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Three new species of Alpova from Japan: new insights into biogeography in Alpova
2025 年 66 巻 5 号 p. 272-281
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2025 年 66 巻 5 号 p. 272-281
Alpova (Paxillaceae, Boletales) is an ectomycorrhizal fungal genus specifically colonizing alder trees. As observed in other hypogeous fungal taxa that depend on land animals for spore dispersal, Alpova shows strong biogeographical patterns. Although more than 10 Alpova species have been identified in Europe and North America, no Alpova species (except for three Chinese species possibly attributable to Melanogaster) has been formally described from East Asia, which has greater diversity of the host Alnus than other areas. Here, we describe three new Alpova species, i.e., A. fujisanensis sp. nov., A. japonicus sp. nov., and A. venosus sp. nov., collected under Japanese alder trees. Phylogenetic analysis using three DNA loci showed that these three Japanese Alpova species belong to distinctive clades and are distantly related to European and North American species. Both A. fujisanensis and A. japonicus were associated only with the host subgen. Alnobetula, while A. venosus was associated with the subgen. Alnus, indicating host specificity at the subgenus level.
The genus Alpova C. W. Dodge belongs to the Paxillaceae within Boletales, Basidiomycota. The type species, Alpova cinnamomeus C.W. Dodge, was collected under an Alnus tree and is characterized by the presence of pseudoparenchymatous cells in the peridium, gelatinous gleba, lack of a hymenial palisade, and small, pale, ellipsoidal spores (Dodge, 1931). Subsequently, Trappe (1975) expanded the genus Alpova to include species with gelatinous gleba and pale spores regardless of host, leading to the description of several species from Asia and Australia under this broadened definition (e.g., Beaton et al., 1985; Liu et al., 1990). However, subsequent studies using molecular data showed that Alpova sensu Trappe (1975) is polyphyletic and some species were transferred into other genera including Rhizopogon and Amarrendia (Bougher & Lebel, 2002; Grubisha et al., 2001). Recently, molecular phylogenetic studies of hypogeous Boletales have re-defined Alpova as a monophyletic group that forms ectomycorrhizal associations specifically with Alnus (Nouhra et al., 2005; Vizzini et al., 2010). Species belonging to Alpova sensu stricto share common morphological characteristics, including a pseudoparenchymatous structure of peridium, buffer cells in the gleba, and pale spores (Moreau et al., 2013).
According to Index Fungorum, the genus Alpova includes 16 accepted species, most of which were described from Europe and North America (http://www.indexfungorum.org/; accessed on Oct 10, 2024). This number could be reduced to eight under the re-definition of Alpova sensu stricto confirmed by molecular phylogenetic evidence (Crous et al., 2022; Hayward et al., 2014; Moreau et al., 2011, 2013; Nouhra et al., 2005; Vizzini et al., 2010). Among species currently classified as Alpova, Alpova trappei Fogel and A. pseudostipitatus Calonge & Siquier have been assigned to a different taxon based on molecular data (Alvarado et al., 2021; Grubisha et al., 2001). Furthermore, Moreau et al. (2011) pointed out that several Alpova species from Europe are of doubtful status. Indeed, the original descriptions of the remaining six species contradict the common traits of Alpova sensu stricto, exhibiting either black gleba, subpellis composed of interwoven hyphae without inflated cells, or associations with non-Alnus trees (Liu et al., 1990; Trappe, 1975). Alpova mollis (Lloyd) Trappe and A. yangchengensis B. Liu, K. Tao & Ming C. Chang, both reported from China, are also included in these ambiguous taxa having contradicting traits to Alpova sensu stricto (Liu et al., 1990; Liu & Tao, 1996). Another Chinese species Alpova shanxiensis (B. Liu) B. Liu, K. Tao & Ming C. Chang (≡ Rhizopogon shanxiensis B. Liu), currently treated in Rhizopogon, also differs from Alpova sensu stricto, exhibiting black gleba and subpellis composed of interwoven hyphae (Liu, 1985; Liu et al., 1990). Since these three ambiguous Chinese species represent the only records of Alpova in Asia, no unequivocal evidence exists for Alpova sensu stricto in the region. Given that Asia is the center of Alnus (host tree) diversity, which has 9 spp. in North and South America, 4-5 spp. in Europe, and 18-23 spp. in Asia (Chen & Li, 2004 and references therein), there are presumably many more Alpova species remaining to be found in Asia than in other regions.
In accordance with other hypogeous sporocarp genera, all Alpova species are supposed to depend on land animals for spore dispersal (Elliott et al., 2022; Vašutová et al., 2019). As the migration of such land animals is restricted by geographical barriers, strong biogeographical patterns have been observed in numerous hypogeous fungal genera including Tuber (Bonito et al., 2013; Kinoshita et al., 2011) and Rhizopogon (Grubisha et al., 2007; Mujic et al., 2019). Recent molecular phylogenetic studies of Alpova also confirmed that European and North American Alpova species are unique to those regions (Hayward et al., 2014; Moreau et al., 2011, 2013). Although no Alpova species have been formally described from Asia except the uncertain Chinese species (A. mollis, A. shanxiensis, A. yangchengensis), the presence of Alpova is indisputable based on reports of environmental sequences, specifically ectomycorrhizal (ECM) root tips from Japan (Ishikawa & Nara, 2023; Põlme et al., 2013; Yamanaka & Okabe, 2006; Yamanaka et al., 2022). Given the strong biogeographical pattern of Alpova, these sequences likely belong to new Alpova species. To advance our understanding of the diversity and phylogeny of Alpova, taxonomic information about these potentially new Alpova species in Asia based on sporocarp specimens is needed.
In this study, we collected 16 Alpova sporocarp specimens from various locations in Japan. Based on morphological observation and molecular phylogenetic analysis, we propose three new species, namely Alpova fujisanensis sp. nov., and Alpova japonicus sp. nov., and Alpova venosus sp. nov., all of which are associated with Alnus species in Japan.
Basidiocarps of Alpova were collected under alder species including Alnus sieboldiana Matsum., A. firma Siebold et Zucc., A. alnobetula (Ehrh.) K.Koch subsp. maximowiczii (Callier) Chery and, A. hirsuta (Spach) Fisch. ex Rupr. var. hirsuta from various sites in Japan (Table 1). These specimens were collected between 2002-2023. We photographed the surface and cross-section of each basidiocarps. After recording their macroscopic features, all specimens were cut and subsequently freeze-dried (FDU-810; EYELA, Tokyo, Japan) or heat-dried under unspecified conditions.
| Species | Locality | Voucher | INSDC accession no. | ||
| ITS | RPB2 | GPD | |||
| Alpova alpestris | France | PAM07082629a | HQ714696 | ||
| A. alpestris | France | PAM07090501 | HQ714692 | HQ714828 | HQ714620 |
| A. alpestris | France | PAM08090302 | HQ714721 | HQ714853 | HQ714629 |
| A. alpestris | France | PAM07082632 | HQ714697 | HQ714832 | HQ714621 |
| A. alpestris | France | PAM09082802 | HQ714776 | HQ714898 | |
| A. austroalnicola | Argentina | LSD_2290 | HQ714793 | ||
| A. cinnamomeus | USA | Brown_FP73a | KF835996 | KF835990 | |
| A. cinnamomeus | Canada | Hayward_K8 | KF835998 | ||
| A. cinnamomeus | Canada | HRL1384 | MN594282 | MN594770 | |
| A. cinnamomeus | Canada | HRL2671 | MN594283 | ||
| A. cinnamomeus | Canada | HRL2671-2 | MN594284 | ||
| A. concolor | USA | OSC65696a | KF835994 | KF835991 | |
| A. concolor | USA | UBC_F14673 | KF835997 | ||
| A. corsicus | France | PAM07090803 | HQ714699 | HQ714834 | HQ714624 |
| A. corsicus | France | FR2 | HQ714766 | HQ714890 | HQ714649 |
| A. corsicus | France | FR27 | HQ714768 | HQ714892 | HQ714651 |
| A. corsicus | France | FR28 | HQ714769 | HQ714893 | HQ714652 |
| A. corsicus | France | FR29 | HQ714770 | HQ714894 | HQ714653 |
| A. diplophloeus | USA | BPI_737239a | KF835993 | ||
| A. diplophloeus | USA | Hayward1 | KF835992 | KF835988 | |
| A. diplophloeus | USA | Hayward 2 | KF836001 | ||
| A. diplophloeus | Canada | UBC_F15285 | DQ384577 | ||
| A. fujisanensis | Shizuokab | TNS-F-108818a | LC848174 | LC848190 | LC848206 |
| A. fujisanensis | Shizuokab | CBM-FB-80001 | LC848175 | LC848191 | |
| A. fujisanensis | Shizuokab | M013 | LC848176 | LC848192 | LC848207 |
| A. fujisanensis (mycorrhiza) | Shizuokab | S1065 | HE979127 | ||
| A. japonicus | Tokyob | TNS-F-108819a | LC848177 | LC848193 | LC848208 |
| A. japonicus | Tokyo | M008 | LC848178 | LC848194 | LC848209 |
| A. japonicus | Tokyo | K299 | LC848179 | LC848195 | LC848210 |
| A. japonicus | Miyagib | K329 | LC848180 | LC848196 | |
| A. japonicus | Gunmab | CBM-FB-80003 | LC848181 | LC848197 | LC848211 |
| A. japonicus | Shizuokab | M027 | LC848182 | LC848198 | LC848212 |
| A. japonicus | Okayamab | M017 | LC848183 | LC848199 | LC848213 |
| A. japonicus | Oitab | K309 | LC848184 | LC848200 | LC848214 |
| A. japonicus (mycorrhiza) | Tokyob | 6_Alpova | LC646347 | ||
| A. japonicus (mycorrhiza) | Tokyob | As_oshima_Alp | LC739393 | ||
| A. komovianus | Montenegro | PAM10081201a | JQ436850 | JQ436862 | JQ436861 |
| A. larskersii | Sweden | SF413318a | OM892057 | ||
| A. larskersii | France | PAM09082702 | HQ714779 | HQ714901 | |
| A. larskersii | Sweden | OSC59767 | DQ989497 | ||
| A. larskersii | Sweden | SF410846 | OM892058 | ||
| A. larskersii | Sweden | SF410837 | OM892059 | ||
| A. larskersii (mycorrhiza) | Germany | a001GERM | MK285737 | ||
| A. larskersii (mycorrhiza) | - | L7109 | JN197936 | ||
| A. venosus | Fukushimab | TNS-F-108820a | LC848185 | LC848201 | LC848215 |
| A. venosus | Fukushimab | CBM-FB-80005 | LC848186 | LC848202 | LC848216 |
| A. venosus | Shizuokab | F160 | LC848187 | LC848203 | LC848217 |
| A. venosus | Kanagawab | K084 | LC848188 | LC848204 | LC848218 |
| A. venosus | Kanagawab | K313 | LC848189 | LC848205 | LC848219 |
| A. venosus (mycorrhiza) | Tochigib | S1005 | HE979095 | ||
| A. venosus (mycorrhiza) | Shizuokab | S1029 | HE979107 | ||
| Melanogaster ambiguus | Spain | JC180719NR | MN594286 | MN594771 | |
| M. luteus | France | PAM09082801 | HQ714780 | HQ714902 | |
| M. rivularis | France | PAM08090514 | HQ714767 | HQ714891 | HQ714650 |
a holotype
b Prefecture in Japan
All microscopic features were observed from hand-cut sections of dried specimens rehydrated in 3% KOH for a few minutes. Peridium and gleba structures were observed with a light microscope (Eclipse E600; Nikon, Tokyo, Japan) mounted in 3% KOH and Melzer’s reagent. We measured the peridium thickness from radial cut sections and the length and width of 100 spores using ImageJ software (Schneider et al., 2012). Spore dimensions are given as follows: (minimum value) 1st decile - 9th decile (maximum value) (Moreau et al., 2011, 2013). Portions of each sample were deposited in the National Science Museum, Tsukuba, Japan (accession numbers TNS-F-108818-108820) and the Natural History Museum and Institute, Chiba, Japan (accession numbers CBM-FB-80001-80005).
2.3. Molecular phylogeny2.3.1. DNA extraction, amplification, sequencingTotal DNA was extracted from the inner tissues of dried specimens using a modified version of the previously described cetyl trimethyl ammonium bromide method (Nara et al., 2003). We amplified three DNA loci, namely, the nuclear ribosomal internal transcribed spacer (ITS) regions (ITS1-5.8S-ITS2), RNA polymerase II second largest subunit (RPB2) gene, and glyceraldehyde-3-phosphate dehydrogenase (GPD) gene, through polymerase chain reaction (PCR). PCR amplification was performed using an Emerald Amp PCR Master Mix Kit (Takara Bio, Shiga, Japan) with the following primer pairs: ITS1F/LB-W for the ITS region (Gardes & Bruns, 1993; Tedersoo et al., 2008), fRPB2-5F or bRPB2-6F/bRPB2-7.1R for RPB2 gene (Matheny, 2005) and GPD-f´/GPD-R´ for GPD gene (Jargeat et al., 2010). PCR reactions targeting the ITS were conducted under the following conditions: 30 cycles of 98 °C for 10 s, 56 °C for 30 s, and 72 °C for 60 s. PCR reactions targeting the RPB2 and GPD were conducted under the following conditions: initial denaturation of 95 °C for 3 min, followed by 25 cycles of 95 °C for 45 s, 52 °C for 40 s, and 74 °C for 2 min; then 25 further cycles of 95 °C for 50 s, 52 °C for 45 s, and 74 °C for 2 min 5 s; and a final extension at 72 °C for 10 min (Tochihara & Hosoya, 2019). DNA amplification was confirmed through 1.2% agarose gel electrophoresis.
Amplified products were purified using ExoSAP-IT (Applied Biosystems, Forester City, CA, USA) and subjected to direct sequencing on a 3730xl DNA Analyzer (Applied Biosystems). Sequencing reactions were performed using the BigDye Termination v 3.1 Cycle Sequencing Kit (Applied Biosystems) with the primers ITS1, ITS2, ITS3 or ITS4 (ITS), bRPB2-6F or bRPB2-7.1R (RPB2), GPD-f´ or GPD-R´ (GPD). Chromatograms were manually trimmed and corrected using ATGC ver. 7 (Genetyx, Tokyo, Japan). The sequences were deposited in the DNA Data Bank of Japan under accession numbers LC848174- LC848219.
2.3.2. Phylogenetic analysisThe sequences obtained in this study were compiled into separate datasets representing individual loci. To infer the phylogenetic relationships with known Alpova species, related sequences of eight species available in the International Nucleotide Sequence Database Collaboration (INSDC) were also included in the dataset. These sequences were primarily obtained from Europe, and North and South America (Crous et al., 2022; Hayward et al., 2014; Moreau et al., 2011, 2013; Nouhra et al., 2005). We also downloaded sequences of Melanogaster ambiguus (Vittad.) Tul. & C. Tul., M. luteus Zeller and M. rivularis P.-A. Moreau & F. Rich. as the outgroup for the phylogenetic analysis (Table 1).
For the ITS dataset, sequences from environmental samples (ECM root tips) with >97% identity to our specimens were retrieved from the INSDC or UNITE database and incorporated into the dataset. Identical sequences to those of the environmental samples from the same sampling area and host were removed from the dataset. Sequences of adjacent sites (18S and 28S) were trimmed with ITSx (Bengtsson-Palme et al., 2013).
Multiple sequence alignment was performed using MAFFT v7.490 with the L-INS-i strategy (Katoh & Standley, 2013), followed by manual correction and trimming of unaligned ends with Seaview software ver. 5.0.5 (Gouy et al., 2010). Additionally, poorly aligned sites within the ITS sequences were eliminated using Gblocks (Castresana, 2000) with less stringent settings. Multiple alignment files for individual loci were concatenated into a single alignment file prior to phylogenetic analyses. To estimate the optimal base substitution model for each partition (ITS, RPB2, and GPD), MrModeltest v. 2.4 (Nylander, 2004) was used with PAUP* 4a168 (Swofford, 2003). The GTR+G, SYM+I, and HKY+G models were selected based on the Akaike Information Criterion for the ITS, RPB2, and GPD regions, respectively. Bayesian phylogeny was inferred with MrBayes v.3.2.7 (Ronquist et al., 2012) using Markov chain Monte Carlo (MCMC) iterations in two independent runs with four chains, from which every 100th tree was sampled over one million generations after the initial 10% of iterations were discarded as burn-in. Convergence of the MCMC analysis was assessed by ensuring the average standard deviation of split frequencies (ASDSF) was below 0.01. The parameter file from MrBayes was further analyzed using Tracer 1.7 (Rambaut et al., 2018) to assess effective sample sizes, which were consistently greater than 200. Additionally, a maximum likelihood (ML) tree was constructed using IQ-TREE v.2.3.5 (Minh et al., 2020) with 1000 bootstrap replicates. The optimal base substitution model for ML tree construction was selected by Model Finder (Kalyaanamoorthy et al., 2017), with the TNe+G4, TNe+I, and HKY+F+G4 models applied to the ITS, RPB2, and GPD, respectively. ML trees were also constructed for the individual loci separately using the same models and conditions for topological comparison. Significance thresholds were set to > 0.95 and > 70% for Bayesian posterior probabilities (PP) and ML bootstrap (BS) values, respectively. The concatenated alignment data were deposited in Figshare repository (doi: 10.6084/m9.figshare.29275364).
The ITS data contain 54 sequences of 828 aligned bases, of which 156 bp were identified as poorly aligned by Gblocks. The final alignments included 210/664 (from 54 ITS sequences), 129/786 (from 31 RPB2 sequences), and 147/699 (from 27 GPD sequences) variable sites/total sites. Excluding the outgroups, mean pairwise distances for ITS, RPB2, and GPD were 4.8%, 2.4%, and 4.5%, respectively. The Bayesian consensus tree was generated from the 18002 sampled trees and was merged into the ML tree, as the two trees had the same topology.
Our specimens formed three distinct clades that differed from previously described Alpova species in both ML and Bayesian analyses (Fig. 1). Alpova venosus (see Taxonomy for details) formed a monophyletic clade, basal to most European and American species, with strong branching support from BS (71) and PP (0.97). Alpova fujisanensis was a sister to the North American A. cinnamomeus with strong branching support (BS/PP values = 98/1.0). Alpova japonicus was placed in a clade composed of Argentina A. austroalnicola L.S. Domínguez, European A. larskersii Jeppson & E. Larss., and A. corsicus P.-A. Moreau & F. Rich. with strong branching support (100/1.0), though these species were clearly separated into individual sub-clades (>83/0.85 branching supports). Distinct clades of the three Japanese species were also supported in phylogenetic trees based on the individual loci (Supplementary Fig. S1), although the topology of the trees was not fully consistent as the available sequences for RPB2 and GPD were far fewer than ITS. These three Japanese species were therefore considered new species based on morphological observations (see Taxonomy for details) and molecular phylogeny.
Japanese, North American, and European sequences were separated into geographically distinct clades in the phylogenetic trees (Fig. 1), indicating a strong biogeographic pattern at the intercontinental scale. Additionally, A. japonicus and A. venosus collected from multiple locations within Japan exhibited intraspecific sequence variations (Table 1), which placed geographically close specimens in phylogenetically close positions (Fig. 1), exemplifying minor biogeographical patterns even within Japan.
Each species-level Alpova clade in the phylogeny exhibited host preference toward either subgen. Alnus or subgen. Alnobetula (Fig. 1). All A. venosus specimens examined in this study were collected under subgen. Alnus, whereas an environmental sequence from ectomycorrhizal roots of subgen. Alnobetula (HE979107; see Table 1) was also included in the same clade. The other two Japanese species were solely associated with the subgen. Alnobetula, representing two of the three Alnobetula clades confirmed in the entire genus Alpova.
The greatest mean similarity values to known species in the ITS region prior to application of Gblocks were 96.0% between A. venosus and A. diplophloeus (Zeller & C.W. Dodge) Trappe & A.H. Sm., 97.4% between A. japonicus and A. larskersii and 96.8% between A. fujisanensis and A. cinnamomeus. Additionally, all the environmental ITS sequences with > 99% identity to our specimens were detected from ECM root tips of Alnus spp. in Japan and belonged to the clades of the three Alpova species described in this study.
3.2. TaxonomyAlpova fujisanensis A. Ishikawa, E. Fujii & K. Nara, sp. nov. Fig. 2.
MycoBank no.: MB856217.
Diagnosis: Distinct from other Alpova species in the combination of small oval spores (3.3-5.5 × 1.6-2.5 µm) and thromboplerous hyphae in the subpellis and gleba.
Type: JAPAN, Shizuoka Prefecture, Suntou District, Oyama town, 35°21'35.7"N 138°46'19.5"E, under Alnus alnobetula subsp. maximowiczii, 29 Sep 2023, A. Ishikawa, M025, (holotype, TNS-F-108818).
Gene sequences ex-holotype: LC848174 (ITS), LC848190 (RPB2), LC848206 (GPD).
Etymology: fujisanensis (Lat.), referring to the type locality “Mt. Fuji”.
Basidiomata hypogeous to emergent, globose to subglobose or irregularly lobed, 0.8-1.5 × 0.6-0.7 cm in diam. Peridial surface smooth, wrinkled when young, initially light to pastel orange, reddish brown spots expanding gradually, entire surface becoming reddish brown with age. Fresh peridium turning reddish brown on handling. Peridium tissue 0.2-0.4 mm thick in section when fresh, light to pastel orange, turning slightly ochre on exposure. Gleba gelatinized, chambers 0.15-0.86 mm in diam, divided by distinct intermixed white veins apparently forming irregular reticulation, pastel orange when immature and yellowish ochre at maturity, waxy/cheesy when dried. Rhizomorphs inconspicuous. Odor and flavor not distinctive.
Peridium 2-layered, 180-340 µm thick in total. Peridiopellis 20-40 µm thick, amber-brown in 3% KOH, composed of inflated cells 9-27 µm in diam and interwoven cylindrical hyphae 4-8 µm in width, with amber-yellow pigment, occasional clamps, dermatocystidia 20-30 × 5-10 µm, cylindrical, attenuate to subcapitate. Subpellis 140-300 µm thick when rehydrated in 3% KOH, composed of yellowish ochre, round to elliptical cells, 6-50 × 6-34 µm, forming a pseudoparenchymatous structure, frequent cylindrical cells parallel to the peridial surface near gleba, with occasional thromboplerous hyphae 5-8 µm in width. Gleba composed of hyaline hyphae 1.5-4.0 µm in width with occasional clamps, intermixed with thromboplerous hyphae 4-5 µm in width containing brown pigment, frequent buffer cells 19-33 × 23-35 µm. Basidia 4.1-9.6 × 1.0-4.6 µm, club-shaped, with up to eight basidiospores developing on narrow and short sterigmata. Basidiospores (100 measurements) (3.5-)3.9-4.7(-5.0) × (1.6-)1.8-2.1(-2.3) µm, mean (± SD) 4.3 (± 0.3) × 2.0 (± 0.1) µm, Q = (1.8-)1.9-2.5(-3.0), oval to elongated oval and thick-walled, biguttulate, pale brown when suspended in 3% KOH, inamyloid.
Habitat and distribution: Only collected under A. alnobetula subsp. maximowiczii growing on lava flow or tephra deposits at 2000 m elevation on Mt. Fuji. The climate type is Dfb in the Köppen-Geiger climate classification (Beck et al., 2023). The habitat is an ephemeral stream where mosses grow, and the surrounding environment is relatively wet.
Additional specimens examined: JAPAN, Shizuoka Prefecture, Suntou District, Oyama Town, under A. alnobetula subsp. maximowiczii, 25 Sep 2022, leg. A. Ishikawa, M013; ibid., 22 Oct 2022, leg. E. Fujii, M014 (CBM-FB-80001).
Alpova japonicus A. Ishikawa, H. Sasaki & K. Nara, sp. nov. Fig. 3.
MycoBank no.: MB856218.
Diagnosis: Distinct from other Alpova species in the combination of rhizomorphs usually being present around the basal spot and small spores (3.8-5.6 × 1.6-2.7 µm). Also characteristic in being associated with Alnus subgen. Alnobetula spp. and distributed in various environments including non-riparian areas such as alpine and primary successional sites.
Type: JAPAN, Tokyo, Oshima Town, 34°43'18.0"N 139°24'37.8"E, under Alnus sieboldiana, 21 Jul 2022, A. Ishikawa, M004, (holotype, TNS-F-108819; isotype, CBM-FB-80002).
Gene sequences ex-holotype: LC848177 (ITS), LC848193 (RPB2), LC848208 (GPD).
Etymology: japonicus (Lat.), referring to the species’ occurrence across a broad range within Japan.
Basidiomata hypogeous to emergent, globose to subglobose, often irregular unevenness, 0.6-2.8 × 0.6-1.6 cm in diam. Peridial surface smooth, light to pastel orange, reddish brown spots expanding gradually, entire surface becoming reddish brown with age, reddish ochre when dried. Peridium tissue 0.2-0.5 mm thick in section when fresh, pastel orange, turning slightly reddish brown on exposure. Gleba gelatinized, chambers 0.13-0.97 mm in diam, pastel orange when immature and olivaceous to yellowish ochre at maturity, reddish ochre to ochre after few minutes when cut, intermixed with white veins forming irregular reticulation, brown to dark brown when dried. Rhizomorphs distinct around the basal part, adpressed to peridium, dark brown. Odor and flavor not distinctive.
Peridium 2-layered, 210-350 µm thick in total. Peridiopellis 30-50 µm thick, dark brown in 3% KOH, composed of cylindrical to catenulate hyphae 3-6 µm in width, interwoven with inflated cells 8-30 µm in diam, with brown pigment, frequent clamps, dermatocystidia 20-27 × 5-10 µm, cylindrical, attenuate to subcapitate. Subpellis 180-300 µm thick, composed of hyaline, round to elliptical cells, 6-46 × 6-28 µm, forming a pseudoparenchymatous structure, smaller and denser cells toward gleba. Gleba composed of hyaline hyphae 2.5-3.5 µm in width with occasional clamps, thromboplerous hyphae 3-5 µm in width containing brown pigment, occasional buffer cells 12-27 × 10-19 µm. Basidia, 4.1-6.0 × 1.5-3.3 µm, eight basidiospores developing on narrow and short sterigmata. Basidiospores (100 measurements) (3.9-)4.2-5.0(-5.5) × (1.7-)2.0-2.4(-2.5) µm, mean (± SD) 4.6 (± 0.3) × 2.2 (± 0.2) µm, Q = (1.7-)1.9-2.4(-2.6), oval, smooth and thick-walled, biguttulate, pale brown when suspended in 3% KOH, inamyloid.
Habitat and distribution: Associated with Alnus subgen. Alnobetula (i.e. Alnus firma, A. sieboldiana, and A. alnobetula subsp. maximowiczii) in a wide range of environments, including planted trees in parks, lowland forests (Cfa in the Köppen-Geiger climate classification; Beck et al., 2023), subalpine zones, and volcanic successional sites.
Additional specimens examined: JAPAN, Miyagi Prefecture, Sendai City, Izumi Ward, under A. firma, 29 Jul 2006, leg. Masahito Taniguchi, K329; Gunma Prefecture, Tone District, Katashina Town, under A. alnobetula subsp. maximowiczii, 6 Sep 2023, leg. E. Fujii, M022 (CBM-FB-80003); Shizuoka Prefecture, Suntou District, Oyama Town, under A. alnobetula subsp. maximowiczii, 29 Sep 2023, leg. A. Ishikawa, M027; Okayama Prefecture, Kurashiki City, Kojimakamino Town, under A. firma, 18 Jun 2023, leg. Tomoya Hirao, M017; Oita Prefecture, Bungo-ono City, Ogata Town, 19 Oct 2008, under A. subgen. Alnobetula spp., leg. Atsuko Hadano, K309.
Alpova venosus A. Ishikawa, H. Sasaki & K. Nara, sp. nov. Fig. 4.
MycoBank no.: MB856219.
Diagnosis: Distinct from other Alpova species in the combination of oblong ellipsoid spores (4.1-6.6 × 1.5-2.6 µm), the presence of subcapitate dermatocystidia and frequent thromboplerous hyphae in the subpellis. Also characteristic in being associated mostly with Alnus subgen. Alnus.
Type: JAPAN, Fukushima Prefecture, Futaba District, Kawauchi Village, under Alnus hirsuta, 27 Jul 2024, H. Sasaki, M030, (holotype, TNS-F-108820; isotype, CBM-FB-80004).
Gene sequences ex-holotype: LC848185 (ITS), LC848201 (RPB2), LC848215 (GPD).
Etymology: venosus (Lat.), referring to the frequent presence of thromboplerous hyphae in the subpellis, resembling veins.
Basidiomata hypogeous, globose to subglobose or irregularly oblong to constricted, 1.2-1.8 × 0.7-1.4 cm in diam. Peridial surface smooth to finely felted when dry, initially whitish light orange, turning yellow brown to reddish brown with age. Fresh peridium turning reddish brown on handling. Peridium tissue 0.2-0.6 mm thick when fresh, yellowish brown, turning slightly ochre on exposure. Gleba gelatinized, chambers 0.18-1.54 mm in diam, yellowish ochre with occasional olive when fresh, becoming reddish brown to ochre after few minutes when cut, chambers divided by distinct, whitish to yellowish brown veins, brown to dark brown when dried. Rhizomorphs inconspicuous. Odor and flavor slightly sweet like Melanogaster when mature.
Peridium 2-layered, 220-450 µm thick in total. Peridiopellis 20-50 µm thick, yellowish brown in 3% KOH, composed of inflated cells 9-23 µm in diam and interwoven cylindrical hyphae 2-8 µm in width, with occasional inflated hyphae up to 8-15 μm in width, dermatocystidia frequent, 17-25 × 2-10 µm, cylindrical, subcapitate. Subpellis 200-400 µm thick when rehydrated in 3% KOH, composed of yellowish ochre irregularly round to cylindrical cells, 7-42 × 5-37 µm, forming a pseudoparenchymatic structure, with a compact hyphal layer 10-25µm thick next to gleba, composed of compressed hyphae 3-5 µm in width, abundant thromboplerous hyphae 5-9 µm in width. Gleba composed of hyaline hyphae 1.5-3.5µm in width with occasional clamps, occasional thromboplerous hyphae 4-10 µm in width with brown contents, occasional buffer cells 19-39 × 10-27 µm. Basidia 9-15 × 3-5 µm, with eight to ten basidiospores developing on narrow and short sterigmata. Basidiospores (100 measurements) (4.7-)5.1-6.1(-6.4) × (1.9-)2.0-2.3(-2.5) µm, mean (± SD) 5.6 (± 0.4) × 2.1 (± 0.1) µm, Q = (2.2-)2.3-2.9(-3.2), oblong to ellipsoid, smooth and thick-walled, biguttulate, pale brown when suspended in 3% KOH, inamyloid.
Habitat and distribution: Associated with Alnus subgen. Alnus in a mountain forest mixed with oak trees. The climate type is Cfa in the Köppen-Geiger climate classification (Beck et al., 2023). The basidiomata occur beneath the litter on mineral soil where the humus layer is not well-developed.
Additional specimens examined: JAPAN, Yamanashi Prefecture, Gotenba City, under A. hirsuta, 27 Aug 2002, leg. K. Nara, F160; Kanagawa Prefecture, Aiko District, Kiyokawa Village, under A. hirsuta, 5 Jul 2003, leg. H. Sasaki, K084; Fukushima Prefecture, Futaba District, Kawauchi Village, under A. hirsuta, 27 Jul 2003, leg. H. Sasaki, K093 (CBM-FB-80005).
Two of the three Alpova species described in this study, i.e., A. fujisanensis and A. venosus, correspond to the unknown lineages suggested from the environmental sequences in previous studies (Alvarado et al., 2021; Hayward et al., 2014). Here, we provide the morphological information about the sporocarps of those lineages and formally describe them as new Alpova species. We identified an additional novel lineage, A. japonicus, from sporocarps (this study) and ECM sequences obtained recently (Ishikawa & Nara, 2023). All of these species have pseudoparenchymatous structure in the peridium, the presence of buffer cells in the gleba, small, pale spores, and an ECM association with alder trees, corresponding to the common traits of Alpova sensu stricto (Moreau et al., 2013). Thromboplerous hyphae, previously observed only in the gleba of A. komovianus B. Perić & P.-A. Moreau, were confirmed in all Japanese species described here, especially for A. fujisanensis and A. venosus that have thromboplerous hyphae even in the subpellis. Thus, the thromboplerous hyphae are the unique traits that distinguish them from all the currently accepted Alpova sensu stricto species when combined with smaller spore sizes, as detailed in the diagnosis for the Taxonomy of each species. Needless to say, these morphological traits fundamentally differ from the currently accepted but ambiguous Alpova species, because original descriptions of these ambiguous taxa do not accord with the common traits of Alpova sensu stricto, as mentioned in the Introduction.
The Japanese Alpova species were placed into unique phylogenetic clades, which are distantly related to European and North American clades. No phylogenetic clades at the species level comprised specimens from different continents. These results support the existence of strong biogeographical boundaries in Alpova species distribution, as reported in other hypogeous taxa with limited spore dispersal ability across geographical barriers (Bonito et al., 2013; Grubisha et al., 2007). The continental-scale separation in the distribution of Alpova species also indicate that the geographic origin of sporocarps provide critical information for their identification.
Because A. alpestris P.-A. Moreau & F. Rich. is distributed across both continental Europe and the non-continuous French island Corsica Island (see Supplementary Table S1), the three Japanese species described here could potentially be found in other Asian regions. The currently recorded Alpova species in Asia are A. mollis, A. shanxiensis, and A. yangchengensis, all reported from China (Liu et al., 1990; Liu & Tao, 1996). These ambiguous species, however, differ from Japanese species morphologically. Based on the original descriptions (Liu et al., 1990; Liu & Tao, 1996), subpellis tissues of these Chinese species were composed of interwoven hyphae. Gleba of A. shanxiensis and A. mollis was dark brown. These morphological traits are common to Melanogaster (Moreau et al., 2011) and contradict Alpova sensu stricto including Japanese species. Furthermore, A. yangchengensis and A. mollis were found under Ulmus and Quercus variabilis trees, respectively, while no host information is available for A. shanxiensis (Liu et al., 1990; Liu & Tao, 1996). Thus, based on taxonomic re-examination with molecular phylogenetic analyses, the three Chinese species assigned to Alpova should be transferred to other genera, probably Melanogaster. Additionally, these Chinese species either have larger spores (1.4-1.8 times the size of the Japanese species in A. shanxiensis) or broader spores (1.2-2.1 times the width of the Japanese species in A. yangchengensis and A. mollis) than Japanese species, unequivocally discriminating them from the Japanese species described here.
The type specimen of A. japonicus was collected on Izu-Oshima Island, an oceanic island located at 22 km distance from mainland Japan. Although the process that brought the initial spores of this hypogeous species to the island remains unknown, spore-carrying animals may have migrated from the nearby mainland. Because the distance from the mainland is relatively short, lizards and birds can migrate to the island (Fujita et al., 2023; Motokawa & Hikida, 2003) and can potentially carry the spores either by consuming the sporocarps directly or by feeding on primary consumers, such as invertebrates (Caiafa et al., 2021; Elliott et al., 2022). Moreover, the migration of the Japanese field mouse, Apodemus speciosus, occurred multiple times during the last 250,000 y, as revealed by population genetic structures (Tomozawa et al., 2014). Mice frequently feed on hypogeous sporocarps and play important roles in the spore dispersal of the hypogeous fungal species (Johnson, 1996; Komur et al., 2021), potentially including A. japonicus. Population genetic studies using microsatellites or single nucleotide polymorphism would clarify how the island population of this hypogeous fungus was established and maintained.
Almost all Alpova species are considered to exhibit host specificity at the subgenus level (Hayward et al., 2014; Moreau et al., 2011, 2013). Indeed, ECM roots and sporocarps of A. fujisanensis and A. japonicus were detected only in association with subgen. Alnobetula, while A. venosus was only confirmed from subgen. Alnus in this study. Rochet et al. (2011) hypothesized that ancestors of Alpova had the specificity for the subgen. Alnus and extended their specificity to the subgen. Alnobetula. Our phylogenetic analysis shows that the earliest diverging lineages such as A. komovianus and A. venosus are associated with subgen. Alnus, while all the species associated with subgen. Alnobetula are scattered within more terminal branches (Fig. 1). These results support the hypothesis of the host transition from the Alnus to Alnobetula by Rochet et al. (2011) and suggest that the transition occurred multiple times in different geographical regions during the diversification of Alpova species.
In the ITS region, which is frequently used in fungal barcoding and ECM community studies, A. venosus and A. fujisanensis exhibited more than 3% divergence from all known sequences registered in GenBank, except the conspecific sequences obtained from ECM tips in Japan. Although A. japonicus is similar to A. larskersii and A. austroalnicola, with 2.6% and 2.7% ITS dissimilarity, respectively, the genetic distance between A. corsicus and the recently described A. larskersii is at the same level (2.6%). While many ECM fungal community studies use the 3.0% dissimilarity threshold for species delimitation, previous studies have shown that it is too conservative for some fungal taxa (Nilsson et al., 2008) and recommend a threshold of 2% or less dissimilarity threshold (Kõljalg et al., 2013; Tedersoo et al., 2014). In previous studies, ECM fungal communities associated with Alnus have often used phylogenetic analysis to distinguish operational taxonomic units by strict dissimilarity thresholds (Põlme et al., 2013; Tedersoo et al., 2009). Given that A. japonicus, A. larskersii, and A. austroalnicola are distinguishable morphologically and occupy geographical areas far beyond the range of natural fertilization, the latter strict threshold may be needed for Alpova.
This study describes three new Alpova species from Japan, based on the morphological characteristics of the sporocarps and molecular data from three loci, representing the first Asian species in Alpova sensu stricto. Given that more than half of global Alnus species are distributed in Asia (Chen & Li, 2004; Furlow, 1979), in combination with the strong host specificity of Alpova (Molina, 1979; Pozzi et al., 2018), there are likely far more Alpova species remaining to be found in Asia. In previous studies on ECM communities associated with mature Alnus species, Alpova has rarely been detected (Põlme et al., 2013; Roy et al., 2013), but this genus is dominant in seedlings and young trees (Ishikawa & Nara, 2023). Therefore, additional sampling efforts aimed at discovery novel Alpova species should prioritize young Alnus forests. Alpova species play important roles in host seedling establishment (Ishikawa & Nara, 2023; Yamanaka et al., 2022) and, thus, more attention should be paid to taxonomic analysis and species description of Alpova in the future.
The authors declare no competing interests
We thank Tomoya Hirao, Yoshinori Kaneko, Masahito Taniguchi, Atsuko Hadano for collecting samples. We also thank the Ministry of the Environment, Japan, for the permissions of field survey. This study was supported, in part, by JST SPRING, Grant Number JPMJSP2108.
