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Research Article
Molecular phylogeny and taxonomy of the Hydrangea serrata complex (Hydrangeaceae) in western Japan, including a new subspecies of H. acuminata from Yakushima
expand article infoShun K. Hirota‡, Tetsukazu Yahara§, Kengo Fuse§, Hiroyuki Sato§, Shuichiro Tagane|, Shinji Fujii, Tadashi Minamitani#, Yoshihisa Suyama‡
‡ Tohoku University, Osaki, Japan
§ Kyushu Open University, Fukuoka, Japan
| Kagoshima University, Kagoshima, Japan
¶ University of Human Environments, Okazaki, Japan
# Tsunehisa 5-4-7, Miyazaki, Japan

Corresponding author: Tetsukazu Yahara ( tet.yahara@gmail.com )

Academic editor: Sandy Knapp
© 2022 Shun K. Hirota, Tetsukazu Yahara, Kengo Fuse, Hiroyuki Sato, Shuichiro Tagane, Shinji Fujii, Tadashi Minamitani, Yoshihisa Suyama.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation: Hirota SK, Yahara T, Fuse K, Sato H, Tagane S, Fujii S, Minamitani T, Suyama Y (2022) Molecular phylogeny and taxonomy of the Hydrangea serrata complex (Hydrangeaceae) in western Japan, including a new subspecies of H. acuminata from Yakushima. PhytoKeys 188: 49-71. https://doi.org/10.3897/phytokeys.188.64259
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Abstract

According to the contemporary classification of Hydrangea native to Japan, H. serrata is a polymorphic species including six varieties. We discovered a plant identified as H. serrata, but morphologically distinct from previously known varieties, in Yakushima island where approximately 50 endemic species are known. To determine the relationship of this plant with previously known varieties, we examined morphology and constructed a highly resolved phylogeny of H. serrata and its relatives using three chloroplast genomic regions, rbcL, trnL intron, psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, and Multiplex ISSR genotyping by sequencing (MIG-seq). Based on these morphological and phylogenetic observations, we describe Hydrangea acuminata subsp. yakushimensis subsp. nov. as a newly discovered lineage in Yakushima, Japan and propose Hydrangea minamitanii stat. nov. and Hydrangea acuminata subsp. australis stat. nov. which were previously treated as varieties of H. serrata.

Keywords

cpDNA, DNA barcoding, F ST, island, ITS, MIG-seq, threatened plants

Introduction

Hydrangea L. s. lat. is a genus of Hydrangeaceae, comprising approximately 200 species distributed in East and Southeast Asia and the New World (De Smet et al. 2015). Based on molecular phylogenetic studies, De Smet et al. (2015) proposed a broad circumscription of Hydrangea by absorbing the other eight genera of tribe Hydrangeeae. Under this proposal, Cardiandra Siebold & Zucc., Deinanthe Maxim., Pileostegia Hook. f. & Thomson, Platycrater Siebold & Zucc., and Schizophragma Siebold & Zucc., which have been recognized in the representative flora of Japan (Kitamura and Murata 1979; Satake et al. 1999; Ohba 2017), are reduced to Hydrangea s. lat. In contrast, Ohba and Akiyama (2016) preferred to retain these genera and proposed generic segregation of most of the sections and subsections of Hydrangea s. lat. proposed by De Smet et al. (2015). In this study, we follow the broad circumscription of Hydrangea by De Smet et al. (2015) that retains species widely known as “hydrangea,” including H. macrophylla (Thunb.) Ser. and H. serrata (Thunb.) Ser., under the genus name of Hydrangea.

In 2005, we discovered a plant of the genus Hydrangea from a mountain-top area of the Yakushima Island, a small island with an area of 504.88 km2 and a maximum peak of 1,936 m in elevation, part of which is designated as a UNESCO Man and the Biosphere Reserve (Okano and Matsuda 2013). The Yakushima Island is a center of plant endemism in Japan, harboring approximately 45 endemic species, including Hydrangea grosseserrata Engl. (Masamune 1934; Hotta 1974; Yahara et al. 1987). Whereas H. grosseserrata grows in evergreen forests at lower elevations, the newly discovered plant of Hydrangea is restricted to the mountain-top. In addition, they are morphologically distinct from H. grosseserrata. Although the flora of Yakushima has been well studied by the classic work of Masamune (1934) and a subsequent work of Yahara et al. (1987), recent field surveys discovered six additional new species endemic to this island: Oxygyne yamashitae Yahara & Tsukaya (Burmanniaceae, Yahara and Tsukaya 2008), Carex mochomuensis Katsuy. (Katsuyama 2009), Haplopteris yakushimensis C.W. Chen & Ebihara (Pteridaceae, Chen et al. 2014), Dryopteris protobissetiana K. Hori & N. Murak. (Dryopteridaceae, Hori et al. 2015), Lecanorchis tabugawaensis Suetsugu & Fukunaga (Orchidaceae, Suetsugu and Fukuhara 2016), and lastly Sciaphila yakushimensis Suetsugu, Tsukaya & H. Ohashi (Triuridaceae, Suetsugu et al. 2016). Considering the high endemism of the flora of Yakushima, we suspected that the plant of Hydrangea could be a new taxon. In this study, we compared the newly discovered plant with a morphologically similar species by molecular phylogenetic analysis and morphological observations.

The newly discovered plant is morphologically identified as Hydrangea serrata in having ovate-oblong petals, distinct peduncles, and oblong leaves, based on the key and description of Ohba (2017). According to Yamazaki (2001) and Ohba and Akiyama (2013), H. serrata is a polymorphic species, including six varieties, but the plant discovered from a mountain-top area of the Yakushima Island appeared to be different from those varieties. Among these six varieties, the following three varieties are distributed on the main island of Kyushu located 60 km north of Yakushima: H. serrata var. acuminata (Siebold & Zucc.) Nakai, var. australis T. Yamaz., and var. minamitanii H. Ohba. To examine the genetic divergence of the newly discovered plant from the three varieties of H. serrata distributed on the Kyushu Island, we reconstructed phylogenetic trees of H. serrata and its relatives using three chloroplast genomic regions, rbcL, trnL intron, psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, and Multiplex ISSR genotyping by sequencing (MIG-seq; Suyama and Matsuki 2015).

A previous molecular phylogenetic study was performed on H. serrata and its relatives using rbcL, matK, and Random Amplified Polymorphic DNA (RAPD) markers (Uemachi et al. 2014), but this study did not examine var. australis and var. minamitanii. Uemachi et al. (2014) revealed that H. serrata var. serrata diverged to the western and eastern groups in Japan, corresponding to H. serrata var. acuminata and H. serrata var. serrata s. str., respectively.

Our new molecular phylogenetic analysis covered all the lineages distributed in Kyushu, including the newly discovered lineage from Yakushima, H. serrata var. acuminata , var. australis , and var. minamitanii from western Japan, as well as var. angustata (Franch. & Sav.) H. Ohba and var. serrata s. str. from eastern Japan. The results supported the treatment of the former three varieties as H. acuminata subsp. acuminata, H. acuminata subsp. australis, and H. minamitanii, respectively, and treating the newly discovered lineage as a new subspecies of H. acuminata.

Materials and methods

Field surveys

We carried out field studies in Yakushima Island of Kagoshima Prefecture and five additional prefectures, including Fukuoka, Miyazaki, Kochi, Mie, and Shizuoka. In total, we collected 24 samples consisting of 10 species with five infraspecific taxa of Hydrangea for DNA isolation (Table 1): H. acuminata subsp. acuminata from four localities (Fig. 1), H. acuminata subsp. australis from two localities (Fig. 1), H. acuminata subsp. yakushimensis described below (Fig. 1), H. macrophylla, H. minamitanii, H. serrata var. angustata, and H. serrata var. serrata of sect. Macrophyllae (E. M. McClint.) Y. De Smet & Samain (De Smet et al. 2015); H. grosseserrata, H. kawagoeana, H. luteovenosa, and H. scandens of sect. Chinenses Y. De Smet & Samain; and H. hirta of sect. Hirtae Y. De Smet & Samain. These three sections belong to the monophyletic group Hydrangea II (De Smet et al. 2015). As outgroups, we included H. davidii Franch., H. indochinensis Merr., and H. febrifuga (Lour.) Y. De Smet & Granados (Dichroa febrifuga Lour.) collected in Vietnam (Table 1), where we carried out a series of field studies (Middleton et al. 2019; Nagahama et al. 2021). In each sample, a small leaf piece was cut out, placed in a tea bag, and dried with silica gel in a zip-lock bag.

Table 1.
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Samples used in molecular phylogenetic analyses.

Scientific name Voucher ID Locality Coordinates
Hydrangea (Dichroa) sp. V8372 Bidoup Nui Ba, Vietnam 12.16016944, 108.5364333
Hydrangea acuminata [Shikoku lineage] TGK0472 Ino, Kochi 33.781458, 133.188252
Hydrangea acuminata [Shikoku lineage] JPN3301 cultivated, Fukuoka 33.55545001, 130.1939861
Hydrangea acuminata ssp. acuminata JPN0330 Mt. Ihara, Fukuoka 33.48363400, 130.2638410
Hydrangea acuminata ssp. acuminata JPN0433 Mt. Raizan, Fukuoka 33.48293333, 130.2204444
Hydrangea acuminata ssp. acuminata JPN2336 Mt. Oyaji, Miyazaki 32.77326944, 131.3367306
Hydrangea acuminata ssp. acuminata JPN2063 Mt. Shiraiwa, Miyazaki 32.56233100, 131.1113540
Hydrangea acuminata ssp. australis JPN0908 Mt. Karakuni, Miyazaki 31.93438888, 130.8600000
Hydrangea acuminata ssp. australis JPN3192 Miyakonojyo, Miyazaki 31.78877222, 130.9603278
Hydrangea acuminata ssp. yakushimemsis JPN1708 Yakushima, Kagoshima 30.372031, 130.504266
Hydrangea acuminata ssp. yakushimemsis JPN1799 Yakushima, Kagoshima 30.34255555, 130.4810000
Hydrangea davidii V4997 Fansipan, Vietnam 22.34225, 103.7764167
Hydrangea grosseserrata JPN0528 Yakushima, Kagoshima 30.34619444, 130.3918750
Hydrangea grosseserrata JPN0652 Yakushima, Kagoshima 30.26264444, 130.5800944
Hydrangea hirta JPN2415 Mt. Amagi, Shizuoka 34.86201944, 139.0215139
Hydrangea indochinensis V4959 Fansipan, Vietnam 22.34755555, 103.7721944
Hydrangea kawagoeana TG00879 Suwanose-jima, Kagoshima 29.62290600, 129.69778900
Hydrangea luteovenosa JPN0378 Mt. Ihara, Fukuoka 33.48294444, 130.2541972
Hydrangea luteovenosa JPN0901 Mt. Karakuni, Miyazaki 31.93438888, 130.8600000
Hydrangea luteovenosa JPN1982 Mt. Osuzu, Miyazaki 32.29758800, 131.4459520
Hydrangea macrophylla JPN3302 cultivated, Fukuoka 33.55545001, 130.1939861
Hydrangea macrophylla JPN3303 cultivated, Fukuoka 33.55545001, 130.1939861
Hydrangea minamitanii JPN1983 Mt. Osuzu, Miyazaki 32.29758800, 131.4459520
Hydrangea minamitanii TG01200 Aya, Miyazaki 32.03053900, 131.21502800
Hydrangea scandens JPN1980 Mt. Osuzu, Miyazaki 32.29758800, 131.4459520
Hydrangea scandens JPN2931 Kihoku, Mie 34.18644999, 136.1858528
Hydrangea serrata var. angustata JPN2404 Izu City, Shizuoka 34.96862800, 138.8459450
Hydrangea serrata var. serrata JPN2980 Osugi-dani, Mie 34.21346388, 136.1650250
Figure 1. 

Localities of Hydrangea acuminata subsp. acuminata (including Shikoku lineage), subsp. australis , and subsp. yakushimensis where DNA samples and voucher specimens were collected in this study. The map was produced from Chiriin Chizu Vector (https://maps.gsi.go.jp/vector/).

DNA isolation, genome-wide Single Nucleotide Polymorphism (SNP) genotyping, and construction of phylogenetic trees

Total DNA was extracted from the dried leaves using the cetyl trimethyl ammonium bromide (CTAB) method (Doyle and Doyle 1990). Multiplex ISSR genotyping by sequencing (MIG-seq, Suyama and Matsuki 2015) was used for de novo SNP detection. Briefly, a MIG-seq library was prepared by a two-step PCR amplification process based on the protocol detailed by Suyama et al. (2022). The amplicons in the size range of 300–800 bp were purified and sequenced on an Illumina MiSeq platform (Illumina, San Diego, CA, USA) using an MiSeq Reagent Kit v3 (150 cycles, Illumina). We skipped the sequencing of the first 17 bases of reads 1 and 2 (SSR primer regions and anchors) using “DarkCycle”. Low-quality reads and extremely short reads containing adapter sequences were removed using Trimmomatic 0.39 (Bolger et al. 2014). Stacks 2.41 pipeline software (Catchen et al. 2013; Rochette et al. 2019) was used to obtain individual genotypes with the following parameters: minimum depth of coverage required to create a stack (m) = 3, maximum distance between stacks (M) = 2, maximum mismatches between loci when building the catalog (n) = 2. Three different filtering criteria were applied for quality control of the SNP data. First, any SNP site where one of two alleles had less than three counts was filtered out because it is difficult to distinguish polymorphisms from sequencing errors when the minor allele count of SNPs is too low (Roesti et al. 2012). Second, loci containing SNPs with high heterozygosity (Ho ≥ 0.6) were removed because excess heterozygosity may have resulted from artifactual loci built from several paralogous genomic regions. Third, SNPs with a genotyping rate of < 50% were eliminated. Using the third criterion, the SNPs that were retained by 14 or more samples remained in the SNP dataset.

Maximum likelihood phylogeny based on SNPs was inferred using software RAxML 8.2.10 (Stamatakis 2014). We used a GTRCAT model and performed 1,000 replicates of parallelized tree search bootstrapping. Based on the clades of the MIG-seq tree, we estimated pairwise FST values for each clade using the POPULATIONS program in Stacks.

Sequencing and phylogenetic analysis of chloroplast and nuclear genomic regions

The chloroplast and nuclear genomic regions were sequenced using the next generation sequencing (NGS) technique (Suyama et al. 2022). First, three chloroplast genomic regions, rbcL, trnL intron, and psbA-trnH, and two nuclear genomic regions, ITS1 and ITS2, were simultaneously amplified using the Multiplex PCR Assay Kit Ver. 2 (Takara Bio, Kusatsu, Japan) (first PCR reaction). The first primers consisted of tail sequences and locus-specific primers (Suyama et al. 2022). Second, the products from the first PCR reaction were purified and used for the second PCR. The second PCR was conducted using primer pairs including tail sequences, adapter sequences for Illumina sequencing, and the index sequence to identify each individual sample. Third, the second PCR products from each sample were mixed, and sequencing was performed using an Illumina MiSeq platform with an MiSeq Reagent Nano Kit v2 (500 cycles, Illumina). We skipped the sequencing of the first three bases of reads 1 and 2 (anchor region for the 2nd PCR primer) using the “DarkCycle” option of the MiSeq system. Both ends of the fragments and index sequences were read by paired-end sequences (reads 1 and 2) and index sequencing. The number of bases read was 251 bases for both read 1 and read 2.

The sequences of the five regions were determined using Claident pipeline (Tanabe and Toju 2013, http://www.claident.org/, Tanabe, A.S., Claident, Date of access: 05/01/2021). First, raw MiSeq BCL data were converted into FASTQ data using the BCL2FASTQ program provided by Illumina, and raw FASTQ data were demultiplexed based on index and primer sequences using the clsplitseq program in Claident. Subsequent analysis was performed per region per individual. In ITS1 and ITS2, we merged paired-end reads because reads 1 and 2 overlapped. In rbcL, trnL intron, and psbA-trnH, we independently analyzed reads 1 and 2 because the length of the sequenced reads was too short to merge reads 1 and 2. Second, the low-quality 3’ tails were trimmed and the low-quality sequences were filtered out using the clfilterseq program. Third, the noisy and chimeric sequences were removed using the clcleanseqv program. Fourth, the remaining reads were clustered with a cut-off sequence similarity of 99%. An operational taxonomic unit (OUT) that had the most observed reads within the individual was treated as a representative OTU sequence.

Multiple alignments of the chloroplast and nuclear genomic regions were performed using the program MAFFT 7.313 (Katoh and Standley 2013), and alignment columns containing gaps were trimmed using a heuristic selection method based on similarity statistics of trimAl 1.4.rev15 (Capella-Gutiérrez et al. 2009). We used Kakusan 4.0 (Tanabe 2011) to find suitable nucleotide substitution models and partitioning strategies for the nucleotide datasets. The chloroplast and nuclear genomic regions were independently run through Kakusan. The corrected Akaike Information Criterion (AICc; Sugiura 1978) was used to compare nonpartitioned, partitioned _ equal _ mean _ rate, and separate models. The nonpartitioned model (GTR + Γ) proved optimal for both the chloroplast and nuclear genomic regions. Maximum likelihood phylogenies were inferred using RAxML 8.2.10 (Stamatakis 2014), whereby 1,000 replicates of parallelized tree search bootstrapping were conducted.

Morphological observations

Using the specimens listed in Table 2, we measured the following leaf traits using the largest leaf: leaf blade length, leaf blade width, petiole length, leaf apex length, leaf teeth length, and the number of teeth on one side of the leaf margin. Leaf teeth length was measured as the height from the line between two bases of a tooth to the tip of the tooth, for the highest tooth of the largest leaf. We also measured the corymb length, corymb width, and capsule length for fruiting specimens.

Table 2.
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The specimens used for measurements of nine morphological traits.

Taxa Specimen ID Herbaria
Hydrangea acuminata ssp. acuminata KAG161334, KAG161335, KAG161336, KAG161337, KAG161338, KAG161344, KAG161345, KAG161348, KAG161349, KAG161350 KAG
Hydrangea acuminata ssp. acuminata Fujii 117037 KYO
Hydrangea acuminata ssp. australis KAG023305, KAG083840, KAG083882, KAG086731, KAG161312, KAG161315, KAG161327, KAG161377 KAG
Hydrangea acuminata ssp. australis Fujii 18200, Fujii 178001 KYO
Hydrangea acuminata ssp. yakushimensis Yahara et al. 791, 792, 793–1, 793–2, 793–3, 793–4, 1103, 1104, 1105, JPN1799 FU

Data resources

All raw MIG-seq data were deposited at the DDBJ Sequence Read Archive (DRA) with accession number DRA011509. The demultiplexed raw reads of ITS and cpDNA regions were deposited at the DDBJ Sequence Read Archive (DRA) with accession number DRA011510. All sequences of ITS and cpDNA regions were registered to DNA Data Bank of Japan (DDBJ) under accession nos. LC657594–LC657817.

Results

Phylogenetic and population genetic analyses using MIG-seq

A total of 22,106,838 raw reads (789,530 ± 47,627 reads per sample) were obtained, and after quality control, 20,944,147 reads (748,005 ± 45,296 reads per sample) remained. After de novo SNP detection and filtering, the dataset had 1,746 SNPs from 685 loci.

In the MIG-seq tree (Fig. 2), nine Hydrangea species were clustered into three clades corresponding to sect. Macrophyllae (H. acuminata, H. macrophylla, H. minamitanii, and H. serrata), sect. Chinenses (H. grosseserrata, H. kawagoeana, H. luteovenosa, and H. scandens), and sect. Hirtae (H. hirta). In the Macrophyllae clade, H. minamitanii was sister to the clade including the other three species and monophylies of both H. minamitanii and the latter clade were supported by 100% bootstrap values. Among the latter three species, the clade including H. macrophylla and H. serrata was supported by a 96% bootstrap value and sister to the clade of H. acuminata supported by an 85% bootstrap value. Within H. acuminata, the Shikoku lineage was sister to a clade supported by a 99% bootstrap value including subsp. acuminata, subsp. australis, and subsp. yakushimensis, and the sister relationship of subsp. australis and subsp. yakushimensis was supported by a 90% bootstrap value. Even after the separation of H. acuminata, H. serrata was not monophyletic. The samples of H. serrata from Mie (JPN2980; var. serrata) and Shizuoka (JPN2404; var. angulata) were clustered with H. macrophylla but not sister to each other, and the sister relationship of H. serrata var. angulata and H. macrophylla was supported by a 100% bootstrap value. Similarly, H. luteovenosa was not monophyletic. Whereas H. luteovenosa 1 was sister to a clade including H. kawagoeana and H. grosseserrata, H. luteovenosa 2 was sister to a clade including all the other samples of sect. Chinenses.

Figure 2. 

Molecular phylogenetic tree reconstructed using MIG-seq. Bootstrap values are shown on the nodes, and branch lengths are shown on the internodes. Branch length represents the average number of substitutions per SNP site.

The degree of genetic differentiation measured by FST (Table 3) was 0.251 between H. acuminata subsp. acuminata and subsp. australis , 0.316 between subsp. acuminata and subsp. yakushimensis , and 0.437 between subsp. australis and subsp. yakushimensis. Among the closely related species of sect. Macrophyllae, FST was 0.553 between H. macrophylla and H. serrata, 0.317–0.514 between H. acuminata and H. serrata, and 0.452–0.652 between H. acuminata and H. macrophylla. Hydrangea minamitanii is differentiated from H. acuminata subsp. acuminata , subsp. australis , subsp. yakushimensis, Shikoku lineage, H. serrata, and H. macrophylla in FST values of 0.340, 0.470, 0.546, 0.439, 0.480, and 0.657, respectively. Between species of sect. Chinenses, FST varied from 0.395 (H. kawagoeana vs. H. grosseserrata) to 0.632 (H. grosseserrata vs. H. scandens). Between sections, FST varied from 0.454 (H. luteovenosa 2 of sect. Chinenses vs. H. acuminata subsp. acuminata) to 0.814 (H. hirta vs. H. macrophylla).

Table 3.
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The degrees of genetic differentiation between taxa measured by FST.

Hirtae Chinenses Macrophyllae
H. grosseserrata H. kawagoeana H. luteovenosa 1 H. scandens H. luteovenosa 2 H. minamitanii H. acuminata ssp. acuminata H. acuminata ssp. australis H. acuminata ssp. yakushimensis Shikoku lineage H. serrata H. macrophylla
Hirtae H. hirta 0.739 0.696 0.700 0.693 0.624 0.724 0.511 0.654 0.720 0.637 0.715 0.814
Chinenses H. grosseserrata – 0.395 0.580 0.632 0.616 0.749 0.590 0.705 0.769 0.711 0.735 0.808
Chinenses H. kawagoeana – – 0.473 0.561 0.524 0.768 0.578 0.697 0.736 0.695 0.734 0.792
Chinenses H. luteovenosa 1 – – – 0.544 0.510 0.729 0.574 0.681 0.728 0.687 0.702 0.775
Chinenses H. scandens – – – – 0.551 0.722 0.574 0.684 0.759 0.695 0.711 0.787
Chinenses H. luteovenosa 2 – – – – – 0.501 0.454 0.594 0.679 0.565 0.598 0.720
Macrophyllae H. minamitanii – – – – – – 0.340 0.470 0.546 0.439 0.480 0.657
Macrophyllae H. acuminata ssp. acuminata – – – – – – – 0.251 0.316 0.257 0.317 0.452
Macrophyllae H. acuminata ssp. australis – – – – – – – – 0.437 0.405 0.441 0.606
Macrophyllae H. acuminata ssp. yakushimensis – – – – – – – – – 0.453 0.514 0.652
Macrophyllae Shikoku lineage – – – – – – – – – – 0.364 0.585
Macrophyllae H. serrata – – – – – – – – – – – 0.553

Phylogenetic tree reconstructed using ITS sequences

A total of 111,216 reads (3,972 ± 299 reads per sample, ITS1) and 81,988 reads (2,928 ± 155 reads per sample, ITS2) were obtained. After gaps were trimmed, the total length of the sequences was 635 bp (ITS1: 267 bp, ITS2: 368 bp). In the ITS tree (Fig. 3), sect. Macrophyllae was supported by a 92% bootstrap value, and sect. Chinenses was supported by an 85% bootstrap value. In sect. Macrophyllae, only three branches were supported by bootstrap values larger than 80%: a clade including H. acuminata and H. minamitanii was supported by an 88% bootstrap support, H. acuminata subsp. yakushimensis was supported by 97%, and H. macrophylla was supported by 94%. In sect. Chinenses, a clade including H. kawagoeana and H. grosseserrata was supported by a 91% bootstrap value, and another clade including H. luteovenosa 1 and H. scandens was supported by a 90% bootstrap value. Hydrangea luteovenosa 1 and H. luteovenosa 2 were not sister to each other.

Figure 3. 

Molecular phylogenetic tree reconstructed using ITS sequences. Bootstrap values are shown on the nodes. Nodes supported by less than 70% bootstrap values are not shown.

Phylogenetic tree reconstructed using cpDNA sequences

A total of 20,290 reads (725 ± 68 reads per sample, rbcL), 18,724 reads (669 ± 68 reads per sample, trnL intron), and 20,194 reads (721 ± 72 reads per sample, psbA-trnH) were obtained. After gaps were trimmed, the total length of the sequences was 1,354 bp. The sequenced lengths of each region were 222 bp and 227 bp (read 1 and 2 of rbcL), 228 bp and 228 bp (read 1 and 2 of trnL intron), and 226 bp and 223 bp (read 1 and 2 of psbA-trnH). In the cpDNA tree reconstructed using these sequences (Fig. 4), the monophyly of sect. Macrophyllae was supported by a 96% bootstrap value, and the two lineages of sect. Chinenses and H. hirta were polychotomous.

Figure 4. 

Molecular phylogenetic tree reconstructed using cpDNA sequences. Bootstrap values are shown on the nodes. Nodes supported by less than 60% bootstrap values are not shown.

Morphological observations

Morphologically, H. acuminata subsp. yakushimensis is similar to subsp. acuminata in having blue-colored flowers: fertile flowers with blue-colored petals, stamens, and sterile flowers with blue-colored calyces (Fig. 5). However, H. acuminata subsp. yakushimensis is distinct from subsp. acuminata in that the upper leaf surface is glabrous except on veins (vs. sparsely hairy), the lower leaf surface is glabrous or only slightly hairy except for tufted hairs at axils of lateral veins (vs. sparsely hairy), and capsules are shorter than 2.7 mm (vs. 3.2 mm or longer; Fig. 6; Table 4; Table 5). In addition, H. acuminata subsp. yakushimensis is different from subsp. acuminata in that the number of teeth along each margin of the largest leaf exceeds 27 (vs. 27 or fewer in subsp. acuminata), the length of leaf serrations of the largest leaf exceeds 3 mm (vs. 1.0–2.9 mm), and the width of infructescence attains to 7–12 cm (vs. 3.2–8.7 cm).

Table 4.
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Measurements for nine morphological traits of H. acuminata ssp. acuminata , ssp. australis , ssp. yakushimensis, and H. minamitanii.

H. a. ssp. yakushimensis H. a. ssp. acuminata H. a. ssp. australis H. minamitanii
Leaf length 12.5±1.5 (10.2–14.4) cm 10.4±2.8 (10.4–15.6) cm 13.4±1.6 (10.1–15.4) cm 12.6±1.1 (12–14) cm
Leaf width 6.2±0.9 (4.6–7.5) cm 4.3±1.1 (2.8–6.2) cm 7.7±1.4 (5.4–9.7) cm 6.2±0.9 (5.0–6.9) cm
Petiole length 2.8±1.0 (1.2–5.3) cm 2.1±0.8 (0.9–3.5) cm 2.8±1.3 (0.9–5.0) cm 3.0±1.2 (2.2–4.5) cm
Leaf apex length 1.7±0.5 (1.0–2.4) cm 1.8±0.7 (0.9–2.9) cm 1.7±0.5 (0.7–2.5) cm 1.5±0.5 (0.8–1.9) cm
Leaf teeth length 2.8±1.1 (1.0–5.0) mm 1.8±0.6 (1.0–2.9) mm 3.0±1.0 (1.0–5.0) mm 3.1± 0.8 (2.2–4.2) cm
No of teeth 28.5±7.1 (15–42) 21.2±6.1 (9–27) 28.5±7.1 (15–42) 28.8±4.9 (23–35)
Corymb length 4.3±1.5 (2.0–7.0) cm 4.1±1.5 (1.9–6.3) cm 5.8±2.2 (3.0–9.2) cm 5.1±0.9 (4.5–6.3) cm
Corymb width 6.7±2.7 (3.9–11.8) cm 5.5±1.9 (3.2–8.7) cm 8.9±2.5 (6.2–12.8) cm 7.9±0.9 (7.2–9.2) cm
Capsule length 2.4±0.2 (2.2–2.7) mm 3.9±0.6 (3.2–5.1) mm 4.3±0.4 (3.8–4.9) mm 4.6±1.0 (3.9–5.3) mm
Figure 5. 

Hydrangea acuminata subsp. yakushimensis Yahara & Tagane A a tree growing on cliff along stream B a fruiting twig of the specimen JPN1799 (holotype) C lower leaf surface of the specimen JPN1799. Scale bars: 20 cm (A); 10 cm (B); 2 cm (C).

Phylogenetically, H. acuminata subsp. yakushimensis is sister to subsp. australis. Morphologically, H. acuminata subsp. yakushimensis is similar to subsp. australis in having leaves larger than subsp. acuminata but distinguished with leaves glabrous adaxially except veins (vs. sparsely hairy in subsp. australis; Table 5) and capsule less than 3 mm long (Table 4).

Table 5.
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Morphological comparison between H. acuminata ssp. acuminata , ssp. australis , ssp. yakushimensis, and H. minamitanii.

H. a. ssp. yakushimensis H. a. ssp. acuminata H. a. ssp. australis H. minamitanii
Upper surface of lamina glabrous sparsely hairy sparsely hairy glabrous
Upper surface of veins hairy hairy hairy hairy
Lower surface of lamina glabrous sparsely hairy densely curled hairy glabrous
Lower survace of veins glabrous or glabrescent sparsely hairy densely curled hairy glabrous or glabrescent
Axils of lateral veins hairs densely tufted hairs not densely tufted hairs not densely tufted hairs densely tufted
Petiole glabrous hairy densely hairy glabrous
Young shoot glabrous hairy densely hairy glabrous
Calyx of showy flower blue blue blue pink or white
Figure 6. 

Fruits of Hydrangea acuminata subsp. yakushimensis Yahara & Tagane A and subsp. acuminata B Specimen: JPN1799 (holotype) A JPN2063 B. Scale bars: 3 mm.

Discussion

The discovery of H. acuminata subsp. yakushimensis is surprising because Yakushima is a well-botanized island, and H. acuminata subsp. yakushimensis has conspicuous, blue-colored flowers. This discovery illustrates that botanical surveys in the mountain-top area of Yakushima still remain insufficient, most likely because of its steep topography. In fact, our recent surveys resulted in the discovery of not only H. acuminata subsp. yakushimensis but also an additional new taxon of Stellaria (Caryophyllaceae) (Yahara et al. 2021b). Further field surveys including researchers who have more experience climbing mountains and steep cliffs could result in the discovery of even more undescribed taxa from the mountain-top area of Yakushima.

Using RAPD and the sequences of rbcL and matK, Uemachi et al. (2014) showed that H. serrata var. serrata s. lat. diverged to western and eastern groups, corresponding to H. acuminata and H. serrata var. serrata s. str., respectively. However, Uemachi et al. (2014) did not examine H. acuminata subsp. australis, H. acuminata subsp. yakushimensis, and H. minamitanii. The MIG-seq tree (Fig. 2) revealed that H. minamitanii is sister to the clade including H. acuminata, H. serrata, and H. macrophylla. Hydrangea minamitanii is differentiated from the other species of sect. Macrophyllae with FST values from 0.340 to 0.657, and this difference was equivalent to the FST variation between the species of sect. Chinenses from 0.395 (H. kawagoeana vs. H. grosseserrata) to 0.632 (H. grosseserrata vs. H. scandens). These findings support the treatment of H. minamitanii as a distinct species.

In contrast, the FST between H. acuminata subsp. acuminata and subsp. australis (0.251) is lower than the above values (0.340 to 0.657) observed between the species, supporting the treatment as two subspecies. Similarly, the FST between H. acuminata subsp. acuminata and subsp. yakushimensis was 0.316, which is considered to be at the subspecies level, and the FST between subsp. australis and subsp. yakushimensis (0.437) was slightly higher. Differences between H. acuminata subsp. acuminata and subsp. australis are smaller, not only genetically, but also morphologically: JPN0908 collected at 1700-m elevation on Mt. Karakuni was identified as subsp. australis in the MIG-seq tree, but is morphologically very similar to subsp. acuminata, suggesting hybridization or intergradation between subsp. acuminata and subsp. australis.

In the MIG-seq tree (Fig. 2), H. acuminata subsp. yakushimensis was sister to H. acuminata subsp. australis distributed in the southern part of Kyushu mainland (Kagoshima Pref. and the southern part of Miyazaki Pref.). There are other cases where the endemic plants of Yakushima have related taxa in southern Kyushu. For example, Asarum (Araceae, Okuyama et al. 2020), Mitella (Saxifragaceae, Okuyama et al. 2005), and Rhododendron (Ericaceae, Minamitani et al. 2018) have all been reported as showing this pattern. The sister relationship between H. acuminata subsp. yakushimensis and subsp. australis provided another case which supported the phytogeographical similarity between the endemic flora of Yakushima and the flora of the southern Kyushu mainland.

The MIG-seq tree (Fig. 2) showed that the Shikoku lineage was distinct from a clade including three subspecies of H. acuminata distributed in Kyushu. This finding agrees with the results of Uemachi et al. (2014), showing that the samples from Shikoku were distinct for both rbcL and matK sequences from other “western subgroups” corresponding to H. acuminata. We did not find differences in rbcL sequences between the Shikoku lineage and other samples of H. acuminata, which is most likely because we determined shorter sequences of rbcL than did Uemachi et al. (2014): 449 bp. vs 1257 bp. The MIG-seq tree and the results described by Uemachi et al. (2014) suggest that the Shikoku lineage may be treated as a fourth subspecies of H. acuminata. However, further morphological and molecular phylogenetic studies, using more samples from Shikoku, are needed to conclude the taxonomic treatment of the Shikoku lineage.

The MIG-seq tree (Fig. 2) also showed that H. serrata was not monophyletic if H. macrophylla was separated as a species; the sample of H. serrata var. angulata was sister to H. macrophylla, and the sample of H. serrata var. serrata was basal to this sister pair. This result suggests that H. serrata includes multiple species even after H. acuminata and H. minamitanii are separated. Hydrangea serrata s. lat. is widely distributed from Kyushu to Hokkaido, the northern-most island of Japan. Our samples were limited to the area of western Japan on the Pacific side and did not include H. serrata var. yezoensis. Further studies of populations in central and northern Japan, including more samples of H. serrata var. angulata , var. serrata , and var. yezoensis, are needed to revise the taxonomy of the complex that has been treated as H. serrata s. lat.

It is notable that H. luteovenosa 1 and H. luteovenosa 2 were not sister to each other in both MIG-seq and ITS trees. In the MIG-seq tree which has a higher resolution than the ITS tree, H. luteovenosa 2 (JPN1982 collected from Mt. Osuzu, Miyazaki Pref.) was basal to a clade including H. scandens, H. luteovenosa 1 (JPN0378 collected from Mt. Ihara, Fukuoka Pref.), H. kawagoeana, and H. grosseserrata. It is likely that H. luteovenosa contains two cryptic species. To test this possibility, further studies with more samples of H. luteovenosa are needed.

This study demonstrated the usefulness of MIG-seq to obtain finely resolved phylogenetic trees for closely related species and infraspecific taxa in taxonomically complicated groups such as Hydrangea. Compared with the ITS and cpDNA trees, where only a few branches were supported by bootstrap values larger than 90%, most branches in the MIG-seq tree were supported by bootstrap values larger than 90%. In the ITS tree, the monophyly of H. acuminata subsp. yakushimensis was supported by the 97% bootstrap value, but the monophyly of H. acuminata subsp. acuminata and subsp. yakushimensis was ambiguous; the cluster of H. acuminata subsp. acuminata and H. minamitanii with the bootstap value 88% was weakly consistent with the MIG-seq tree topology. The resolution of the MIG-seq tree is even higher than that of the RAPD tree for the H. serrata complex obtained by Uemachi et al. (2014). Other recent studies using MIG-seq on Hosta (Yahara et al. 2021a) and Stellaria (Yahara et al. 2021b) have also demonstrated its usefulness in resolving taxonomic complexity and describing new taxa. As this method is more applicable to a small number of poor-quality samples than RAD-seq (Binh et al. 2018; Strijk et al. 2020; Zhang et al. 2020), it is expected to be used for taxonomic studies of many groups for which reliable phylogenetic relationships could not be reconstructed by conventional molecular phylogenetic methods.

1 Calyces of marginal showy flowers, petals of fertile flowers, and stamens always pink or white 2
– Calyces of marginal showy flowers, petals of fertile flowers, and stamens light blue when flowering 3
2 Leaves glabrous adaxially except veins and glabrous abaxially except for tufted hairs at axils of lateral veins. Distributed in Kyushu H. minamitanii
– Leaves more or less hairy adaxially and abaxially. Distributed in Honshu H. serrata
3 Leaves glabrous adaxially except veins. Capsules 2.7 mm or shorter H. acuminata subsp. yakushimensis
– Leaves hairy adaxially. Capsules 3.2 mm or longer 4
4 Leaves usually sparsely hairy abaxially, hair not curled. Leaf width less than 6.2 cm H. acuminata subsp. acuminata
– Leaves usually densely hairy abaxially, hair curled. Leaf width often 6.2 cm or larger H. acuminata subsp. australis

Taxonomy

Hydrangea acuminata

Hydrangea acuminata Siebold & Zucc., Fl. Jap. 1: 110, t. 56, 57-I (1839); Ohba & Akiyama in Bull. Natl. Mus. Nat. Sci., Ser. B, 39: 178 (2013).

Type

Japan, Higo Province, Kyushu (L0043373, the lectotype designated by Ohba and Akiyama (2013)).

Hydrangea acuminata subsp. acuminata

Hortensia serrata var. acuminata (Siebold & Zucc.) H. Ohba & S. Akiyama, J. Jap. Bot. 91: 347 (2016).

Hydrangea macrophylla (Thunb.) Ser. var. acuminata (Siebold & Zucc.) Makino, Ill. Fl. Nippon: 484, f. 1451 (1940), nom. tant.

Japanese name

Sawa-ajisai, Nishino-yama-ajisai.

Distribution and habitats

Hydrangea acuminata subsp. acuminata is widely distributed on the main island of Kyushu, and usually grows on the soil near streams and often on cliffs, and sometimes in disturbed habitats.

Note

Ohba and Akiyama (2016) treated this species as a variety of Hortensia serrata. However, our phylogenetic analysis described below supports the treatment of it as a distinct species.

Hydrangea acuminata subsp. yakushimensis Yahara & Tagane, subsp. nov.

urn:lsid:ipni.org:names:77248597-1
Figures 4, 5

Diagnosis

Hydrangea acuminata subsp. yakushimensis is different from subsp. acuminata in that it has smaller capsules, 2.2–2.7 mm long with calyx tube 1.2–1.4 mm long and projected apical part including persistent style 1.0–1.3 mm (vs. capsules 3.2–5.1 mm long with calyx tube 1.6–3.4 mm and projected apical part including persistent style 1.5–2.0 mm), a larger infructescence attaining to 7 × 12 cm (vs. attaining to 6.3 × 8.7 cm), leaves glabrous adaxially except veins (vs. hairy) and glabrous or only slightly hairy abaxially except for tufted hairs at axils of lateral veins (vs. hairy overall on abaxial surface).

Type

Japan. Kagoshima Pref.:Yakushima Migitani, on cliff along stream, 30.34255555°N, 130.48100000°E, 1520 m elevation, 9 September 2020, with fruits, K. Fuse JPN1799 (holotype: KYO!).

Description

Shrubs 1–1.5 m tall. First year’s twigs green when fresh, with dark purple lenticels, glabrous, terete. Old twigs pale brown; bark not peeled off. Leaves opposite; petioles purplish green, 1.7–3 cm long, glabrous; leaf blade adaxially green, abaxially light green when fresh, pale green when dried, elliptic, 9–12 × 4.6–6.4 cm, papery, adaxially glabrous except veins which are covered with minute hairs, abaxially glabrous or only sparsely hairy except for tufted hairs at axils of lateral veins, secondary veins 6–9 on each side of midvein, adaxially slightly sunken, abaxially slightly elevated, base broadly cuneate, apex long acuminate, margin serrate, teeth 2–3 mm high, 13–31 along each side of the margin. Inflorescences corymbose cymes, 2–7 cm long, 4–12 cm in diam., densely pubescent, apex flat to slightly arcuate, 3–5-branched; the longest internode of each branch 1.5–2.5 cm long, densely pubescent; infructescence attaining to 7 × 12 cm. Marginal showy flowers light blue, on pedicel 1–2 cm long; sepals 3 to 5, rhomboid-elliptic, 0.8–1.4 × 0.5–1.1 cm, glabrous, apex obtuse, base rounded to cuneate, margin entire. Fertile flowers protandrous, light blue. Male-stage flowers on pedicel 1–1.8 mm long; calyx tube funnel-shaped, ca. 1 mm long, 0.8 mm in diam., lobes 5, triangular, 0.5 × 0.4 mm, apex acute; petals 5, light blue, elliptic, 2–2.2 × 1 mm, glabrous, apex acute; stamens 10, light blue, subequal, filaments 1.5–3 mm long, glabrous, anthers white, globular, 0.6 mm in diam.; ovary nearly 1/2 superior, style 3, connate at base, slightly spreading, dark blue, ca. 0.7 mm long, stigma flat. In female-stage flowers, petals and stamens fallen off; ovary nearly 1/2 superior; calyx tube light blue, ca. 1 mm long; style darker blue, spreading, ca. 1 mm long; capsules 2.2–2.7 mm long; calyx tube subglobose, 1.2–1.4 mm long, 1.5–2 mm in diam., projected apical part including persistent styles 1.5 mm long. Seeds light brown, elliptic, 0.8 × 0.5 mm, not winged.

Japanese name

Yakushima-ruri-ajisai.

Phenology

Flowers were collected in July and August, and fruits were collected in September.

Distribution and habitat

Yakushima (Yaku Island), Japan (endemic). The distribution of H. acuminata subsp. yakushimensis is restricted to cliffs along streams at Yakushima. It mainly grows in the mountain-top area from 1520 to 1750 m, but one population occurs at an elevation of 575 m, along the Miyanoura River.

Etymology

The specific epithet is derived from the type locality, Yakushima.

IUCN Conservation status

Endangered (EN) based on criterion D; the population size is above 50, but less than 250.

Additional specimens examined

Japan. Kagoshima Pref., Yakushima: Mt. Nagata, on cliff, 30.343799°N, 130.492056°E, 1750 m elevation, 2 August 2005, with flowers, T. Yahara, S. Tagane, K. Fuse & T. Saito 0791 (FU!); Kamisamano-kubo, on cliff, 30.343799°N, 130.492056°E, 1750 m elevation, 2 August 2005, with flowers, T. Yahara, S. Tagane, K. Fuse & T. Saito 0792 (FU!); ditto, with flowers, T. Yahara, S. Tagane, K. Fuse, T. Saito 0793 (FU!); Nemachino-kubo, on cliff, 30.345465°N, 130.49468230°E, 1740 m elevation, 12 July 2006, sterile, S. Tagane & K. Fuse 1065 (FU!); Migitani, on cliff along stream, 30.34255555°N, 130.48100000°E, 1520 m elevation, 13 July 2006, with flowers, S. Tagane & K. Fuse 1103, 1104, 1105 (FU!); Sensuikyo, 30.372031°N, 130.504266°E, 575 m elevation, 31 August 2020, sterile, K. Fuse JPN1708 (FU!).

Hydrangea acuminata subsp. australis (T. Yamaz.) Yahara, stat. nov.

urn:lsid:ipni.org:names:77248598-1

Hydrangea serrata var. australis T. Yamaz., J. Jap. Bot. 76: 175 (2001). Type. Japan. Kagoshima Pref., Mt. Takakuma, 11 August 1942, T. Yamazaki s.n. (TI).

Hortensia serrata var. australis (T. Yamaz.) H. Ohba & S. Akiyama in Ohashi et al., Wild Fl. Jap. rev. ed. 4: 166 (2017), comb. nud.

Japanese name

Nangoku-yama-ajisai.

Distribution and habitats

Hydrangea acuminata subsp. australis is widely distributed at lower elevations in the Kagoshima Prefecture and the southern part of the Miyazaki Prefecture of the Kyushu Island and usually grows in disturbed places along the margins of evergreen forests or Cryptomeria plantations. JPN0908 was collected on a volcanic cliff at 1700 m elevation on Mt. Karakuni and was identified as subsp. australis in the MIG-seq tree (Fig. 2).

Note

Hydrangea acuminata subsp. australis is distinguished from subsp. acuminata mainly by its larger and wider leaves often exceeding 6.2 cm wide (vs. not exceeding 6.2 cm), having more serrations along margin (22–43 vs. 9–27) and dense curled hair on the lower surface of lamina. However, JPN0908, was identified as subsp. australis in the MIG-seq tree, which is morphologically similar to subsp. acuminata in having smaller leaves, fewer serrations, and sparser pubescence on the lower surface of the leaf. This specimen might be of hybrid origin between subsp. australis and subsp. acuminata.

Representative specimens examined

Japan. Kagoshima Pref.: Kagoshima City, 22 July 2002, with flowers, K. Maruno s.n. (KAG 083840!); Shibushi City, 4 June 2002, with fruits, K. Maruno s.n. (KAG 083882!); Aira City, 11 July 2004, K. Maruno s.n. (KAG 086731!); Kimotsuki Town, 300 m elevation, 20 July 1986, with fruits, S. Hatusima 41199 (KAG 161312!); KHydrangeahima City, 450 m elevation, 22 November 1986, with fruits, S. Hatusima 41920 (KAG 161315!); Mt. Nokubi, 700 m elevation, 12 July 1987, with flowers, S. Hatusima 42447 (KAG 161327!).

Hydrangea minamitanii (H. Ohba) Yahara, stat. nov.

urn:lsid:ipni.org:names:77248599-1

Hydrangea serrata (Thunb.) Ser. var. minamitanii H. Ohba in J. Jap. Bot. 64: 199 (1989); Ohba & Akiyama, Bull. Natl. Mus. Nat. Sci., Ser. B, 39: 179 (2013). Type. Japan. Miyazaki Pref., Saito City, T. Minamitani 26304 (TI).

Hortensia serrata var. minamitanii (H. Ohba) H. Ohba & S. Akiyama, J. Jap. Bot. 91: 347 (2016).

Japanese name

Hyuga-ajisai.

Note

Hydrangea minamitanii and H. acuminata ssp. acuminata often grow close, within 100 m of each other, but the former grows on wet cliffs along streams, and the latter grows on soil along forest margin. Hydrangea minamitanii is distinct from H. acuminata in having leaves glabrous abaxially except tufted hairs at axils of lateral veins, glabrous petioles, and glabrous young shoots (Table 5). No intermediates have been discovered in localities where two species grow. Hydrangea minamitanii is similar to H. acuminata subsp. yakushimensis in growing on cliffs along streams and having leaves glabrous on both surfaces except veins and tufted hairs at axils of lateral veins, but they are distinguished by their capsule size (3.9–5.3 mm or longer in H. minamitanii vs. 2.2–2.7 mm in H. acuminata subsp. yakushimensis) and flower colors (pink or white flowers vs. blue flowers). Whereas H. acuminata subsp. yakushimensis is endemic to the Yakushima island, H. minamitanii is restricted to the mountains of central and eastern Kyushu, mainly in the Miyazaki Prefecture.

Additional specimens examined

Japan. Miyazaki Pref.: Mt. Osuzu, 500 m elevation, 20 October 1960, with fruits, S. Sako 3285 (KAG 161375!); ditto, 500 m elevation, 28 July 1971, with flowers, S. Hatusima & S. Sako 32643 (KAG 161376!); ditto, 11 July 1976, with flowers, T. Minamitani 22630 (KAG 161378!); Aya Town, 73 m elevation, 24 October 2019, with fruits, S. Tagane 1200 (KAG 128616!).

Acknowledgements

We thank Toshihiro Saito for his help with our fieldwork in Yakushima. Specimens of H. acuminata subsp. yakushimensis were collected in the protected area of the Yakushima National Park with permission from the Ministry of Environment, Yakushima office of Forestry Agency, and Kagoshima office of Agency for Cultural Affairs. Specimens of H. minamitanii, H. luteovenosa, and H. scandens were collected in the protected area of Osuzu Prefectural Natural Park with permission from the Miyazaki Prefecture and Forestry Agency. In addition, specimens of other species were collected in the following national parks with permission from the Ministry of Environment and the prefectures for both Kokuritu and Kokutei Park and in the national forests with permission from the local offices of Forestry Agency: Mt. Shiraiwa of Kyushu-chuo-sanchi National (Kokutei) Park, Mt. Oyaji of Sobo-katamuki National (Kokutei) Park, Mt. Karakuni of KHydrangeahima National (Kokuritsu) Park, Osugi-dani of Yoshino-kumano National (Kokuritsu) Park, and Mt. Amagi of Fuji-hakone National (Kokuritu) Park. We thank the Ministry of Environment’s Rare Species Conservation Promotion Office for their help in obtaining collection permits. We would like to thank Editage (www.editage.com) for English language editing. This study was supported by the Environment Research and Technology Development Fund (JPMEERF20204001) of the Ministry of the Environment, Japan, and partly by JSPS KAKENHI grant number 21K06307.

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