Amanita Satotamagotake Sp. Nov., A Cryptic Species Formerly Included in A. Caesareoides, Has Invaded The Natural Habitat of A. Caesareoides

1 Full Paper
ed
2 Amanita satotamagotake sp. nov., a cryptic species formerly included in A. caesareoides, has
3 invaded the natural habitat of A. caesareoides
iew
5 Miyuki Kodaira a, Wataru Aoki b, Naoki Endo c, Daisue Sakuma d, Eiji Hadano e, Atsuko Hadano
6 e, Yasushi Hashimoto f, Seiki Gisusi g, Masaki Fukuda a, b, Akiyoshi Yamada a, b, h*
v
8 a Department of Agriculture and Life Science, Graduate School of Science and Technology,
re
9 Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan
10 b Department of Science and Technology, Graduate School of Medicine, Science and
11
er
Technology, Shinshu University, 8304, Minami-minowa, Nagano, 399-4598, Japan
12 c Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University,

pe
13 4-101 Koyama, Tottori, 680-8553, Japan
14 d Osaka Museum of Natural History, 1-23, Nagai-koen, Higashisumiyoshi-ku, Osaka, Osaka,
15 546-0034, Japan.
ot
16 e 1-1-36, Ryougo, Oita, Oita, 870-0883, Japan.
17 f Department of Agro-environmental Science, Obihiro University of Agriculture and Veterinary

tn
18 Medicine, Obihiro 080-8555, Japan
19 g Forest Products Research Institute, Hokkaido Research Organization, Nishikagura 1-10,

rin
20 Asahikawa, Hokkaido 071‒0198, Japan
21 h Institute for Mountain Science, Shinshu University, 8304, Minami-minowa, Nagano, 399-
ep
22 4598, Japan
23
24 *Corresponding author
Pr
25 akiyosh@shinshu-u.ac.jp
This preprint research paper has not been peer reviewed. Electronic copy available at: https://ssrn.com/abstract=4261673
1 Abstract
ed
2 We evaluated the inclusion of a cryptic species in a Japanese A. caesareoides population. We
3 sampled A. caesareoides specimens under various vegetation and climate, then conducted
4 phylogenetic analyses of six loci. The A. caesareoides population showed two distinct clades,
iew
5 except when the ITS phylogeny was considered. These two phylogroups showed different
6 distribution: subalpine–cool temperate and temperate–subtropical areas, respectively. Although
7 these two populations overlapped in terms of basidiospore size, the latter tended to exhibit a
v
8 smaller basidiospore. Additionally, only the former showed mycelial growth on agar. Based on
re
9 these phylo-morpho-ecophysiological characteristics, we separated A. caesareoides into two
10 species. Because the lectotype of A. caesareoides showed similarity to the former by the DNA
11
er
analysis, the latter was described as Amanita satotamagotake. Based on the geographic patterns
12 of the two species, A. satotamagotake has invaded the natural habit of A. caesareoides, possibly
pe
13 because of natural forest disturbance and global warming.
14
15
ot
16 Keywords: Biogeography, Caesar’s mushroom, Competition, Ectomycorrhizal association,
17 Fungal resource conservation, Invasiveness, Multi-locus phylogenetic analyses, Speciation,

tn
18
19
rin
ep
Pr
1 1. Introduction
ed
2 Amanita caesareoides Lj.N. Vassiljeva, belonging to the section Caesareae, is associated with
3 Pinaceae and Fagaceae trees as an ectomycorrhizal symbiont (Endo 2015; Endo et al. 2013).
4 Species in the sect. Caesareae (“Caesar’s mushrooms”) are highly edible and are consumed
iew
5 globally. The sect. Caesareae in the genus Amanita is estimated to have originated around 60
6 Mya (Paleocene) in the southwest of the Laurasia Continent (present southeast Asia), then
7 expanded to Africa, Europe, Australia, and North and Central America (Sánchez-Ramírez et al.
v
8 2015). In Japan and the surrounding far East Asian region, the reddish-pileus Caesar’s
re
9 mushrooms, represented by A. caesareoides, are estimated to have expanded from the ancestral
10 clade around 8–6 Mya (late Miocene; Tortonian–Messinian) (Sánchez-Ramírez et al. 2015).
11
er
A. caesareoides was first described from the Kamchatka Peninsula, Russia (Vassiljeva,
12 1950) and was regarded as A. hemibapha (Berk. & Broome) Sacc. or A. caesarea (Scop.) Pers.
pe
13 in East Asia (Hennings 1900; Kawamura 1913, 1930, 1954; Imazeki & Hongo 1957, 1987;
14 Hongo 1975; Oda et al. 1999; Zhang et al. 2004; Yang 2005, 2015; Cho et al. 2015; Yang et
15 al. 2018). In Japan, A. caesareoides was initially identified as A. caesarea by Hennings (1900)
ot
16 with the common name Obenitake, which means “large crimson mushroom” in Japanese
17 (Matsumura 1904; Shirai 1905). The specimen was collected by Mitsutaro Shirai in 1894 in a
tn
18 Quercus crispula Blume forest under a cool-temperate climate at the lakeside of Chuzenji-ko,
19 Nikko, Tochigi Prefecture; its external morphology was illustrated in color (Shirai & Hennings
rin
20 1899). In 1913, Seiichi Kawamura described Japanese A. caesarea with a color drawing of
21 basidiomata, a description of its local name “Tamagotake” (“egg mushroom” in Japanese), and
ep
22 its consumption by the local population. Subsequently, Tamagotake was accepted as the
23 Japanese common name of A. caesarea (Sirai & Miyake 1917; Yasuda 1920; Kawamura 1930).
24 Kawamura (1954) suggested that Tamagotake includes three varieties distinguished by pileal
Pr
25 color: yellow, reddish orange (vermilion), and deep red (crimson). Yasuda (1920) described the
1 microscopic features of Japanese A. caesarea, which corresponds to the yellowish-pileus type
ed
2 of Tamagotake reported by Kawamura (1954). The specimen of Yasuda (1920) was sampled
3 in 1919 in Kiso-fukushima, Nagano Prefecture, presumably under an intermediate-temperate
4 climate. The yellowish-pileus type of Tamagotake reported by Kawamura (1954) was sampled
iew
5 on July 13, 1928, in Matsudo, Chiba Prefecture, under a warm-temperate climate. The crimson-
6 pileus type corresponds to the specimen of Kawamura (1913), which was sampled on October
7 30, 1910, in southwest Iizuna (at the foot of Mt. Reisenji-yama), Nagano Prefecture (Kawamura
v
8 1930, 1954), presumably under a cool-temperate climate. Although the sampling site of the
re
9 other vermilion-pileus type of Tamagotake reported by Kawamura (1954) was not described, a
10 corresponding specimen was sampled in October 1930, in Sanbu, Chiba Prefecture (warm-
11
er
temperate climate) (Kawamura 1931). Hongo (1975) identified the reddish-pileus type(s) of
12 Tamagotake as A. hemibapha (Berk. & Broome) Sacc. and the yellowish-pileus type (newly
pe
13 named Kitamagotake) as A. hemibapha subsp. javanica Corner & Bas, based on the following
14 morphological differences: Tamagotake has a thinner pileus with striation, a slender stipe with
15 orangish stripes on the surface, and rounder spores, compared with Mediterranean A. caesarea.
ot
16 Imazeki & Hongo (1987) regarded Japanese A. hemibapha as conspecific to A. caesareoides.
17 However, Hongo (1975) and Imazeki & Hongo (1987) did not observe type specimens of A.
tn
18 hemibapha or A. caesareoides. Hongo (1975) inspected a Tamagotake specimen sampled in
19 Nango-imodani, Kusatsu, Shiga Prefecture, under a warm-temperate climate; this specimen

rin
20 may correspond to the vermilion-pileus type reported by Kawamura (1954). Oda et al. (1999)
21 described Kitamagotake as a new species, A. javanica (Coner & Bas) T. Oda, C. Tanaka &
ep
22 Tsuda, based on a phylogenetic analysis of the internal transcribed spacer (ITS) region of the
23 nuclear rDNA operon (nuc rDNA). However, they did not examine the type specimens of A.
24 hemibapha or A. hemibapha subsp. javanica (Endo et al. 2016, 2017). Kitamagotake was
Pr
25 recently distinguished from the type specimen of A. hemibapha subsp. javanica (distributed in
1 the south and southeast Asian regions) and newly described as A. kitamagotake N. Endo & A.
ed
2 Yamada (Endo et al. 2017).
3 The reddish-pileus type(s) Tamagotake was compared with its Asian relatives,
4 including the lectotype of A. caesareoides, and identified as A. caesareoides, based on
iew
5 morphological characteristics and a phylogenetic analysis of the nuc rDNA ITS region (Endo
6 2015; Endo et al. 2016). Notably, Japanese A. caesareoides exhibits variations in spore size and
7 culture characteristics, suggesting two ecological groups: subalpine–cool-temperate and warm-
v
8 temperate–subtropical (Endo 2015). This is reminiscent of the different color types of
re
9 Tamagotake (i.e., vermilion- and crimson-pileus types) described by Kawamura (1954).
10 Therefore, we hypothesized that the Japanese A. caesareoides population consists of two
11
er
independent species. To test this hypothesis, we conducted phylogenetic analyses of six DNA
12 loci—the ITS and intergenic spacer 1 (IGS1) regions of nuc rDNA, tef1, rpb2, cox3, and atp6—
pe
13 and morphological observations of the Japanese A. caesareoides and other species in the sect.
14 Caesareae. Additionally, we established cultures of Tamagotake and performed comparisons
15 of its colony morphology and vegetative mycelial structure. Based on the resulting data, we
ot
16 describe a new Amanita species. Because the taxonomic positions of these closely related two
17 species suggested that they have distinct geographic distributions, we discuss their ecological
tn
18 significance in relation to land use in Japan and climate change. We also address the
19 phylogenetic and evolutionary background of the section Caesareae.

rin
20
21 2. Materials and Methods

ep
22 2-1. Sampling and morphological observation of Tamagotake
23 Specimens of A. caesareoides were collected in various forests in Japan from 2008 to 2020
24 (Table 1). The following macroscopic characteristics of basidiomata were recorded: pileal color,
Pr
25 shape, and diameter; lamellar width and color; stipe length, width, and surface ornamentation;
1 and volval shape, width, length, and color. Young, fresh basidiomata were collected for tissue
ed
2 isolation (see below). Subsequently, basidiomata were freeze-dried, oven-dried at 60°C
3 overnight, and stored in the laboratory as dried specimens. Where necessary, specimens were
4 deposited in the National Museum of Nature and Science (TNS), Japan. The warmth index (WI)
iew
5 (Kira 1948) of each sampling site was estimated to characterize the vegetational zone and
6 habitat properties: 15–45, subarctic (subalpine) evergreen coniferous forests; 45–85, cool-
7 temperate deciduous broad-leaved forests; ~85, intermediate-temperate deciduous broad-
v
8 leaved forests; 85–180, warm-temperate evergreen broad-leaved forests; and 180–240,
re
9 subtropical evergreen broad-leaved forests. The coldness index (CI) (Kira 1948) and mean
10 annual temperature (MAT) were estimated for each sampling site. These indices were
11 calculated based on AMEDAS

er data (Japan Meteorological Agency;
12 http://www.jma.go.jp/jma/index.html) of the one to three points nearest each sampling site,

pe
13 using a lapse rate of 0.55°C/100 m around the Japanese Archipelago (Yoshino 1986).
14 2-2. Tamagotake tissue isolation
15 Because the culture characteristics of Japanese A. caesareoides differ between specimens

ot
16 sampled from subalpine–cool temperate and warm temperate forests (Endo 2015), we subjected
17 young basidiomata to tissue isolation using the method reported by Endo et al. (2013) with
tn
18 minor modification. The basidiomata surface was wiped with absorbent cotton soaked in 70%
19 ethanol; pileus inner tissue was axenically cut into several 2 × 2-mm mycelial pieces using a
rin
20 scalpel, then inoculated on modified Norkran’s C (MNC) agar (Yamada & Katsuya 1995). The
21 specimens were incubated for 4 months in an air-conditioned room (~20–25°C), and mycelium
ep
22 growth was observed under a stereoscopic microscope (Stemi 2000-C, Carl Zeiss AG, Jena,
23 Germany). Mycelia and colonies were also observed under a differential interference contrast
24 microscope (AXIO Imager A1, Carl Zeiss AG) and hyphal width was measured.
Pr
25 2-3. Microscopic observation
1 Small pieces of dried specimens were rehydrated in 70% ethanol for 1 min, transferred to
ed
2 distilled water to fully rehydrate mycelia (~1–2 h), and mounted with 100% lactic acid on a
3 glass slide. If necessary, basidia were stained with acid fuchsin. Hyphae and basidiospores were
4 observed under a differential interference contrast microscope with a 40× or 100× oil-
iew
5 immersion objective lens (AXIO Imager A1, Carl Zeiss AG), then photographed. The sizes of
6 50 basidiospores, 30 basidia, 10 sterigmata, and 10 cheilocystidia-like terminally swollen
7 hyphae at the lamellae edge were measured for each specimen. Pileipellis and stipitipellis
v
8 hyphae were observed in several selected specimens.
re
9 2-4. DNA analysis
10 Genomic DNA was extracted from dried specimens of A. caesareoides, A. caesarea, A.
11
er
kitamagotake N. Endo & A. Yamada and A. chatamagotake N. Endo & A. Yamada by the
12 cetyltrimethylammonium bromide method (Gardes & Bruns 1993; Endo et al. 2016) with minor
pe
13 modification. We performed DNA analyses of the ITS and intergenic spacer 1 (IGS1) regions
14 of nuc rDNA, translation elongation factor 1α gene (tef1), RNA polymerase II subunit 2 gene
15 (rpb2), cytochrome c oxidase subunit 3 gene (cox3), and ATPase subunit 6 gene (atp6). The
ot
16 following primer pairs were used for PCR amplification: ITS1-F/LB-W (Gardes & Bruns 1993;
17 Tedersoo et al. 2008) for ITS, CNL12/5s-Anderson (Anderson & Stasovski 1992; Duchesne &
tn
18 Anderson 1990) for IGS1, tef-1F/tef-2218R (first PCR; Morehouse et al. 2003; Rehner &
19 Buckley 2005) and tef-983F/tef1-R (second PCR; Rehner & Buckley 2005; Morehouse et al.
rin
20 2003) for tef1, bRPB2-6F/bRPB2-7.1R (Matheny 2005) for rpb2, and COX3-1/COX3-2
21 (Kretzer and Bruns, 1999) for cox3. The primer pair ATP6-AmF (5'-
ep
22 GGTTTAAATGCTCCTATTTTAGGTC-3') and TP6-AmR 5'-
23 GRAAATAATCTAACTCCTAATGA-3') for atp6 was designed based on the genome
24 sequence of Amanita muscaria (L.) Lam (Amanita muscaria Koide BX008; Kohler et al. 2015).
Pr
25 For DNA amplification from old specimens (i.e., A. caesareoides LE203026 [lectotype;
1 Vassiljeva 1950] and Hongo 5297 [Hongo 1975]), the following primers were used: IGS1-
ed
2 AcoidesF (5’-GAGCTTTGGGATGTGTTGTGGAGA-3’), IGS1-AcoidesR (5’-
3 CTTGGGTTCAGGCAGAGTCAGAGG-3’), rpb2-AcoidesF (5’-
4 CGTCAACGGAGTATGGATG-3’), rpb2-AcoidesR (5’-TCTGAACTCTTCGCCTTCGT-3’),
iew
5 rpb2-AcoidesR’ (5’-TCTGAACTCTTCRCCTTCGT-3’), tef1-AcoidesF (5’-
6 CATCTCAAGCTGATTGTGCCAT-3’), tef1-Acoides2R (5’-TCAATGGCATCAAGAAGG-
7 3’), tef1-UF(5’-AGCTGGTATCTCCAAGGACGG-3’), tef1-AcoidesR’ (5’-
v
8 GAAKCGATCTTCGRTCCACT-3’), cox3-AcoidesF (5’-
re
9 GCTGGAAATAGAAAAGCTGCAA-3’), and cox3-AcoidesR (5’-
10 TAAGCCGTGAAGACCCGTAG-3’) for the first and second rounds of PCR. PCR
11
er
amplification was conducted using Dream Taq DNA polymerase (Thermo Fisher Scientific Inc.,
12 USA) or Tks Gflex™ DNA Polymerase (TaKaRa, Kusatsu, Japan) and the ProFlex PCR
pe
13 System 3  32-well (Applied Biosystems, USA) or GeneAmpⓇPCR System 2700 (Applied
14 Biosystems). First-round PCR for ITS, IGS1, atp6, and cox3 was conducted with the following
15 protocol: denaturation at 95°C for 3 min; 40 cycles of denaturation at 95°C for 30 s, annealing
ot
16 at 52°C for 30 s, and extension at 72°C for 1 min (ITS, cox3, atp6) or 90 s (for IGS1); and final
17 extension at 72°C for 7 min (ITS, cox3, atp6) or 10 min (IGS1). To amplify tef1, touchdown
tn
18 PCR was conducted with the following protocol: denaturation at 95°C for 3 min; 40 cycles of
19 denaturation at 95°C for 15 s, annealing at 65°C decreasing by 1°C per cycle (cycles 1–10) or
rin
20 52°C (cycles 12–40), and extension at 72°C for 1.5 min; and final extension at 72°C for 10 min.
21 To amplify tef1 from the Hongo 5297 specimen, PCR was conducted using the tef1-
ep
22 AcoidesF/tef1-Acoides2R primers with the following protocol: denaturation at 95°C for 3 min;
23 40 cycles of denaturation at 95°C for 30 s, annealing at 50°C for 30 s, and extension at 72°C
Pr
24 for 1 min; and final extension at 72°C for 7 min. To amplify rpb2 from the Hongo 5297
25 specimen, the following protocol was used: denaturation at 95°C for 3 min; 40 cycles of
1 denaturation at 95°C for 30 s, annealing at 60–54°C (cycles 1–5 at 60°C, 6–10 at 58°C, 11–20
ed
2 at 56°C, and 21–35 at 54°C) for 30 s, and extension at 72°C for 1 min; and final extension at
3 72°C for 7 min. Second-round PCR was conducted using the following primer pairs and
4 annealing temperatures: IGS1, IGS1-AcoidesF/IGS1-AcoidesR (57°C); rpb2, rpb2-
iew
5 AcoidesF/rpb2-AcoidesR or rpb2-AcoidesF/rpb2-AcoidesR’ (both 51°C); tef1, tef1-UF/tef1-
6 AcoidesR’ (53°C); and cox3, cox3-AcoidesF/cox3-AcoidesR (50°C). PCR amplicons were
7 subjected to electrophoresis on 1.5–2.0% agarose gels to confirm amplification specificity, then
v
8 purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Bands for non-
re
9 specific amplicons were excised from the gel and extracted using the QIAquick Gel Extraction
10 Kit (Qiagen).
11
er
The BigDye Terminator v. 3.1 Cycle Sequence Kit (Thermo Fisher Scientific, Inc.)—
12 with primers identical to the sequences used for first- or second-round PCR—was used for cycle
pe
13 sequencing with the ProFlex PCR System 3x32-well (Applied Biosystems). The amplicons
14 were purified by ethanol precipitation and sequenced directly using the ABI 3130 Genetic
15 Analyzer (Applied Biosystems). The sequences were edited using SeqScanner software v. 2.0,
ot
16 then assembled by CLUSTALW (http://www.genome.jp/tools-bin/clustalw). We confirmed
17 nucleotide complementarity between the two strands and registered the sequences in the DNA
tn
18 Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/index.html). Because the IGS1 region
19 of A. caesareoides was not directly sequenced, DNA cloning was performed using the Mighty
rin
20 TA-cloning Kit (TaKaRa) in accordance with the manufacturer’s recommendation.
21 2-5. Phylogenetic analyses

ep
22 The A. caesareoides, A. caesarea, A. kitamagotake, and A. chatamagotake N. Endo & A.
23 Yamada sequences (Supplementary Table S1) were subjected to phylogenetic analyses.
24 Additionally, sequences of A. caesareoides, A. caesarea, A. jacksonii Pomerl., A. vernicoccora

Pr
25 Bojantchev & R.M. Davis, A. basii Guzmán & Ramírez-Guillén, A. calyptroderma G.F. Atk.
1 & V.G. Ballen, A. rubroflava Yang-Yang Cui, Qing Cai & Zhu L. Yang, A. subhemibapha Zhu
ed
2 L. Yang, Yang-Yang Cui & Qing Cai, A. fuscoflava Zhu L. Yang, Y.Y. Cui & Q. Cai, A.
3 kitamagotake, and A. chatamagotake in GenBank were included in the phylogenetic analyses
4 (Supplementary Table S1). For the six loci, the following datasets were constructed: A.
iew
5 caesareoides, A. jacksonii, A. caesarea, A. basii, A. calyptroderma, A. kitamagotake, and A.
6 chatamagotake for ITS; A. caesareoides, A. jacksonii, A. caesarea and A. chatamagotake for
7 IGS1; A. caesareoides, A. jacksonii, A. basii, A. caesarea, and A. chatamagotake for tef1; A.
v
8 caesareoides, A. jacksonii, A. caesarea, A. kitamagotake, A. vernicoccora, A. rubroflava, A.
re
9 subhemibapha, and A. chatamagotake for rpb2; and A. caesareoides, A. jacksonii, A. caesarea,
10 A. kitamagotake, and A. chatamagotake for atp6 and cox3.
11
er
DNA sequences were aligned using MUSCLE (Edgar 2004) in MEGA v. 7.0 (Kumar
12 et al. 2016) and manually edited. Phylogenetic trees were constructed based on the maximum-
pe
13 likelihood (ML) and Bayesian methods using RAxML v. 8.2.4 (Stamakis 2014) and MrBayes
14 v. 3.2.6., respectively. For ML analysis, GTRGAMMA was selected as the substitution model.
15 Branch support was evaluated by 1000 replicates of bootstrap analysis using the rapid bootstrap
ot
16 option. Because we compared two similar populations of A. caesareoides, we selected A.
17 chatamagotake as the outgroup taxon for all datasets. For the Bayesian method, PAUP* v.
tn
18 4.0a164 (Swofford 2002) and MrModeltest v. 2.3 were used to estimate the substitution model
19 based on the Akaike Information Criterion (Nylander 2004). Markov chain Monte Carlo
rin
20 analysis was run for 1,000,000 generations with trees sampled at each 100-generation interval.
21 Sampled trees were summarized after omission of the first 25% of trees. Phylogenetic trees
ep
22 were edited by TreeGraph v. 2 (Stöver & Müller 2010).
23 2-6. Divergence time estimation
24 The alignments of nuc rDNA ITS and IGS1, tef1, atp6, rpb2, and cox3 for the two phylogenetic
Pr
25 clades of A. caesareoides—as well as the North American A. jacksonii and the Mediterranean
10
1 A. caesarea—were used for divergence time estimation. Bayesian estimation of species
ed
2 divergence time was performed using BEAST v. 2.6.2 and calibrated using BEAUti
3 (Drummond & Bouckaert, 2015), with a birth-death model. The section Caesareae in the genus
4 Amanita lineage was assumed to have originated 25 Mya, and A. jacksonii and the Japanese A.
iew
5 hemibapha lineages branched from their ancestral lineage approximately 6 Mya (Sanchèz-
6 ramiréz et al. 2015). The clock model was run as a relax clock log normal model; the GTR
7 model was used for all genes and regions with a gamma distribution. The posterior distributions
v
8 of parameters were obtained by Markov chain Monte Carlo analysis for 10 million generations
re
9 with a burn-in percentage of 10%. The Markov chain Monte Carlo result was confirmed using
10 Tracer v. 1.6. The relationships among samples from the posterior distributions were
11
er
summarized as a maximum clade credibility tree with the maximum sum of posterior
12 probabilities listed on the internal nodes using TreeAnnotator v. 2.6.2 (Drummond & Bouckaert
pe
13 2015); the posterior probability limit was set to 0.5 to summarize the mean node heights.
14 FigTree v. 1.4.2 was used to visualize the constructed tree, and mean divergence times were
15 calculated using 95% highest posterior density intervals. For expected geological age, we
ot
16 referred to the international chronostratigraphic chart (Cohen et al. 2013).
17 3. Results
tn
18 3.1. Phylogenetic position of Tamagotake specimens
19 The aligned datasets of ITS, IGS1, tef1, atp6, rpb2, and cox3 comprised 644, 1204, 706, 489,
rin
20 795, and 735 bp, respectively. After the exclusion of ambiguous aligned sites from the
21 phylogenetic analyses, these datasets comprised 235, 849, 475, 408, 455 and 497 bp,
ep
22 respectively (TreeBase accession nos. S29648).
23 The ITS phylogenetic tree (Fig. 1) showed that all tested A. caesareoides specimens
24 consisted of a single clade with the A. caesareoides lectotype, which was separated from closely
Pr
25 related A. caesarea and A. jacksonii with strongly supported values. This is in agreement with
11
1 the findings by Endo et al. (2016). In the phylogenetic trees of tef1 (Fig. 2), rpb2 (Fig. 3), atp6
ed
2 (Supplementary Fig. S1), and cox3 (Supplementary Fig. S2), A. caesareoides specimens were
3 separated into clades A and B with strongly supported values. The IGS1 phylogenetic tree (Fig.
4 4) also showed two clades of A. caesareoides (clades A and B) with moderately supported
iew
5 values. Although the tef1 and rpb2 phylogenies indicated independent positionings of A.
6 caesareoides clades A and B from A. caesarea and A. jacksonii, the atp6 and cox3 trees
7 exhibited a clade A–A. caesarea continuum (Supplementary Figs. S1 and S2) and clade B–A.
v
8 jacksonii continuum (Supplementary Fig S2), respectively. No probable hybrid specimen
re
9 between clades A and B was detected in the A. caesareoides specimens.
10 Although the lectotype LE203026 of A. caesareoides was only obtained partial
11
er
sequences of rpb2, cox3, IGS1 (and no sequences in atp6 and tef-1), these sequences showed
12 higher homologies to the sequences of clade A (99.3–100%) than to the sequences of clade B
pe
13 (94.7–99.6%) (Table 2). The A. caesareoides Hongo 5297 specimen was only obtained the
14 partial sequence of tef1, which showed higher homology to clade B (100%) than to clade A
15 (97.0%) (Table 2). Therefore, A. caesareoides clade A was regarded as true A. caesareoides. In
ot
16 contrast, A. caesareoides clade B was presumed to correspond to A. hemibapha sensu Hongo
17 (Hongo 1975, 1982).

tn
18 Two distinct phylogroups of A. caesareoides showed different host associations (Figs.
19 2–4, Supplementary Figs. S1 and S2, Table S1). Specimens in clade A generally showed
rin
20 associations with subalpine and cool-temperate Pinaceae, Betulaceae, and Fagaceae (deciduous
21 species) as canopy trees. In contrast, specimens in clade B typically showed associations with
ep
22 temperate-to-subtropical Pinaceae, Betulaceae, and Fagaceae (deciduous and evergreen
23 species) as canopy trees.
24 3.2. Evolution of the two phylogroups of A. caesareoides

Pr
25 We analyzed the evolution of the two A. caesareoides phylogroups, based on the global
12
1 evolutionary pattern of the section Caesareae reported by Sánchez‐Ramírez et al. (2015). The
ed
2 six-gene phylogenetic tree showed that the ancestral lineage of the two A. caesareoides
3 phylogroups branched from the A. jacksonii lineage at around 6 Mya (Fig. 5). The ancestral
4 lineage of A. caesareoides branched into two (lineages I and II) at around 4.8 Mya, one of which
iew
5 (lineage I) further branched into two (i.e., A. caesareoides clade A and A. caesarea) at around
6 2.7 Mya.
7 3.3. Geographic distributions and habitats of the two A. caesareoides phylogroups
v
8 Based on the phylogenetic analyses, Japanese A. caesareoides specimens were divided into
re
9 population A (phylogenetic clade A) and population B (clade B). The distributions of these two
10 populations differed. Population A was sampled from subalpine–cool-temperate areas of
11
er
Hokkaido and higher elevation areas of Honshu Island (840–2052 m above sea level), whereas
12 population B was sampled from cool-temperate to subtropical areas of Hokkaido, Honshu,

pe
13 Kyushu, and Okinawa Islands. To assess the abilities of WI, CI, and MAT to explain the
14 different geographic distributions of populations A and B, regression lines were generated
15 between altitude and the temperature indices in samples from Hokkaido, Honshu and Kyushu
ot
16 Islands (Fig. 6, Supplementary Fig. S3). The regression coefficient (R) tended to be higher in
17 WI (Fig. 6). The slopes of regression lines slightly differed according to topography: population
tn
18 A in Honshu was distributed in mountainous areas and showed a steep slope, whereas
19 population B (Honshu and Kyushu) was distributed in both mountainous and plains areas.
rin
20 Furthermore, population A in Hokkaido was distributed in both mountainous and plains areas,
21 and its slope was similar to the slope of population B. The habitats of populations A and B had
ep
22 WI values of 37.0–71.0 and 57.3–188.2, respectively (Table 1, Fig. 6). Therefore, the two
23 Japanese A. caesareoides populations had overlapping distributions in central Honshu (WI
24 57.3–71.0; within the cool-temperate range). No sample of population A was collected in a

Pr
25 warm-temperate area, and no sample of population B was collected in a subalpine area.
13
1 Population B was sampled from a single site at Atsubetsu, Sapporo in Hokkaido. No sample of
ed
2 population A was collected in western Japan.
3 3.4. Sympatric distribution and competition of the two A. caesareoides populations in a
4 mountain area
iew
5 To assess the importance of the probable absence of a hybrid between populations A and B of
6 A. caesareoides, as well as the distinct vertical patterns within each local region, we compared
7 the two populations in (1) the Myoko Volcano Group (Hayatsu et al. 1994), (2) Yatsugatake
v
8 Mountains, and (3) Ina Mountains and the marginal ranges of the Akaishi Mountains. In these
re
9 three regions, population A was distributed in higher-elevation areas, whereas population B
10 was distributed in lower-elevation areas (Fig. 7). The probable sympatric distributions of the
11
er
two populations in these three areas had WI values of 67.9–71.5, 61.5–68.4, and 57.3–67.9,
12 respectively. In the Myoko Volcano Group, the slopes of the regression lines slightly differed
pe
13 between the two populations (Fig. 7A–C). Therefore, in higher elevation areas within the
14 overlapped distribution, population B was expected to be distributed at higher WI sites, whereas
15 population A was expected to be distributed at lower WI sites. However, in the other two
ot
16 regions, the slopes of regression lines did not exhibit large differences (Fig. 7D–I), suggesting
17 that populations A and B were equally competitive in the sympatric areas. Four forest sampling
tn
18 sites (i.e., Mt. Madarao-san and the lakesides of Reisenji-ko, Matsubara-ko, and Komade-ike)
19 harbored both populations sympatrically. At these sites, the WI values were 65–66, 65–66, 67–
rin
20 68, and 59–60, respectively.
21 3.5. Physiological characteristics of the two A. caesareoides populations

ep
22 Samples of A. caesareoides population A showed mycelial growth on MNC agar, and their
23 mycelia were subcultured on MNC agar. However, samples of population B showed limited
24 mycelial growth, typically only on the surface of the mycelial inoculum (basidioma tissue).
Pr
25 Therefore, mycelium of population B could not be subcultured on MNC agar. The mycelial
14
1 growth rate from the mycelial inoculum onto MNC agar was significantly different between the
ed
2 populations (P < 0.05; Table 3, Supplementary Table S2).
3 Mycelial colonies of A. caesareoides population A on MNC agar were white to light
4 yellow, irregular in shape, and had a filamentous margin (Fig. 8A–C). Generative hyphae of
iew
5 the colony margin were straight or interwoven, 1.6–5.6 µm in diameter, sometimes inflated
6 intercalary or terminally with a chlamydospore- or monilioid-cell-like structure (Fig. 8G, H).
7 Although knobs of hyphae were sometimes present, no clamp connections were observed.
v
8 Mycelial colonies of A. caesareoides population B on MNC agar were white to ocher-yellow
re
9 in color, irregular in shape, and had a flat and undulate margin (Fig. 8D–F). Generative hyphae
10 of the colony margin were straight and 1.7–5.7 µm in diameter. Additionally, chlamydospore-
11
er
or monilioid-cell-like swellings were observed at the termini of generative hyphae, which were
12 8.6–46.3 µm in length and 7.5–31.7 µm in width (Fig. 8I, J). Some terminal swellings burst
pe
13 (Fig. 8I). No clamp connections were observed.
14 3.6. Morphology of basidiomata of the two A. caesareoides populations
15 The lengths of 50 spores, 30 basidia, 10 sterigmata, and 10 cells on the lamellar margin of 26
ot
16 specimens of A. caesareoides population A (including the lectotype), 29 specimens of
17 population B (including Hongo 5297—used in the taxonomic study to describe Japanese A.

tn
18 caesareoides [Hongo 1975]) were measured (Supplementary Table S3). Based on the species
19 delimitation criterion for the section Caesareae (Yang 2005; Endo et al. 2016, 2017), the two
rin
20 A. caesareoides populations did not show substantial morphological differences. However, the
21 mean spore size was significantly larger in population A (P < 0.01; Table 3) and the mean
ep
22 sterigmata length tended to be greater in population A (P = 0.0579; Table 3).
23 Based on the phylogenetic positions and the ecological, physiological, and
24 morphological differences of the two A. caesareoides populations, we regard them as two

Pr
25 distinct species. Because A. caesareoides population A included the A. caesareoides lectotype,
15
1 we describe population B as a new Amanita species.
ed
2 4. Taxonomy
3 Amanita satotamagotake M. Kodaira, N. Endo & A. Yamada, sp. nov.
4 Fig. 9A-D, F-L
iew
5 MycoBank no.: MB 845966
6 Diagnosis: This species is cryptic against A. caesareoides but can be distinguished by
7 the tef1, rpb2, atp6, cox3, and IGS of nuc rDNA phylogenies.
v
8 Type: JAPAN, Miyagi Pref., Shichigahama, Hanabushi-shrine, on the ground in a
re
9 forest of Abies firma, 14 Oct 2017, coll. N. Endo (specimen ID: AC-36; TNS-F-82293).
10 Gene sequences ex-holotype: LC723298 (ITS), LC723439–LC723441 (IGS1), LC723398
11
er
(tef1), LC723328 (rpb2), LC723507 (atp6), LC723491 (cox3).
12 Etymology: satotamagotake is the set of “sato” and “tamagotake,” the former means
pe
13 “secondary forests and the surrounding village areas at the foot of mountains” in Japanese.
14 Description: Pileus 34–107 mm in diam, ovate when young or convex, then convex-
15 umbonate to plano-convex-umbonate; surface red or reddish orange, sometimes orange, smooth,

ot
16 glabrous, viscid when moist; margin striation 12–26 mm in length, diam ratio 0.15–0.28(–0.38)
17 mm, sulcate–striate; color gradient between center to margin absent or slightly darker in center.
tn
18 Lamellae 6–10 mm in width at the center, dense; surface pale yellow, edge yellow, pale yellow,
19 or orange. Stipe 106–232 mm in length, upper portion 6–13 mm in width, basal portion 10–
rin
20 14(–17) (–18) mm in width, cylindrical or tapering upward, stuffed when young, then hollow;
21 surface pale yellow, yellow or cream, mostly covered with orangish or pale orangish, ﬁber-like
ep
22 patches toward the base, sometimes lacking in the patch. Annulus membranous, upper surface
23 sulcate–striate, concolorous or slightly deeper than the stipe, lower surface smooth, slightly
24 lighter than upper. Volva 32–59(–81) mm in height, 12–31 mm in width, saccate, ellipsoid,
Pr
25 elongate or cylindrical, membranous; outer surface whitish; inner surface whitish or pale yellow.
16
1 Basidiospores [1000 spores/10 basidiomata/9 specimens] 6.9–10.9 × 5.5–8.8 [Lm × Wm = 7.9–
ed
2 8.6 × 6.6–7.2], Q = 1.03–1.46, Qm = 1.18–1.23, subglobose, broadly ellipsoid, thin-walled, with
3 one or several oily droplets, hyaline, non-amyloid; apiculus cubic. Basidia [270/9/8] 32.2–67.7
4 µm × 6.4–13.8(–15.0) µm [Lm × Wm = 38.5–48.2 × 8.2–10.9(–12.0)], cylindrical or clavate, 4-
iew
5 spored, rarely 1 or 2-spored, thin-walled, containing some oily droplets, hyaline; sterigma 1.3–
6 6.5×1.0–2.7 µm [Lm × Wm = 2.7－4.3 × 1.5–2.0], thin-walled, hyaline. Cells at lamellar edge
7 [60/7/6] 19.4–53.3 × 9.4–31.6 µm [Lm × Wm = 23.4–43.3 × 10.5–20.6 µm], globose–subglobose
v
8 or pyriform, thin-walled, hyaline.
re
9 Ecology: Under temperate–subtropical forests, dominated by Quercus serrata, Q.
10 glauca, Q. acutissima, Castanopsis sieboldii, Fagus crenata, Betula platyphylla var. japonica,
11
er
Pinus densiflora, Tsuga sieboldii, and Abies firma. Fruiting season in Jun to Oct.
12 Known distribution: Japan, from the southwest of Hokkaido Island as the northern
pe
13 limit to Okinawa Island as the southern limit. It is presumably distributed in China.
14 Additional specimens examined in Japan: Hokkaido Pref., S-257 (= TNS-F-61979; N.
15 Endo); Aomori Pref., AC-24-2 (M. Kodaira); Gunma Pref., AC-37-2 (R. Nagumo); Niigata
ot
16 Pref., AC-41-2 (M. Kodaira); Nagano Pref., AC-32 (R. Nagumo), AC-70-5 (M. Kodaira), AC-
17 43-1 (M. Kodaira), AC48 (M. Kodaira), Okaya201108 (A. Yamada), AC-23 (A. Yamada), AC-
tn
18 34-1 (M. Kodaira), AC-35-3 (M. Kodaira), Ahem080927 (N. Endo), AC-28-2 (M. Kodaira),
19 AC-30 (M. Kodaira), AC-45 (M. Kodaira), 2070921Y (N. Endo), S-108 (= TNS-F-61969; N.
rin
20 Endo), Ahem080907 (= TNS-F-61982; N. Endo); Yamanashi Pref., AC-39-4 (M. Kodaira);
21 Tokyo Metropolis, S-325 (N. Endo), S-327 (=TNS-F-61983; N. Endo); Shiga Pref., Hongo
ep
22 5297 (= OSA-MY100271; T. Hongo); Wakayama Pref., AC-14-1 (M. Kodaira), AC-17 (M.
23 Kodaira); Tottori Pref., AC-22 (N. Endo); Oita Pref., S-78 (A. Hadano); Miyazaki Pref., S-70
Pr
24 (T. Katayama); Okinawa Pref., Mar20170515-01 (N. Endo).
25 Commentary: Most specimens of A. satotamagotake correspond to A. hemibapha
17
1 sensu Hongo (Hongo 1975; Imazeki & Hongo 1987) in their macromorphology and the shape
ed
2 and size of basidiospores. Basidiospores of A. satotamagotake were significantly smaller than
3 basidiospores of A. caesareoides at the population level (Table 3). Therefore, it is difficult to
4 distinguish A. caesareoides and A. satotamagotake based on morphological characteristics.
iew
5 However, phylogenetic data (IGS1 of nuc rDNA, rpb2, tef1, cox3, and atp6), geographic
6 distribution (habitat), and culture characteristics show substantial differences between A.
7 satotamagotake and A. caesareoides, and no intermediate specimens were found. Because the
v
8 type specimen AC-36 showed a macroscopically younger trend (Fig. 9), we evaluated spore
re
9 maturity. However, basidiospores were fully developed and the measured size was ordinary in
10 the A. satotamagotake specimens (Supplementary Table S2).
11
er
A. caesarea and A. jacksonii, red and orange pileal Caesar’s mushrooms related to A.
12 caesareoides, were morphologically distinguishable from A. satotamagotake; A. caesarea is

pe
13 distinguished from A. satotamagotake by the non-umbonate pileus and larger and more
14 elongated spores (Table 4; Nevile & Poumarat 2004). A. jacksonii is similar to A.
15 satotamagotake in macromorphology, but its spores are more elongated than the spores of A.
ot
16 satotamagotake (Table 4; Guzmán & Ramírez-Guillén 2001; Tulloss & Yang 2009). A.
17 subhemibapha and A. rubroflava, both of which are yellow- to reddish-pileal species in the
tn
18 section Caesareae recently described from China (Yang et al. 2018), are similar to A.
19 satotamagotake in terms of spore size (Table 4). However, A. subhemibapha has a non-
rin
20 umbonate pileus, whereas A. satotamagotake has an umbonate pileus. A. rubroflava shows a
21 distinct color gradient on the pileal surface (from red in the center to yellow in the margin),
ep
22 whereas A. satotamagotake has an evenly reddish or orangish pileal surface. Both Chinese
23 species had phylogenetic positions distinct from the A. satotamagotake and A. caesareoides
24 clades (Fig. 1).

Pr
25
18
1 5. Discussion
ed
2 The Japanese reddish pileus Tamagotake consists of two species (A. caesareoides and A.
3 satotamagotake) based on the tef1, rpb2 atp6, and cox3 phylogenies (Figs. 2 and 3,
4 Supplementary Figs. S1 and S2), although they have long been regarded as a single species
iew
5 (Hennings 1900; Kawamura 1913, 1954; Hongo 1975; Imazeki & Hongo 1987; Endo 2015;
6 Endo et al. 2016). However, they converged in a single clade in the ITS phylogeny (Fig. 1).
7 Notably, the two species are difficult to distinguish based on morphological characteristics.
v
8 Phylogenetic trees of tef1, atp6, rpb2, and cox3 revealed minimal topological differences
re
9 between these species. Such phylogenetic relationships enabled estimation of the divergence
10 time between A. caesareoides and A. satotamagotake in relation to their closest relatives, A.
11
er
jacksonii and A. caesarea. Sánchez-Ramírez et al. (2015) used a Japanese reddish-pileus
12 Tamagotake specimen included in A. satotamagotake, but they did not use a specimen included
pe
13 in A. caesareoides. Because A. satotamagotake is closer to A. jacksonii, the previously
14 estimated divergence time of these species (around 6 Mya, according to Sánchez-Ramírez et al.
15 (2015)) is reasonable. However, the notion of divergence of A. caesarea directly from the
ot
16 ancestral lineage in the southeastern-southwestern Asian region (Sánchez-Ramírez et al. 2015)
17 should be reconsidered because our data indicated that A. caesarea, in parallel with A.
tn
18 caesareoides, diverged from A. satotamagotake, presumably in eastern Asia around 4.8 Mya
19 (Fig. 6). This divergence issue presumably can be resolved by examining A. caesareoides
rin
20 specimens from Siberia and from the expected A. caesareoides–A. caesarea continuum in
21 central Eurasia (e.g., western Russia and north-central Asia).

ep
22 Analyses of nuclear and mitochondrial DNA markers indicated that there were no
23 hybrids between A. caesareoides and A. satotamagotake. This finding suggests reproductive
24 isolation even if both species are concurrently present in the same forest site. Indeed, the
Pr
25 lakesides of Reisenji-ko, Matsubara-ko, and Komade-ike harbored both species. Notably, in the
19
1 lakesides of Reisenji-ko and Komade-ike, A. satotamagotake fruited in the warmer season (late
ed
2 summer) and A. caesareoides fruited in the cooler season (early summer or autumn) (Table 1,
3 Supplementary Table 1). Although these distinct fruiting periods suggest a mechanism for
4 reproductive isolation, both species fruited in late summer at Matsubara-ko. Thus, there must
iew
5 be a more robust genetic mechanism for reproductive isolation. Because mating-factor genes
6 control cell fusion and the subsequent nuclear transfer events (Moor et al. 2020), genetic
7 analyses for such loci are expected. Reproductive isolation has been examined in cultivated
v
8 mushrooms by crossbreeding experiments (e.g., de Mattos-Shipley et al. 2016). However, it is
re
9 difficult to perform mating tests with Caesar’s mushrooms. Indeed, homokaryotic
10 (monokaryotic) mycelial culture has not yet been reported for Caesar’s mushrooms, and most
11
er
established cultures of A. caesarea and A. caesareoides are dikaryotic (Daza et al. 2006; Endo
12 et al. 2013; Endo 2015). Analysis of the IGS1 region of the nuc rDNA operon indicates that the
pe
13 mechanism of rDNA sequence conservation may differ between A. caesareoides and A.
14 satotamagotake. Direct sequencing of IGS1 is possible in A. caesareoides but requires cloning
15 in A. satotamagotake because of numerous mutations in the tandem repeat region (Fig. 4).
ot
16 Indeed, 34 point mutations were detected in the IGS1 region of A. satotamagotake specimen S-
17 70, and 3–4 cytosine insertions were detected at nucleotides 518–521 in the 1003 bp alignment
tn
18 of nine cloned sequences (data not shown).
19 Phylogenetic data suggest that the ITS region of nuc rDNA is not capable of
rin
20 discriminating cryptic species of Caesar’s mushrooms, despite its usefulness in DNA barcoding
21 of diverse fungal taxa at the species level; this discrepancy has also been reported for Hebeloma
ep
22 (Aanen et al. 2000; Vesterholt et al. 2014; Eberhardt et al. 2015), Serpula (Balasundaram et al.
23 2015), and Tricholoma (Aoki et al. 2022). It is difficult to separate recently diverged species
24 on a geochronological age scale (e.g., 2.6 to 10.6 Mya) by ITS phylogeny, which may
Pr
25 incorrectly group cryptic species (Ryberg 2015; Ryberg & Matheny 2012). The estimated
20
1 divergence time of around 4.8 Mya for A. satotamagotake and A. caesareoides is thus feasible,
ed
2 but the divergence times of around 6 Mya for A. jacksonii and A. satotamagotake and around
3 2.7 Mya for A. caesarea and A. caesareoides are not feasible.
4 We discovered physiological differences between A. satotamagotake and A.
iew
5 caesareoides: only colony mycelia of A. caesareoides could be subcultured on MNC agar.
6 Notably, all Tamagotake isolates (S-46, S-48, S-125, S-248) reported as A. caesareoides based
7 on the ITS sequence (Endo 2015; Endo et al. 2013) were identified as A. caesareoides; no
v
8 isolate was identified as A. satotamagotake. Additionally, colony morphology and related
re
9 hyphal structure differed between these two species. The probable differences in nutrient
10 availability between these two species suggest substantial differences in ecophysiological
11
er
properties. Because the subculture of A. satotamagotake mycelia on MNC agar failed, we could
12 not compare the optimal temperatures for mycelial growth. We suspect that A. caesareoides
pe
13 and A. satotamagotake mycelia have different optimal growth temperatures because of the
14 different climates in which they are distributed.
15 There was no distinct macromorphological difference between A. caesareoides and A.

ot
16 satotamagotake, although A. caesareoides tended to have larger basidiomata and greater
17 variation in stipe ornamentation. The only distinguishable morphological characters at the

tn
18 population level were spore size and sterigma length (smaller in A. satotamagotake than in A.
19 caesareoides), suggesting that A. satotamagotake is a truly cryptic species relative to A.

rin
20 caesareoides. Similar cases of morphologically non-distinguishable cryptic species are the
21 Suillus pictus–S. phylopictus relationship (Zhang et al. 2017) and the two phylogenetically
ep
22 distinguishable populations of Tricholoma matsutake in Eurasia (i.e., B/C and A/E clades; Aoki
23 et al. 2022). The first report of Tamagotake under the name “A. caesarea” based on
24 macroscopic data (Kawamura 1913) with supportive microscopic data (Kawamura 1954) was
Pr
25 presumably A. caesareoides based on its morphological characteristics, fruiting habitat, and
21
1 phenology. The first report of “A. caesarea” from Japan (Hennings 1900) was also presumably
ed
2 A. caesareoides based on its macroscopic characteristics (crimson type; Shirai & Hennings
3 1899) and habitat (WI 56.2; Supplementary Table S1). In A. hemibapha sensu Hongo (Hongo
4 1975, 1982), the partial tef1 sequence of the Hongo 5297 specimen and A. satotamagotake clade
iew
5 (including holotype AC-36) showed 100% similarity (Table 1.5), suggesting that Hongo 5297
6 is A. satotamagotake; this is confirmed by the habitat data (WI 119.7). Considering these
7 taxonomic histories, the Hongo 5297 specimen could be a holotype of A. satotamagotake.
v
8 Because DNA degradation hindered full phylogenetic analysis of this specimen, no such
re
9 designation was assigned.
10 A. satotamagotake and A. caesareoides are largely distributed in different climatic
11
er
regions (Figs. 6 and 7). However, several forest sites harbored both species in the central
12 Japanese Archipelago, such as Mt. Madarao-yama and the lakesides of Reisenji-ko and
pe
13 Komade-ike, where the WI values were 68–69.5, 70.7–71.5, and 61–62.1, respectively. This
14 finding suggests that both species are sympatrically present and equally competitive at the forest
15 sites, but there is no hybridization in terms of interspecific crossing. The minimum WI values
ot
16 in A. satotamagotake habitats were 57.3 at Mt. Togurayama (AC-45) and 58.0 at Mikuni-touge
17 pass (AC-37-2), where A. caesareoides was strongly expected to be naturally present because
tn
18 of these lower WI values. In contrast, the maximum WI values in A. caesareoides habitats were
19 70.9 at the lakeside of Reisenji-ko, Iizuna, Nagano (AC-66-1), 68.0 at Mt. Madarao-san, Iiyama,
rin
20 Nagano, (AC-72-1, AC-72), and 67.9 at Fujisawa, Ina, Nagano (AC-28-2). Therefore, areas
21 with WI values of 57–71 may harbor both A. satotamagotake and A. caesareoides. Because Mt.
ep
22 Togurayama, Nagano, Mikuni-touge pass, and Gunma lacked subalpine–cool temperate conifer
23 vegetation in high-elevation areas, A. caesareoides might have ceased occupation of those areas,
24 leading to occupation by A. satotamagotake that ascended from low-elevation areas. A.

Pr
25 caesareoides is often associated with conifers in high-elevation (and lower WI) areas. These
22
1 data suggest that the different geographic distributions of A. satotamagotake and A.
ed
2 caesareoides are primarily determined by temperature and secondarily determined by forest
3 vegetation. Similarly, large-scale analyses of soil ectomycorrhizal fungi suggested that
4 temperature (MAT) and vegetation are important (van der Linde et al. 2018; Miyamoto et al.
iew
5 2018).
6 In the analysis of temperature effect on the fungal habitat and distribution, our data
7 suggested the advantage adopting WI, rather than MAT and CI (Figs. 7 and 8, Supplementary
v
8 Fig. S3). Therefore, we analyzed the adaptation and competition of A. satotamagotake and A.
re
9 caesareoides in relation to WI. In the Myoko Volcano Group, Yatsugatake Mountains, and Ina
10 Mountains and the marginal ranges of the Akaishi Mountains, A. satotamagotake and A.
11
er
caesareoides exhibited different vertical distributions (Fig. 8). In the Myoko Volcano Group,
12 the habitats of these two species overlapped at approximately 800–1200 m. However, in the
pe
13 overlap region, the two species showed different WI trends; at the same elevation, A.
14 satotamagotake and A. caesareoides preferred sites with high and low WI values, respectively.
15 These findings suggest that, at the same elevation, A. satotamagotake prefers open forest sites
ot
16 or southern slopes, whereas A. caesareoides prefers closed forest sites or northern slopes. If the
17 WI value were to increase by 5–10, the habitat of A. caesareoides would retreat to areas of
tn
18 approximately 100–200 m asl, and the habitat of A. satotamagotake would expand into that area.
19 Additionally, if land use changed from deep forest (canopy closure) to open forest, the area of
rin
20 A. caesareoides habitat would presumably decrease because of excess irradiation and an
21 increased WI value.
ep
22 Based on the scenario described above, the isolated population of A satotamagotake at
23 Atsubetsu, Sapporo, Hokkaido (specimens S-257 and S-320) may have invaded or been
24 artificially introduced from Honshu Island and subsequently established a habitat (WI 70.1).
Pr
25 Thus, global warming may decrease the area of A. caesareoides habitat and lead to replacement
23
1 with A. satotamagotake. Because WI has been increasing by approximately 5–15/century
ed
2 because of the increasing annual mean temperature since the beginning of the 20th century in
3 temperate areas of the Japanese Archipelago (Supplementary Table S4), forests in the southern
4 coastal region of Hokkaido may alternate between A. caesareoides and A. satotamagotake.
iew
5 Currently, there is a lack of data regarding the rate and magnitude of changes in the habitat and
6 distribution of ectomycorrhizal fungi associated with global warming. The differences in
7 habitat among cryptic species observed in this study will enable investigation of these
v
8 environmental issues. Conservation of fungal diversity requires an accurate taxonomy of
re
9 similar cryptic species.
10
11 Declaration of competing interest

er
12 There is no conflict of interests in the conducting the study and writing the paper.
pe
13
14 Acknowledgement
15 We thank the staff of the Research Center for Human and Environmental Sciences,
ot
16 Shinshu University for the DNA sequencing. This study was supported in part by a Grant-in-
17 Aid for Scientific Research (15H01751) from the Ministry of Education, Culture, Sports,
tn
18 Science, and Technology of Japan.
19
rin
20 References
21 Aanen DK, Kuyper TW, Boekhout T, Hoekstra RF, 2000. Phylogenetic relationships in the
ep
22 genus Hebeloma based on ITS1 and 2 sequences, with special emphasis on the
23 Hebeloma crustuliniforme complex. Mycologia 92: 269–281.
24 https://doi.org/10.2307/3761560.
Pr
25 Anderson JB, Stasovski E, 1992. Molecular phylogeny of Northern hemisphere species of
24
1 Armillaria. Mycologia 84: 505–516. https://doi.org/10.2307/3760315.
ed
2 Aoki W, Bergius N, Kozlan S, Fukuzawa F, Okuda H, Murata H, Ishida TA, Vaario LM,
3 Kobayashi H, Kalmiş E, Fukiharu T, Gisusi S, Matsushima K, Terashima Y,
4 Narimatsu M, Matsushita N, Ka KH, Yu F, Yamanaka T, Fukuda M, Yamada A, 2022.
iew
5 New findings on the fungal species Tricholoma matsutake from Ukraine, and revision
6 of its taxonomy and biogeography based on multilocus phylogenetic analyses.
7 Mycoscience 63: 197–214.
v
8 Balasundaram SV, Engh IB, Skrede I, Kauserud H, 2015. How many DNA markers are needed
re
9 to reveal cryptic fungal species? Fungal Biology 119: 940–945.
10 https://doi.org/10.1016/j.funbio.2015.07.006.
11
er
Cho HJ, Park MS, Lee H, Oh SY, Jang Y, Fong JJ, Lim YW, 2015. Four new species of Amanita
12 in Inje County, Korea. Mycobiology 43: 408–414; doi:10.5941/MYCO.2015.43.4.408.

pe
13 Cohen KM, Finney SC, Gibbard PL, Fan J-X, 2013. The ICS international
14 chronostratigraphic chart. Episodes 36: 199–204.
15 https://stratigraphy.org/ICSchart/Cohen2013_Episodes.pdf
ot
16 Daza A, Manjón JL, Camacho M, De La Osa, LR, Aguilar A, Santamaría C, 2006. Effect of
17 carbon and nitrogen sources, pH and temperature on in vitro culture of several isolates
tn
18 of Amanita caesarea (Scop.:Fr.) Pers. Mycorrhiza 16: 133–136.
19 https://doi.org/10.1007/s00572-005-0025-6.
rin
20 de Mattos-Shipley KMJ, Ford KL, Alberti F, Banks AM, Bailey AM, Foster GD, 2016. The
21 good, the bad and the tasty: The many roles of mushrooms. Studies in Mycology 85:
ep
22 125–157. https://doi.org/10.1016/j.simyco.2016.11.002.
23 Drummond AJ, Bouckaert RR, 2015. Bayesian Evolutionary Analysis with BEAST.
24 Cambridge University Press, Cambridge.

Pr
25 https://doi.org/10.1017/CBO9781139095112
25
1 Duchesne LC, Anderson JB, 1990. Location and direction of transcription of the 5S rRNA gene
ed
2 in Armillaria. Mycological Research 94: 266–269. https://doi.org/10.1016/S0953-
3 7562(09)80626-6.
4 Eberhardt U, Beker HJ, Vesterholt J, 2015. Decrypting the Hebeloma crustuliniforme complex:
iew
5 European species of Hebeloma section Denudata subsection Denudata (Agaricales).
6 Persoonia 35: 101–147. https://doi.org/10.3767/003158515X687704.
7 Edgar RC, 2004. MUSCLE: a multiple sequence alignment method with reduced time and
v
8 space complexity. BMC Bioinformatics 5: 113. https://doi.org/10.1186/1471-2105-5-
re
9 113.
10 Endo N, 2015. Cultivation studies in the edible Caesar’s mushrooms “Tamagotake” (Amanita
11
er
caesareides and its relatives). PhD dissertation, Shinshu University.
12 Endo N, Gisusi S, Fukuda M, Yamada A, 2013. In vitro mycorrhization and acclimatization of

pe
13 Amanita caesareoides and its relatives on Pinus densiflora. Mycorrhiza 23: 303–315.
14 https://doi.org/10.1007/s00572-012-0471-x.
15 Endo N, Fanfuk W, Sakuma D, Phosri C, Matsushita N, Fukuda M, Yamada A, 2016.

ot
16 Taxonomic consideration of the Japanese red-cap Caesar's mushroom based on
17 morphological and phylogenetic analyses. Mycoscience 57: 200–207.

tn
18 https://doi.org/10.1016/j.myc.2016.01.005.
19 Endo N, Fangfuk W, Kodaira M, Sakuma D, Hadano E, Hadano A, Murakami Y, Phosri C,

rin
20 Matsushita N, Fukuda M, Yamada A, 2017. Reevaluation of Japanese Amanita section
21 Caesareae species with yellow and brown pileus with descriptions of Amanita
ep
22 kitamagotake and A. chatamagotake spp. nov. Mycoscience 58: 457–471.
23 https://doi.org/10.1016/j.myc.2017.06.009.
24 Gardes M, Bruns TD, 1993. ITS primers with enhanced specificity for basidiomycetes:
Pr
25 application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–
26
1 118. https://doi.org/10.1111/j.1365-294X.1993.tb00005.x.
ed
2 Guzmán G, Ramírez-Guillén F, 2001. The Amanita caesarea-complex. Bibliotheca
3 Mycologica 187. J Cramer, Berlin.
4 Hayatsu K, Shimizu S, Itaya T, 1994. Volcanic history of Myoko volcano group, central
iew
5 Japan –poly-generation volcano–. Journal of Geography 103: 207–220.
6 https://www.jstage.jst.go.jp/article/jgeography1889/103/3/103_3_207/_pdf
7 Hennings P, 1900. Fleischige Pilze aus Japan. Hedwigia Beiblatt 39: 155–157.
v
8 https://www.zobodat.at/pdf/Hedwigia_Beiblatt_39_1900_0155-0157.pdf
re
9 Hongo T, 1975. Notulae Mycologicae 14. Memories of the Faculty of Education Shiga
10 University Natural Science 25: 56–63.
11
er
Hongo T, 1982. The Amanitas of Japan. Acta Phytotaxonomica et Geobotanica 33: 116–126;
12 https://doi.org/10.18942/bunruichiri.KJ00001079145
pe
13 Imazeki R, Hongo T, 1957. Colored illustrations of fungi of Japan I (in Japanese). Hoikusha,
14 Osaka.
15 Imazeki R, Hongo T, 1987. Colored illustrations of mushrooms of Japan I (in Japanese).

ot
16 Hoikusha, Osaka.
17 Kawamura S, 1913. Illustrations of Japanese fungi (in Japanese). 2nd edn. The Bureau of
tn
18 Forestry, Ministry of Agriculture and Commerce, Tokyo.
19 Kawamura S, 1930. The Japanese Fungi. Daichi-shoin, Tokyo.

rin
20 Kawamura S, 1931. Edible and poisonous mushrooms. Iwanami-shoten, Tokyo.
21 Kawamura S, 1954. Icons of Japanese fungi (in Japanese). Kazama-shobo, Tokyo.

ep
22 Kira T, 1948. On the altitudinal arrangement of climate zones in Japan: a contribution to the
23 rational land utilization in cool highlands (in Japanese). Agricultural Science of the
24 North Temperate Region 2: 143-173.

Pr
25 Kohler A, Kuo A, Nagy LG, Morin E, Barry KW, Buscot F, Canbäck B, Choi C, Cichocki, N,
27
1 Clum A, Colpaert J, Copeland A, Costa, MD, Doré J, Floudas D, Gay G, Girlanda M,
ed
2 Henrissat B, Herrmann S, Hess J, Högberg N, Johansson T, Khouja HR, LaButti K,
3 Lahrmann U, Levasseur A, Lindquist EA, Lipzen A, Marmeisse R., Martino E, Murat
4 C, Ngan CY, Nehls U, Plett JM, Pringle A, Ohm RA, Perotto S, Peter M, Riley R,
iew
5 Rineau F, Ruytinx J, Salamov A, Shah F, Sun H, Tarkka M, Tritt A, Veneault-Fourrey
6 C, Zuccaro A, Mycorrhizal Genomics Initiative Consortium, Tunlid A, Grigoriev IV,
7 Hibbett DS, Martin F, 2015. Convergent losses of decay mechanisms and rapid
v
8 turnover of symbiosis genes in mycorrhizal mutualists. Nature Genetics 47: 410–415.
re
9 https://doi.org/10.1038/ng.3223.
10 Kretzer AM, Bruns TD, 1999. Use of atp6 in fungal phylogenetics: an example from the
11 Boletales. Molecular
er
Phylogenetics and Evolution 13: 483–492.
12 https://doi.org/10.1006/mpev.1999.0680.
pe
13 Kumar S, Stecher G, Tamura K, 2016. MEGA7: molecular evolutionary genetics analysis
14 version 7.0 for bigger datasets. Molecular Biology and Evolution 33: 1870–1874.
15 https://doi.org/10.1093/molbev/msw054.
ot
16 Matheny PB, 2005. Improving phylogenetic inference of mushrooms with RPB1and RPB2
17 nucleotide sequences (Inocybe; Agaricales). Molecular Phylogenetics and Evolution

tn
18 35: 1–20. https://doi.org/10.1016/j.ympev.2004.11.014.
19 Matsumura J, 1904. Index plantarum japonicum I. Cryptogamae. Maruzen, Tokyo.

rin
20 Miyamoto Y, Terashima Y, Nara K, 2018. Temperature niche position and breadth of
21 ectomycorrhizal fungi: Reduced diversity under warming predicted by a nested

ep
22 community structure. Global Change Biology 24: 5724–5737.
23 https://doi.org/10.1111/gcb.14446
24 Morehouse EA, James TY, Ganley AR, Vilgalys R, Berger L, Murphy PJ, Longcore JE, 2003.
Pr
25 Multilocus sequence typing suggests the chytrid pathogen of amphibians is a recently
28
1 emerged clone. Molecular Ecology 12: 395–403. https://doi.org/10.1046/j.1365-
ed
2 294X.2003.01732.x.
3 Moore D, Robson GD, Trinci APJ, 2020. 21st century guidebook to fungi, 2nd edn. Cambridge
4 University Press, Cambridge.
iew
5 Nylander JAA, 2004. MrModeltest v2. Program distributed by the author. Evolutionary Biology
6 Centre, Uppsala University.
7 Oda T, Tanaka C, Tsuda M, 1999. Molecular phylogeny of Japanese Amanita species based on
v
8 nucleotide sequences of the internal transcribed spacer region of nuclear ribosomal
re
9 DNA. Mycoscience 40: 57–64. https://doi.org/10.1007/BF02465674.
10 Rehner SA, Buckley E, 2005. A Beauveria phylogeny inferred from nuclear ITS and EF1-a
11
er
sequences: evidence for cryptic diversification and links to Cordyceps teleomorphs.
12 Mycologia 97: 84–98. https://doi.org/10.1080/15572536.2006.11832842.

pe
13 Ryberg M, 2015. Molecular operational taxonomic units as approximations of species in the
14 light of evolutionary models and empirical data from Fungi. Molucular Ecology 24:
15 5770–5777. https://doi.org/10.1111/mec.13444.
ot
16 Ryberg M, Matheny PB, 2012. Asynchronous origins of ectomycorrhizal clades of Agaricales.
17 Proceedings of the Royal Society B 279: 2003–2011; doi: 10.1098/rspb.2011.2428.

tn
18 Sánchez-Ramírez S, Tulloss RE, Amalfi M, Moncalvo JM, 2015. Palaeotropical origins,
19 boreotropical distribution and increased rates of diversification in a clade of edible

rin
20 ectomycorrhizal mushrooms (Amanita section Caesareae). Journal of Biogeography
21 42: 351–363. https://doi.org/10.1111/jbi.12402.

ep
22 Shirai M, 1905. A list of Japanese fungi hitherto known. Nihon-engei-kennkyukai, Tokyo.
23 Shirai M, Hennings P, 1899. Berlin-kinpu, Hekisui Shirai, Tokyo (in Japanese).
24 Shirai M, Miyake I, 1917. A list of Japanese fungi hitherto known, 2nd edn, Nihon-engei-
Pr
25 kennkyukai, Tokyo.
29
1 Stöver, BC, Müller KF, 2010. TreeGraph 2: Combining and visualizing evidence from different
ed
2 phylogenetic analyses. BMC Bioinformatics 11: 7. https://doi.org/10.1186/1471-2105-
3 11-7.
4 Stamakis A, 2014. RAxML version 8: a tool for phylogenetic analysis and post-analysis of
iew
5 large phylogenies. Bioinformatics 1: 1312–1313.
6 https://doi.org/10.1093/bioinformatics/btu033.
7 Swofford DL, 2002. PAUP*, phylogenetic analysis using parsimony (*and other methods),
v
8 Version 4. Sinauer Associates, Sunderland
re
9 Tedersoo L, Jairus T, Horton BM, Abarenkov K, Suvi T, Saar I, Kõljalg U, 2008. Strong host
10 preference of ectomycorrhizal fungi in a Tasmanian wet sclerophyll forest as revealed
11
er
by DNA barcoding and taxon-specific primers. New Phytologist 180: 479–490.
12 https://doi.org/10.1111/j.1469-8137.2008.02561.x
pe
13 Tulloss RE, Yang ZL, 2009. Notes on Amanita section Caesareae, Torrendia, and
14 Amarrendia (Agaricales, Amanitaceae) with provisional division into stirpes and
15 annotated world key to species of the section.

ot
16 http://www.amanitaceae.org/content/uploaded/pdf/hemibkey.pdf.
17 van der Linde S, Suz LM, Orme CDL, Cox F, Andreae H, Asi E, Atkinson B, Benham S, Carroll
tn
18 C, Cools N, De Vos B, Dietrich H-P, Eichhorn J, Gehrmann J, Grebenc T, Gweon HS,
19 Hansen K, FJacob F, Kristöfel F, Lech P, Manninger M, Martin J, Meesenburg H,

rin
20 Merilä P, Nicolas M, Pavlenda P, Rautio P, Schaub M, Schröck H-W, Seidling W,
21 Šrámek V, Thimonier A, Thomsen IM, Titeux H, Vanguelova E, Verstraeten A,

ep
22 Vesterdal L, Waldner P, Wijk S, Zhang Y, Žlindra D, Bidartondo MI, 2018.
23 Environment and host as large-scale controls of ectomycorrhizal fungi. Nature 558:
24 243–248. https://doi.org/10.1038/s41586-018-0189-9
Pr
25 Vassiljeva LN, 1950. Species novae fungorum. Notulae Systematicae e Sectione Cryptogamica
30
1 Instituti Botanici Nomeine V.L. Komarovii Academiae Scientificae USSR 6: 188–200.
ed
2 Vesterholt J, Eberhardt U, Beker HJ, 2014. Epitypification of Hebeloma crustuliniforme.
3 Mycological Progress 13: 553–562; doi: 10.1007/s11557-013-0938-y.
4 Yamada A, Katsuya K, 1995. Mycorrhizal association of isolates from sporocarps and
iew
5 ectomycorrhizas with Pinus densiflora seedlings. Mycoscience 36: 315–323.
6 https://doi.org/10.1007/BF02268607.
7 Yang ZL, 2005. Amanitaceae. Flora fungorum sinicorum 27. Science Press, Beijing.
v
8 Yang ZL, 2015. Atlas of the Chinese species of Amanitaceae (in Chinese). Science Press,
re
9 Beijing.
10 Yang YC, Qing C, Li PT, Jian WL, 2018. The family Amanitaceae: molecular phylogeny,
11
er
higher-rank taxonomy and the species in China. Fungal Diversity 91: 5–230.
12 https://doi.org/10.1007/s13225-018-0405-9.
pe
13 Yasuda A, 1920. Notes on fungi (95) (in Japanese). The Botanical Magazine 34: 67–68.
14 Yoshino M, 1986. Climate in a small area. Chijin-shokan, Tokyo
15 Zhang L, Yang J, Yang Z, 2004. Molecular phylogeny of eastern Asian species of Amanita
ot
16 (Agaricales, Basidiomycota): taxonomic and biogeographic implications. Fungal
17 Diversity 17: 219–238. http://www.fungaldiversity.org/fdp/sfdp/17-13.pdf.

tn
18 Zhang R, Muller GM, Shi XF, Liu PG, 2017. Two new species in the Suillus spraguei
19 complex from China. Mycologia 109: 296–307.

rin
20 http://dx.doi.org/10.1080/00275514.2017.1305942.
21
ep
22
23 Figure Legends
24 Fig. 1. Maximum-likelihood (ML) tree of Amanita section Caesareae based on ITS sequences.
Pr
25 Values at nodes are percentages of ML bootstrap support (BS) and Bayesian posterior
31
1 probability (PP). Thick-line node indicates significantly supported branch (BS ≥ 70%, PP ≥
ed
2 0.90). Sample names after species epithets are specimen IDs in this study or DDBJ accession
3 numbers and localities. JP, Japan; SK, South Korea; RU, Russia; US, United States of America;
4 CA, Canada; MX, Mexico; IT, Italy; ES, Spain; BG, Bulgaria. Closed circle: ever-green
iew
5 Fagaceae tree, open circle: deciduous Fagaceae tree, close triangle: Pinaceae tree. Abi: Abies,
6 Lar: Larix, Pic: Picea, Pin: Pinus, Bet: Betula, Cae: Castanea, Cao: Castanopsis, Fag: Fagus,
7 Que: Quercus.
v
8
re
9 Fig. 2. ML phylogenetic tree of Amanita section Caesareae based on tef1 sequences. Notation
10 as in Fig. 1.
11
er
12 Fig. 3. ML phylogenetic tree of Amanita section Caesareae based on rpb2 sequences. Notation
pe
13 as in Fig. 1.
14
15 Fig. 4. ML phylogenetic tree of Amanita section Caesareae based on IGS1 sequences. Notation
ot
16 as in Fig. 1.
17
tn
18 Fig. 5. Chronogram and estimated divergence times of Amanita section Caesareae by
19 molecular clock analysis of six loci: nuc rDNA ITS and IGS1, tef1, cox3, rpb2, and atp6. The
rin
20 chronogram calibration point (arrow) is based on the divergence times of A. jacksonii and the
21 A. caesareoides–A. satotamagotake–A. caesarea lineages at 6 Mya. Notation as in Fig. 1.

ep
22
23 Fig. 6. Distribution pattern of two A. caesareoides populations based on warmth index (WI)
24 and altitude. Open and closed symbols, sampling sites of populations A and B, respectively.
Pr
25 Square: Hokkaido Island; triangle, Tohoku Region of Honshu Island; circle, central and western
32
1 Honshu Island; trapezoid, Kyushu Island. A sample from Okinawa Prefecture (WI 188.2) is not
ed
2 shown. Regression lines are shown for population A from Hokkaido and central Honshu Island,
3 and for population B from central and western Honshu Island.
iew
5 Fig. 7. Distribution pattern of two A. caesareoides populations based on warmth index (WI; A,
6 D, G), annual mean temperature (MAT; B, E, H), and coldness index (CI; C, F, I) in the Myoko
7 Volcano Group (A–C), Yatsugatake Mountains (D–F), and Ina Mountains and the marginal
v
8 ranges of the Akaishi Mountains (G–I). Open and closed symbols, sampling sites of populations
re
9 A and B, respectively. Triangle, conifer forest; square, mixed forest; circle, broad-leaf forest
10 (oak or birch). Regression lines are shown for population A (dotted, blue) and population B
11 (solid, red).
er
12
pe
13 Fig. 8. Cultured mycelium of Tamagotake populations A and B on MNC agar. A–F, Colony
14 morphology under a dissection microscope. A, AC-8 (population A); B, AC-47 (population A);
15 C, AC-67-1 (population A); D, AC-26 (population B); E, AC-66-11 (population B); F, AC-
ot
16 70-1 (population-B). G–J, hyphal structure under a light microscope; G, monilioid-cell-like
17 hyphae on the surface of MNC agar in AC-47; H, submerged hyphae with monilioid-cell-like
tn
18 enlargement in AC-67-1; I, aerial hyphae with terminal enlargement in AC-66-7, some of which
19 have ruptured, on MNC agar (arrow); J, aerial hyphae with terminal enlargement in AC-70-1.
rin
20 Bars in A–F, 5 mm; bars in G–J, 20 µm.
21
ep
22 Fig. 9. Macro- and microscopic characteristics of A. satotamagotake. A–D, External
23 morphology of a basidioma (A), sectioned view (B), stria of the pileal margin (C), and yellowish
24 lamellae with reddish margin at the boundary with the pileal surface (D) of AC-36 specimen.
Pr
25 E, Growth of mature and young basidiomata of specimen AC-70-5. F–L, Basidiospores (F–H),
33
1 basidia (I–K), and cells on the lamellar edge (L) of AC-36. M, Cells on the lamellar edge of
ed
2 AC-28-2. N, External morphology of a basidioma of Hongo 5297. Bar in A, 10 cm; bars in F–
3 M,10 µm.
v iew
re
er
pe
ot
tn
rin
ep
Pr
34

Amanita Satotamagotake Sp. Nov., A Cryptic Species Formerly Included in A. Caesareoides, Has Invaded The Natural Habitat of A. Caesareoides

Uploaded by

Document Information

Original Title

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Amanita Satotamagotake Sp. Nov., A Cryptic Species Formerly Included in A. Caesareoides, Has Invaded The Natural Habitat of A. Caesareoides

Uploaded by

Copyright:

Available Formats

1 Full Paper

3 invaded the natural habitat of A. caesareoides

6 e, Yasushi Hashimoto f, Seiki Gisusi g, Masaki Fukuda a, b, Akiyoshi Yamada a, b, h*

10 b Department of Science and Technology, Graduate School of Medicine, Science and

12 c Fungus/Mushroom Resource and Research Center, Faculty of Agriculture, Tottori University,

14 d Osaka Museum of Natural History, 1-23, Nagai-koen, Higashisumiyoshi-ku, Osaka, Osaka,

16 e 1-1-36, Ryougo, Oita, Oita, 870-0883, Japan.

17 f Department of Agro-environmental Science, Obihiro University of Agriculture and Veterinary

18 Medicine, Obihiro 080-8555, Japan

19 g Forest Products Research Institute, Hokkaido Research Organization, Nishikagura 1-10,

20 Asahikawa, Hokkaido 071‒0198, Japan

6 distribution: subalpine–cool temperate and temperate–subtropical areas, respectively. Although

16 Keywords: Biogeography, Caesar’s mushroom, Competition, Ectomycorrhizal association,

17 Fungal resource conservation, Invasiveness, Multi-locus phylogenetic analyses, Speciation,

3 in 1919 in Kiso-fukushima, Nagano Prefecture, presumably under an intermediate-temperate

16 Imazeki & Hongo (1987) regarded Japanese A. hemibapha as conspecific to A. caesareoides.

18 hemibapha or A. caesareoides. Hongo (1975) inspected a Tamagotake specimen sampled in

19 Nango-imodani, Kusatsu, Shiga Prefecture, under a warm-temperate climate; this specimen

4 including the lectotype of A. caesareoides, and identified as A. caesareoides, based on

7 culture characteristics, suggesting two ecological groups: subalpine–cool-temperate and warm-

10 Therefore, we hypothesized that the Japanese A. caesareoides population consists of two

14 Caesareae. Additionally, we established cultures of Tamagotake and performed comparisons

19 phylogenetic and evolutionary background of the section Caesareae.

21 2. Materials and Methods

22 2-1. Sampling and morphological observation of Tamagotake

7 temperate deciduous broad-leaved forests; ~85, intermediate-temperate deciduous broad-

11 calculated based on AMEDAS

12 http://www.jma.go.jp/jma/index.html) of the one to three points nearest each sampling site,

14 2-2. Tamagotake tissue isolation

15 Because the culture characteristics of Japanese A. caesareoides differ between specimens

25 2-3. Microscopic observation

6 50 basidiospores, 30 basidia, 10 sterigmata, and 10 cheilocystidia-like terminally swollen

10 Genomic DNA was extracted from dried specimens of A. caesareoides, A. caesarea, A.

22 GGTTTAAATGCTCCTATTTTAGGTC-3') and TP6-AmR 5'-

23 GRAAATAATCTAACTCCTAATGA-3') for atp6 was designed based on the genome

3 CTTGGGTTCAGGCAGAGTCAGAGG-3’), rpb2-AcoidesF (5’-

4 CGTCAACGGAGTATGGATG-3’), rpb2-AcoidesR (5’-TCTGAACTCTTCGCCTTCGT-3’),

6 CATCTCAAGCTGATTGTGCCAT-3’), tef1-Acoides2R (5’-TCAATGGCATCAAGAAGG-

7 3’), tef1-UF(5’-AGCTGGTATCTCCAAGGACGG-3’), tef1-AcoidesR’ (5’-

10 TAAGCCGTGAAGACCCGTAG-3’) for the first and second rounds of PCR. PCR

4 annealing temperatures: IGS1, IGS1-AcoidesF/IGS1-AcoidesR (57°C); rpb2, rpb2-

6 AcoidesR’ (53°C); and cox3, cox3-AcoidesF/cox3-AcoidesR (50°C). PCR amplicons were

7 subjected to electrophoresis on 1.5–2.0% agarose gels to confirm amplification specificity, then

16 then assembled by CLUSTALW (http://www.genome.jp/tools-bin/clustalw). We confirmed

18 Data Bank of Japan (DDBJ; https://www.ddbj.nig.ac.jp/index.html). Because the IGS1 region

20 TA-cloning Kit (TaKaRa) in accordance with the manufacturer’s recommendation.

21 2-5. Phylogenetic analyses

22 The A. caesareoides, A. caesarea, A. kitamagotake, and A. chatamagotake N. Endo & A.

23 Yamada sequences (Supplementary Table S1) were subjected to phylogenetic analyses.

24 Additionally, sequences of A. caesareoides, A. caesarea, A. jacksonii Pomerl., A. vernicoccora

3 kitamagotake, and A. chatamagotake in GenBank were included in the phylogenetic analyses

6 chatamagotake for ITS; A. caesareoides, A. jacksonii, A. caesarea and A. chatamagotake for

7 IGS1; A. caesareoides, A. jacksonii, A. basii, A. caesarea, and A. chatamagotake for tef1; A.

10 A. kitamagotake, and A. chatamagotake for atp6 and cox3.

16 option. Because we compared two similar populations of A. caesareoides, we selected A.

22 were edited by TreeGraph v. 2 (Stöver & Müller 2010).

23 2-6. Divergence time estimation

16 referred to the international chronostratigraphic chart (Cohen et al. 2013).

18 3.1. Phylogenetic position of Tamagotake specimens

22 respectively (TreeBase accession nos. S29648).

10 Although the lectotype LE203026 of A. caesareoides was only obtained partial

16 contrast, A. caesareoides clade B was presumed to correspond to A. hemibapha sensu Hongo

17 (Hongo 1975, 1982).