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Research Article
Morphological and molecular evidence reveals three new species of Lithocarpus (Fagaceae) from Bidoup-Nui Ba National Park, Vietnam
expand article infoNguyen Van Ngoc‡, Hoang Thi Binh‡, Ai Nagahama§, Shuichiro Tagane|, Hironori Toyama, Ayumi Matsuo#, Yoshihisa Suyama#, Tetsukazu Yahara¤
‡ Dalat University, Dalat, Vietnam
§ Kyushu University, Fukuoka, Japan
| Kagoshima University, Kagoshima, Japan
¶ Biodiversity Division, National Institute for Environmental Studies, Ibaraki, Japan
# Tohoku University, Miyagi, Japan
¤ Kyushu Open University, Fukuoka, Japan

Corresponding author: Nguyen Van Ngoc ( mr.ngocnv@gmail.com )

Academic editor: Norbert Holstein
© 2021 Nguyen Van Ngoc, Hoang Thi Binh, Ai Nagahama, Shuichiro Tagane, Hironori Toyama, Ayumi Matsuo, Yoshihisa Suyama, Tetsukazu Yahara.
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: Ngoc NV, Binh HT, Nagahama A, Tagane S, Toyama H, Matsuo A, Suyama Y, Yahara T (2021) Morphological and molecular evidence reveals three new species of Lithocarpus (Fagaceae) from Bidoup-Nui Ba National Park, Vietnam. PhytoKeys 186: 73-92. https://doi.org/10.3897/phytokeys.186.69878
Open Access

Abstract

Three new species, Lithocarpus bidoupensis Ngoc & Tagane, L. congtroiensis Ngoc & Yahara, and L. hongiaoensis Ngoc & Binh are described from Bidoup-Nui Ba National Park, Central Highland of Vietnam. Morphological analyses and Maximum likelihood tree based on genome-wide SNPs support the distinction of those species from the previously known taxa in the region. The three new species are considered to be endemic to the Bidoup-Nui Ba National Park and the preliminary conservation status for each species is evaluated as Critically Endangered.

Keywords

Fagales, Lam Dong Province, MIG-seq, phylogeny, taxonomy

Introduction

Fagaceae are highly diversified in Vietnam and 216 species of 6 genera have been reported in various forest types, from dry evergreen forest at lowlands to montane evergreen forest in the higher elevation (Ho 2003; Ban 2005; Ngoc et al. 2016). Recently, 10 species of Fagaceae were newly described from Vietnam: Castanopsis grandicicatricata N.H.Xia & D.H.Vuong, C. multiporcata N.H.Xia & D.H.Vuong (Vuong and Xia 2014), Lithocarpus dahuoaiensis Ngoc & L.V.Dung (Ngoc et al. 2016), L. vuquangensis Ngoc & V.H.Nguyen (Ngoc et al. 2018), Quercus baolamensis H.T.Binh & Ngoc (Binh et al. 2018a), Q. bidoupensis H.T.Binh & Ngoc (Binh et al. 2018a), Q. honbaensis H.T.Binh, Tagane & Yahara (Binh et al. 2018a), Q. trungkhanhensis H.T.Binh & Ngoc (Binh et al. 2018b), Q. xuanlienensis H.T.Binh, Ngoc & T.N.Bon (Binh et al. 2018c), and Q. ngochoaensis Binh & Son (Binh et al. 2021).

Lithocarpus Blume is the largest genus of the family Fagaceae in Vietnam, including 119 species and two varieties, among which 44 species are endemic (Ho 2003; Ban 2005; Linh et al. 2013; Ngoc et al. 2016, 2018). The previous taxonomic treatments of Lithocarpus in Vietnam were mostly based on Camus’ studies using the specimens collected by French botanists, the results of which were documented in the part of Flore générale de l’Indo-Chine (Hickel and Camus 1930) or Chênes Atlas (Camus 1948). All these early studies relied only on morphological features to identify and construct the keys to species level, so the taxonomic circumscriptions were sometimes inaccurate, leading to continued uncertainty in the taxonomic status and relationship among species.

The phylogenetic approach has become a widespread and efficient way to identify and delimit species, but there is only one study for Lithocarpus in Vietnam (Ngoc et al. 2018). The MIG-seq is a PCR-based method used to identify large numbers of genetic markers throughout the genome (Suyama and Matsuki 2015) that is highly applicable for use in phylogenetic studies (Binh et al. 2018a; Okabe et al. 2021). Recently, a growing number of taxonomic studies of Lithocarpus have yielded new species (Ngoc et al. 2016, 2018), but we often find Lithocarpus material that is difficult to identify to species level. Here we applied MIG-seq for phylogenetic reconstruction to accurately assess the diversity and taxonomy of Lithocarpus species.

Bidoup-Nui Ba National Park (Fig. 1) is in the core zone of UNESCO Langbiang Biosphere Reserve, which is in Lam Dong Province in the central highland of Vietnam. The national park, with the area of 70,038 ha covering almost the entire Langbiang Plateau, harbors 1933 species of vascular plants (Bidoup-Nui Ba National Park 2021) including 62 threatened species (Ban et al. 2007; IUCN 2012), and 42 endemic species (Tagane et al. 2017, 2020; Bidoup-Nui Ba National Park 2021). For Fagaceae, 25 species of Lithocarpus, nine species of Castanopsis, eleven species of Quercus, and one species of Trigonobalanus have been recorded from Bidoup-Nui Ba National Park (Dung 2005; Ngoc et al. 2016; Binh et al. 2018a).

Figure 1. 

Type locality of the new species (Black dots): L1: L. bidoupensis, L2: L. congtroiensis, and L3: L. hongiaoensis.

During our floristic research in Bidoup-Nui Ba National Park from 2015 to present, we found some individuals of the genus Lithocarpus that could not be identified to species level. We here describe them as Lithocarpus bidoupensis Ngoc & Tagane, sp. nov., Lithocarpus hongiaoensis Ngoc & Binh, sp. nov., and Lithocarpus congtroiensis Ngoc & Yahara, sp. nov., based on comparisons of morphology with related species and provide molecular phylogenetic evidence using the MIG-seq method (Suyama and Matsuki 2015).

Materials and methods

Taxon sampling

In the present study, we conducted botanical inventories in Bidoup-Nui Ba National Park and the other protected areas in Vietnam and collected a total of 63 samples consisting of 23 species including sixteen samples of three unknown species. Five samples of Lithocarpus balansae (Drake) A.Camus, which is morphologically distinct from the other species of the genus, were included as an outgroup for the phylogenetic analysis. Localities and voucher specimens of these materials are listed in Table 1.

Table 1.
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List of vouchers specimen that were used in this study.

Species Vouchers Localities
L. annamitorus Nguyen et al. V3214 Bidoup-Nui Ba NP
L. bidoupensis Tagane et al. V4320, Yahara et al. V7850, V8190, V8417, V9940 (DLU, FU, KAG); Ngoc et al. NAF122, NAF185 (DLU) Bidoup-Nui Ba NP
L. blaoensis Nguyen et al. V3176, V3176A (DLU, FU) Bidoup-Nui Ba NP
L. coalitus Tagane et al. V4191, Yahara et al. V10140 (DLU, FU), Bidoup-Nui Ba NP
L. congtroiensis Ngoc et al. NAF200 (DLU); Nguyen et al. V3205, Tagane et al. V9102, Tagane et al. V9470, Tagane et al. V9492, Yahara et al. V9555 (DLU, FU) Bidoup-Nui Ba NP
L. encleisocarpus Tagane et al. V1627; Nguyen et al. V3263 (DLU, FU) Bidoup-Nui Ba NP
L. dahuoaiensis Nguyen et al. V3194 (DLU, FU) Bidoup-Nui Ba NP
Ngoc et al. V5404, V5404A (DLU, FU) Dong Nai NR
L. dalatensis Tagane et al. V9106 (DLU, FU, KAG) Bidoup-Nui Ba NP
L. dealbatus Ngoc et al. V3258, Tagane et al. V4357 (DLU, FU) Bidoup-Nui Ba NP
L. hancei Ngoc et al. V5111, V4918, SP008 (DLU, FU) Hoang Lien NP
L. honbaensis Tagane et al. V0003, V207; Ngoc et al. V5540 (DLU, FU) Hon Ba NR
L. hongiaoensis Nguyen et al. V3235 (DLU, FU); Ngoc et al. NAF123, NAF192 (DLU) Bidoup-Nui Ba NP
L. laoticus Nguyen et al. V3193 (DLU, FU) Bidoup-Nui Ba NP
L. lemeeanus Tagane et al. V4523 (DLU, FU) Bidoup-Nui Ba NP
L. licentii Tagane et al. V6261, V6400 (DLU, FU) Ngoc Linh NR
L. sp1 Nguyen et al. V3171, V3171A (DLU, FU) Bidoup-Nui Ba NP
L. parvulus Yahara et al. V8636, V9720, V10068, V10077, V10164 (DLU, FU, KAG) Bidoup-Nui Ba NP
L. pseudomagneinii Nguyen et al. V3183, V3223 (DLU, FU) Bidoup-Nui Ba NP
L. syncarpus Nguyen et al. V3188, V3188A, V3246, V3250 (DLU, FU) Bidoup-Nui Ba NP
L. vinhensis Nguyen et al. V3591, V3787 (DLU, FU) Vu Quang NP
L. vuquangensis Yahara et al. V5743, V5938 (DLU, FU) Vu Quang NP
L. xylocarpus Tagane et al. V4337; Ngoc et al. V8464 (DLU, FU) Bidoup-Nui Ba NP
L. balansae (outgroup) Yahara et al. V2938 (DLU, FU) Bach Ma NP
Nguyen et al. V3177, Ngoc et al. V8467 (DLU, FU) Bidoup NP
Nguyen et al. V5447, V5512 (DLU, FU) Pu Mat NP

NP = National Park; NR = Nature Reserve.

Morphological analysis

We compared morphological traits of three unknown species with those of related species using taxonomic literature (Camus 1931, 1938, 1942, 1943, 1945, 1948; Huang et al. 1999; Ho 2003; Ban 2005; Phengklai 2008), specimens kept in the herbaria ANDA, BKF, DLU, HN, KAG, KYO, P, and VNM, and digitized plant specimen images available on the web of JSTOR Global Plants (https://plants.jstor.org/) and Chinese Virtual Herbarium (http://www.cvh.org.cn/).

The ImageJ software (Schneider et al. 2012) was used to measure the following characters of the new species and related species based on images of type specimens: length, width, aspect ratio and circularity of leaf blade, petiole length, and size of cupules. Aspect ratio and circularity are defined as length/width of leaf blade and 4π × (area/perimeter squared), respectively. Analysis of variance (ANOVA) and post hoc Tukey’s honestly significant difference test (Tukey’s HSD) (Tukey 1953) were applied to reveal the mean difference among species. All statistical analyses were performed in R version 4.0.5 (R Core Team 2021) with R-Sutido ver. 1.4.1106 (R-Studio Team 2021).

DNA extraction and sequencing

Leaf pieces were dried using silica-gel in the field, and DNA was isolated with the CTAB method (Doyle and Doyle 1987) with minor modifications described in Toyama et al. (2015). The extracted DNA was diluted to 10 ng/µl and used as templates to amplify thousands of short sequences (loci) from a wide variety of genomes with a standard PCR protocol according to Suyama and Matsuki (2015). MIG-seq library was constructed as described in Suyama and Matsuki (2015) with a minor update by using dual-indexed primers (Suyama et al. 2021). The 1st PCR, multiple non-repetitive regions from various inter-simple-sequence repeats (ISSRs) were amplified from genomic DNA by multiplexed PCR with tailed ISSR forward and reverse primers sets. The first PCR products were diluted and used as the templates for the 2nd PCR with dual indexed primers sets. Then, 3 µl of each 2nd PCR product was pooled in equimolar concentrations as single mixture library. The mixture was then purified and the size range of 350–800 bp were isolated by a Pippin Prep DNA size selection system (Sage Science, Beverly, MA, USA). Quantitative PCR was performed to measure final concentration of size-selected library with approximately 10 pM and then used for sequencing on an Illumina MiSeq Sequencer (Illumina, San Diego, CA, USA), using a MiSeq Reagent Kit v3 (150 cycle, Illumina).

Phylogenetic analysis

A total of 50 samples of 22 species of Lithocarpus including samples of unknown species were sequenced (except NAF122, NAF123, NAF185, NAF192, V3205, V9470, V9492, V9555), of which five samples of L. balansae were used as an outgroup. The low-quality reads and primer sequences were eliminated from raw data by using the trimmomatic software version 0.40 (Bolger et al. 2014). The quality-filtered sequence data were demultiplexed and filtered through the software Stacks v1.46 (Catchen et al. 2011; Catchen et al. 2013) following the parameters set as described by Takata et al. (2019) with minor modifications: in the U-stacks, the option settings of ‘maximum distance allowed between stacks (M)’ = 4,‘maximum distance allowed to align secondary reads to primary stacks (N)’ = 4; in the population program, the minimum percentage of individuals required to process a locus across all data (r) was set at 10% and the minimum minor allele frequency required to process a nucleotide site at a locus (min_maf) = 0.005, the maximum observed heterozygosity required to process a nucleotide site at a locus (max_obs_het) = 0.6.

Phylogenetic analyses were conducted using maximum likelihood method on SNPs data set. The model of sequence evolution was set to GTR+G as selected by jMrModeltest 2.1.10 (Darriba et al. 2012). Maximum Likelihood analyses were implemented using the RAxML ver. 8.2 (Stamatakis 2014). The topological reliability of the maximum likelihood tree was evaluated with 1000 bootstrap replicates.

Results

Morphological analyses

After the morphological examination and taxonomic review in Lithocarpus of Vietnam and its surrounding countries, sixteen unknown samples of Lithocarpus were not assignable to any of the species recognized in the region. Hence, hereafter we named these samples as (1) Lithocarpus bidoupensis Ngoc & Tagane, sp. nov. for Ngoc et al. NAF125, NAF185; Tagane et al. V4320; Yahara et al. V7850, V8190, V8417, and V9940; (2) Lithocarpus congtroiensis Ngoc & Yahara, sp. nov. for Ngoc et al. NAF200; Nguyen et al. V3205, Tagane et al. V9102, V9470, V9492; Yahara et al. V9555; and (3) Lithocarpus hongiaoensis Ngoc & Binh, sp. nov. for Nguyen et al. V3235; Ngoc et al. NAF123, NAF192.

Lithocarpus bidoupensis is most similar to L. blaoensis in having completely entire leaf margin, leaf blade width 3–5 cm, 10–12 pairs of secondary veins, cupules clustered in sets of three, and a concave nut scar, but ANOVA with a post-hoc Tukey HSD test showed significant differences (p < 0.05) between species (Table 2 and Table 3). Specifically, L. bidoupensis significantly differed in much shorter petioles (0.5 ± 0.1 cm long in L. bidoupensis vs. 1.89 ± 0.23 cm long in L. blaoensis), shorter leaf blades (9.74 ± 1.12 cm long vs. 13.66 ± 1.89 cm long), bigger cupules (0.98 ± 0.19 cm high, 2.47 ± 0.2 cm in diam. vs. 0.64 ± 0.06 cm high, 1.58 ± 0.11 cm in diam.) Lithocarpus bidoupensis also has a larger scar of the nut than L. blaoensis (1.4–1.9 cm in diam. vs. 1–1.2 cm in diam.). Lithocarpus bidoupensis is also similar to L. licentii. Both have a completely entire leaf margin, glossy green leaf blades, a leaf blade length of 7.2–11.6 cm, and a cupule height of 0.7–1.4 cm. However, L. bidoupensis has a significantly shorter and wider leaf blade (9.74 ± 1.12 cm × 4.5 ± 0.59 cm in L. bidoupensis vs. 10.76 ± 2.01 cm × 3.46 ± 0.53 cm in L. licentii), lower leaf blade aspect ratio (2.17 ± 0.15 vs. 3.1 ± 0.31), higher leaf blade circularity (0.71 ± 0.03 vs. 0.55 ± 0.05), shorter petioles (0.5 ± 0.1 vs. 0.81 ± 0.14), and bigger cupules size (0.98 ± 0.19 cm cm high, 2.47 ± 0.2 cm in diam. vs. 0.88 ± 0.15 cm cm high, 2.17 ± 0.13 cm in diam.) (Table 2 and Table 3). It also has fewer secondary veins (10–12 pairs in L. bidoupensis vs. 12–15 pairs in L. licentii), much shorter infructescences (8.4–11.5 cm long vs. 15–20 cm long), cupules clustered in sets of three (vs. solitary in L. licentii), a cupule covering less than 1/3 of the nut (vs. covering 1/2–2/3 of the nut), and a concave basal scar (vs. convex).

Table 2.
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The comparisons of mean (X) and standard deviation (SD) value of the leaf blade and cupule size between L. bidoupensis, L. congtroiensis, and L. hongiaoensis with related species.1Derived from type specimens, 2Derived from this study collections, n = number of leaf or cupule were measured in this study.

Parameters (cm) L. bidoupensis1 L. blaoensis1,2 L. licentii1,2
X SD n X SD n X SD n
Leaf blade length 9.74 1.12 23 13.66 1.89 22 10.76 2.01 20
Leaf blade width 4.5 0.59 23 4.41 0.51 22 3.46 0.53 20
Leaf blade aspect ratio 2.17 0.15 23 3.11 0.36 22 3.1 0.31 20
Leaf blade circularity 0.71 0.03 23 0.58 0.05 22 0.55 0.05 20
Petiole length 0.5 0.1 23 1.89 0.23 22 0.81 0.14 20
Cupule high 0.98 0.19 27 0.64 0.06 22 0.88 0.15 16
Cupule diameter 2.47 0.2 27 1.58 0.11 22 2.17 0.13 16
L. congtroiensis1 L. dahuoaiensis1,2 L. honbaensis1,2
X SD n X SD n X SD n
Leaf blade length 14.83 1.6 22 19.4 3.45 22 20.39 3.44 20
Leaf blade width 5.3 0.84 22 8.06 1.48 22 8.84 1.66 20
Leaf blade aspect ratio 2.86 0.34 22 2.41 0.17 22 2.32 0.16 20
Leaf blade circularity 0.59 0.06 22 0.7 0.04 22 0.69 0.04 20
Petiole length 1.42 0.19 22 1.4 0.14 22 2.11 0.36 20
Cupule high 1.10 0.18 21 1.23 0.14 28 - - -
Cupule diameter 2.99 0.28 21 2.24 0.19 28 - - -
L. hongiaoensis1 L. vinhensis1,2 L. vuquangensis1,2
X SD n X SD n X SD n
Leaf blade length 10.81 1.93 29 8.42 2.26 22 7.49 1.32 25
Leaf blade width 3.26 0.6 29 2.97 0.95 22 2.39 0.32 25
Leaf blade aspect ratio 3.33 0.33 29 2.9 0.4 22 3.13 0.33 25
Leaf blade circularity 0.49 0.04 29 0.57 0.07 22 0.5 0.05 25
Petiole length 2.59 0.49 29 0.74 0.12 22 1.3 0.23 25
Cupule high 1.01 0.15 25 0.57 0.05 19 0.86 0.26 18
Cupule diameter 2.06 0.28 25 1.31 0.14 19 1.63 0.18 18

Lithocarpus congtroiensis is morphologically similar to L. dahuoaiensis in having a completely entire leaf margin, blade broadly elliptic, glabrous adaxially, petioles length 1–1.8 cm long, and concave nut scar, but significantly differed in shorter leaf blades (14.83 ± 1.6 cm × 5.3 ± 0.84 cm in L. congtroiensis vs. 19.4 ± 3.45 cm × 8.06 ± 1.48 cm in L. dahuoaiensis), higher leaf blades aspect ratio (2.86 ± 0.34 vs. 2.41 ± 0.17), lower leaf blade circularity (0.59 ± 0.06 vs. 0.7 ± 0.04), bigger cupules (2.99 ± 0.28 vs. 2.24 ± 0.19) (Table 2 and Table 3), and also differs in having more secondary veins (13–15 pairs vs. 11–12 pairs), shorter infructescences (10–15 cm long vs. 20–25 cm long), cupule clustered of three (vs. solitary). Lithocarpus congtroiensis is also similar to L. honbaensis in having completely entire leaf margin, adaxially glabrous lamina, long petioles, cupule clustered of three, and concave nut scar, but significantly differed in smaller leaf blades (14.83 ± 1.6 cm × 5.3 ± 0.84 cm in L. congtroiensis vs. 20.39 ± 3.44 cm × 8.84 ± 1.66 cm in L. honbaensis), higher leaf blade aspect ratio (2.86 ± 0.34 vs. 2.32 ± 0.16), lower leaf blade circularity (0.59 ± 0.06 vs. 0.69 ± 0.04), shorter petioles (1.42 ± 0.19 vs. 2.11 ± 0.36) (Table 2 and Table 3), and also differed in having more secondary veins (13–15 pairs vs. 10–11 pairs), shorter infrutescences (10–15 cm long vs. 15–24 cm long), and shorter acorn-stalk (0.2–0.4 cm long vs. 0.5–0.8 cm long).

Table 3.
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Differences between the species for morphological characters and their levels of significance determined by TukeyHSD Test.

Comparisons Leaf blade length Leaf blade width Aspect ratio Circularity Petiole length Cupule high Cupule diameter
diff. P diff. P diff. P diff. P diff. P diff. P diff. P
L. bidoupensis – L. blaoensis 3.92 0 -0.09 0.85 0.93 0 -0.13 0 1.39 0 -0.35 0 -0.89 0
L. bidoupensis – L. licentii 1.01 0.13 -1.04 <0.001 0.90 0 -0.16 0 0.31 0 -0.1 0.07 -0.29 0
L. congtroiensis – L. dahuoaiensis 4.57 <0.001 2.76 0 -0.45 <0.001 0.11 0 -0.02 0.97 0.13 <0.01 -0.75 0
L. congtroiensis – L. honbaensis 5.56 <0.001 3.53 0 -0.09 0 0.09 0 0.69 0 - - - -
L. hongiaoensis – L. vinhensis -2.39 <0.001 -0.29 0.26 -0.43 <0.001 0.08 <0.001 -1.85 0 -0.44 0 -0.75 0
L. hongiaoensis – L. vuquangensis -3.33 0 -0.58 <0.001 -0.20 0.09 0.02 0.55 -1.29 0 -0.15 0.012 -0.43 <0.001

Note: diff. = mean difference; Bold font indicates statistically significant differences, (-) not available.

Lithocarpus hongiaoensis is most similar to L. vinhensis in having blades narrowly elliptic to lanceolate, completely entire leaf margins, adaxially glabrous and abaxially hairy leaf surface, solitary cupules, and concave nut scar, but ANOVA analysis with post-hoc Tukey HSD test showed that L. hongiaoensis significantly differed from L. vinhensis in having longer leaf blade length (10.81 ± 1.93 cm long in L. hongiaoensis vs. 8.42 ± 2.26 cm long in L. vinhensis), higher leaf blade aspect ratio (3.33 ± 0.33 vs. 2.9 ± 0.4), lower leaf blade circularity (0.49 ± 0.04 vs. 0.57 ± 0.07), much longer petioles (2.59 ± 0.49 vs. 0.74 ± 0.12), bigger cupules (1.01 ± 0.15 cm high, 2.06 ± 0.28 cm in diam. vs. 0.57 ± 0.05 cm high, 1.31 ± 0.14 cm in diam.) (Table 2 and Table 3). Lithocarpus hongiaoensis is morphologically almost identical with L. vuquangensis such as blade narrowly elliptic to lanceolate, completely entire leaf margin, midrib flat or slightly prominent, adaxially glabrous and abaxially hairy leaf surface, solitary cupules, and concave nut scar, but the new species significantly differed in having bigger leaf blades (10.81 ± 1.93 cm × 3.26 ± 0.6 cm in L. hongiaoensis vs. 7.49 ± 1.32 cm × 2.39 ± 0.32 cm in L. vuquangensis), longer petioles (2.59 ± 0.49 vs. 1.3 ± 0.23), bigger cupules (1.01 ± 0.15 cm high, 2.06 ± 0.28 cm in diam. vs. 0.86 ± 0.26 cm high, 1.63 ± 0.18 cm in diam.), and also differs in having longer infrutescences (12.5–16.5 cm long vs. 5–7 cm long), larger nut scar (1.2–1.4 cm in diam. vs. 1–1.1 cm in diam.) (Table 2 and Table 3).

Phylogeny inference

The Maximum likelihood tree based on data set of 4962 genome-wide SNPs strongly supports two sister clades, clade 1 and 2, with 100% bootstrap value (Fig. 2). Clade 1 is divided into two subclades, 1a and 1b, each with 100% bootstrap value. Clade 2 is divided into four subclades of 2a, 2b, 2c, and 2d, each with 100% bootstrap value. The three candidates for new species were included in the clade 2 and supported the monophyly of L. congtroiensis and L. bidoupensis with 100% bootstrap value. The specimen here described as Lithocapus hongiaoensis is sister to two specimens of L. vuquangensis.

Figure 2. 

Maximum likehood tree of three new species (Bold) with their related based on SNPs data from MIG-seq.

Lithocarpus bidoupensis (clade 2a) was clearly separated from the morphologically similar species of L. licentii (Clade 1a) and L. blaoensis (Clade 1b) and is sister to many different species in clade 2b. Lithocarpus congtroiensisis is included in a clade with L. encleisocarpus, L. lemeeanus and L. pseudomagneinii (clade 2.b2) with 97% bootstrap support, but it is well supported as monophyletic The morphologically similar L. honbaensis is in a different clade (clade 2.b1).

Lithocarpus hongiaoensis (clade 2.c1) was clearly separated from the morphologically similar species of L. vinhensis (clade 2.b3) but showed a sister relationship with L. vuquangensis in the clade 2.c1 with a strongly bootstrap value (100%). Those three species share the character of solitary cupules but the distribution of L. hongiaoensis is narrowly restricted and apart from the two species: L. vinhensis and L. vuquangensis are distributed in Nghe An and Ha Tinh Provinces, which are located in the north of the Central Coast of Viet Nam, whereas L. hongiaoensis was found only in Bidoup-Nui Ba National Park, Lam Dong Province about 1000 km further south.

Discussion

The morphological comparison and phylogenetic analysis provided evidence of the validity of three new species. Lithocarpus bidoupensis is most similar to L. blaoensis that occurred in the same locality with L. bidoupensis, and also similar to L. licentii that was collected in Kon Tum Province, which is the type locality of L. licentii. However, the new species is clearly different from both in many morphological traits (Table 2 and Table 3) as well as shown in the phylogenetic results (Fig. 2). The molecular phylogenetic tree strongly supports this disjunction in that the monophyly of L. bidoupensis was supported by 100% bootstrap value, while L. blaoensis and L. licentii were placed in another clade.

Lithocarpus congtroiensis is placed in the same clade with L. encleisocarpus and L. dahuoaiensis (2.b2), but the morphology was clearly distinct. Lithocarpus congtroiensis is distinguished from L. encleisocarpus by its greater number of secondary vein (13–15 pairs in L. congtroiensis vs. 8–10 pairs in L. encleisocarpus), cupules with tiny imbricate scales, enclosing 1/3–1/2 of the nut (vs. the scales forming 5–7 dimly concentric flanges, cupules completely enclosing the nut). Especially, the cupule of L. congtroiensis usually clustered of three, while the cupules of L. encleisocarpus is solitary. The morphological distinctness between L. congtroiensis and L. dahuoaiensis is clearly shown in Tables 2 and 3.

Although we could not collect any specimens of L. honbaensis with mature fruits, the morphological analysis of leaf and cupule characters provided enough evidence to distinguish species from L. congtroiensis. In addition, the molecular phylogenetic tree showed that L. honbaensis has a close genetic relationship with L. lemeeanus and L. pseudomagneinii than L. congtroiensis (clade 2b).

Lithocarpus hongiaoensis is most similar to L. vinhensis and L. vuquangensis, of which the latter showed the sister relationship to L. hongiaoensis in the molecular phylogenetic tree (Fig. 2, clade 2.c1). However, L. hongiaoensis is narrowly endemic to the Hon Giao area of Bidoup-Nui Ba National Park, Lam Dong Province, in the southern part of Vietnam while L. vuquangensis is endemic to Vu Quang National Park of Ha Tinh Province, north-central coast of Vietnam. From 2015 to 2017 we conducted three field trips at Bach Ma National Park, Ba Na Nature Reserve and Ngoc Linh National Park. These protected areas are located between Lam Dong and Ha Tinh Province, but we did not find any individual of Lithocarpus similar to L. hongiaoensis or L. vuquangensis. Also, morphological differences are distinct enough to distinguish them as different species (Table 2 and 3). While L. hongiaoensis was collected in Lam Dong Province, L. vinhensis occurred in Nghe An Province, the province located in the north central coast of Vietnam. The genetic differences between L. hongiaoensis and L. vinhensis were presented in the phylogenetic tree, L. hongiaoensis and L. vuquangensis a sister to each other (2.c1), while the two samples of L. vinhensis formed a clade not closely related to these two species (2.b3).

Taxonomic treatments

Lithocarpus bidoupensis Ngoc & Tagane, sp. nov.

urn:lsid:ipni.org:names:77234073-1
Fig. 3

Type

Vietnam. Lam Dong Province: Bidoup-Nui Ba National Park, in hill evergreen forest dominated by the species of Fagaceae, 1698 m elev., 12°09'52.95"N, 108°32'00.38"E, 24 February 2016, S. Tagane, H. Toyama, H. Nagamasu, A. Naiki, V.S. Dang, N.V. Ngoc, J. Wai V4320 [fr.] (holotype DLU!, isotypes FU!, HN!, KYO!, VNM!).

Diagnosis

Lithocarpus bidoupensis is most similar to L. blaoensis but differs in its shorter leaf blades, petioles and infructescences, and bigger cupules and nuts. The new species is also similar to L. licentii but distinguished from L. licentii by its shorter petioles, fewer secondary veins (10–12 pairs in L. bidoupensis vs. 12–15 pairs in L. licentii), much shorter infructescences, clustered cupules (vs. solitary), cupule covering less than 1/3 of the nut (vs. 1/2–2/3 of the nut), and concave basal scar of the nut (vs. convex) (Table 2).

Figure 3. 

Lithocarpus bidoupensis Ngoc & Tagane A leafy twig B abaxial leaf surface C holotype (Tagane et al. V4320, DLU) D mature fruit E cupule F mature nut G section of mature nut.

Description

Evergreen tree, up to 27 m tall. Branches yellowish green when young, turning greyish brown when old, glabrous, sparsely lenticellate. Terminal and lateral buds ovoid, up to 7 mm long. Leaves alternate; petiole 0.3–0.4 cm long, glabrous; blade elliptic to oblong-elliptic, obovate-elliptic, 6–11.6 × 2.8–5.3 cm, coriaceous, glabrous, glossy green on both surfaces, apex acuminate or attenuate, acumen up to 0.9 cm long, base cuneate, rarely obtuse, margin completely entire, midrib prominent on both surfaces, secondary veins 10–12 pairs, prominent abaxially, at an angle of 55–70° from the midrib, tertiary veins scalariform-reticulate, visible abaxially. Inflorescences not seen. Infructescences a woody spike, 8.4–11.5 cm long. Cupules sessile, usually in cluster of 3, fused at the base each other, depressed obconical or saucer-shaped, 0.71–1.40 cm high, 2.13–2.80 cm in diam., enclosing 1/4–1/3 of the nut, pubescent with short grayish indumentum outside; wall woody, ca. 2 mm thick, with brown triangular scales outside, the scales up to 4 × 4 mm, imbricate, arranged in 3 or 4 interrupted concentric rings. Nut broadly ovoid-conical to depressed ovoid-globose, 1.5–1.6 cm high, 2.1–2.3 cm in diam., glabrous, brown to blackish brown; basal scar slightly concave, 1.4–1.9 cm in diam.

Phenology

Unknown. Fallen fruits were collected in February.

Distribution

Vietnam (Khanh Hoa and Lam Dong provinces) (Fig. 1).

Etymology

The specific epithet is derived from the type locality, Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam.

Vernacular name

Dé đá Bidoup

Additional specimens examined

Vietnam. Lam Dong Province: Bidoup-Nui Ba National Park; 1602 m elev., 12°09'27.6"N, 108°32'06.6"E, 24 Mar. 2018, T. Yahara, H. Nagamasu, H. Toyama, M. Zhang, A. Nagahama, N.V. Ngoc, K. Tsuchiya V7850 [ster.] (DLU!, FU!); ibid., 1656 m elev., 12°09'36.61"N, 108°32'11.16"E, 24 Mar. 2018, T. Yahara, S. Tagane, M. Zhang, A. Nagahama, K. Tsuchiya, N.V. Ngoc, H.T. Binh, T.Q. Cuong V8190, V8417 [ster.] (DLU!, FU!); ibid., 1669 m elev., 12°09'36.65"N, 108°32'11.18"E, 29 Apr. 2019, N.V. Ngoc, H.T. Binh, N.V. Duy, T.T. Nhung NAF122 [ster.] (DLU!); ibid., 1669 m elev., 12°09'36.62"N, 108°32'11.25"E, 29 Apr. 2019, N.V. Ngoc, H.T. Binh, N.V. Duy, T.T. Nhung NAF185 [ster.] (DLU!). Khanh Hoa Province: Son Thai Commune, Khanh Vinh District, in evergreen; 1430 m elev., 12°10'42.09"N, 108°43'32.59"E, 23 Apr. 2019, T. Yahara, S. Tagane, A. Nagahama, N. Komada, V.N. Ngoc, H.V. Thanh V9940 [ster.] (DLU!, FU!).

Conservation status

Critically Endangered (CR). From our intensive field survey in Bidoup-Nui Ba Naitonal Park and its vicinities from 2015 to present (Tagane et al. 2017 & 2020, Binh et al 2018b), Lithocarpus bidoupensis was found only in a narrow area within the protected areas of Bidoup-Nui Ba National Park, and its adjascent area of Son Thai Commune at the elevation range between 1400 and 1669 m. In the area, we observed fewer than 50 mature individuals. Based on criterion D of the IUCN Red List criteria (IUCN 2012), this species is qualified as CR.

Lithocarpus congtroiensis Ngoc & Yahara, sp. nov.

urn:lsid:ipni.org:names:77234074-1
Fig. 4

Type

Vietnam. Lam Dong Province, Bidoup-Nui Ba National Park: Cong Troi, at edge of evergreen forest, roadside, 1752 m elev., 12°05'37.3"N, 108°22'38.8"E, 11 July 2018, S. Tagane, A. Nagahama, K. Tsuchiya, N.V. Ngoc., T.Q. Cuong V9102 [fr.] (holotype DLU!; isotypes FU!, HN!, KYO!, VNM!).

Diagnosis

Lithocarpus congtroiensis is most similar to L. dahuoaiensis but differs by its smaller leaf blades, more secondary veins, shorter infructescences, cupules clustered of three (vs. solitary), and bigger nut size. It is also similar to L. honbaensis but distinguished mainly by its shorter petioles, smaller leaf blade, more secondary veins, shorter infructescences, and shorter fruiting stalk (Table 2).

Figure 4. 

Lithocarpus congtroiensis Ngoc & Yahara A twig with infructescence B infructescence C, D leaves adaxially and abaxially, respectively E side view of mature cupule and nut F botton view of nut with basal scar.

Description

Evergreen tree, up to 25 m tall. Twigs blackish gray, glabrescent. Stipules caducous, not seen. Leaves alternate; petioles 1.1–1.8 cm long, glabrous; blades broadly elliptic, 12–18 × 4.2–7.2 cm, coriaceous, glabrous on both sides, apex acuminate, acumen 0.7–1.2 cm long, base acute to cuneate, margin entire, midrib flat or slightly prominent adaxially, strongly prominent abaxially, yellowish in vivo, brown in sicco, secondary veins 13–15 pairs, prominent abaxially, at an angle of 40–50° from the midrib, tertiary veins parallel, faintly visible abaxially. Inflorescence a terminal or axillary spike, 8–12 cm long, erect, male and female flowers separate or female below. Male flowers white, in 1–3-flowered cluster, calyx 6-lobed, lobes ovate, 0.4–0.7 mm × 0.5–0.7 mm; stamens 12, 1–1.2 mm long, anthers 0.15–0.20 mm long. Female flower always cluster of three, styles 3, stigmata pointed. Infructescences terminal, erect, 10–15 cm long, rachis grayish brown, lenticellate. Cupules clustered of three, 0.2–0.4 cm long stalked, bowl-shaped, 0.7–1.4 cm high, 2.5–3.6 m diam., enclosing 1/3–1/2 of the nut; wall woody, with tiny imbricate scales; scales triangular, obscure, covered with white-grayish indumentum. Nut broadly conical or globose, 2.1–2.6 cm high, 2.3–3.1 cm in diam., outer surface densely white tomentose; wall woody, crackled; apex shortly acuminate; basal scar slightly concave, 1.5–1.8 cm in diam.

Phenology

Flowers were collected in December and mature fruits were collected from June to July.

Distribution

Vietnam (so far known only from Mt. Cong Troi and Mt. Langbiang of Bidoup-Nui Ba National Park, Lam Dong Province). (Fig. 1)

Etymology

The specific epithet is derived from the type locality, Mt. Cong Troi of Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam.

Vernacular name

Dé đá Công TrÓi.

Additional specimens examined

Vietnam. Lam Dong Province: Bidoup-Nui Ba National Park, Cong Troi area; 1750 m elev., 12°04'08.5"N, 108°21'55.5"E, 18 June 2015, N. Nguyen, D. Luong, B. Hoang V3205 [fr.] (DLU!, FU!); ibid., 1860 m elev., 12°06'06.85"N 108°23'00.32"E, S. Tagane, T. Yahara, A. Nagahama, M. Zhang, K. Tsuchiya, T. Nguyen, C.T. Nguyen V9470 [fr.] (DLU!, FU!); 1790 m elev., 12°06'03.86"N, 108°23'39.73"E, 20 Dec. 2018, T. Yahara, S. Tagane, A. Nagahama, K. Tsuchiya, C.T. Quong, P. Chhang V9555 [male and female fl.] (DLU!, FU! KAG [KAG127308]!); ibid., 1864 m elev., 12°04'08"N, 108°21'54.5"E, 15 Jun. 2019, N.V. Ngoc, H.T. Binh, N.V. Duy, T.T. Nhung NAF200 [fr.] (DLU!); Mt. Langbiang; 1918 m elev., 7 Oct. 2018, 12°02'48.13"N, 108°26'06.67"E, S. Tagane, T. Yahara, A. Nagahama, M. Zhang, K. Tsuchiya, T. Nguyen, C.T. Nguyen V9492 [ster.] (DLU!, FU!).

Conservation status

Critically Endangered (CR). We found around ten individuals of L. congtroiensis along the road and inside the permanent plot at Cong Troi area, and three individuals in Mt. Langbiang, both located inside the protected area of Bidoup-Nui Ba National Park. Based on criterion D of the IUCN Red List criteria (IUCN 2012), this species is qualified as CR. The new species is endemic to Bidoup-Nui Ba National Park, Lam Dong Province.

Lithocarpus hongiaoensis Ngoc & Binh, sp. nov.

urn:lsid:ipni.org:names:77234075-1
Fig. 5

Type

Vietnam. Lam Dong Province, Bidoup-Nui Ba National Park, Hon Giao, at edge of evergreen forest, roadside, 1580 m elev., 12°10'35.9"N, 108°42'25.1"E, 19 June 2015, N. Nguyen, D. Luong, B. Hoang V3235 [young male fl. and fruits] (holotype DLU!; isotypes FU!, HN!, KYO!, VNM!).

Diagnosis

Lithocarpus hongiaoensis is similar to L. vinhensis but differs in having much longer petioles, fewer secondary veins, longer infructescences, bigger cupules, and bigger nuts. It is also similar to L. vuquangensis but differs in having much longer petioles, more secondary veins, longer infructescences, and bigger cupules (Table 2).

Figure 5. 

Lithocarpus hongiaoensis Ngoc & Binh A habit B twig with young inflorescences C leaves D young male inflorescence E infructescence F inside of mature cupule.

Description

Evergreen tree, up to 25 m tall. Twigs blackish gray, glabrescent, densely lenticellate. Stipules narrowly triangular, ca. 5 × 1 mm, densely covered with indumentum abaxially, almost glabrous adaxially. Leaves alternate; petioles 1.7–3.5 cm long, glabrous; blades narrowly elliptic to lanceolate, 7.6–14.7 × 2.3–4.6 cm, coriaceous, glabrous adaxially, covered with adherent waxy scales abaxially, apex acuminate, acumen up to 1.5 cm long, base attenuate and decurrent on petiole, margin entire, midrib flat or slightly prominent near base adaxially, prominent abaxially, greenish yellow in vivo, reddish brown in sicco; secondary veins 8–11 pairs, prominent abaxially, at an angle of 35–45° from the midrib, tertiary veins scalariform, faintly visible or invisible on both sides. Young male inflorescences terminal, ca. 5–7 cm long, densely covered with white indumentum. Infructescences terminal, erect, 12.5–16.5 cm long, rachis 0.4–0.6 cm thick at base, grayish brown, lenticellate, covered with indumentum. Cupules solitary, sessile, obconical to saucer-shaped, 0.8–1.2 cm high, 1.7–2.6 cm in diam., enclosing 1/3–1/2 of the nut; wall woody, ca. 2 mm thick, with triangular scales not united into concentric rings; scales up to 4 mm long, apex shortly acuminate, covered with dense grayish indumentum outside. Nut strongly depressed ovoid, 0.6–1.1 cm high, 1.2–1.5 cm in diam., glabrous, reddish brown to grayish brown in sicco, tomentose with soft white hairs near apex; basal scar slightly concave, 1.2–1.4 cm in diam.

Phenology

Young male flowers and mature fruits were collected in May and June.

Distribution

Vietnam (so far known only from Hon Giao area of Bidoup-Nui Ba National Park, Lam Dong Province). (Fig. 1).

Etymology

The specific epithet is derived from the type locality, Hon Giao area of Bidoup-Nui Ba National Park, Lam Dong Province, Vietnam.

Local name

Dé đá Hòn Giao.

Conservation status

The new species is narrowly endemic to Hon Giao area of Bidoup-Nui Ba National Park, Lam Dong Province. During our floristic expedition from 2015 to present, we found only five mature individuals of Lithocarpus hongiaoensis at the road in montane evergreen forest which is located within the protected area of the national park. According to criterion D of the IUCN Red List criteria (IUCN 2012), this species is qualified as CR.

Additional specimens examined

Vietnam. Lam Dong Province: Bidoup-Nui Ba National Park, Hon Giao, 1574 m elev., 12°10'34.5"N, 108°42'25.5"E, 15 May 2019, N.V. Ngoc, H.T. Binh, N.V. Duy, T.T. Nhung NAF122 [fr.] (DLU!); ibid., 1574 m elev., 12°10'35.5"N, 108°42'25.9"E, 11 June 2020, N.V. Ngoc, H.T. Binh, N.V. Duy, T.T. Nhung NAF192 [fr.] (DLU!).

Acknowledgements

The authors thank the colleagues from the Bidoup-Nui Ba National Park for their help in collecting samples, and the directors and staff of the following herbaria ANDA, BKF, DLU, FOF, HN, HNL, KAG, KYO, P, and VNM for allowing us to examine their collections. This research is funded by Vietnam National Foundation for Science and Technology Development (NAFOSTED) under grant number 106.03-2018.325.

References

  • Ban NT (2005) Fagaceae. In: Ban NT (Ed.) Checklist of plant species of Vietnam 2. Agricultural Publishing House, Hanoi, 227–271.
  • Ban NT, Ly DT, Tap N, Dung VV, Thin NN, Tien VN, Khoi KN (2007) Vietnam Red Data Book Part II. Plants. Natural Sciences and Technology Publishers, Hanoi, 563 pp.
  • Binh HT, Ngoc NV, Tagane S, Toyama H, Mase K, Mitsuyuki C, Strijk JS, Suyama Y, Yahara T (2018a) A taxonomic study of Quercus langbianensis complex based on morphology and DNA barcodes of classic and next generation sequences. PhytoKeys 95: 37–70. https://doi.org/10.3897/phytokeys.95.21126
  • Binh HT, Ngoc NV, Tai VA, Son HT, Tagane S, Yahara T (2018c) Quercus trungkhanhensis (Fagaceae), a New Species from Cao Vit Gibbon Conservation Area, Cao Bang Province, northeastern Vietnam. Acta Phytotaxonomica et Geobotanica 69(1): 53–61.
  • Binh HT, Ngoc NV, Son HT, Tagane S, Yahara T (2021) Quercus ngochoaensis (Fagaceae), a new species from Ba Vi National Park, northern, Vietnam. Phytotaxa 516(3): 283–288. https://doi.org/10.11646/phytotaxa.516.3.7
  • Camus A (1931) Sur quelques genres de Fagacees. Riviera Scientifique 18: 37–42.
  • Camus A (1938) Fagacées nouvelles de l’asie orientale. Notulae systematicae (Paris) 6(4): 178–185.
  • Camus A (1942) Fagacées asiatiques nouvelles. Bulletin du Muséum National d’Histoire Naturelle Series II 14(5): 357–360.
  • Camus A (1948) Les Chênes: Monographie du genres Quercus et Lithocarpus. Chênes Atlas Volume 3. Paul Lechevalier & fils, 1314 pp.
  • Catchen JM, Amores A, Hohenlohe PA, Cresko WA, Postlethwait JH (2011) Stacks: build- ing and genotyping loci de novo from short-read sequences. G3: Genes, Genomes. Genetics 1: 171–182. https://doi.org/10.1534/g3.111.000240
  • Catchen JM, Hohenlohe PA, Bassham S, Amores A, Cresko WA (2013) Stacks: An analysis tool set for population genomics. Molecular Ecology 22(11): 3124–3140. https://doi.org/10.1111/mec.12354
  • Darriba D, Taboada GL, Doallo R, Posada D (2012) jModelTest 2: More models, new heuristics and parallel computing. Nature Methods 9(8): e772. https://doi.org/10.1038/nmeth.2109
  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.
  • Dung LV (2005) Fagacae in Bidoup-Nui Ba National Park. Published by author, Lam Dong, 68 pp.
  • Hickel R, Camus A (1930) Fagaceae. In: Lecomte PH, Gagnepain F (Eds) Flore Générale de l’Indo-Chine 5. Masson, Paris, 962–1007.
  • Ho PH (2003) An Illustrated Flora of Vietnam Vol. 2. Young Publishing House, Ho Chi Minh City, 951 pp.
  • Huang CC, Chang YT, Bartholomew B (1999) Fagaceae. In: Wu ZY, Raven PH (Eds) Flora of China 4. Science Press, Beijing & Missouri Botanical Garden Press, Saint Louis, 314–400.
  • IUCN (2012) IUCN Red List Categories and Criteria: Version 3.1. 2nd edn. Gland, Switzerland and Cambridge, UK, [iv +] 32 pp.
  • Linh DT, Thanh NT, Cuong NT, Hai DV, Hoan DT (2013) Basis of taxonomy for Lithocarpus Blume (Fagaceae Dumort.) in Vietnam. Proceeding of the 5th National conference on Ecology and Biological resources. Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, Hanoi, 127–131.
  • Ngoc NV, Dung LV, Tagane S, Binh HT, Son HT, Trung VQ, Yahara T (2016) Lithocarpus dahuoaiensis (Fagaceae), a new species from Lam Dong Province, Vietnam. PhytoKeys 69: 23–30. https://doi.org/10.3897/phytokeys.69.9821
  • Ngoc NV, Hung NV, Hoang Thi B, Tagane S, Toyama H, Son HT, Ha TV, Yahara T (2018) Lithocarpus vuquangensis (Fagaceae), a new species from Vu Quang National Park, Vietnam. PhytoKeys 95: 15–25. https://doi.org/10.3897/phytokeys.95.21832
  • Okabe N, Yahara T, Tagane S, Mitsuyuki C, Matsuo A, Sasaki T, Moritsuka E, Fuse K, Shimizu-Kaya U, Sang JA, Pungga RS (2021) A new species of Actinodaphne (Lauraceae), A. lambirensis from Sarawak, Malaysia, and an analysis of its phylogenetic position using MIG- seq and ITS sequences. Acta Phytotaxonomica et Geobotanica 72(1): 43–59. https://doi.org/10.18942/apg.202005
  • Phengklai C (2008) Fagaceae. In: Santisuk T, Larsen K (Eds) Flora of Thailand 9(3). The Forest Herbarium, Bangkok, 179–410.
  • RCore Team (2021) R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/
  • Suyama Y, Matsuki Y (2015) MIG-seq: An effective PCR-based method for genome-wide single-nucleotide polymorphism genotyping using the next-generation sequencing platform. Scientific Reports 5: e16968. https://doi.org/10.1038/srep16963
  • Suyama Y, Hirota SK, Matsuo A, Tsunamoto Y, Mitsuyuki C, Shimura A, Okano K (2021) . Complementary combination of multiplex high-throughput DNA sequencing for molecular phylogeny. Ecological Research: 1–11. https://doi.org/10.1111/1440-1703. 12270
  • Tagane S, Dang VS, Ngoc NV, Binh HT, Komada N, Wai JS, Naiki A, Nagamasu H, Toyama H, Yahara T (2017) Macrosolen bidoupensis (Loranthaceae), a new species from Bidoup Nui Ba National Park, southern Vietnam. PhytoKeys 80: 113–120. https://doi.org/10.3897/phytokeys.80.13338
  • Tagane S, Ngoc NV, Binh HT, Nagahama A, Zhang M, Cuong TQ, Son LV, Dang VS, Toyama H, Komada N, Nagamasu H, Yahara T (2020) Fifteen new species of angiosperms from Bidoup-Nui Ba National Park, southern highlands of Vietnam. Acta Phytotaxonomica et Geobotanica 71(3): 201–229. https://doi.org/10.18942/apg.202002
  • Takata K, Taninaka H, Nonaka M, Iwase F, Kikuchi T, Suyama Y, Nagai S, Yasuda N (2019) Multiplexed ISSR genotyping by sequencing distinguishes two precious coral species (Anthozoa: Octocorallia: Coralliidae) that share a mitochondrial haplotype. PeerJ 7: e7769. https://doi.org/10.7717/peerj.7769
  • Toyama H, Kajisa T, Tagane S, Mase K, Chhang P, Samreth V, Ma V, Sokh H, Ichihasi R, Onoda Y, Mizoue N, Yahara T (2015) Effects of logging and recruitment on community phylogenetic structure in 32 permanent forest plots of Kampong Thom, Cambodia. Philosophical Transactions of the Royal Society B: Biological Sciences 370(1662): 1–13. https://doi.org/10.1098/rstb.2014.0008
  • Tukey JW (1953) The problem of multiple comparisons. Unpublished manuscript, Princeton University.
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