A new species and two new records of Quercus (Fagaceae) from northern Vietnam

Abstract A new species, Quercus xuanlienensis Binh, Ngoc & Bon, is described from Xuan Lien Nature Reserve, Vietnam. The new species is morphologically similar to Q. edithiae Skan, in having 8–11 pairs of secondary veins, bowl-shaped cupules and ellipsoid to cylindrical-ellipsoid and basally convex nuts. It differs in having serrulate leaf margins only at apical 1/5–1/7, almost entire margins of bracts on cupule and much longer nuts. The species is also similar to Q. fleuryi Hickel & A. Camus in having leaves glabrous on both surfaces with only an apically serrulate margin but differs in having shorter petioles, cupules enclosing 1/5 of the nut and much longer nuts. In addition, Q. disciformis Chun & Tsiang. and Q. bella Chun & Tsiang., previously known from China, are newly recorded from Ba Vi National Park, Vietnam.


Introduction
Quercus L. comprises ca. 400-500 species (Nixon 1993, Valencia-A et al. 2016 and has been divided into two subgenera, Quercus subgenus Cyclobalanopsis (Oerst.) Schneider (ring-cup oaks) characterised by stigma capitate to subcapitate or discoid stigma, cupule bracts being connate or forming concentric or spiral rings and Quercus subgenus Quercus (scale-cup oaks) characterised by usually linear ampliate or broadly ampliated stigma, free and imbricate cupule bracts (Nixon 1993, Manos et al. 1999. In Vietnam, according to Ho (2003) and Ban (2005), a total of 43 Quercus species were recorded, amongst which 37 species belong to subgenus Cyclobalanopsis and six species belong to subgenus Quercus. Recently, the following two species were reported and the species of Quercus in Vietnam rose to 45 species: Q. lineata Blume of subgenus Cyclobalanopsis (Li et al. 2016) and Q. trungkhanhensis Binh & Ngoc of subgenus Quercus (Binh et al. in press).
To widen our knowledge on the Fagaceae of Vietnam, field surveys were undertaken by the authors for 13 conservation areas (national parks, nature reserves and conservation area) in Vietnam and a total of 105 Quercus samples were collected. Amongst them, during the field surveys in Xuan Lien Nature Reserve and Ba Vi National Park ( Fig. 1), we discovered three unknown species of the subgenus Cyclobalanopsis which were not identical to any of the 38 species of Cyclobalanopsis previously recorded from Vietnam (Ho 2003, Ban 2005, Li et al. 2016. Xuan Lien Nature Reserve, Thuong Xuan District, Thanh Hoa Province, North Central Coast of Vietnam, was established in 1999 with a total area of 21,000 ha. Until now, 1,142 species of vascular plants belonging to 620 genera and 180 families have been recorded (Xuan Lien Nature Reserve 2017). In Fagaceae, 31 species including 17 Lithocarpus species (55%), 10 species of Castanopsis (32%) and four species of Quercus (13%) have been recorded (Xuan Lien Nature Reserve 2017). Ba Vi National Park, Ha Noi Capital, northern Vietnam was established in 1991 with a total area of 7,377 ha ( Fig. 1). In this national park, located in the Ba Vi mountain range, 1,201 vascular plant species of 649 genera and 160 families including 19 species of Fagaceae are recorded (Ba Vi National Park 2008).
In this study, a new species is reported from Xuan Lien Nature Reserve and two species are newly recorded from Ba Vi National Park. A new species is described as Quercus xuanlienensis Binh, Ngoc & Bon. The two newly recorded species to the country are Q. disciformis Chun & Tsiang. and Q. bella Chun & Tsiang. In addition to the morphological examination, DNA sequences and phylogenetic analyses are helpful for delimiting species (Hebert andGregory 2005, Dick andWebb 2012). Here, DNA sequences of the three species were compared with those of 20 species in Vietnam to confirm that the three species are divergent and thus distinct from the other species. First, two DNA barcode regions were sequenced, the partial genes for the large subunit ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL) and maturase K (matK) as basic DNA barcodes (CBOL Plant Working Group 2009). However, those sequences show limited divergence in the genus Quercus and thus multiple gene markers (Hubert et al. 2014, Simeone et al. 2016, RAD-seq (Cavender-Bares et al. 2015, Fitz-Gibbon et al. 2017) and MIG-seq (Suyama and Matsuki 2015, Binh et al. in review) have been used to determine phylogenetic relationships in Quercus. In particular, Binh et al. (in review) successfully used MIG-seq to determine the phylogenetic relationship in the Quercus langbianensis complex in Vietnam and revise its taxonomy. In this study, the authors compared the MIG-seq of Q. xuanlienensis, Q. disciformis and Q. bella with those of 18 Quercus species studied by Binh et al. (in review) and two additional species Q. platycalyx Hickel & A.Camus and Q. quangtriensis Hickel & A.Camus that have cupules similar to Q. disciformis and Q. bella, to determine their identities and phylogenetic relationships.

Morphological observations
The validity of a new species and the identities of newly recorded species were examined based on literature of the genus Quercus in Vietnam and its surrounding countries (Camus 1936-1954, Soepadmo 1972, Ho 2003, Huang et al. 1999, Ban 2005, Phengklai 2008, Li et al. 2016, authentic specimens including types by visiting the herbaria DLU, HN, FU, P and VNM and using images available on the web of JSTOR Global Plants (https://plants.jstor.org/) and Chinese Virtual Herbarium (http://www.cvh.org.cn/).

DNA extraction
DNA was isolated from silica-gel dried leaf materials following the CTAB method (Doyle and Doyle 1987) with minor modifications, as in Toyama et al. (2016). Before the DNA extraction, dry leaf material was milled by QIAGEN TissueLyser to obtain fine powder and the powder was washed up to five times by 1 ml buffer (0.1 M HEPES, pH 8.0; 2% Mercaptoethanol; 1% PVP; 0.05 Ascorbic acid).

DNA barcoding
DNA regions of the large subunit of ribulose-1,5-bisphosphate carboxylase oxygenase (rbcL) and maturase K (matK) were amplified and sequenced following the protocols of Kress et al. (2009) and Dunning and Savolainen (2010), respectively.
Next generation DNA sequencing -MIG-seq DNA products were used from 105 Quercus spp. as templates to amplify thousands of short sequences (loci) from a wide variety of genomes using primers designed for "multiplexed ISSR genotyping by sequencing" (MIG-seq, Suyama and Matsuki 2015). Then presence/absence of each locus in each sample was used for phylogenetic tree reconstruction regardless of whether it has SNP or not. According to the MIG-seq protocol of Suyama and Matsuki (2015) with minor modifications as in Binh et al. (in review), the 1st PCR, multiple non-repetitive regions from various inter-simple-sequence repeat (ISSR) are amplified from genomic DNA by multiplexed PCR with tailed ISSR primers. The 2nd PCR step was performed based on products from the 50 times dilution for each 1st PCR product with deionised water. Then, 3 µl of each 2nd PCR product was pooled as a single mixture library and purified. Subsequently, the Pippin Prep DNA size selection system (Sage Science, Beverly, MA, USA) was used to selected fragments in the size range 350-800 bp. A SYBR green quantitative PCR assay (Library Quantification Kit; Clontech Laboratories, Mountain View, CA, USA) was used to measure the concentration of the size-selected library with approximately 10 pM of libraries. Finally, 10 pM of libraries were used for sequencing on an Illumina MiSeq Sequencer (Illumina, San Diego, CA, USA), using a MiSeq Reagent Kit v3 (150 cycle, Illumina).

Phylogenetic analyses
In MIG-seq, raw data from 105 samples were pretreated and quality control completed following Suyama and Matsuki (2015) as described in Binh et al. (in review). Subsequently, a list of loci obtained was used for the next steps. This list of loci was detected at least in one individual (1/105=0.01) with the following settings: all samples belong to the same population and threshold frequency of haplotype count in a population (r) = 0.001, a threshold one-order higher than 0.01. Presence/absence (1/0) data of loci were used to compute a distance matrix, construct a neighbour-joining (NJ) tree and the reliability of the tree topology was examined by bootstrapping with 1000 replicates using PHYLYP ver. 3.695 (Shimada and Nishida 2017) as follows; 1000 times re-sampling with Seqboot, distance computation with Restdist, tree construction with NJ and consensus tree construction with Censense. In addition, FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/) was used to visualise the resulting tree. A phylogenetic tree for 105 samples including 43 Quercus species amongst 44 species previously recorded in Vietnam (data not shown) was constructed and subsequently reduced to 28 samples by focusing on the clades containing Q. xuanlienensis, Q. disciformis, Q. bella and the additional 20 Quercus species following Binh et al. (in review). A total of 19,916 loci were used for the final phylogenetic tree.
According to the key and descriptions in the Flora of China (Huang et al. 1999), the other two unknown taxa from Ba Vi National Park were identified as Q. disciformis and Q. bella. Excluding slightly thinner leaves and lower teeth, one species is identical with Q. disciformis in the following diagnostic characters: leaf blade oblong to obovate-elliptic (6-13 × 2.5-4 cm), margin serrate in the upper 2/3, glabrous on both surfaces when ma-ture; lateral veins 11-13 pairs; petiole ca. 2 cm long; cupule discoid, rim flat when ripe, 3-4 cm in diam., covering base of the nut, scales arranged in 8-10 rings, margin of rings denticulate except apical 2 or 3 entire; nuts oblate 1.5-2 cm high, 2 cm in diam., apex flattened densely appressed hairy. Another species was identified as Q. bella having the following characteristics: leaf blade oblong-elliptic to lanceolate (8-15 cm × 2-3.5 cm), base slightly oblique, margin serrate in the upper 1/2; lateral veins 12 pairs of lateral veins; petiole 1-2 cm long; cupule discoid (ca. 0.5 cm × 2.5-3 cm), covering base of the nut, scales arranged in 6-8 rings, margin of rings irregular denticulate; nut oblate nut 1.5-2 cm high and 2.2-3 cm in diam.
A phylogenetic tree, inferred using MIG-seq, showed that Q. xuanlienensis, Q. disciformis and Q. bella are not identical with any of the 20 species from Vietnam. The neighbour-joining (NJ) tree based on MIG-seq data for 28 sample of Quercus recognised two major clades using Trigonobalanus as an outgroup (Fig. 2). Clade 1 with 82% bootstrap value consists of three species of subgenus Quercus (Q. lanata, Q. setulosa and Q. trungkhanhensis) and Clade 2 with 99% bootstrap value consists of 20 species of subgenus Cyclobalanopsis including Q. bella, Q. disciformis and Q. xuanlienensis. These three species were clustered with Q. quangtriensis, Q. neglecta and Q. platycalyx and a clade of those six species was strongly supported (74% bootstrap value). Amongst the six species, Q. xuanlienensis was separated from the other five species forming a clade with a 74% bootstrap value. Four samples of Q. disciformis and three samples of Q. bella formed two distinct clades, each supported by 100% bootstrap value. Quercus disciformis was sister to Q. bella and the clade of those two species had an 84% bootstrap value.

Discussion
The results of the NJ tree, based on MIG-seq data, showed that Q. disciformis is sister to but well differentiated from Q. bella. These two species were collected in Ba Vi National Park where they co-occur with similar in leaf and nut morphologies, but differ in the coverage of the cupule (Fig. 4, less than 1/8 in Q. disciformis vs. Fig. 5, basal 1/8 to 1/4 in Q. bella). According to the Flora of China (Huang et al. 1999), Q. bella is recorded from Guangdong, Guangxi and Hainan provinces, whereas Q. disciformis is distributed in SW Guangdong, Guangxi, Guizhou, Hainan and Hunan provinces. Ba Vi National Park is located in northern Vietnam, neighbouring Guangxi province and therefore the occurrences of Q. disciformis and Q. bella there are understandable.  (Huang et al. 1999) The two species are similar to Q. platycalyx and Q. quangtriensis in having oblong to oblong-elliptic leaves, usually serrate along leaf margins in the upper 1/2 to 2/3, glabrous on both surfaces when mature, and cupules covering less than 1/3 of the nut and oblate to ovoid nuts (Huang et al. 1999, Phengklai 2008, Ho 2003. The MIGseq tree showed that those four species are related; the monophyly of a clade including the four species and Q. neglecta was supported by a 77% bootstrap value, although the support for the monophyly of the four species is weaker. The affinity of the four species and Q. neglecta was unexpected because Q. neglecta is an easily distinguished species having linear leaves and small nuts (Huang et al. 1999, Ho 2003. The MIG-seq tree showed that Q. xuanlienensis is related to the above four species and Q. neglecta that is morphologically distinct from the other Quercus species. From the four species (Q. disciformis, Q. bella, Q. quangtriensis and Q. platycalyx), Q. xuanlienensis is clearly distinguished by the leaf margin (leaf margin serrulate only at apical 1/5-1/7 in Q. xuanlienensis vs. serrate in upper 1/2 to 2/3 in the four species) and nut shape (ellipsoid to cylindrical-ellipsoid vs. oblate to ovoid). Thus, Q. xuanlienensis is separated as a distinct species from them.
Whereas Q. edithiae is morphologically most similar to Q. xuanlienensis, the type specimens of Q. edithiae collected from Hong Kong (type: Ford 623, K [K000832101, K000832102]) are morphologically distinct from Q. xuanlienensis in having distinct serrations, denticulate cupule bracts and smaller nuts and the description of Q. edithiae in Flora of China (Huang et al. 1999) agrees with the type specimen. The morphological differences between Q. edithiae and Q. xuanlienensis are as distinct as those between related species of Quercus in Vietnam and its surrounding countries. Huang et al. (1999) recorded Q. edithiae in Guangdong, Guangxi, Hainan and Vietnam, but no specimen could be found of Q. edithiae collected from Vietnam in any herbarium in Vietnam or on the Chinese Virtual Herbarium website (http://www.cvh.org.cn/). Further studies are needed to confirm the occurrence of Q. edithiae itself in Vietnam.
The MIG-seq tree (Fig. 2) was very helpful in deriving the conclusions contained in this paper. As Q. disciformis and Q. bella are morphologically similar and were collected from the same locality, it was difficult to ascertain whether these are in fact two distinct species and not variants of a single species without the support of the MIG-seq data. Also, the separation of Q. xuanlienensis from the other species in Fig. 2 supported the conclusion that it is a new species. The authors also obtained sequences data of rbcL and matK but the informative content of those sequences was too low to resolve the relationships amongst such closely related species of Quercus. Difficulties were faced in determining the sequences of ITS for Q. disciformis, Q. bella and Q. xuanlienensis, most likely due to the low quality of the authors' samples. MIG-seq is applicable to low quality samples and provides finer resolution of the relationship amongst closely related species (Suyama and Matsuki 2015, Binh et al. in review). Further studies using MIG-seq would be fruitful to elucidate the diversity of Quercus in Vietnam, a centre of oak species richness in SE Asia.
Ecology in Vietnam. At present, only one individual was found in evergreen forest, at 810 m altitude.  Ecology in Vietnam. In the field survey, only three individuals were found at an altitude of 737 m and one at 1172 m; in evergreen forest.
Phenology. Flowering from March to April, fruiting from August to September in China (Huang 1999 (2017). The Vietnamese population in Ba Vi National Park extends its distribution range, representing the south western limit. Given the situation, the population in Vietnam is locally important but the category Least Concern (LC) (IUCN 2012, Ban et al. 2007) would be appropriate for this species.  Distribution. China (Guangdong, Guangxi, Hainan) and Vietnam (Ba Vi National Park, Fig. 1).

Quercus bella
Ecology in Vietnam. Quercus bella was found on the slopes in evergreen forests in Ba Vi National Park: at alt. 600-1172 m.
Phenology. Flowering from February to April, fruiting from October to December (Huang et al. 1999). Flowering and fruiting specimens were collected from Vietnam in September.
GenBank accession no. Binh et al. V6038: LC331259 (rbcL), LC331256 (matK). Preliminary conservation status. Quercus bella was only previously known as an endemic species to China and distributed in Guangdong, Guangxi and Hainan. The species is not recorded as a threatened species in IUCN (2017). Although only three fruiting individuals of Q. bella were collected in Ba Vi National Park, more individuals are expected to occur there and the habitat in the Ba Vi National Park is currently wellprotected from anthropogenic activities under the law. Thus, it is appropriate to place this species under the category Least Concern (LC) following IUCN Red List (IUCN 2012) and Vietnam Red Data book (Ban et al. 2007).