Fungal Biology 123 (2019) 318e329 Contents lists available at ScienceDirect Fungal Biology journal homepage: www.elsevier.com/locate/funbio The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota Kanami Yoshino a, Kohei Yamamoto b, Kojiro Hara c, Masatoshi Sonoda a, * Yoshikazu Yamamoto c, Kazunori Sakamoto a, a Division of Environmental Horticulture, Graduate School of Horticulture, Chiba University, 648 Matsudo, Matsudo, Chiba, 271-0092, Japan b Tochigi Prefectural Museum, 2-2 Mutsumi-cho, Utsunomiya, Tochigi, 320-0865, Japan c Faculty of Bioresource Sciences, Akita Prefectural University, 241-438 Kaidobata-nishi, Shimoshinjo-nakano, Akita, 010-0195, Japan article info abstract Article history: In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. Received 4 June 2018 However, the existence of polyol transporter genes and their correlation with lichenization remain un- Received in revised form clear. Here, we report candidate polyol transporter genes selected from the genome of the lichen- 30 December 2018 forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and Accepted 21 January 2019 monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and Available online 31 January 2019 various ascomycetous fungi suggested that the characterized yeast’ polyol transporters form multiple Corresponding Editor: Martin Grube clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In Keywords: addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated Gene duplication proteins in each cluster. Consequently, the number of sequences similar to characterized yeast’ polyol Genome transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, Lecanoromycetes LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, Lichen-forming fungi various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have Phylogenetic analysis expanded during the evolution of Lecanoromycetes. Ramalina conduplicans © 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved. 1. Introduction Only one order in Coniocybomycetes, Coniocybales (Prieto et al., 2013), is entirely lichenized and associates with green algae Lichen-forming fungi (LFFs) have evolved in various classes, (Rikkinen et al., 2016). On the basis of recent classifications, Lücking including Arthoniomycetes, Coniocybomycetes, Dothideomycetes, et al. (2016) suggested that 14e23 independent lichenization Eurotiomycetes, Lecanoromycetes, Leotiomycetes and Lichinomy- events occurred during the evolution of Ascomycota. Approxi- cetes, in Ascomycota (James et al., 2006; Schoch et al., 2009a; mately 85 % of LFFs have green algae as their primary photosyn- Lumbsch and Rikkinen, 2017; Prieto et al., 2018). Lecanor- thetic partners, and 3%e4 % are associated with both green algae omycetes is the largest class to which LFFs belong, with over 95 % of and Cyanobacteria (Honegger, 2008). Although LFFs belong to many the species being lichenized (Miadlikowska et al., 2014). Although genera in seven classes of Ascomycota, their green algal partners Dothideomycetes and Eurotiomycetes include mainly non- belong to a more limited number of genera, including Trebouxia, lichenized species, some classes, such as Trypetheliales (Dothi- Asterochloris and Diplosphaera in the class Trebouxiophyceae and deomycetes) and Verrucariales (Eurotiomycetes), include LFFs Trentepohlia in the class Ulvophyceae (Friedl and Büdel, 2008). (Spatafora et al., 2006; Gueidan et al., 2007; Schoch et al., 2009b). Thus, the fungal partners may diversify if they receive particular advantages from specific green algae, such as the acquisition of carbon sources. A stable supply of carbon from a photosynthetic partner is a * þ Corresponding author. Fax: 81 47 308 8720. fi E-mail addresses: [email protected] (K. Yoshino), [email protected]. bene t of LFFs and probably the main reason why lichens expanded jp (K. Yamamoto), [email protected] (K. Hara), [email protected] worldwide and retain their symbiotic relationships for long periods (M. Sonoda), [email protected] (Y. Yamamoto), [email protected] of time. Heterotrophs, such as fungi, cannot survive or grow in (K. Sakamoto). https://doi.org/10.1016/j.funbio.2019.01.006 1878-6146/© 2019 British Mycological Society. Published by Elsevier Ltd. All rights reserved. K. Yoshino et al. / Fungal Biology 123 (2019) 318e329 319 nature without a carbon source, and, in fact, the green algal part- activity studies of extracts and isolated compounds (reviewed in ners release acyclic polyols, such as ribitol, sorbitol, and erythritol Moreira et al., 2015). Additionally, it is distributed worldwide (Kirk (Richardson et al., 1968), when they associate with a fungal partner et al., 2008). Some metabolites of R. conduplicans have possible (Smith, 1968; Green and Smith, 1974). For example, ribitol, which is industrial, pharmaceutical and agricultural uses (Oh et al., 2006; absent in almost all plants (Negm and Marlow, 1985), is released Wei et al. 2008; Vinayaka et al., 2009; Luo et al., 2010), and from Trebouxia spp., the most common photosynthetic partners in R. conduplicans is associated with Trebouxia spp., the most common lichens (Honegger, 2008). Observations of Xanthoria aureola photosynthetic partner in lichens (Honegger, 2008). Therefore, (Richardson and Smith, 1968a, 1968b) and Xanthoria calcicola (Lines R. conduplicans could be a lichen model for studying secondary et al., 1989), which associate with green algae (Trebouxia), indicated metabolites and polyol utilization. that ribitol is converted to mannitol and arabitol through the pentose phosphate pathway of fungal metabolism. The other algal 2. Materials and methods partners also release polyols, and polyols are present in many li- chens (Lindberg et al., 1953; Lewis and Smith, 1967). Therefore, the 2.1. Collection, isolation and culture conditions polyol transport pathway is most likely essential to LFFs associated with green algae. However, the mechanisms of polyol transfer from On 15 July 2014, R. conduplicans was collected from the shore of green algae to fungi have not been elucidated, and, so far, a polyol Lake Towada (Aomori, Japan, 40260N140550E). Fungal cultures transporter has not been isolated from LFFs, even though its func- (strain ID: KY1401) were isolated from thallus fragments using the tion is required for symbiotic associations. methodology of Yamamoto et al. (1985). The specimens were Polyols are widely present in diverse organisms (Lewis and deposited into the herbarium of the Natural History Museum and Smith, 1967; Pfyffer and Rast, 1980; Honegger et al., 1993; Institute, Chiba, Japan (CBM) under the accession number CBM-FL- Gustavs et al., 2011), and they function to remove reactive oxygen 204754. The culture strain KY1401 was preserved in the Biological species (Parida and Das, 2005), to adjust the osmotic pressure of Resource Center, NITE (NBRC) under the accession number 113405. compatible solutes (Wegmann, 1986; Reed et al., 1987), and to store The culture was incubated in Malt/Yeast extract agar medium the reducing power provided by NADH or NADPH (Jennings, 1985; (Yoshimura et al., 2002) for DNA extraction and modified Czapek-Dox Williamson et al., 2002). Polyol transporters have been reported in liquid medium [3 % ribitol as a sole carbon source, 0.2 % NaNO3,0.1% bacteria, such as D-arabinitol and ribitol transporters from Klebsi- K2HPO4,0.05%MgSO4$7H2O, 0.05 % KCl, 0.001 % FeSO4$7H2O] for RNA ella pneumoniae (Heuel et al., 1997), in red alga, such as sugar and extraction with shaking at 200 rpm at 19 Cfor3m. mannitol transporters from Galdieria sulphuraria (Schilling and Oesterhelt, 2007), in plants, such as the first reported mannitol 2.2. DNA extraction and primers for molecular identifications transporter from celery (Noiraud et al., 2001), and in humans, such as glycerol, mannitol, sorbitol and the other neutral solute channels Genomic DNA was extracted from the culture strain KY1401 and (Tsukaguchi et al.,1999). The orthologs or paralogs of these proteins the thalli of specimens. The culture was ground with a mortar and have also been characterized as polyol transporters. Recently, some pestle in Buffer T1 from the NucleoSpin Tissue XS kit (TaKaRa Bio, þ transporters (or H symporters), having the capabilities to trans- Shiga, Japan), and we followed the kit manufacturer's instructions port acyclic polyols, were reported in ascomycetous yeast, such as after grinding. For the thalli, the standard cetyltrimethylammonium Debaryomyces hansenii (Syl1 and 2; Pereira et al., 2014), Ambro- bromide method was used. Extracted genomic DNA was used as the siozyma monospora (Lat1 and 2; Londesborough et al., 2014) and PCR template for the amplification of internal transcribed spacer Saccharomyces cerevisiae (Hxt11, 13, 15, 16 and 17; Jordan et al., (ITS) regions of nuclear ribosomal DNA (nrDNA) and 5.8S nrDNA with 2016). Some of these yeast proteins have no ability to transport the primer pair, ITS5 and ITS4 (White et al., 1990). sugars, even though their encoding genes were paralogous to monosaccharide transporters.