fungal biology 121 (2017) 939e955
journal homepage: www.elsevier.com/locate/funbio
Out of Asia: Biogeography of fungal populations reveals Asian origin of diversification of the Laccaria amethystina complex, and two new species of violet Laccaria
Lucie VINCENOTa,*,1, Flavius POPAb,c,1, Francisco LASOd, Kathrin DONGESb, Karl-Heinz REXERb, Gerhard KOSTb, Zhu L. YANGe, Kazuhide NARAf, Marc-Andr e SELOSSEd,g aNormandie Univ, UNIROUEN, IRSTEA, ECODIV, Batiment^ Blondel, F-76821 Mont Saint Aignan Cedex, France bPhilipps-Universitat€ Marburg, Department of Systematic Botany and Mycology, Karl-von-Frisch Straße 8, 35039 Marburg, Germany cBlack Forest National Park, Department of Ecosystem Monitoring, Research & Conservation, Kniebisstraße 67, 77740 Bad Peterstal-Griesbach, Germany dMus eum National D’Histoire Naturelle, Institut de Syst ematique, Evolution, Biodiversit e (UMR 7205 ISYEB), CP 50, 45 Rue Buffon, 75005 Paris, France eKey Laboratory for Plant Diversity and Biogeography of East Asia, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China fDepartment of Natural Environmental Studies, The University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa, Chiba 277-8563, Japan gDepartment of Plant Taxonomy and Nature Conservation, University of Gdansk, Poland article info abstract
Article history: Purple Laccaria are ectomycorrhizal basidiomycetes associated with temperate forests all over Received 1 March 2017 the Northern Hemisphere in at least two taxa: Laccaria amethysteo-occidentalis in North Amer- Received in revised form ica, and L. amethystina complex in Eurasia, as shown by Vincenot et al. (2012). Here, we 28 July 2017 combine a further study of the genetic structure of L. amethystina populations from Europe Accepted 9 August 2017 to southwestern China and Japan, using neutral Single Sequence Repeat (SSR; microsatellite) Available online 24 August 2017 markers; and a systematic description of two novel Asian species, namely Laccaria moshuijun Corresponding Editor: and Laccaria japonica, based on ecological, morphological, and molecular criteria (rDNA se- Kentaro Hosaka quences). Population genetics provides evidence of the ancient isolation of three regional groups, with strong signal for speciation, and suggests a centre of origin of modern popula- Keywords: tions closest to present-day Chinese populations. Phylogenetic analyses confirm speciation Cryptic geographic species at the molecular level, reflected in morphological features: L. moshuijun samples (from Fungal species complex Yunnan, China) display strongly variable cheilocystidia, while L. japonica samples (from Japan) Hydnangiaceae present distinctive globose to subglobose spores and clavate cheilocystidia. This study of Phylogeography a species complex primarily described with an extremely wide ecological and geographical Purple Laccaria range sheds new light on the biodiversity and biogeography of ectomycorrhizal fungi. Taxonomy ª 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved.
* Corresponding author. Tel.: þ33 232769434. E-mail address: [email protected] (L. Vincenot). 1 Authors equally contributed to the experiments, data analyses and preparation of the manuscript. http://dx.doi.org/10.1016/j.funbio.2017.08.001 1878-6146/ª 2017 British Mycological Society. Published by Elsevier Ltd. All rights reserved. 940 L. Vincenot et al.
Introduction in association with coniferous (Pinaceae) and deciduous (Betulaceae, Fagaceae, Salicaceae) hosts (Mueller 1984; Roy Ectomycorrhizal fungi (EMF) play a major role in ecosystems, et al. 2008). Therefore, a northern hemisphere distribution especially in temperate and boreal forests (Smith & Read of L. amethystina was assumed, although several different 2008), through their symbiotic association with the roots of violet species may exist worldwide (Wilson et al. 2013; numerous tree species. Most studies of EMF were done in ear- Wilson et al. 2017a; Fig 1).Onepossiblereasonforthissim- lier decades in Europe and North America, and this has plistic picture of global distribution could be difficulty in resulted in a geographic bias in the history of fungal systemat- delineating species within a global L. amethystina species ics (Dickie & Moyersoen 2008). complex, as suggested by Vincenot et al. (2012).Fewmor- Mycologists began to explore biogeographical patterns phological and systematically informative characters have and speciation processes of fungi in recent decades (Kohn been described, whereas more conspicuous characters 2005; Giraud et al. 2008), with case studies mostly focussing such as the colour of the different fruitbody parts show on pathogenic and saprotrophic species (Brown & broad phenotypic variation (Mueller 1992; Wang et al. Hovmøller 2002; Giraud et al. 2010; Stukenbrock 2013). Speci- 2004; Popa et al. 2014). Nevertheless, Mueller (1984) distin- ation and historical distribution of EMF species have been ex- guished the North American violet Laccaria amethysteo-occi- plored mainly at the local scale, and data remain scarce at the dentalis from the Eurasian L. amethystina. With a first continental level (Douhan et al. 2011; Vincenot & Selosse continental-scale characterization of spatial genetic struc- 2017). Yet today, many EMF species are still named world- ture of populations, Vincenot et al. (2012) showed that L. wide and across a variety of ecosystems using European or amethystina from Western Europe and Scandinavia belongs North American names, due to the above-mentioned geo- to a single, panmictic metapopulation, whose dispersal and graphic bias, and based on superficially similar morphology genetic homogenization were possibly favoured by host (Hawksworth 2001; Schmit & Mueller 2007; Feng et al. 2012). generalism (Roy et al. 2008)andtheabsenceofmajorgeo- The common assumption that some EMF species show graphic barriers. But we also demonstrated reproductive a worldwide distribution (Taylor et al. 2006)isreminiscent isolation between European and Japanese populations of of the Beijerinckian view that in the microbial world ‘every- L. amethystina, which is strongly supported by phylogenetic thing is everywhere’ (O’Malley 2007). Assumptions of world- analysis (Vincenot et al. 2012). This raised the hypothesis of wide distributions imply not-too-ancient, long-distance a cryptic species complex over Eurasia, and the need for in- gene flow, a long history of repeated diffuse gene flow over vestigation of more populations between the two investi- small to medium distances that prevent parapatric specia- gated Eurasian range limits. tion, or a combination of both processes (de Queiroz 2005; In the present study, we sought a finer understanding of Lomolino et al. 2010). The continental scale continuity of ecto- the biogeography of Eurasian L. amethystina and of the taxo- mycorrhizal host forests without major mountain barriers, nomic relationships within the L. amethystina species com- for example over Europe, may allow such gene flow plex. We used a larger set of neutral markers for population (Douhan et al. 2011). Nevertheless, phylogeographic analyses genetics, combined with phylogenetics, and completed mo- based on molecular data often distinguish cryptic biological lecular data with ecological, macro and micromorphological species, which have remained hidden so far because of the observations. Most importantly, we examined genotypic and limited availability (or investigation) of morphological char- phenotypic variation among populations from 12 sites over acters. In fact, recent studies of various fungal species, EMF three regions: Europe, China (Yunnan province, one of the or not, have highlighted a disjunctive population structure most important biodiversity hotspots in Asia; Myers et al. suggestive of distinct biological or phylogenetic species (e.g. 2000) and Japan. Based on these populations, we offer an Geml et al. 2006, 2010; O’Donnell et al. 2011; Grubisha et al. updated view of the L. amethystina complex over Eurasia, de- 2012; Zhao et al. 2013; Merenyi et al. 2014; Jargeat et al. 2016). lineate two new species, and propose biogeographic scenarios In particular, range disjunction can be related to global, an- relating their histories to that of the amended European cient geographic patterns, as observed between Old and L. amethystina sensu stricto. New World taxa (Geml et al. 2006, 2010; Garnica et al. 2011; O’Donnell et al. 2011). One of the most documented model species in ectomy- Material and methods corrhizal population genetics is Laccaria amethystina Cooke (Cooke 1884), because of its unique appearance, seemingly Collection and field sites wide geographic distribution and large host range (Gherbi et al. 1999; Wadud et al. 2006b; Roy et al. 2008; Vincenot Fruitbodies of Laccaria amethystina were collected at forest et al. 2012), and because of the early availability of molecu- sites in three regions: five sites in Europe, two in Japan (from lar resources for sister species L. bicolor (Martin et al. 2008). Vincenot et al. 2012) and five new sites in China (Table 1; It belongs to an ECM genus distributed worldwide (Wilson Fig 1). This sampling scheme covered regional Euclidian dis- et al. 2017a,b). The typical violet fruitbodies of L. amethys- tances of 2340 km (Europe), 370 km (China) and 960 km (Japan). tina are described as widely distributed from temperate As in Vincenot et al. (2012), all fruitbodies were collected at and boreal regions of the northern hemisphere to moun- least 1.5 m away from each other to avoid collecting the tain areas (above 2000 m a.s.l.) of subtropical regions, and same genet twice. The ecology of sampling sites, mainly Asian origin of Laccaria amethystina species complex 941
Fig 1 e Map of worldwide distribution of described violet Laccaria species, Section Amethystinae Bon. Smaller maps on the sides display the location of sampling sites of populations included in this study in Europe (five sites), southwest China (five sites) and Japan (two sites).
dominant vegetation, climate and altitude, varied amongst nuclei per basidiospore of Laccaria moshuijun. Samples were sites (Table 1). Populations from Europe and Japan were for- treated with a solution of WGA-AF 488 (Invitrogen, Karlsruhe, merly studied by Vincenot et al. (2012). The holotype of newly Germany) and propidium iodine (Merck Schuchardt OHG, described Laccaria moshuijun was collected in a mixed forest Hohenbrunn, Germany), following protocols from mainly populated with Pinus yunnanensis in Yunnan Province, Dohlemann€ et al. (2011) to prepare for Confocal Laser Scanning near Lufeng (Lat.: 25.119, Long.: 102.081), 2200 m a.s.l. on Spectral microscope (CLSM, Leica, Germany) observations. Fi- 10.VIII.2013 by F. Popa and G. Kost [HKAS93732, Holotype]. nally, fragments of lamellae from the dried collection [MB- The holotype of Laccaria japonica, the second species described 001174] were sputtered with gold (King & Brown 1983) to ob- in this study, was collected by K. Nara [TNS-F64167] in Japan serve spores of L. moshuijun with a scanning electron micro- on Honshu Island, Mount Fuji (Lat.: 35.340 Long: 138.799) on scope (Hitachi S530, Hitachi, Japan). 19.VIII.2000, on volcanic soil nearby Salix reinii. Population genetics analyses Microscopy Sample total genomic DNA was newly extracted from fruit- Eight fruitbodies, also used for population genetics and phylo- bodies for five populations from China as in Vincenot et al. genetic analyses, were examined microscopically: two from (2012), and DNA extracted for a former study (Vincenot et al. Japan and six from China (names and population of origin in 2012) was re-used to genotype samples from five European Table 1). Light microscopy examinations were done using and two Japanese populations (Table 1). Twelve SSR markers a Laborlux D device (Leica, Wetzlar, Germany) on material were amplified to characterize genetic diversity of popula- mounted in 5 % KOH. Spore sizes and length/width ratio (Q) tions: eight markers (La03, La06, La12, La14, La17, La21, La23, were measured excluding ornamentation and apiculus. Fluo- La33) were originally designed for Japanese Laccaria amethys- rescence microscopy was used to investigate the number of tina populations and one marker (Ll35) for Japanese Laccaria 942 L. Vincenot et al.
Table 1 e Populations and geography and ecology of sampling sites. Number of samples for each site are displayed in Table 3. Population Locality Elevationa Climate Dominant vegetation Further use of samplesb
EurEst Tartu, Jarvselja€ (Estonia) 30 m Continental Mixed Picea abies, Tilia e 58 1805200N, 27 1502300E cordata, Populus tremula forest EurSpa Basque Country, Guipuscoa (Spain) 600 m Maritime Fagus sylvatica e dominated Phylogeny e GQ406451 43 0701600N, 2 1503800W mature forest EurSwe Blekinge, Olofstrom€ (Sweden) 100 m Temperate Temperate F. sylvatica e Phylogeny e GQ406455 56 2002900N, 14 2600600E dominated mature EurSwi Luzern, Eschenbach (Switzerland) 700 m Temperate Temperate P. abies e e 47 0902400N, 8 1205300E dominated mature coniferous forest EurUK Aberdeenshire, Kirkhill 120 m Temperate to Temperate F. sylvatica & e (United Kingdom) maritime Larix decidua e dominated 57 2103000N, 2 4604700W forest ChiGa Yunnan, Lijiang Gaomeigu 3200 m Temperate Mountainous Pinus armandii e village (PRC) e dominated forest, with 26 420200N, 100 105200E sparse Quercus spp. ChiKu Yunnan, Kunming Qiongzhu 2170 m Subtropical Mountainous mixed Microscopy e MB-005258 temple (PRC) broadleaved forest Phylogeny e KU962969; 25 701200N, 102 506000E dominated by KU962972 Cyclobalanopsis glaucoides, Lithocarpus sp., Quercus spp., sparse Pinus yunnanensis ChiLi Yunnan, Lijiang Alpine 3900 m Temperate Mountainous mixed forest e Botanical Garden (PRC) dominated by C. glaucoides, 27 002100N, 100 1004800E Lithocarpus sp., Quercus spp. ChiBi Yunnan, Bitahai (PRC) 3650 m Temperate Mountainous coniferous Microscopy e MB-005259 27 430600N, 99 580700E forest dominated by Picea Phylogeny e KU962978 spp., Abies spp., Larix sp., with some Betula spp., Rhododendron spp., Quercus aquifolioides ChiAi Yunnan, Ailao Shan Field Station 2500 m Subtropical Mountainous broadleaved Microscopy e LaMB126; of Xishuangbanna evergreen forest dominated LaMB146 Tropical Botanical Garden (PRC) by Lithocarpus sp., Quercus Phylogeny e KU962973; 24 330000N, 101 104800E spp. KU962974 JapMF Shizuoka, Mount Fuji (Japan) 1381 m Temperate Sparse vegetation patches Microscopy e TNS-F64167 35 2002400N, 138 4705500E dominated by Salix reinii Phylogeny e KU962975; KU962976; KU962988; GQ406444; GQ406445 JapMT Hokkaido, Mount Tokachi (Japan) 766 m Temperate Sparse vegetation patches Microscopy e MB-005257 43 2703100N, 142 3800600E established after the 1926 Phylogeny e KU962977 eruption; mainly Salix bakko
All samples were used for population genetics. a, elevation above sea level. b, further use: samples mentioned here were also included in phy- logenetic analyses (followed by GenBank accession number) or microscopic observations (followed by specimen Collection number).
laccata populations by Wadud et al. (2006a,b), while three from the 12 previous ones (1209M5, 1195M13, La12, La14, markers (Lv257, Jl1195M13, Jl1209M5) were originally designed Ll35) for which at least 50 % of individuals within at least for European L. amethystina populations by Vincenot et al. one population presented null alleles in the homozygous (2012). As loci La03, La17, La23, Ll35, Lv257, Jl1195M13, and state. Jl1209M5 had already been genotyped for Japanese and Euro- Departure from HardyeWeinberg equilibrium and linkage pean populations by Vincenot et al. (2012), these former data disequilibrium were tested using the software Genetix 4.05 were incorporated into the present study. Amplification of (Belkhir et al. 1996e2004), which was also used to estimate al-
SSR loci, detection of amplicons and analyses of fragment lelic richness Ar and an unbiased genic diversity Hn.b. (Nei sizes were conducted as in Vincenot et al. (2012). SSR data 1978). Genepop 4.5.1 (Rousset 2008) was used to calculate alle- were produced for a total of 199 European, 165 Chinese and lic frequencies, heterozygosities, and fixation indices within
73 Japanese samples and were used for population genetics and among populations based on allele identity (FIS,FST) and analyses. All population genetics analyses were run indepen- on allele size (RST) to detect possible discrepancies from an dently (i) on the whole 12-SSR dataset and (ii) on a subset of SSR stepwise mutation model. Spatial genetic structure seven SSR loci (thereafter, 7-SSR dataset), excluding five loci within and among populations was further investigated Asian origin of Laccaria amethystina species complex 943
through correspondence analysis conducted with Genetix (including the outgroup Mythicomyces corneipes) with 604 posi- 4.05. Genetic structure without prior assumption of geo- tions was used for phylogenetic analyses using two indepen- graphic origin was examined by cluster analysis with Struc- dent methods: maximum likelihood (ML), and Bayesian ture 2.3.4 (Pritchard et al. 2000), for two models: one analyses, through the bioinformatics platform CIPRES Science assuming admixture and correlated allele frequencies, the Gateway (Miller et al. 2010). ML analyses were run in RAxML- other assuming no admixture and independent allele fre- HPC2 8.2.3 (Stamatakis 2014) using the GTRCAT model. Sup- quencies. A total of 500 000 simulations (plus 50 000 for burn- port values for best tree topology were assessed by nonpara- in) were run to explore a range of possible cluster numbers metric bootstrap (Felsenstein 1985) based on 1000 replicates. from K ¼ 1toK ¼ 12, and each run was repeated 10 times. Bayesian analysis was performed using MrBayes 3.2.6 Mean ln(likelihood) for each K was calculated from ten out- (Ronquist et al. 2012) with two independent runs of 107 gener- puts, and DK was calculated and plotted along K values ations (25 % burn-in) and four chains per run, with sampling (Evanno et al. 2005) to determine the most plausible values every 100 generations, with number of substitution of K. An analysis of molecular variance (AMOVA) with 1000 types ¼ 1, rates ¼þG (gamma-distributed rate model). permutations was run in Arlequin 3.5.2.2 (Excoffier & Lischer 2010) to hierarchize the participation of structure within and between populations and regional groups in the total vari- Results ance. To investigate the demographic history of regional groups, we examined the possible occurrence of recent bottle- Evidence for three distinct regional population groups necks (including founder events) by calculating the M ratio as described by Garza & Williamson (2001), with M ¼ k/r, k being Correspondence analysis based on SSR datasets revealed the number of allele sizes in the population, and r the total al- three conspicuous clusters of populations corresponding to lele size range. The rationale of this method is that a bottle- the geographic origins of samples, namely Europe, China or Ja- neck event would cause an abrupt population size drop with pan: this was observed based on the 12-SSR dataset (the two a random sampling of allele sizes (k) in the population, while principal components accounted for 73.3 % of variance, the total range of allele sizes (r) would decrease less abruptly: Fig 2) and on the 7-SSR dataset (81.2 % of variance, Sup. thus, populations having experienced a recent bottleneck Fig 1). No clustering of samples from a given population was have a lower M value. Finally, to assess whether genetic diver- observed within any region. AMOVA carried out on the 12- gence between populations or regional groups of populations SSR dataset also highlighted the strong differentiation be- was related to geographic distance, we ran Mantel tests with tween regional groups: 58.93 % of total variance was explained 1000 permutations in Genepop, looking for correlation be- by the structure in three regions, whereas structure among tween genetic distance [FST/(1 FST)] and geographic distance populations within regions accounted for only 3.53 % and [ln(Euclidian distance)]. structure within populations for 37.54 % of variance. Cluster analysis without prior assumption of geographic origin also Phylogenetic tree construction highlighted three distinct genetic groups in a model allowing admixture between groups and correlated allelic frequencies The sequence of internal transcribed spacer (ITS) of the nu- with 12-SSR dataset (Sup. Fig 2). Those genetic groups corre- clear ribosomal DNA was amplified for phylogenetic analyses spond exactly to three regional metapopulations, entirely ex- using nested PCR amplification, for 30 Laccaria spp. samples clusive of each other and without any internal structure (Sup. (Sup. Table 1). Total DNA was extracted by using the High Fig 2). In the model with the second set of simulation priors Pure PCR Template Preparation Kit (Roche, Mannheim, Ger- (no admixture between clusters and independent alleles fre- many). Primer combination NSI1-NLB4 (Kendall & Rygiewicz quencies), cluster analysis revealed seven clusters with the 2005) was used for the first PCR reaction and primers ITS1F 12-SSR dataset (Sup. Fig3; the latter increase of clusters num- (Gardes & Bruns 1993) and ITS4 (White et al. 1990) for the sec- ber could be explained by the fact that the second STRUCTURE ond, specific reaction. PCR amplicons were kit-purified (Invi- model was more stringent than the first one). Those seven tek AG, Berlin, Germany), then both strands were Sanger- clusters corresponded to the three regional metapopulations, sequenced with an ABI capillary sequencer 3730XL by LGC with substructure within Europe and China: Japan had no in- (LGC Genomics GmbH, Berlin, Germany). Raw sequences ternal structure while Europe displayed two genetic groups were manually edited and consensus sequences retrieved present in every population and China showed signal of inter- for each sample using CodonCode Aligner 6.0.1 (CodonCode nal diversity, with four non-homogeneously distributed ge- Corporation 2013). These sequences are available in GenBank netic groups. For example, in China, populations ChiGa and database under accession numbers KU962960 to KU962989. ChiKu were dominated (>60.0 % membership coefficient) by For phylogenetic analyses, 33 other purple Laccaria se- two distinct genetic groups, present in lower proportions in quences, including ten sequences produced by Vincenot the other Chinese populations (Sup. Fig 3). With the 7-SSR et al. (2012), were retrieved from GenBank (Benson et al. 2005) dataset, cluster analysis with either simulation priors sets and UNITE (Abarenkov et al. 2010) and added to our dataset revealed three genetic clusters, again corresponding to the (Sup. Table 1). A sequence alignment was produced using three regional metapopulations (data not shown) that were Mafft version 7.0 (Katoh & Standley 2013) and corrected using further investigated. Finally, we note that some of our primers Gblocks 0.91b tool (Castresana 2000), which allowed smaller fi- for SSR amplification display problems of portability among nal blocks and gaps within the final blocks, as well as less regions, which justified the delineation of the 7-SSR dataset strict flanking positions. The final alignment of 64 sequences for markers performing well everywhere: markers 1209M5 944 L. Vincenot et al.
Fig 2 e Evidence for isolation of the three regional metapopulations. (A) Correspondence analysis based on genotypes from the 12-SSR dataset. Green dots, populations from Europe; blue dots: populations from China; purple dots: populations from Japan. (B, C) Venn diagrams of alleles private to regional metapopulations or shared between them, (B) based on actual allele counts or (C) allele counts weighted by sample size (Europe, n [ 199; China, n [ 165; Japan, n [ 73) (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).
and 1195M13, designed on European samples, failed to am- divergence between Europe and both China and Japan. In con- plify for 50 % of samples in populations ChiAi and JapMT; trast, although RST between China and Japan was still moder-
La12, La14 and Ll35, designed on Japanese Laccaria laccata sam- ately high (RST: 0.28), it was lower than FST. This means that ples, showed similar defects in EurEst, EurSwe, EurSwi, and Chinese and Japanese populations are less divergent when EurUK. considering allele sizes rather than allele identities and points
FST values over 12 loci were extremely high and equivalent to restricted gene flow or genetic drift rather than to the occur- between the three regions (FST from 0.44 to 0.50), meaning rence of allelic stepwise mutation within either of these two a lack of gene flow between regional groups (Table 2). RST groups (Hardy et al. 2003). FST within Europe (from 0.01 to values were even higher than FST between Europe and China 0.07) and within Japan (0.07) were low, supporting recent
(RST: 0.53) or Japan (RST: 0.60), which also support complete gene flow within these regions. Within China, some spatial Asian origin of Laccaria amethystina species complex 945
or within China (Fig 3) and, notably, genetic distances within Table 2 e Fixation indices between pairs of regional metapopulations. these regions were lower than would be expected from the slopes between regions, further suggesting that different Europe China Japan mechanisms operated within and between regions. (L. amethystina (L. moshuijun) (L. japonica) sensu stricto) Similar structures within populations Europe e 0.440 0.497 China 0.533 e 0.442 Japan 0.600 0.277 e Tests for linkage disequilibrium did not show any significant correlation ( p ¼ 10 4) between pairs of the 12 SSR loci over Calculations were based on the 12-SSR loci dataset, either on SSR the 12 populations. None of the 12 populations departed sig- allele identities (F , upper diagonal in shaded cells) or on allele ST nificantly from HardyeWeinberg equilibrium when consider- sizes (RST, lower diagonal). ing all loci (data not shown). Genic diversity in Chinese
populations (Hn.b. ranging from 0.36 to 0.54, Table 3) tended e genetic structure appeared, since FST and RST were heteroge- to be higher than in Europe (Hn.b.: 0.34 0.40) and Japan (Hn.b.: ¼ neous and ranged from very low (FST ChiBi-ChiLi 0.02) to 0.32e0.39). Fixation indices within populations highlighted ¼ e moderate values (e.g. FST ChiAi-ChiBi 0.21; FST ChiAi- a higher trend of inbreeding in Chinese (FIS: 0.26 0.32) and ¼ e ChiLi 0.15; Sup. Table 2). Similar patterns were observed Japanese populations (FIS: 0.29 0.32) than in European ones e with the 7-SSR dataset (Sup. Table 3). (FIS: 0.17 0.24, Table 3). Similar but less marked trends were Isolation by distance (IBD) between regions was significant observed with the 7-SSR dataset (Sup. Table 4). and displayed high R2 values (Fig 3), but because slopes were different (Fig 3) it is hard to conclude that genetic distance is Contrasted genetic structures among regions the result of geographic distance alone. Indeed, comparison be- tween correlation slopes showed similar IBD between Europe Fine examination of genic diversity within regional groups and China (0.217) and between China and Japan (0.279), while gave further evidence for isolation. Genetic diversity over the rate of IBD between Europe and Japan was 1.4e1.8 times China was markedly higher (Hn.b.: 0.55) than over Europe higher (0.400), suggesting a discontinuous isolation from (0.39) and Japan (0.37; Table 3). Estimated allelic richness over Europe to Japan. No IBD pattern was observed within Europe, China (5.05; Table 3)wasca. 1.5 times higher than over Japan
Fig 3 e Patterns of isolation by distance amongst and within regional groups. Correlation coefficients (R2) and regression lines are displayed for significant Mantel tests only. Green dots: distances within Europe (no IBD, p [ 0.506); blue dots: distances within China (no IBD, p [ 0.279); purple dot: distances within Japan (IBD not testable); empty circles: distances between populations from two different regions (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). 946 L. Vincenot et al.
resolved group with the European Laccaria amethystina cluster Table 3 e Diversity indices of populations based on the 12-SSR loci dataset. and the American species Laccaria amethysteo-occidentalis, L. trullisata, and L. ochropurpurea. In accordance with cladistic Population Sample nb. H F Ar n.b. IS species concept, we decided to describe the Chinese and Jap- Europe 199 0.388 0.383 3.050 anese metapopulations as species distinct from the European EurEst 37 0.343 0.181 2.917 L. amethystina. EurSpa 37 0.377 0.221 3.000 EurSwe 41 0.381 0.226 3.167 EurSwi 43 0.353 0.170 3.000 Taxonomy EurUK 41 0.399 0.242 3.167 China 165 0.548 0.417 5.050 ChiGa 26 0.498 0.312 4.833 Laccaria moshuijun Popa & Yang, sp. nov. Fig 4, Sup. Fig 5 ChiKu 37 0.533 0.319 5.833 ETYMOLOGY: Laccaria moshuijun after the local name used for this ChiLi 50 0.537 0.316 6.250 species by people from Yunnan. ChiBi 39 0.505 0.306 5.500 HOLOTYPUS: China: Yunnan Province, ‘near Lufeng’ (Lat.: 25.119, ChiAi 13 0.358 0.256 2.833 Japan 73 0.372 0.151 3.451 Long.: 102.081), 2200 m a.s.l., collected in a mixed forest with JapMF 36 0.386 0.316 3.818 Pinus yunnanensis, Cunninghamia lanceolata and undetermined JapMT 37 0.316 0.291 3.083 broadleaved trees species, on soil. 10 Aug. 2013, F. Popa (HKAS93732, Holotype). The isotype is at Herbarium Marbur- H , unbiased genic diversity (Nei 1978). F , fixation index within n.b. IS gense (MB-301397), paratypes are in the Herbarium Marbur- populations. Ar, allelic richness. In bold: estimates for regional metapopulations. gense and the Herbarium Kunming. Herbarium Marburgense number: MB-301397, MycoBank accession number: (3.45) and over Europe (3.05). Within China, most alleles (46/ MB816291; GenBank accession number: KU962989. 111 ¼ 41.4 %; Fig 2B) were private to this region, while only DIAGNOSIS: Fruitbodies (Fig 4) small to medium-sized. Pileus up 16.4 % of European alleles (10/61) and 18.3 % of Japanese alleles to 30 mm in diameter, initially convex to plano-convex, flat- (11/60) were private to their respective regions. Nearly half of tening with age, centrally depressed, glabrous, translucent- all European (47.5 %) and Japanese alleles (48.3 %) were shared striate, hygrophanous, violet to bluish (colour codes 2.5P 2/2, e by all three regions: the European metapopulation shared 10PB 3/4 to 10PB 5/6 5/8) with brown tinge at the stipe and 31.1 % and 28.3 % of its alleles with Chinese and Japanese meta- in the middle of the pileus, light-coloured when drying. Pileus populations, respectively, while European and Japanese meta- thin, at margin very thin. Lamellae densely close, adnexed to populations shared only 4.9 % and 5.0 % of their alleles, adnate, deep violet like the pileus, in age brighter, fertile. e respectively, with each other (Fig 2B). More detailed examina- Lamellettes present. Stipe up to 100 3 5 mm, cylindrical, fis- tion of each locus (Sup. Fig 4) showed that three loci (La06, tulose, glabrous, longitudinally striate, especially in old fruit- e La14, Lv257) have no allele specific to Japan; three loci (La17, bodies with brownish complexions. Basidia (25) 30 40 (50) m e m La21, La33) have no allele specific to Europe; and three loci m (8.5) 10 12 (15) m, clavate, hyaline, 4-spored, sterig- e m e (1209M5, La03, La12) have no allele specific to Europe or Japan, mata 8 10 m long. Basidiospores (Fig 4) (7.5) 8 9 (8) e m ¼ e whereas all loci had private alleles in China. Only the three last 9 10 m, globose to subglobose, Q 1.0 1.1 (1.25), hyaline, e m loci (1195M13, La23, Ll35) have specific alleles in the three re- echinulate, echinulae (0.7) 1 1.3 (1.5) m, hilar appendix up m e e m gions. When taking into account the sampling effort, the num- to 2 m long. Pleurocystidia (25) 30 50 3 6 m clavate, ber of alleles weighted by sample size was significantly 1.8 thin-walled, hyaline, not as variable as cheilocystidia. Cheilo- times lower in Japan than in China (c2, p ¼ 0.019), and 3.0 lower cystidia clavate, flexuose dichotomy or trichotomy up to cap- e e m in Europe than in Japan (Fig 2C), further supporting a higher di- itate (25) 30 50 5 8 (12) m, thin-walled, hyaline. Clamp versity in Chinese metapopulations. GarzaeWilliamson ratios connections present. Pileipellis consisting of interwoven radi- e m provided indications regarding demographic histories: China ating hyphae (6 8 m in diameter) with scattered perpendic- displayed a value close to that expected at equilibrium ular clusters of hyphae, with intracellular colouration; some ¼ hyphae with oily content, smooth, thin-walled, and clamp (MChina 0.67; Garza & Williamson 2001). Whereas drastically ¼ connections are present. Stipitipellis consisting of radial hy- lower values for Europe (MEurope 0.45) and Japan ¼ phae, hyphae becoming broader towards the centre of the (MJapan 0.40) indicate either a single, harsh bottleneck event or a milder and more diffuse process of population size reduc- stipe, thin-walled, smooth, clamp connections present, tion. In both scenarios, the present low Ar values for these re- some hyphae with clavate outgrowings. Caulocystidia absent. gions (Table 3) suggest a drop to an extremely small population Basal mycelium fresh violet, with age whitish (Fig 4). size or a limited time of recovery since reduction, or both. GEOGRAPHIC DISTRIBUTION: Laccaria moshuijun is widely distributed in Yunnan (PRC) and was found from south-western subtropical regions in the Ailao Shan Mountains at approximately 2500 m Phylogenetic analysis a.s.l. elevation up to the Tibetan border in northwestern Yunnan. In subtropical regions the collections were found Phylogenetic analysis based on ITS showed three distinct, within mixed forest with different Quercus spp., Eucalyptus strongly supported clades (bootstrap values > 0.95), grouping spp., Lithocarpus spp., and Ficus spp. In northwestern Yunnan, respectively all samples from Europe, China, and Japan (Sup. from Lijiang to Shangrila the species was found in pure and Fig 5). Based on ITS, Chinese and Japanese clusters were in sis- mixed Pinus sp., Picea sp. and Quercus aquifolioides forests. Lac- ter positions, in a well-supported clade, which formed a poorly caria moshuijun fruitbodies are present under various possible Asian origin of Laccaria amethystina species complex 947
Fig 4 e Macroscopic and microscopic morphological characters of Laccaria moshuijun sp. nov. (A) fruitbody of [MB-101186] sampled under its common host Lithocarpus sp.; (B) fruitbody of [HKAS93732, Holotype]; (C) confocal laser scanning micros- copy photograph of stained mature basidiospores; (D) scanning electron microscopy (SEM) photograph of young basidio- spore (10003) from [MB-001174]. Micromorphological observations of [HKAS93732, Holotype]: (E) hymenium; (F) spores; (G) cheilocystidia; (H) stipitipellis; (I) pileipellis. Specific scales are indicated by the bars in the figure. 948 L. Vincenot et al.
host trees and therefore the species can be interpreted as 001001], Paratypes are deposited in the Herbarium Marburgense a generalist with respect to the plant host range. Laccaria mosh- and the Herbarium of National Science Museum, Tokyo. Her- uijun adds to other recently described Chinese species (Wang barium Marburgense number: MB-001001, MycoBank accession et al. 2004) that differ by fruitbodies’ colours. number: MB816292; GenBank accession number: KU962988. DISCUSSION: Macromorphologically, L. moshuijun can be placed DIAGNOSIS: Fruitbodies (Fig 5) small to medium-sized. Pileus in the Section Amethystinae Bon. The most related species 10e30 mm in diameter, initially convex to plano-convex, cen- are L. amethystina, L. japonica,andL. amethysteo-occidentalis. trally depressed, smooth, glabrous, hygrophanous, bright violet Laccaria moshuijun can be differentiated from L. japonica by to purple (colour codes 7.5RP 5/4, 7.5RP 3/4, 10P 4/4) in young globose spores, and different cheilocystidia morphology. stages, then becoming paler, sometimes mixed with pale tan Whereas L. amethystina and L. amethysteo-occidentalis char- to flesh colour (2.5YR 6/4, 10RP 5/4). Lamellae adnexed to adnate, acteristically lack cheilocystidia. Furthermore, the colour fertile, bright violet, lamellettes present. Stipe 30e80 of the fruitbody has more bluish tints compared with the (100) 5 mm, cylindrical. Basidia 30e40 10e12 mm, clavate, hy- other more violet or reddish, flesh-coloured to violet spe- aline, 4-spored, sterigmata 8e10 mm long, occasionally stalked. cies. The macro- and micromorphological differences can Basidiospores (Fig 5)9e10 (11) 7e9 mm, globose to subglobose, be confirmed using molecular markers for phylogeny (Sup. Q ¼ 1.1e1.2 (1.4), hyaline, thin-walled, echinulate, echinulae (0.7) Fig 5). 0.8e1 (1.5) mm, hilar appendix up to 1.5 mm long. Pleuro- and chei- locystidia cylindrical, clavate to flexuose (25) 30e50 3e7(9)mm. SPECIMEN COLLECTIONS: China: Yunnan Province. ‘Ailao Shan’, 2400 m a.s.l., within Clamp connections can be extended. Pileipellis consists of inter- a mixed forest dominated by Quercus spp. and Lithocarpus spp., on soil, GPS (Lat.: m 25.022, Long.: 101.400), 12 Jul. 2007, G. Kost, K.H. Rexer, K. Donges (MB-001113, woven radiating hyphae (up to 8 m) with scattered perpendic- MB-001115, MB-001119, Paratypes); China: Yunnan Province. ‘Ailao Shan’, 2497 m ular clusters of hyphae, smooth, thin-walled, with intracellular a.s.l., within a mixed forest dominated by Quercus spp. and Lithocarpus spp., on colouration. Stipitipellis consist of radial hyphae, hyaline, soil, GPS (Lat.: 24.118, Long.: 101.030), 14 Jul. 2007, G. Kost, K.H. Rexer, K. Donges smooth, thin-walled, clamp connections present. Caulocystidia (MB-001126, Paratype); China: Yunnan Province. ‘Ailao Shan’, 2500 m a.s.l., within absent. Basal mycelium violet, in age whitish (Fig 5). a mixed forest dominated by Quercus spp., on soil, GPS (Lat.: 24.057, Long.: 101.031), 15 Jul. 2007, G. Kost, K.H. Rexer, K. Donges (MB-001146, MB-001147, Para- GEOGRAPHIC DISTRIBUTION: Laccaria japonica usually appears in early type); China: Yunnan Province. ‘Ailao Shan’, 2468 m a.s.l., within a mixed forest dom- successional settings, including volcanic deserts, nurseries, inated by Quercus spp. and Lithocarpus spp., on soil, GPS (Lat.: 24.533, Long.: 101.033), sand dunes, gardens, parks, and forest edges, but is rare inside China 16 Jul. 2007, G. Kost, K.H. Rexer, K. Donges (MB-301397, Paratype); : Yunnan deep old growth forests. It is associated with many tree species, Province, Kunming. ‘near Lufeng’, 2200 m a.s.l., collected in a mixed forest with Pinus yunnanensis, Cunninghamia lanceolata and undetermined broadleaved tree species, such as Pinaceae, Salicaceae, Fagaceae, and possibly Betula- on soil, GPS (Lat.: 25.119, Long.: 102.081), 10 Aug. 2013, F. Popa, G. Kost ceae, virtually all ectomycorrhizal trees in Japan, and thus ap- (HKAS93732, Holotype); China: Yunnan Province, Lijiang County. ‘Yulong Xue Shan’, pears as a typical generalist. Wadud et al. (2014) showed that 3200 m a.s.l., mixed Pinus spp., Picea spp. and Quercus aquifolioides forest, on soil, the genet size of L. japonica is very small, less than 1 m, which GPS (Lat.: 27.001, Long.: 100.169), 14 Aug. 2013, F. Popa, G. Kost (MB-301547, MB- was only known before from L. amethystina in Europe (Gherbi 301548, MB-301549, MB-301550, MB-301582, MB-301589); Yunnan Province, Lijiang County. ‘Yulong Xue Shan’, 3200 m a.s.l., mixed Picea sp. and Q. aquifolioides forest, et al. 1999; Fiore-Donno & Martin 2001). Laccaria japonica fruits on soil, GPS (Lat.: 27.001, Long.: 100.169), 15 Aug. 2013, F. Popa, G. Kost (MB-301628, from May to November, with a peak of abundance in rainy sea- MB-301633, MB-301665, MB-301667); China: Yunnan Province, Lijiang County. ‘Yulong son in June and September. Xue Shan, near Baishui’, 3300 m a.s.l., mixed forest with Quercus sp., Picea sp., Pinus DISCUSSION: Laccaria japonica is very similar in macromorphology sp. and Sorbus sp., on soil, GPS (Lat.: 27.134, Long.: 100.240), 17 Aug. 2013, F. Popa, G. to L. amethystina, L. moshuijun,andL. amethysteo-occidentalis,but Kost (MB-301702, MB-301703, MB-301704, MB-301798); China: Yunnan Province, Lijiang County. ‘Yulong Xue Shan’, 3500 m a.s.l., Q. aquifolioides and P. yunnanensis can be well distinguished using micromorphological charac- mixed forest, on soil, GPS (Lat.: 27.001, Long.: 100.168), 19 Aug. 2013, F. Popa, G. ters, especially the subglobose spores, which separate L. japon- Kost (MB-301872, MB-301919); China: Yunnan Province, Lijiang County. ‘Yulong Xue ica from others. Furthermore, the species is cultivable in vitro Shan’, 3500 m a.s.l., Q. aquifolioides and P. yunnanensis mixed forest, on soil, GPS (Teramoto et al. 2012), unlike L. amethystina, so far not culti- (Lat.: 27.001, Long.: 100.168), 20 Aug. 2013, F. Popa [MB-301967]; China: Yunnan Prov- vated in vitro in spite of our efforts (M.-A. Selosse, unpublished ince, Lijiang County. ‘Yulong Xue Shan, near Baishui’, 3300 m a.s.l., mixed forest with Quercus sp., Picea sp., Pinus sp. and Sorbus sp., on soil, GPS (Lat.: 27.134, Long.: data). The macro- and micro-morphological, as well as the 100.240), 21 Aug. 2013, F. Popa, G. Kost (MB-302016, MB-302017); China: Yunnan Prov- physiological, differences were validated using molecular ince, Lijiang County. ‘Yulong Xue Shan’, 3500 m a.s.l., Q. aquifolioides intermixed with markers used for phylogeny (Sup. Fig 5). P. yunnanensis, on soil, GPS (Lat.: 27.001, Long.: 100.169), 11 Aug. 2014, F. Popa, G. SPECIMEN COLLECTIONS: Japan: Honshu Island. ‘Mont Fuji’, on volcanic soil together with Kost (MB-304938); China: Yunnan Province, Lijiang County. ‘Yulong Xue Shan’, Salix reinii, on soil, GPS (Lat.: 35.340 Long.: 138.799), 19 Aug. 2000, K. Nara (MB- 3700 m a.s.l., mixed coniferous forest with Pinus armandii and P. yunnanensis,on 001001, Isotype, TNS-F64167, Holotype). Japan: Hokaido Island. ‘Mount Tokachi’, on soil, GPS (Lat.: 26.998, Long.: 100.192), 12 Aug. 2014, F. Popa, G. Kost (MB-305001, volcanic soil together with Salix bakko, on soil, GPS (Lat.: 43.420 Long.: 142.680), 09 MB-305004, MB-305051); China: Yunnan Province, Lijiang County. ‘Yulong Xue Aug. 2009, K. Nara (MB-005257). Shan’, 2900 m a.s.l., P. armandii mixed with P. yunnanensis forest, on soil, GPS (Lat.: 27.003, Long.: 100.185), 16 Aug. 2014, F. Popa, G. Kost (MB-305244); China: Yunnan Province, Shangrila. ‘Baimang Snow Mountain’, 3800 m a.s.l., collected in a mixed forest with Picea sp. and Q. aquifolioides, under Q. aquifolioides, on soil, GPS (Lat.: 28.305, Long.: 99.153), 27 Aug. 2014, F. Popa, G. Kost (MB-305755). Discussion
Laccaria japonica Popa & Nara, sp. nov. Fig 5, Sup. Fig 5 Evidence for differentiation of three species ETYMOLOGY: Laccaria japonica is named after the collecting area, Japan. In population analyses, extremely high values of fixation indi- Japan HOLOTYPUS: : Honshu Island, ‘Mount Fuji’ (Lat.: 35.340 Long.: ces, correspondence analysis, AMOVA, cluster analyses, ex- 138.799), on volcanic soil together with Salix reinii, on soil, 19 treme differentiation values (FST, RST), SSR portability, and Aug. 2000, K. Nara (TNS-F64167, Holotype). Isotype at MB [MB- analysis of private alleles all clearly pointed to three Asian origin of Laccaria amethystina species complex 949
Fig 5 e Laccaria japonica sp. nov. (A) fruitbodies of Laccaria japonica sp. nov. at Mount Fuji together with host tree Salix reinii; (B) holotype collection [TNS-F64167, Holotype]; (C, D) SEM photographs of basidiospore of (60003) young (C) and mature (D) from [TNS-F64167, Holotype]. Micromorphological observations of [TNS-F64167, Holotype] (E); hymenium; (F) spores; (G) cheilocystidia, note distinctive clavate shape; (H) pileipellis; (I) stipitipellis. 950 L. Vincenot et al.
genetically distinct metapopulations, corresponding to three preference for pioneer or ruderal areas. Thus, host spectrum geographic origins (Europe, China, and Japan). Moreover, in and ruderality level slightly differ between the three species, the AMOVA, nearly two-thirds of the genetic diversity was dis- even if in these respects, the Asian species tend to be more tributed between regions, in an unexpected situation for ecto- similar. Finally, L. japonica is cultivable in vitro (Teramoto mycorrhizal basidiomycetes with aerial spore dispersal et al. 2012), while we are only aware of failed attempts for Eu- (Carriconde et al. 2008; Engh et al. 2010; Douhan et al. 2011) ropean L. amethystina s.s. (M.-A. Selosse pers. obs.). where intra-population diversity normally dominates (as re- ported in Laccaria amethystina at a European scale; Roy et al. Evidence in favour of an origin genetically close to present 2008; Vincenot et al. 2012). Vincenot et al. (2012) formerly Chinese population showed a differentiation between European and Japanese L. amethystina populations, and hypothesised the occurrence of Phylogenetic analysis and portability of most SSR markers a ring species over the Northern Hemisphere (i.e. a set of con- suggest a recent common ancestry, and the genetic features nected populations, each breeding with closely related popu- of the metapopulation provide clues about an Asian origin of lations, but with two ends of the continuum distantly the three species, although the representation of the Japanese related and not interbreeding, namely Europe and North population remains limited. First, the discontinuity of IBD pat- America here; Martins et al. 2013), although they acknowl- tern from Europe to Japan, but following similar slopes be- edged that IBD between Japan and Europe was more marked tween Europe and China and between China and Japan, than expected by extrapolating IBD within each of these re- suggests a central position of origin of modern European and gions. We expected that somewhat genetically intermediate Japanese metapopulations, i.e. an ancestral position closer populations would be found in Yunnan, perhaps with greater to the present Chinese populations. Second, allelic richness, similarity to either the European or the Japanese genetic pool along with allelic frequencies and shared allele patterns, if IBD rate was not continuous. Our findings in effect support show that (i) the Chinese metapopulation harbours a higher a gradual pattern of differentiation over Eurasia, but also sup- allelic richness than the European or Japanese; (ii) its many port very distinctive features of the Chinese metapopulation private alleles and GarzaeWilliamson’s M-ratio suggest that in terms of allelic richness, abundance of private alleles, pop- the Chinese metapopulation is ancient and at equilibrium rel- ulations close to mutationedrift equilibrium, and regional ative to the others; (iii) the European and the Japanese allele structure as compared with the Japanese and European meta- pools each evolved independently from a subset of a pool ge- populations. Even though the present sampling design suffers netically close to the modern Chinese allelic pool, and these from geographic gaps, especially in Russia and China, our new two pools share a very limited overlap with each other Chinese samples, in addition to our original European and Jap- (<5.0 %). We hypothesize independent divergences of two anese samples, reveal three distinct metapopulations. populations from a paleo-population close to the modern Chi- The distinction between metapopulations was further sup- nese one (Fig 6). At first glance, this appears to be at odds with ported by micromorphological analyses, which allowed for- the phylogenetic position of European Laccaria amethystina as mal morphological description of two additional species, a clade basal to an Asian clade comprising Chinese and Japa- Laccaria moshuijun and Laccaria japonica, setting L. amethystina nese species (Sup. Fig 5), but lineage sorting of an ancestral s.s. apart for European samples (where the type is from; polymorphism independently from speciation for this locus Hudson 1778; Cooke 1884). The most distinctive micromor- may explain this (Stewart et al. 2014); more loci should be stud- phological characters were subglobose spores distinguished ied before a reliable phylogenetic background can be the Japanese L. japonica, while globose spores and different considered. cheilocystidia differentiated the Chinese L. moshuijun. The Importantly, Chinese populations show larger regional ge- macromorphology was very similar for all three species (ex- netic differentiation than European populations, in spite of cept the bluish tints in L. moshuijun), placing all species in a similar sampling effort (five populations) and smaller area the Section Amethystinae Bon and reflecting a close phyloge- covered (Figs 1 and 3). Furthermore, genetic structure among netic relationship, illustrated in our phylogenetic analysis Chinese populations does not display a clear spatial pattern, based on ITS (see also Wilson et al. 2017a). Thus, the superficial and a Mantel test rejected a genetic differentiation solely similarity can be nuanced by closer micromorphological in- driven by distance in Yunnan; similarly, isolation by elevation vestigation, as shown in the description of many ‘cryptic’ spe- was rejected by a Mantel test (analysis not shown). Nor could cies in other taxa (e.g. Wang et al. 2013; Stefani et al. 2014). Chinese genetic diversity be explained by a Wahlund effect at Ecological features can also disentangle ‘cryptic’ species the local scale (that is, no local substructure within popula- (e.g. Sato et al. 2007; Jargeat et al. 2016). Eurasian violet Laccaria tion; PCA analysis, not shown). This heterogeneity is reflected, also display small ecological divergence. Laccaria amethystina for instance, by cluster analysis, where populations from s.s. in Europe forms small genets in mature forests (Gherbi ChiGa and ChiKu appeared more genetically homogeneous et al. 1999; Fiore-Donno & Martin 2001; Hortal et al. 2012) and than the others; similarly, fixation indices (FST,RST) also is generalist in terms of host trees, with the exception of Sal- showed moderate limitation to gene flow between ChiAi and icaceae and Pinaceae (Roy et al. 2008; M.-A. Selosse, pers. obs.). ChiBi, ChiAi and ChiLi (Sup. Table 4). Yunnan’s mountainous Laccaria moshuijun was found in various forest sites together landscape (succession of mountain summits and valleys) with different Pinaceae and Fagaceae, but also in ruderal areas could act as a driver of genetic isolation, as described for the from subtropical to temperate regions. Laccaria japonica can be ectomycorrhizal basidiomycete Tricholoma matsutake in the found together with different host trees from Salicaceae, Pina- Eastern Himalaya (Amend et al. 2010). Moreover, climate, ceae, and Fagaceae in volcanic deserts, forests with a marked available hosts, and ecology also vary between populations Asian origin of Laccaria amethystina species complex 951
(Table 1), hence one cannot exclude that genetic differences Amanita species correlates with a higher species richness in reflect adaptive variations (ecotypes), as shown for Califor- Asia (Zhang et al. 2004). The widely-distributed fly agaric com- nian Suillus brevipes populations (Branco et al. 2015). plex (Amanita muscaria (L.) Lam.) most likely comes from Of course, the restriction of our sampling to Yunnan and northeast Asia and may have colonized the American conti- awareness of possible range displacement related to climatic nent through forest refugia in Beringia (Geml et al. 2006, oscillations (Lomolino et al. 2010) hamper a precise localiza- 2010), a path also suggested to have been followed by Cortinar- tion of an ancestral population of the species complex. Never- ius arcuatorum and C. elegantior (Garnica et al. 2011). Finally, theless, Yunnan is a major biodiversity hotspot in Asia (Myers phylogenetic analyses support an Asian origin for the ectomy- et al. 2000), with 4000 fungal species currently recorded that corrhizal porcini (Boletus edulis and allied species; Feng et al. account for ca. 40 % of the known fungal species of China 2012), other Boletales (Wilson et al. 2012), and several Tuber (Yang 2005). A variety of Laccaria species is found in the clades (Bonito et al. 2013). species-rich forests of south-western China (Wang et al. 2004; Yang 2005; Popa et al. 2014). The topography and land- scape of this region could have preserved a large part of the Elements of demographic history for European and Japanese ancestral gene pool since, during the complex history of Hi- populations malayan glaciations (Xiao et al. 2015), the spatial distribution of mountain belts and valleys allowed species to expand European and Japanese populations have been exposed to northwards during interglacials or retreat towards southern contrasting geographic and climatic constraints that shaped refugia in glacial ages (Qiu et al. 2011). Yunnan is part of the their respective diversities and clearly accumulated distinct Oriental biogeographic region, reported as a centre of diversity divergences from a China-related ancestral polymorphism for plants (Lomolino et al. 2010), but also for fungi (e.g. Feng (as shown, e.g., by the level of shared alleles, which is very et al. 2012), thanks to a spatio-temporal continuity of suitable low between Japan and Europe, and by phylogenetic relation- habitats. Impact of climatic oscillations on the connectivity of ships, Sup. Fig 5 and Vincenot et al. 2012). Correspondence watersheds in the Hengduan Mountains have, for instance, re- analysis, fixation indices, and cluster analyses showed the cently been shown to have shaped the spatial genetic struc- absence of spatial genetic structure within European and Jap- ture of populations of the Tuber indicum species complex anese metapopulations, confirming Vincenot et al.’s (2012) (Feng et al. 2016). conclusion of non-interrupted gene flow at this scale An out-of-Asia hypothesis has been proposed, or seems (1000 km) in Laccaria amethystina and Laccaria japonica. Indeed, likely for other ectomycorrhizal taxa. In Amanita, the strong these regions, which are devoid of major obstacles to North- e relationship between Eurasian and Asian-North American South exchanges (such as mountains or water stretches), are more or less continuously covered by forests harbouring
Fig 6 e Scenarios of demographic history for violet Laccaria Eurasian populations. 952 L. Vincenot et al.
ectomycorrhizal trees e even the Tsugaru Strait separating suggests that the Japanese metapopulation also went through Honshu and Hokkaido Islands does not exceed 50 km in some population size reduction, either by ‘bottleneck’orby width. a milder ‘diffuse reduction’. Despite low allelic richness and lim- In Europe, moderate genetic diversity and low inbreeding ited sampling, the Japanese metapopulation harboured many suggest that populations are now at equilibrium; moreover, private alleles, suggesting a very narrow sampling of alleles a very low IBD is not surprising given the generalist character from the ancestral population, followed by efficient recovery of L. amethystina (Roy et al. 2008), allowing diffuse gene flow and by accumulation of new alleles, which strongly contrasts over European forests. The present European metapopulation with Europe. Thus, gene flow with the ancestral population appears to have diverged from a subset of an ancestral paleo- was likely interrupted after a size reduction event. Similarly, population resembling modern Chinese populations, as 79 % smaller values for RST than for FST between China and Japan of alleles found in Europe are shared with the Chinese meta- (mainly for Chinese populations versus JapMF; Sup. Table 4) in- population (whereas only 43 % of all alleles from China can dicate that differentiation is most likely due to interruption of be found in Europe; Fig 2). The GarzaeWilliamson test sug- gene flow (causing a departure from Stepwise Mutation Model gests a size reduction in the European metapopulation and distribution of alleles being favoured by a drop to a very small this is congruent with the low allelic richness compared effective population size). Thus, we favour a ‘bottleneck’ sce- with China. This size reduction could have occurred either nario (Fig 6), old enough to have permitted the accumulation through a harsh event (‘bottleneck’ scenario), with an abrupt of private alleles and speciation. We disfavour the alternative interruption of gene flow, or through a more progressive pro- possibility of an increased mutation rate in Japan, whose bio- cess of reduction (‘diffuse reduction’ scenario) of gene flow, or logical bases would be unclear to us. The isolation from China even an intermittently interrupted gene flow (Fig 6). could be due to rapid selection (possibly driven by contrasted The low number of private alleles in Europe could support environments and hosts in Japan), to a foundation event by a recent, abrupt reduction to a small effective population size long-distance dispersal of migrants, or a combination of and thus a ‘bottleneck’ scenario, with too short a recovery time both processes. As for the European metapopulation, the ab- to accumulate private alleles. However, European L. amethys- sence of precise dating around speciation prevents linking tina does not display the high inbreeding rate expected in that scenario to historical events; the relative isolation of L. ja- a young, small population, and furthermore, presents a high ponica is also not correlated with a major geographic obstacle diversity. Alternatively, low inbreeding and high diversity to gene flow between Central Asia and Japan. rather suggest an ancient event of size reduction, with subse- Intriguingly, L. japonica populations display, among and quent recovery over generations to reach the modern popula- within the two populations, moderate genetic diversity, low tion stability. The European allelic richness, which is lower allelic richness, and some inbreeding (Table 3), which is some- than in China, and the level of private alleles are in favour of what unexpected in an ancient bottleneck scenario that would this second hypothesis of a slow recovery after a strong reduc- have allowed recovery of populations towards genetic equilib- tion of effective population size. Furthermore, higher values rium. The two Japanese populations were sampled on volca- for RST than for FST between the Europe and China groups sug- nic slopes recurrently disturbed by volcanic activity, and are gest that divergence results more from slow acquisition of part of the pioneer communities in secondary successions new alleles than from interruption of gene flow. Thus, the (Wadud et al. 2014). This pioneer trait, a specificity of L. japonica population genetic recovery could have been associated with amongst the species studied, may have entailed intense size episodic or reduced gene flow after the size reduction event, reduction events (Mount Fuji’s last eruption was in 1707; hampering the accumulation of new and private alleles inde- Mount Tokachi’s in 1926), independently leading to very small pendently from the ancestral gene pool. All these consider- population sizes. Thus, two independent, recent bottlenecks ations support a ‘diffuse reduction’ of European population could explain the peculiar patterns observed for these Japa- size associated with a progressive divergence from an ances- nese populations, and samples from more populations, espe- tral population, with reduced but maintained gene flow. An cially performed in less harsh environments, could test this ancient expansion from Asia could explain such a scenario, possibility and refine the demographic history of the Japanese where few migrants towards the West would have maintained metapopulation. low continuous or intermittent gene flow with the populations of origin (Fig 6). Wilson et al. (2017a) recently estimated that dispersal of Northern Hemisphere Laccaria spp. through Conclusions and perspectives Laurasia would be posterior to late Oligocene e early Miocene, associated with host switches to Pinaceae and Salicaceae that Our study supports that Laccaria amethystina s.l. should now could have accelerated Laccaria diversification. Without more be divided into three geographic species, whose origin is precise dating of the divergence, it is difficult to speculate Asian, close to extant Chinese Laccaria moshuijun populations. what event or period may have favoured the steps described Our study is in line with other published investigations on above. continental or intercontinental species in indicating that The Japanese L. japonica metapopulation also appears to they contain species separable on genetic bases (with DNA have evolved from an ancestral paleo-population resembling sequencing data and isolation by reduced gene flow repre- the present Chinese one, since 77 % of Japanese alleles were senting a proxy to biological species), as well as a posteriori shared with the Chinese metapopulation (whereas only 41 % on the basis of (micro)morphology (e.g. Osmundson & of Chinese alleles were found in Japanese samples; Fig 2). Halling 2010). Yet a lot remains to be done on violet Laccaria The GarzaeWilliamson test, along with low allelic richness, spp. since large parts of Eurasia have yet to be sampled, Asian origin of Laccaria amethystina species complex 953
and such sampling may reveal new species or specify the ex- the Tuberaceae and their newly identified Southern Hemi- act boundaries and reasons for isolation of the species now sphere sister lineage. PLoS One 8: e52765. described. Only then can we anticipate a more precise demo- Branco S, Gladieux P, Ellison CE, Kuo A, LaButti K, Lipzen A, Grigoriev IV, Liao HL, Vilgalys R, Peay KG, Taylor JW, Bruns TD, graphic history, using demographic simulations, and some 2015. Genetic isolation between two recently diverged popu- dating of the events shaping this history. Moreover, many lations of a symbiotic fungus. Molecular Ecology 24: 2747e2758. other areas worldwide are still not or are insufficiently sam- Brown JKM, Hovmøller MS, 2002. Aerial dispersal of pathogens on pled, such as large parts of Asia, South America, and Africa. the global and continental scales and its impact on plant In particular, the exact relationships between North Ameri- disease. 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