trans- formed Daucus carota on modified Strullu–Romand medium (Diop, 1995; Declerck et al., 1996) and in pot cultures as described previously. Single-spore cultures Asymbiotic and symbiotic culture systems inoculated with a sin- gle spore were used to demonstrate the spore dimorphism of R. irregularis. Myristate was used to enable asymbiotic cultivation and spore production for R. irregularis (Sugiura et al., 2020). Specifically, five Petri dishes (VWR, 100 mm9 15 mm) filled with modified minimal (M) medium and myristate (to be described later) were radially inoculated with eight spores each of R. cf fasciculatus from the 562E, 864D, and 1184D cultures, with sufficient distance between spores to avoid physical contact after germination. Spores were surface sterilized as follows: 5 min in sterile water; 3 min in 2% Chloramine T with 2 drops of Tween 20; 5 min in sterile water repeated twice; and 2 min in streptomy- cin (200 mg l�1) and gentamicin (100 mg l�1) antibiotic solu- tion. The culture medium was 2% M medium (B�ecard & Fortin, 1988) solidified with Phytagel and supplemented with New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist2 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 0.5 mM Myr-K (in the form of potassium salt of myristic acid dissolved in alcohol) after autoclaving the M medium. Cultures were incubated for 3 months and checked periodically for con- tamination. Four weeks after the size of the daughter spores had stabilized, the spores were extracted by liquefying the medium with citrate buffer (Doner & B�ecard, 1991). The mother spores of R. cf fasciculatus together with their respective daughter spores were then transferred to microscope slides for observation by light microscopy. The superabsorbent polymer (SAP)-based autotrophic system was used for symbiotic cultures, (Par�e et al., 2022). The polymer was a poly(acrylamide-co-acrylic acid) potassium salt, and it is marketed as HORTA-SORB® MD Granule by Horticultural Alliance (Sarasota, FL, USA). A total of 28 cultures were inocu- lated with a single spore of R. irregularis DAOM 197198 (com- mercial product Agtiv Potato L, Premier Tech, Rivi�ere-du-Loup, QC, Canada). Two Petri dishes were not inoculated as negative controls. After 5 months, colonized grains of SAP were randomly selected from 5/28 cultures and used to inoculate 10 new SAP- based autotrophic cultures. Two Petri dishes were not inoculated as negative controls. After 2 months of growth, spores were har- vested for observations by microscopy. Microscopy and statistical analyses Different microscopy techniques were used to analyse and com- pare the spore surface, number of spore layers, and wall architec- ture of each spore morph. Light microscopy was used to study spore wall structure and the reaction to Melzer’s reagent. We used the terminology proposed by Walker (1983) to describe the wall structure. Scanning and transmission electron microscopy was also used to better characterize the spore wall structure observed by light microscopy. Finally, spores were subjected to confocal microscopy to assess the presence of nuclei in each spore morph. Optical microscopy Crushed spores were mounted on slides in polyvinyl alcohol-lactic acid-glycerol (PVLG) and PVLG + Mel- zer’s reagent (1 : 1). The morphology of the spores was observed and recorded using a Nikon Eclipse 800 (Nikon Instruments Inc., Melville, NY, USA) compound microscope equipped with Nomarski differential interference-contrast optics. Photographs were taken with a Nikon DS-Ri2 high-resolution colour camera and analysed using NIS-ELEMENTS BR v.4.6 analysis software (Nikon Instruments Inc., Tokyo, Japan). Variances of subtend- ing hyphae widths of morph-1 and morph-2 were assessed by F-test, and mean differences between the two morphs were com- pared by Welch’s t-test in R v.4.2.3 (R Core Team, 2023). Scanning electron microscopy (SEM) Forty spores with the R. cf fasciculatus morph per subculture and many spores of the R. irregularis morph were fixed in 2.5% glutaraldehyde and 4% paraformaldehyde in 0.1M Na cacodylate buffer (pH 7.2) for 48 h at 35°C. Spores were then washed in buffer and postfixed in 1% osmium in 0.1M Na cacodylate buffer (pH 7.2) for 90 min at 35°C. Samples were then washed 29 15min in cacodylate buf- fer and dehydrated in 10%, 30%, 50%, 70%, 80%, 90%, 95%, and 29 100% ethanol solution for 1 h at room temperature for Table 1 In vivo and in vitro cultures analysed in this study. Culture no. Collecting no. Collector Collecting date Collection site Subculture starting date Subculture analysis date Field host plant Culture host plant 864D 4397 S. S�eguin, Y. Dalp�e 22 August 2000 Canada, Qu�ebec, Les Îles-de-la-Madeleine, Havre-Aubert 28 June 2018 16 December 2019 Ammophila breviligulata Plantago maritima 1184D 4674–78 S. S�eguin, Y. Dalp�e 2 September 2000 Canada, Prince Edward Island, Chelton Beach National Park 20 July 2017 14 February 2019 Ammophila breviligulata Plantago maritima 562E 4835 S. S�eguin, Y. Dalp�e 9 July 2011 Canada, Qu�ebec, Outaouais, La Petite- Nation 28 June2018 2 November 2019 Pisum sativum Plantago maritima 2253Aa – C. Plenchette, V. Furlan 1987-07 Canada, Qu�ebec, Pont-Rouge 30 April 2015 6 February 2017 – Plantago maritima 57-9b – C. Plenchette, V. Furlan 1987-07 Canada, Qu�ebec, Pont-Rouge 8 September 2021 10 May 2021 – Plantago lanceolata IVT23c – C. Plenchette, V. Furlan 1987-07 Canada, Qu�ebec, Pont- Rouge – – – Carrot ROCd IVT89e 4375 Y. Dalp�e 21 August 2000 Canada, Qu�ebec, Les Îles-de-la-Madeleine – – Ammophila breviligulata Carrot ROC The collecting date corresponds to the field sampling date of the environmental soil sample. The letter following the culture number indicates the subculture, that is ‘D’ means the fourth subculture. aIn vivo culture of R. irregularis DAOM 197198 (MUCL 43203). bIn vivo culture of R. irregularis DAOM 197198 (commercial product Agtiv Potato L, Premier Tech). cIn vitro culture of R. irregularis DAOM 197198 (MUCL 43203). dRoot organ culture. eIn vitro culture of R. irregularis DAOM 234181. � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) www.newphytologist.com New Phytologist Research 3 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense each step and overnight at 4°C for the final 100% ethanol step, and critical point dried (Biodynamics Research Corp., Rockville, MD, USA). During the critical point drying, spores were wrapped in lens optics tissue cleaning paper. After critical point drying, the spores were carefully unwrapped in a glass Petri dish to avoid static electricity during spore handling. Spores were care- fully cracked or broken in half with fine metal tweezers, mounted on carbon double-stick tape on aluminium stubs, and coated with an 8 nm layer of gold in an Emitech K550V sputter coater (EM Technologies Ltd, Ashford, Kent, UK). Samples were imaged on a Quanta 600 SEM operating at 20 kV (FEITM Co., Brno, Czech Republic). Transmission electron microscopy (TEM) Similarly to the first steps of the SEM protocol, 40 spores of the R. cf fasciculatus morph per subculture and many spores of the R. irregularis morph were fixed in 2.5% glutaraldehyde and 4% paraformalde- hyde in 0.1M Na cacodylate buffer (pH 7.2) for 48 h at 35°C. The spores were washed in buffer and postfixed in 1% osmium in 0.1 M Na cacodylate buffer (pH 7.2) for 90 min at 35°C. The spores were then placed in 3% agar solution (1.5 g in 50 ml dis- tilled water) and maintained at 50°C during the process. A fine coating of warm agar was applied to a microscope slide with a Pasteur pipette, and air bubbles were created with a pipette. After solidification, the top layer of each air bubble was removed with a scalpel to create wells for the spores. A few spores were placed in each agar well, and warm 4% agar was poured to coat the spores and seal the wells. After solidification, small squares of agar were cut with a scalpel and each square of agar was transferred to a 2 ml plastic tube. The samples were washed 29 15 min in buf- fer and dehydrated in 10%, 30%, 50%, 70%, 80%, 90%, 95%, and 29 100% ethanol solution for 1 h for each step at room temperature. The samples were then infiltrated with London Resin (LR) White: Ethanol (Electron Microscopy Sciences, Hat- field, PA, USA) 1 : 2 for 24 h, 1 : 1 overnight, 3 : 1 for 24 h, 100% resin for 24 h, and 100% resin overnight and then poly- merized in capsules at 60°C for 48 h. Seventy-nanometre thick sections were cut with a diamond knife and a Leica UC7 ultra- microtome (Leica Microsystems, Vienna, Austria) and collected on formvar and carbon-coated copper grids (Electron Micro- scopy Sciences). Sections were stained with 5% (w/v) uranyl acet- ate for 12 min, rinsed with H2O, blotted dry, stained with Reynold’s lead citrate for 5 min, then rinsed with H2O, and air dried. Sections were analysed with a Hitachi H-7000 TEM (Hitachi, Tokyo, Japan) equipped with an ORIUS SC200 digital camera using DIGITAL MICROGRAPH software v.1.8.3 (Gatan Inc., Pleasanton, CA, USA). Confocal microscopy We examined the nuclear content of R. cf fasciculatus and R. irregularis spores using previously published protocols (Kokkoris et al., 2020, 2021). Briefly, 20 spores with the R. cf fasciculatus morph per subculture and many spores of the R. irregularis were stained with 2% (v/v) SytoGreen 13 live– water solution (Invitrogen) for 1 h at 35°C. The stained spores were mounted in 80% water–glycerol solution and were imaged using a Zeiss LSM800 Airyscan CLSM confocal microscope (Carl Zeiss MicroImaging, G€ottingen, Germany), and the images were processed using ZEN v.3.1 black edition software. For image acquisition, an excitation wavelength of 488 nm and a detection wavelength of 400–600 nm were used with the GaAsP-Pmt1 detector and a Plan-Apochromat 630 NA 1.4 oil objective. For each spore, multiple z-stacks were acquired at 0.35 lm intervals with manually adjusted depth brightness. A pseudocolour along the z-axis was applied to the stacked three-dimensional image of each spore for depth recognition, and the coloured image was finally projected into two dimensions. DNA extraction To rigorously link the spore morphology to DNA sequences, individual spores were simultaneously analysed by DNA sequen- cing and optical microscopy. Fifteen spores of morph-2 were ana- lysed for each in vivo culture 864D, 562E, and 1184D. Specifically, individual spores were crushed in PCR tubes to col- lect the nuclei, and the spore debris were recovered for morpholo- gical analysis. Spores recovered from wet sieving or from the SAP-based autotrophic system were individually transferred to a sterile 0.2 ml microtube containing 5 ll of sterile water. Each spore was gently crushed to release the nuclei using a fine metal needle or a glass Pasteur pipette manually shaped over a flame to fit the shape of the microtube bottom. Tubes were vortexed and centrifuged for 5 s. The disrupted spore was transferred to a glass slide for morphological analysis. The remaining volume (4 ll and 2 ll for each target) was used to amplify the gene encoding the protein glomalin (c. 645 bp) and the SSU-ITS-LSU regions of the rRNA gene (c. 2700 bp). Glomalin amplification and Sanger sequencing Amplification was performed in 10 ll of reaction mix as follows: 6.82 ll PCR-grade water, 1 ll of 109 Titanium Taq PCR Buffer (Takara Bio Inc., Mountain View, CA, USA), 100 lM of each dNTP, 1 lg of bovine serum albumin (Thermo Fisher Scientific, Waltham, MA, USA), 80 nM of each primer, 0.1 ll of Titanium Taq Polymerase (Takara Bio Inc.), and 1 ll of gDNA. A nested PCR was performed to amplify the glomalin gene: the primer sets Glml-116F1 (50-ATCTWCGTCGYGGWGTTC-30)/Glml- 1082R1 (50-GCHGCWCGWGTDGCATT-30) and Glml-265- F2 (50- GCHATGGAAAAAGTHGG -30)/Glml-983-R2 (50- GTDGCATTYAARGCRTC-30) were used for the first round and second round of PCR, respectively. [Correction added on 16 November 2023, after first online publication: the first primer sequence listed for the primer set Glml-116F1 has been updated.] The primer set Glml-116F1/Glml-1082R1 are degenerate ver- sions of the primers Glom-Rout and Glom-Fout, respectively (Magurno et al., 2019). Thermocycling conditions for the first round of PCR were as follows: an initial denaturation step of 94°C for 3 min followed by 40 cycles at 94°C for 30 s, 56°C for 45 s, 72°C for 90 s, and a final extension step performed at 72°C for 7 min. Samples were then held at 10°C. The same thermocy- cling conditions were used for the second round of PCR, except for the annealing temperature, which was lowered to 55°C for New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist4 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense 45 s. Amplicons were separated by electrophoresis on a 1.5% agarose gel stained with GelRed® (1 : 10 000; Biotium Inc., Fre- mont, CA, USA) at 60 V for 40 min and visualized using the Gel-Doc system (Bio-Rad Laboratories, Mississauga, ON, Canada). Big Dye DNA sequencing, reactions were as follows: 1 ll of each amplicon was mixed with 8.5 ll of 1 : 8 BDT sequencing mix (BrilliantDyeTM Terminator (v3.1) Cycle Sequen- cing Kit, NimaGen Inc., Nijmegen, The Netherlands) and 0.5 ll of either primer Glml-265-F2 or Glml-983-R2 (3.2 lM). For each reaction, the 1 : 8 BDT sequencing mix contained 3.75 ll of sterile filtered H2O (BioShop, Burlington, ON, Canada), 2.5 ll of 20% D-(+)-Trehalose (BioShop), 1.75 ll of 59 sequencing buffer (Thermo Fisher Scientific), and 0.5 ll of BIG DYE TERMI- NATOR v.3.1 (Thermo Fisher Scientific). Thermocycling condi- tions were as follows: 95°C for 3 min; 40 cycles at 95°C for 30 s, 45°C for 15 s, and 60°C for 2 min. Samples were then put on hold at 10°C. Sanger sequencing was performed on a 3500xl Genetic Analyzer at the Molecular Technologies Laboratory of Agriculture and Agri-Food Canada in Ottawa (ON, Canada). Partial 45 rRNA gene amplification and PacBio sequencing The PacBio sequencing technology was used to amplify and sequence the SSU-ITS-LSU regions. Four spores of morph-2 were isolated from each in vivo culture 562E, 864D, and 1184D. DNA was isolated from single spores as described previously. Two microliters of the suspension containing the nuclei were used as input for the amplification. We followed a two-step PCR procedure to prepare SMRTbell® libraries using PacBio® Bar- coded Universal Primers (BUP) for multiplexing amplicons (part no. 101-791-800 v.01, June 2019; Pacific Biosciences, Menlo Park, CA, USA). The first PCR reaction was performed with the primer set AML1 (Lee et al., 2008)/wLSUmBr (Schlaeppi et al., 2016). These primers were modified (Table S1) to include universal sequences at the 50 position in order to re-amplify the PCR products with the Barcoded Universal F/R Primers Plate-96 (part no. 101-629-100; Pacific Biosciences) and to prepare the SMRTbell® library for multiplexing amplicons. PCR products were purified and normalized (5 ng ll�1) using the NGS Nor- malization 96-well kit (Norgen Biotek Corp., Thorold, ON, Canada) before and after preparing the multiplexing amplicons. Sequencing was performed at the McGill University and Gen- ome Qu�ebec Innovation Centre (Montr�eal, QC, Canada). Bioinformatic analyses CUTADAPT v.2.8 (Martin, 2011) was used to remove the primers from the PacBio circular consensus sequencing (CCS) reads, filter- ing out sequences > 400 nt longer or shorter than the expected length when necessary. The trimmed sequences were reverse- complemented where required. Next, the sequences were derepli- cated and chimaeras were removed using VSEARCH v.2.22.1 (Rognes et al., 2016). Finally, the dereplicated sequences were clustered using the SWARM v.3 clustering algorithm (Mah�e et al., 2021). Sequence similarity of each cluster was searched using the nucleotide-nucleotide BLASTN program (Altschul et al., 1990) on a custom BLAST (Basic Local Alignment Search Tool) database generated from the 33 chromosomes of R. irregularis DAOM 197198 (Yildirir et al., 2022). GENEIOUS PRIME v.20201.1.0 (Bio- matters Ltd, Auckland, New Zealand) was used to perform the BLAST search, format the custom database, and edit the Sanger sequences. Glomalin sequences and CCS reads were deposited in the NCBI GenBank database under accession nos. OQ628336– OQ628347 and Bioproject PRJNA944584, respectively. Phylogenetic analyses A Bayesian phylogenetic tree based on glomalin sequences was inferred to show the relationships between five sequences obtained from the spores with the morphology of R. cf fasciculatus and seven sequences from spores with the morphology of R. irregu- laris. The alignment included 12 reference glomalin sequences from Magurno et al. (2019), and four glomalin sequences from Kokkoris et al. (2021) from well-identified cultures of AMF. Sequences were aligned using CLUSTAL OMEGA v.1.2.3 (Sievers et al., 2011) as implemented in GENEIOUS with default para- meters. The best-fitting substitution models were determined with MODELTEST-NG v.1.0.0 (Flouri et al., 2015; Darriba et al., 2019) as implemented in CIPRES (Cyberinfrastructure for Phylogenetic Research, Miller et al. (2010)) using the Akaike information criteria corrected for small sample sizes (AICc; Hur- vich & Tsai, 1989). DNA candidate models were set to MRBAYES (three schemes) to ensure that the set of candidate models con- tains only models available in MRBAYES. The following individual models of evolution were selected for each codon position: HKY + I +G4 (first), GTR + I +G4 (second), and HKY + I +G4 (third). The posterior tree simulation was performed using MRBAYES v.3.2.7a (Huelsenbeck & Ronquist, 2001) as implemen- ted in GENEIOUS with the MRBAYES_GENEIOUS v.11 plugin and run on CIPRES. Two simultaneous and independent runs were evaluated for each analysis. Four Markov Chain Monte Carlo (MCMC) runs were performed for 5 million generations. The convergence of the MCMC chains and the effective sample size (ESS) values of the parameters were evaluated using TRACER v.1.7.2 (Rambaut et al., 2018). The number of saved trees was set to 50 000, and the first 10 000 trees were excluded before com- puting consensus trees with Bayesian posterior probabilities (PPs). Results Pure cultures produce the two spore morphotypes with identical gene sequences Over a 3 months’ timeframe, single spores of R. cf fasciculatus asymbiotically cultivated in Petri dishes with 2% Mmedium sup- plemented with Myr-K produced daughter spores with a wall architecture like that of R. irregularis (Fig. 1). Ten out of the 24 spores that were cultured asymbiotically germinated (n = 10), and one of these spores produced five daughter spores. The obser- vation of the wall architecture of the daughter spores by light microscopy showed an outermost evanescent layer (swl1), a unit layer (swl2), and an innermost laminated layer (swl3, Fig. 1a), � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) www.newphytologist.com New Phytologist Research 5 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense info:refseq/OQ628336 info:refseq/OQ628347 https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA944584 and the cracked mother spore showed a spore wall architecture similar to R. cf fasciculatus, with an outermost unit layer, a thick uniform layer and an innermost membranous layer (Fig. 1b). Spores with the morphology of R. cf fasciculatus and R. irregu- laris were simultaneously produced in the superabsorbent poly- mer autotrophic system (SAP-AS) inoculated with a single spore of the commercial strain DAOM 197198 (Fig. 2). In these SAP- AS cultures, the two morphotypes coexisted after 8 wk of propa- gation, orange-brown, and white-yellowish spores corresponding to the morphology of R. irregularis and R. cf fasciculatus, respec- tively (Fig. 2a–d). The spores with the morphotype of R. irregu- laris (Fig. 2c–e) were globose to subglobose, 61.6–829 61.5– 86.6 lm (n = 20), stained faint pink to dark scarlet in Melzer’s reagent, and the spore wall was (3.2–) 4.8 (�6.1) lm thick. Spores with the R. cf fasciculatus morphotype (Fig. 2f–h) were globose to subglobose, 56.6–85.19 56.5–87.5 lm (n = 29), stained dark scarlet to rusty red in Melzer’s reagent, and the spore wall was (4.8–) 6.6 (�9.4) lm thick. The mean width of the subtending hypha was larger (t(41.71) = 8.3026, P = 2.24e-10) for the spores with the morphotype of R. cf fasciculatus, (7–) 9.8– (�12.6) lm, than for those with the morphotype of R. irre- gularis, (4.4–) 6.2 (�9.3) lm. The genetic identity of each morphotype was first validated using CCS reads of the partial SSU-ITS-LSU region (2780 bp). A total of 52 157 CCS reads were obtained from the 11 spores of morph-2 isolated from in vivo cultures 562E (four spores), 864D (three spores), and 1184D (four spores). The CCS reads were grouped in 711 clusters of identical reads. These clusters had a median pairwise similarity of 99.8% (SD = 0.005%) to the RNA operon sequences from chromosomes 9 (n = 15), 18 (n = 419), 23 (n = 80), and 28 (n = 197) of the homokaryotic strain R. irre- gularis DAOM 197198, respectively (Yildirir et al., 2022; Table S2). Validation was also performed using glomalin sequences (645 bp) for 19–45 spores of morph-2 isolated in the three pot cultures. All the sequences had 100% pairwise similarity with the sequences of R. irregularis DAOM 197198 and with the sequences of both morphs isolated from SAP-based autotrophic cultures (Fig. 3). The glomalin gene sequences discriminate closely related strains of the same species as the R. irregularis strains. The strains SL1, A4, and MUCL43196 differ by four to six single nucleotide polymorphisms (SNPs) from other R. irregu- laris strains. SEM and TEM analyses SEM and TEM imaging was used to examine the fine details and ultrastructure of the spore walls of each morph (Figs 4, 5). High-resolution images in TEM revealed a thick and unlayered swl2 and a thin swl3 consisting of three to four thin laminations tightly appressed in R. cf fasciculatus spores (Fig. 4d,f). In light microscopy, these thin and tightly appressed laminations of swl3 had the appearance of a membranous wall, that is a very thin wall that eventually wrinkles (Figs 1b, 6e, S2c). Swl3 was also laminated in R. irregularis DAOM 197198 (Fig. 4c,e), but consisted of more distinctly spaced and thicker laminations, occasionally visible in light microscopy as thin overlapping sub- layers (Fig. S3g,h). The analysis of each spore morphotype by confocal microscopy showed that both morphs are rich in nuclei (Fig. 4g,h). Remarkably, the spores with the morphology of R. cf fascicula- tus were not a transient developmental stage leading to the typical spore morphology of R. irregularis. Specifically, over 12 wk of growth under in vitro conditions, the spore wall architecture of DAOM 197198 never resembled that of spores with the mor- phology of R. cf fasciculatus (Fig. 5). The ultrastructure of the wall of 2-wk-old spores showed the presence of a double fibrillar outer layer (swl1 and 2) adhering to two superimposed opaque layers (swl3) separated by a thin layer of granular material. After 4 wk, the thickness of swl3 increased slightly, mainly due to the accumulation of layers on the inner surface giving rise to the superimposed layers. At this stage, four opaque inner layers sepa- rated by granular material of irregular thickness were visible, giving rise to the laminated swl3. In some spores, the surface of swl1 began to slough off into fibrillar particles and swl2 gradu- ally separated from swl1. After 8 wk of growth, the thickness of the spore wall layers stabilized. Gradual compaction of the swl1 Fig. 1 Spores produced in asymbiotic culture system. (a) Asymbiotic culture, 2% minimum medium enriched with Myr-K, inoculated with a single spore of Rhizophagus cf fasciculatus (‘M’: mother spore) isolated from the pot culture 562E, and observed by light microscopy in PVLG and (b) PVLG +Melzer reagent. The daughter spores were observed 3 months following the inoculation of the culture medium. The outermost spore swl1 was absent and swl2 stained dark scarlet to reddish-pink in PVLG +Melzer buffer. Bars, 25 lm. d, daughter spore; M, mother spore; Myr-K, potassium salt of myristic acid; PVLG, polyvinyl alcohol-lactic acid-glycerol; swl, spore wall layer. New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist6 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense and swl2 was observed in some spores while swl3 showed alter- nating accumulation of opaque and granular sublayers. Up to 12 consecutive laminations could be distinguished in 12-wk-old spores. In addition, both the typical thickness of the subtending hyphae and swl2 were clearly visible in immature and mature morph-2 spores of spore clusters isolated from pot cultures (Figs 6a, S2c,d). Discussion Spore dimorphism in R. irregularis The model species of AMF, Rhizophagus irregularis, produces two distinct glomoid spore morphs, as evidenced by single-spore isolates of each morph producing either both morphs or only one of them. As noted by Błaszkowski et al. (2022), dimorphism is not a stable phenomenon among dimorphic AMF species. For reasons that are not fully understood, some species may produce both morphs, or only one of them, even after many propagation cycles (Bills & Morton, 2015). When produced, the alternative spore morphology to that described in the protologue of R. irre- gularis presents stable morphological and histochemical features (the spore colour, the width of the subtending hyphae, the thick- ness of the second wall layer, the thickness of the laminated innermost layer, and the dextrinoid reaction of the two outermost spore wall layers to Melzer’s reagent). These characters were observed in three cultivation systems (three different in vivo pot cultures, in vitro asymbiotic cultures, and in vivo SAP-based auto- trophic cultures), inoculated with fungal material originally col- lected from different geographical locations spanning over 1000 km. The presence of the two spore morphs observed in this study cannot be explained by the development of spores from an imma- ture to a mature stage. Single-spore cultures of morph-1 can pro- duce both morph-1 and morph-2 daughter spores simultaneously after 2 months of growth, and asymbiotic cultures of morph-2 (a) (b) (c) (f) (d) (g) (e) (h) Fig. 2 Two spore morphotypes observed by light microscopy in PVLG and PVLG +Melzer buffer. Spores were isolated in the superabsorbent polymer-based autotrophic system inoculated with a single spore of the commercial strain DAOM 197198. (a, b) The black and white arrowheads indicate morph- 1 and morph-2 spores, respectively. (c–e) Spores with the morphology of Rhizophagus irregularis (spore morph-1). (f–h) Spores with the morphology of R. cf fasciculatus (spore morph-2). Bars: (a) 100 lm; (b–h) 25 lm. PVLG, polyvinyl alcohol-lactic acid- glycerol; swl, spore wall layer. � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) www.newphytologist.com New Phytologist Research 7 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense (a) (b) (c) (e) (d) (f) (g) (h) (i) Fig. 3 Fifty per cent majority rule consensus phylogram inferred from a Bayesian analysis of 28 arbuscular mycorrhizal fungi (AMF) glomalin gene sequences, including Entrosphospora claroidea as an outgroup. (a) Black dots indicate clades supported with Bayesian posterior probabilities > 0.95. Sequences from morph-2 spores (i.e. morphotype of Rhizophagus cf fasciculatus) are shown in blue. The scale bar at the bottom indicates 0.03 expected change per site per branch. (b–e) Spores observed in light microscopy and mounted in PVLG reagent (bars, 25 lm). The DNA used to produce the sequences 4674_1184D and 4397_864D and 4835_562E was isolated from the spores shown in (b–d). The spores were isolated from the in vivo cultures 1184D, 864D, and 562E. Sequence IVT23A_PMU823 is from spores collected in an in vitro culture of R. irregularis DAOM 197198. Sequences PMU1670 to PMU1677 are from spores isolated in the SAP-based autotrophic cultures of R. irregularis DAOM 197198 (f–i: bars, 100 lm). (g, i) The sequences PMU1675 and PMU1677 are from the spores of morph-1. (f, h) The sequences PMU1670 and PMU1673 are from the spores of morph-2. PVLG, polyvinyl alcohol-lactic acid-glycerol; SAP, superabsorbent polymer; swl, spore wall layer. New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist8 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense can produce morph-1 daughter spores after 3 months of culture. In addition, the consistently 30% larger subtending hyphal size of morph-2 compared with morph-1 indicates that morph-2 can- not be an immature morph-1 spore, as no reduction in subtend- ing hyphal size was ever observed during spore maturation, and immature morph-2 spores already exhibit the typical thick sub- tending hypha and swl2 that are visible in mature morph-2 spores. Finally, the development of the spore wall architecture of DAOM 197198 morph-1 did not resemble that of morph-2 over 3 months of observation. Therefore, morph-1 also has a stable morphology, and the observed morphological differences between morph-1 and morph-2 spores cannot be attributed to differences in spore maturation. Finally, Yamato et al. (2022) showed that R. irregularis produces sporocarps and, that the spores directly connected to the swollen structures of the sporocarp are morphologically very similar to those of morph-2 and reported no morph-1 spores. Taken together, these conclusively demonstrate that morph-2 spores are not a transient developmental (i.e. ontogenetic) stage leading to morph-1 spores, nor are they part of a continuum of phenotypic variation caused by environmental factors (i.e. acclimation). Confusion in culture collections and incorrect taxonomic assignment of DNA sequences in public databases The morph-2 spores can be confused with those of Glomus fasci- culatum (Thaxter sensu Gerd. & Trappe), and it is likely that col- lection curators have already encountered R. irregularis morph-2 spores and misidentified them as R. fasciculatus. For example, the notes to the species description of R. fasciculatus from INVAM report that every attempt to propagate what seemed to be R. fasci- culatus, resulted in cultures of R. intraradices [sic] while the Inter- national Bank for Glomeromycota (IBG) reports a culture (BEG53) of ‘Glomus irregularis originally misidentified as Glomus fasciculatum [sic] ’. This overlooked dimorphism in R. irregularis has also led to incorrect taxonomic assignments of DNA sequences in public databases. A total of 96 sequences are available in NCBI (a) (b) (c) (d) (e) (f) (g) (h) Fig. 4 Two spore morphotypes observed by scanning electron microscopy (SEM), transmission electron microscopy (TEM), and confocal microscopy. (a, b) SEM photographs of intact spores of morph-1 of Rhizophagus irregularis DAOM 234181 and morph-2 isolated from the pot culture 864D. (c–f) TEM photographs of morph-1 spores of R. irregularis DAOM 197198 and morph-2 spores from pot culture 864D. (g, h) Confocal photographs of morph-1 spores of R. irregularis DAOM 197198 and morph-2 spores from pot culture 1184D. The lines visible in the TEM photographs are artefacts from the cutting of thin sections with the diamond knife. swl, spore wall layer. � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) www.newphytologist.com New Phytologist Research 9 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://invam.ku.edu/fasciculatus https://www.i-beg.eu/ GenBank under the name R. fasciculatus or G. fasciculatum (Table S3). Ten sequences (two mitochondrial genomes, four mitochondrial genes, one SSU sequence, two ITS sequences, and one LSU sequence) were associated with an official herbarium nos.: BEG 53, BEG 58, and DAOM 240159. While BEG 58 is not listed on the International Bank for the Glomeromycota (IBG), BEG 53 is still available and is now identified as R. irregu- laris. A note in the culture database indicates that it was originally considered to be G. fasciculatum. Regarding DAOM 240159, the Canadian Collection of Arbuscular Mycorrhizal Fungi has always distributed this in vitro culture as R. irregularis and it was prob- ably renamed later without seeking taxonomic approval. Two identical mitochondrial genomes (KM586389 and NC_029185) were sequenced from DAOM 240159, and these were closely related to the mitochondrial genome HQ189519 from R. irregu- laris DAOM 197198 (pairwise similarity > 98.7%). A total of 78 LSU sequences (133–414 bp) were uploaded to NCBI GenBank under the name R. fasciculatus (Bioproject PRJEB34396). Each sequence has an isolate number and seems to be associated with plants of the genus Tolpis (Asteraceae), growing in the Canary Islands (Spain). However, the lack of spore photographs and the shortness of their LSU fragments make the identification of the samples difficult, as a sequence of at least 1500-bp long covering the end of the SSU to the partial LSU is required to discriminate closely related AMF species (Kr€uger et al., 2009; Stockinger et al., 2010). The distribution of per cent similarity of these 78 pairwise aligned sequences had a median of 88.3% (min = 30.6%, max = 100%), indicating potential taxonomic heterogeneity from the 78 LSU sequences associated with the name R. fasciculatus. As such, most of the R. fasciculatus or G. fas- ciculatum sequences from NCBI GenBank and taxonomic refer- ence databases such as UNITE, GBIF, MaarjAM, and the Global Fungal Red List are unlikely to be from the correct taxon. What induces spore phenotypic plasticity? Spore morphology in fungi is known to correlate with different functions and lifestyles. In agarics, spore wall traits, such as wall thickness and pigmentation, are thought to be associated with different lifestyles (Halbwachs et al., 2015). Some rust species Fig. 5 Transmission electron microscopy (TEM) of morph-spore wall development over 3 months of Rhizophagus irregularis DAOM 197198 spores grown in vitro. Bars, 0.52 lm. swl, spore wall layer. New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist10 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJEB34396 such as Puccinia triticina or P. recondita produce up to five spore morphs depending on the life cycle stage or their host (e.g. Anik- ster et al., 2005). Ascomycetes produce asexual and sexual spores, with each category being morphologically distinct, reflecting eco- logical trade-offs between dispersal and survival (Bertrand & English, 1976). All of these factors could potentially contribute to the evolution of AMF spore dimorphism. From 1975 to 2000, AMF identification and classification relied solely on spore morphological traits (St€urmer, 2012), and spore phenotypes were assumed to be genetically predeter- mined and not influenced by soil abiotic and biotic properties or host identity (Morton, 1985). The current view emphasises that external and epigenetic factors play an important role in glomeromycotan spore phenotypes. For example, Lumini et al. (2007) demonstrated the influence of endobacteria on the spore phenotype by comparing wild-type and cured spores of Gigaspora margarita. In Geosiphon pyriformis, glomoid spores are produced, but in the presence of Nostoc punctiforme (a cya- nobacterium), hyphae extend to produce endocyanoses within bladders that appear aboveground. These bladders do not act as propagules but can fix nitrogen and are photosynthetically active, suggesting that alternative functions can exist in mor- phologically diverse AMF structures (Gehrig et al., 1996; Sch€ußler & Hock, 2012). Although there is limited informa- tion on viruses infecting AMF (Purin & Rillig, 2008; Turina et al., 2018), viral infections can affect spore morphology in other fungal groups, as seen in Aspergillus flavus when infected by a mycovirus (Jiang et al., 2019). Here, spore dimorphism in R. irregularis was mainly observed under in vivo (i.e. nonsterile) conditions (pot cultures and in SAP-based autotrophic systems), whereas spore dimorphism has never been reported for the R. irregularis strain DAOM 197198 since its first propagation in monoxenic culture systems using Ri T-DNA transformed Daucus carota (Chabot et al., 1992). It is thus plausible that biotic and/or abiotic factors induce the pro- duction of spores with two divergent morphologies primarily in vivo, but not under the very stable in vitro conditions com- monly used for the propagation of the strain DAOM 197198 of R. irregularis. In this context, to capture all the intraspecific mor- phological variability, Walker et al. (2021) emphasized the importance of characterizing new species based on analyses of multiple cultures grown long enough and the need for intermit- tent redescriptions of species in the Glomeromycotina, as type specimens may represent only a subset of the phenotype based on variation in anatomical/histochemical characters. Recent analyses of genome organization in R. irregularis have shown that chromosomes are organized into two compartments that differ in gene content, transcription, and regulation (Yildirir et al., 2022; Sperschneider et al., 2023). Since there is compelling evidence that these compartments change depending on the AMF life stage or growth habitat (e.g. extraradical mycelium vs colonized tissue), similar epigenetic variability could produce the two distinct spore morphs described in this study, and future work should determine whether the host- and strain-specific nuclear dynamics recently described in heterokaryotic AMF strains (Ropars et al., 2016; Cornell et al., 2021; Kokkoris (a) (b) (c) (d) (e) (f) Fig. 6 Morph-2 spores observed in light microscopy in PVLG (a, c, e) and PVLG +Melzer reagent (b, d, f). (a, b) Spores were isolated from pot cultures 562E. (c, d) Spores were isolated from pot cultures 864D. (e, f) Spores were isolated from pot cultures 1184D. Note the immature and mature morph-2 spores in (a). The spores have three wall layers (swl1, swl2, and swl3). swl1 and swl2 stained pinkish-grey and dark scarlet in PVLG +Melzer buffer, respectively. Bars, 25 lm. PVLG, polyvinyl alcohol-lactic acid- glycerol; Sh, subtending hypha; swl, spore wall layer. � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. New Phytologist (2023) www.newphytologist.com New Phytologist Research 11 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense et al., 2021; Sperschneider et al., 2023) also contribute to spore morphological changes. Understanding the mechanisms underlying epigenetic changes in AMF may provide new insights into the biology of these plant root symbionts. The presence of a putative fungal mating-type (MAT) locus in most AMF genomes studied so far, as well as homokaryotic and heterokaryotic strains within species of R. irre- gularis (Ropars et al., 2016; Chen et al., 2018), make sexual reproduction a possibility in AMF (Sperschneider et al., 2023). It is also possible that spore morphs specifically related to mating exist in AMF, although no evidence has been found so far that these processes occur in sporocarps of R. irregularis homokaryons (Yamato et al., 2022). Conclusion Here, we show that R. irregularis, the model species in AMF research and a key strain of the biostimulant industry (strain DAOM 197198), is dimorphic, involving two glomoid morphs. The alternative morphology to that described in the protologue of R. irregularis, whose expression we demonstrated here, resem- bles the one of Glomus fasciculatus (Thaxter sensu Gerd. & Trappe). This similarity has caused taxonomic confusion in cul- ture collections and possibly in AMF research by erroneously reporting the use of R. fasciculatus as inoculum or recording it as a morphospecies, or DNA sequence type of known species iden- tity. Our study supports the view that the relationships between phenotypic and molecular diversity are not well understood (Walker, 1992), and that rigorous linkage of spore morphology to DNA sequences, combined with the analyses of single-spore cultures under multiple growth conditions, are essential tools for disentangling AMF phenotypic and molecular diversity and for improving AMF taxonomy and our understanding of AMF biol- ogy. Common garden-type experiments with collections of iso- lates of the same AMF species are warranted to improve our understanding of AMF biology and ecology. Moving forward, it is important to investigate how widespread the observed dimorphisms are throughout the genus Rhizophagus, to under- stand what controls the production of alternative spore morpho- types, and to what extent and under what conditions spore dimorphism is functionally relevant. Acknowledgements Our research is kindly supported by the Natural Sciences and Engineering Research Council (RGPIN2020-05643), a Dis- covery Accelerator Supplements Program (RGPAS-2020- 00033), and by Agriculture and Agri-Food Canada (AAFC) under project J-002272 (Fungal and Bacterial Biosystematics). NC is a Research Chair at the University of Ottawa, and VK was supported by the MITACS Industrial PDF program (IT16902) to NC, and by Agriculture and Agri-Food Canada (AAFC) under project J-002272. We would also like to thank Denise Chabot of the Microscope Laboratory (Ottawa-RDC, AAFC) for excellent training and technical assistance in all microscopy techniques. Competing interests None declared. Author contributions CB recognized the presence of potential dimorphisms within R. irregularis pot cultures. VK and FS conceived and designed the experiments. CB maintained and analysed the in vivo and in vitro cultures and provided the fungal inocula. CB prepared the light microscopy slides. LA, SS and VK performed the molecular work. FS performed the bioinformatic and phylogenetic analyses and microscopy measurements. VK and KH performed the microscopy (SEM, TEM, and confocal). YD performed the spore ontology experiment. WF developed the bioinformatics pipelines to process the PacBio sequences. VK propagated the single spores on myristate. LP propagated the single spores in the superabsor- bent polymer (SAP)-based autotrophic systems, maintained, and analysed the cultures. VK and FS drafted the manuscript. FS, VK, JD, NC and YD discussed the results and contributed to the final version of the manuscript. FS wrote the final version of the manuscript. NC, JD and FS obtained funding for research and salary associated with this study. All authors provided critical review of drafts and gave final approval for publication. FS was responsible for oversight and project management, as well as AAFC funding and resource acquisition. ORCID Lobna Abdellatif https://orcid.org/0000-0001-8221-0770 Nicolas Corradi https://orcid.org/0000-0002-7932-7932 Yolande Dalp�e https://orcid.org/0000-0002-4807-5197 Vasilis Kokkoris https://orcid.org/0000-0002-1667-0493 Franck Stefani https://orcid.org/0000-0002-6025-2192 Data availability The data that support the findings of this study are openly avail- able in the NCBI GenBank database under accession nos. OQ628336–OQ628347 and Bioproject PRJNA944584. References Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. 1990. 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Supporting Information Additional Supporting Information may be found online in the Supporting Information section at the end of the article. Fig. S1 Illustration of the steps taken to demonstrate dimorph- ism in Rhizophagus irregularis. Fig. S2 Spore clusters of morph-1 and morph-2 of Rhizophagus irregularis. Fig. S3Morph-1 spores of Rhizophagus irregularis. Table S1 Primers for amplification of the SSU-ITS-LSU regions of the 45 rRNA gene for PacBio CCS sequencing. Table S2 BLAST results between the CCS reads of the partial SSU-ITS-LSU sequences from single spores of morph-2 and the 33 chromosomes of R. irregularis DAOM 197198. Table S3 Current NCBI records for Rhizophagus fasciculatus/ Glomus fasciculatum. Please note: Wiley is not responsible for the content or function- ality of any Supporting Information supplied by the authors. Any queries (other than missing material) should be directed to the New Phytologist Central Office. New Phytologist (2023) www.newphytologist.com � 2023 His Majesty the King in Right of Canada and The Authors. New Phytologist published by John Wiley & Sons Ltd on behalf of New Phytologist Foundation. Reproduced with the permission of the Minister of Agriculture and Agri-Food Canada. Research New Phytologist14 14698137, 0, D ow nloaded from https://nph.onlinelibrary.w iley.com /doi/10.1111/nph.19121 by C anadian A griculture L ibrary, W iley O nline L ibrary on [22/04/2024]. See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense Summary Introduction Materials and Methods In vivo and in vitro cultures �Single-spore� cultures Microscopy and statistical analyses Optical microscopy Scanning electron microscopy (SEM) Transmission electron microscopy (TEM) Confocal microscopy DNA extraction Glomalin amplification and Sanger sequencing Partial 45 rRNA gene amplification and PacBio sequencing Bioinformatic analyses Phylogenetic analyses Results Pure cultures produce the two spore morphotypes with identical gene sequences SEM and TEM analyses nph19121-fig-0001 Discussion Spore dimorphism in R. irregularis nph19121-fig-0002 nph19121-fig-0003 Confusion in culture collections and incorrect taxonomic assignment of DNA sequences in public databases nph19121-fig-0004 What induces spore phenotypic plasticity? nph19121-fig-0005 nph19121-fig-0006 Conclusion Acknowledgements Competing interests Author contributions The data that support the findings of this study are openly available in the NCBI GenBank database under accession nos. OQ628336-OQ628347 and Bioproject . 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