DNA‐based fungal diversity in Madagascar and arrival of the ectomycorrhizal fungi to the island

Madagascar is known for its high diversity and endemism of fauna flora. Fungi, however, have been largely overlooked in evolution studies on the island, whether fungi exhibit same patterns as animals plants has yet to be further examined. We collected fungal sporocarps ectomycorrhizal (EcM) roots during three opportunistic surveys five forests generated a dataset Internal Transcribed Spacer (ITS) DNA sequences. analyzed them together with all publicly available ITS sequences identified 620 Operational Taxonomic Units (OTUs) from Madagascar, 10% which contained only our surveys. Two hundred ninety-two OTUs belonged EcM species /russula-lactarius, /boletus, /tomentella-telephora, /cortinarius, /amanita being most abundant lineages. Overall, 60% 81% appear endemic. conducted phylogenetic analysis using Amanitaceae, Boletaceae, Russulaceae families elucidate their relative timing arrival Madagascar. found that diverged less than 22 million years ago (mya), long after separation India (88 mya), consistent dispersal mediated process island. Our study provides first comprehensive view overall DNA-based current state knowledge based sequences, useful ecological evolutionary studies. Abstract Malagasy online material. Madagasikara dia fantatra amin'ny habetsahan'ny fahasamihafana sy ny fisian'ny zavamaniry biby tokana an-tany. Na izany aza tena mbola vitsy fikarohana momba olatra natao teto nosy, ary tokony holalinina mba hahafantarana raha mitovy endrika olatra. Nanangona “sporocarpe” na voan'ny hita masosy fakan'ireo miainana miaraka “ectomycorrhizal roots” izahay nandritra fanadihadiana telo tamin'ny ala dimy Madagasikara. Izay nahafahana namoaka angona ADN “Internal sequences”. Natao famakafakana azy ireo niaraka tamin'ireo “ITS sequences” izay fitehirizana hoan'ny rehetra. Araka vokatra azo “Operational Units” nanaovana avy eto ka amin'izy tsy misy afa-tsy filaharan'ny fanadihadinay. Ary roa amby sivy folo roan-jaton'ny OTU an'isan'ireo miaina ara-voajanahary toa an'ny russula-lactarius, /cortinarius ary/amanita manana taranaka indrindra. Amin'ny ankapobeny, 60%'ny rehetra karazana Nanao ara-pirazanana fampiasana fianakaviana Boletaceae hanazavana fotoana nahatongavan'izy Tsapa tamin'izany fa an'i Madagasikara, ao anatin'ireo fianankaviana ireo, efa nisaraka farafahakeliny 34 tapitrisa taona (mye), taloha lavitra nisarahan'I Inde mye), mifanaraka dingan'ny fanaparitahana fahatongavan'ny nosy. Ny fandinihanay manome fijery feno voalohany fahasamihafan'ny ankapobeny toetry fahalalana ankehitriny mifototra ADN, ilaina ho ara-tontolo iainana fiovaovana. well megadiverse country number endemic (Convention Biological Diversity, 2014). This is, entirely plant animal studies, since there very little literature regarding (Buyck, 2008), result, not much about (Ralaiveloarisoa, 2022; Ralaiveloarisoa et al., 2020). The scientific reports Funga (Kuhar 2018)of date back late 19th century (Cooke & Massee, 1890), followed by several French mycologists half 20th century, including those Patouillard (1924, 1928), Romagnesi (1941), Métrod (1949) Heim (1936, 1938a, 1938b, 1945) (Vizzini 2019). It until recently interest resurfaced, new reported, monographs published, high-throughput environmental sequencing projects (Antonín 2005; Antonín Buyck, 2006; Aptroot, 2016; 2008; Buyck 2007; Duhem Eyssartier 2001; 1999a, 1999b; Ghignone 2021; Ralaiveloarisoa, 2020, Shay 2017). varies, 292 reported latest Convention Diversity (CBD) National Report (Madagascar Ministry Environment Forests, 2014), 600 sporocarp-forming 2022), vast majority cannot at level thus could potentially belong new, undescribed taxa (Ralaiveloarisoa 2016). island contains an immense variety ecosystems, ranging dry deciduous West, Central highlands some areas ericoid thicket throughout, evergreen rainforests East, spiny thickets southern part Sambirano rainforest North (Yoder Nowak, 2006). Because organisms evolved isolation, numerous radiations microendemisms emerged many adapted these pronounced gradients (Kleine 2013; Vences 2009). Plants are one such highly diverse groups organisms, 83% (Goodman Benstead, 2005). Notably, Sarcolaenaceae Asteropeiaceae, form obligate relationships (Ducousso 2008). Ectomycorrhizal prominent guilds forest ecosystems (Smith Read, forming symbiotic 30 lineages worldwide (Brundett, 2002; Tedersoo 2020), fungus obtains sugars photosynthesis exchange mineral nutrients water gathered soil lifestyle least 80 times different lineages, proof success this symbiosis (Tedersoo Smith, 2013). Although research tropical increased over past 50 2012; Henry 2017), still poorly In particular, mycorrhizal communities associated native thoroughly studied (Henry 2015). restricted families; other non-endemic Intsia bijuga (Fabaceae) Uapaca (Phyllanthaceae), exotic trees genera Pinus Eucalyptus, also Ducousso 2008, 2012). However, how when arrived remains uncertain 2002). particular geological history; was once supercontinent Gondwana, land mass started drifting 100 mya, final 88 mya led hypotheses speciation events might rates across kingdoms, Theories symbioses history partners, two main proposed so far. hypothesis, al. (2004), suggests vicariance-driven origin habit analyses showing family shared recent common ancestor Dipterocarpaceae, representatives Asia, Africa, South America. A parsimonious reconstruction condition places single gain ancestor, somewhere between 165 155 2004), prior disarticulation Gondwana addition dubious assumptions likelihood independent origins habit, important gaps among dipterocarp clades (Angiosperm Phylogeny Group (APG), Buerki 2013), seem question hypothesis. second hypothesis proposes occurred more recently, continental Africa areas. Several authors suggested occur arose quite (Janssens 2020; Zanne 2014); them, long-distance (Lewis Both however mutually exclusive (Peterson 2010; Sauquet 2012); due polyphyletic nature symbiosis, explained either or both hypotheses, it differ clades. Therefore, fully understand baseline needed accurate reliable inferences can drawn. study, we aimed (a) explore potential field document fungi; (b) compile diversity, performing clustering approach define species; (c) fungi, (d) mechanisms dominant families. Opportunistic sampling macrofungi root samples seven host (the Sarcolaena, Leptolaena Asteropeia, African-Malagasy Uapaca, Indo-Pacific Intsia, pantropical Dalbergia Pacific Eucalyptus) carried out sites trips 2012, 2014, 2017 (Table 1). Sporocarps were photographed (Figure 1), fine nearest tree collected. dried preservation, later accessioned Royal Botanic Gardens Kew Fungarium (K). Mycorrhizal 67 1) tracking lateral trunk stored plastic bags. Roots rinsed 0.5 mm sieve day collection 2× CTAB buffer processing. sp. Asteropeia micraster multiflora rhopaloides littoralis mcphersoni gautieri Sarcolaena oblongifolia thouarsii Eucalyptus Once laboratory, observed under dissecting microscope presence tips. When possible, eight selected each sample ectomycorrhiza randomly analysis. Genomic isolated ~1 mg hymenophore tissue 20 μL Extract-N-Amp (Sigma) 10 individual forward ITS1F (Gardes Bruns, 1993) reverse ITS4 (White 1990) primers used amplify internal transcribed spacer region nrDNA, through PCR. PCR products purified ExoSAP-IT (USB, Cleveland, OH, USA) sequenced bidirectionally BigDye3.1 ABI 3730 (Applied Biosystems, Foster City, CA, USA). obtained manually edited Sequencher v. 4.2. (Gene Codes Corp., Ann Arbor, MI, To retrieve available, Full UNITE + INSDC (version 8.3, 1.039,010 sequences) (Abarenkov, Nilsson, 2010) containing curated assigned Species Hypotheses (SH, Abarenkov 2021a). search PlutoF Tedersoo, last accessed 07/June/2021) GenBank Nucleotide databases (National Centre Biotechnology Information, 1988; 07/June/2021). For PlutoF, filters “Taxon: Fungi R.T. Moore, 1980,” “Country: Madagascar,” “Include subtaxa: Yes”; GenBank, “country = [All Fields] AND “Madagascar“[All fungi[filter]”. standardize sequence lengths remove unwanted LSU (28S) SSU (18S) flanking regions, extracted software ITSx (Bengtsson-Palme duplicate datasets combined dereplicated --derep_fulllength option vsearch (Rognes resulting (PlutoF dataset) then representation Using R Studio Version 1.4.1717 (R Core Team, 2021), taxonomic assignment newly PlutoF+GenBank performed assignTaxonomy function package dada2 (Callahan 2016), General FASTA release 98,084 reference database (Abarenkov 2021b). annotated (all Fungi), 2016) 97% similarity threshold delimit (OTUs). These proxy analyses. metacoder (Foster 2017) graphical expression diversity. status lineage information Hypothesis (SH) FUNGuild (Nguyen rest. confirmed test family-level belonging Russulaceae, (Corrales 2018; Henkel Ramanankierana 2007). dating approach, outgroups basal rest must chosen. Hence, experience authors, chose seventeen Limacella Zhuliangomyces Amanitaceae phylogeny; Chalciporus piperatus chosen outgroup Gloeopeniophorella convolvens phylogeny. After dereplication re-clustering algorithm h-dh-hit-est (three runs, identity cut-off value 0.99, 0.98, 0.97, respectively), CD-HIT Suite (Huang 2010), explored, (potential misidentifications families) removed resulted (one family) representative per OTU. revised merged again order preserve species. aligned FFT-NS-2 MAFFT v7.475 (Katoh Standley, Then, maximum IQ-TREE 2015) partitioned ITS1, 5.8S, ITS2 regions partitions (Chernomor applying ModelFinder find best substitution model partition (Kalyaanamoorthy Ultrafast Bootstrap approximation (Hoang 2018) 1000 replicates. family, ultrametric penalized uncorrelated branch lengths, chronos ape version 5.5 (Paradis, Paradis Schliep, Due lack suitable fossils, mean stem age described He (2019) divergence (125, 98, 97 respectively). 214 57 corresponding 157 ectomycorrhizas. All accession numbers OM219812–OM219816 OM219818–OM220026. Additional Table S1. recovered 2467 1652 database. combining datasets, extracting removing duplicates, GenBank) made up 1179 From global dataset, extraction dereplication, total 735,598 original 1,039,010. present 2; S2). these, 545 previously published Madagascar; remaining 75 (61 study) recorded Basidiomycota phylum (402 OTUs) (87 OTUs), Thelephoraceae (41 (31 Cortinariaceae (22 (15 One seventy-seven Ascomycota Hypocreaceae (29 Lobariaceae (14 Nectriaceae (10 Aspergillaceae abundant. Kickxellomycotina (two Rozellomycotina, Chytridiomycota, Mucoromycotina, Glomeromycotina each) while 35 2a). Only 138 (22%) species-level (either SH, dada2). thirds (405 OTUs, 65% total) referenced therefore considered Some islands Seychelles (seven Réunion OTUs). (43 continent (17 others another continent, but (55 2b). 90 continents, widespread Of novelties 14 elsewhere: species, Americas, America Oceania, Oceania. 61 campaigns mostly ectomycorrhizas (60%; 45 (36%; 27 (4%; (47% Madagascar) classified fungi. Among /russula-lactarius /boletus (a widely documented pattern see Corrales 2018, 2015), 3a). taxa, 236 (81% found) endemic, Thirty-six six Réunion, Oceania (2 (1 OTU), (3 Asia OTU). Six 3b). found, study. those, 12 exclusively roots, sporocarps, sporocarp They (9 (5 /cantharellus, /elaphomyces, /sebacina, each). account 7% analysis, re-clustering, selecting OTU, deleting merging 929, 1207, 3054 respectively. Five 19 public repositories. 4a) showed mya. Seventeen 94 4b) shows closest relatives except clade appears risen 4c) included 76 207 knowledge, data barcode region. data. slightly higher (620 (around morphospecies) results show contributed substantially highlighting sheer documentation (Antonelli case globally. As continuously discovered globe Willis, 2018), well-surveyed (e.g., Ainsworth commercial food trade (Cutler Dentinger Suz, surveyed Castellano continue source contributing world's knowledge. voucher-based morphology, multi-gene phylogenies possible specimens ascertain novelty position gold standard biodiversity will require substantial commitment sustained funded fieldwork. mainly lineages; agree 2015; McGuire 2011); Basidiomycota, /sordariales1, /pyronemataceae1. estimates ranges 84,000 140,000 2% currently catalogued 2022). did include any sequence; therefore, science. indicates likely increase steeply if focusing lichens endophytes, severely understudied Interestingly, detected root-only (60% lower proportion sporocarp-only (36%) 4% (4%). Biodiversity aiming unknown tend focus efforts above-ground (Cazabonne 2012), often officially (Aime 2021). significant belowground; should above belowground. suggest endemisms respect OTUs. previous where percentage morphotypes undiscovered levels align terrestrial (83% average families), amphibians (99%), reptiles (92%), invertebrates (86%) 2005) An issue level, authoritative, unequivocally named Sequencing markers (Andrew fungarium would help overcome limitation. almost India-Madagascar mass, Within phylogeny, even though older, arising around 85 mya; poor close (Dentinger unpublished). make vicariance unlikely primary mechanism part, do support (2004): If partners before expect few early-diverging (close mark) case, later. alternative interpretation, albeit unparsimonious, may existed landmass