Lithocarpus vuquangensis (Fagaceae), a new species from Vu Quang National Park, Vietnam

Abstract Lithocarpus vuquangensis Ngoc & Hung is described from Vu Quang National Park, North Central Vietnam. The morphological comparison and phylogenetic analysis based on rbcL, matK and ITS provided evidence that the new species was not assignable to any of the previously known taxa in Vietnam and its surrounding countries. The description, photographs, preliminary conservation status and DNA barcode sequences are also provided for the new species.


Introduction
It has been known that species richness of the genus Lithocarpus Blume (Fagaceae Dumorier) is high in Vietnam where 120 species and two varieties have been reported including the recently published species, L. dahuoaiensis Ngoc & L. V. Dung (Ban 2003, Ho 2003. Here, an additional new species of Lithocarpus is described from Vu Quang National Park located in Ha Tinh Province, North Central Vietnam (Figure 1). Vu Quang National Park covers an area of ca. 56,000 ha from lowlands (alt. 10-300 m) to the highlands (the highest peak of Rao Co, alt. 2,286 m). Two new species of mammals (Sao La -Pseudoryx nghetinhensis, Artiodactyla and the world's largest muntjac -Muntiacus vuquangensis, Cetartiodactyla) were discovered from this national park in the 1990s (Dung et al. 1993(Dung et al. , 1994. The vegetation is diverse along the elevation gradient and five major forest types are recognised: lowland forests (alt. 10-300 m), hill forest (alt. 300-1,000 m), medium montane forest (alt. 1,000-1,400 m), montane forest (alt. 1,400-1,900 m) and upper montane forest (alt. 1,900-2,100 m) (Kuznetsov 2001, Vu Quang National Park Management Board 2014. Until now, 1,678 species of vascular plants including many endemic and rare species have been reported (Vu Quang National Park Management Board 2014, . As for Fagaceae, one species of Castanea Mill, nine species of Castanopsis (D. Don) Spach., 12 species of Quercus L. and 37 species of Lithocarpus Blume have been recorded from the National Park, amongst which 10 species have been listed in Viet Nam Red Data Book (Ban et al. 2007, Hung et al. 2014). In addition, natural populations of Trigonobalanus verticillata Forman were discovered during the authors' recent botanical surveys in the National Park in 2016 (voucher specimens: Yahara et al. V5764 & V5766, DLU, FU, the herbarium of Vu Quang National Park), which brings the number of Fagaceae genera in the region up to five.
From 2015 to 2016, floristic expeditions were carried out in Vu Quang National Park and trees of the genus Lithocarpus were discovered that did not match any described species. Here, the authors describe and name it as Lithocarpus vuquangensis Ngoc & Hung, sp. nov. accompanied with its photographs and the morphological comparison with related species. In addition to the morphological examination, DNA sequences and phylogenetic analysis are extremely helpful for identifying and delimiting species (Hebert andGregory 2005, Dick andWebb 2012). Here, parts of the DNA barcode regions rbcL, matK (CBOL Plant Working Group 2009) and ITS (China Plant BOL Working Group 2011) were sequenced and the phylogenic relationship of L. vuquangensis and its related taxa were examined.

Morphological observations
The morphological traits of the new species were compared with its putative relatives based on systematic literature (Camus 1948, Huang et al. 1999, Ban 2003, Ho 2003, Phengklai 2008) and more than three hundreds dried specimens kept in the following herbaria were also examined: BKF, DLU, FOF, HN, KYO, P, RUPP, TI and VNM as well as digitised plant specimen images available on the web of JSTOR Global Plants (https://plants.jstor.org/), Muséum National d'Histoire Naturelle (https://science. mnhn.fr/) and Chinese Virtual Herbarium (http://www.cvh.org.cn/).

DNA extraction and sequencing
Total DNA was extracted from 17 silica-gel dried leaf pieces collected in the field. DNA extraction was performed using the CTAB method (Doyle and Doyle 1987) with minor modifications described in Toyama et al. (2015). Two chloroplast DNA barcode regions, rbcL and matK, were amplified and sequenced following published protocols (Kress et al. 2009, Dunning andSavolainen 2010). In addition, the internal transcribed spacer (ITS) region was sequenced using the protocol of Rohwer et al. (2009) with a minor modification in PCR amplification using the Tks GflexTM DNA Polymerase (Takara Bio Inc., Japan).

Phylogenetic analysis
A total of 16 accessions representing 15 species of Lithocarpus, collected throughout Vietnam, were analysed (Table 1). In addition, Trigonobalanus verticillata Forman was used as an outgroup in the phylogenetic analysis. The sequence alignment was performed by ClustalW with default parameters implemented in MEGA v 7.0.25 (Kumar et al. 2016) and subsequently adjusted manually.
Bayesian Inference (BI) of phylogeny was performed on the concatenated data set of three genes (rbcL, matK and ITS) using MrBayes v. 3.2 Ronquist 2001, Ronquist et al. 2012). The hierarchical likelihood ratio test (hLRT) and Akaike Information Criterion (AIC) were used to select the best model of evolution using MrModeltest v. 2.3 (Nylander 2004). The nucleotide substitution model was set to GTR+γ as selected by MrModeltest. Four independent Markov Chain Monte Carlo (MCMC) runs of four chains each were run for 10,000,000 generations sampling every 1,000 generations. The programme Tracer v. 1.6 (Rambaut et al. 2014) was used to examine marginal prior and posterior densities of MCMC outputs. Each run produced 10,001 trees and a relative burnin of 25% was used for diagnostics. Consequently, 7,501 trees of each run were sampled to generate the summary tree and posterior probabilities distributions. The summary tree was visualised and edited with FigTree v1.4.3 (http://tree.bio.ed.ac.uk/software/figtree/).

Results
The morphological comparison showed that Lithocarpus vuquangensis is most similar to L. nantoensis (Hayata) Hayata distributed in Taiwan, in having entire leaf margin, mostly solitary, rarely 2 or 3 clustered cupules, cupules not completely enclosing nut and glabrous nut. The Vietnamese species sharing the above diagnostic feature of L. vuquangensis are L. hongiaoensis, in ined. (Ngoc et al. in review) and L. vinhensis A. Camus. However, the new species is clearly different from all three in the following points: L. vuquangensis is distinguished from L. nantoensis by its fewer secondary veins (7-10 pairs vs. 10-15 pairs), shorter infructescences (4-7 cm long vs. 16 cm long), longer fruiting stalks (4-6 mm long vs. almost sessile), larger nut size (1.7-2.0 cm high by 2.1-2.4 cm in diam. vs. 1.4-1.7 cm high by 1.5-1.6 cm in diam.) and larger basal scar of the nut (ca. 1.1 cm in diam. vs. 0.5-0.8 cm in diam.). Lithocarpus vuquangensis is distinct from L. hongiaoensis by its shorter petioles (1-1.5 cm long vs. 2.1-3 cm long), shorter infructescences (4-7 cm long vs. 10 cm long), longer fruiting stalks (4-6 mm long vs. almost sessile), arrangement of scales on the cupule (scales arranged into concentric rings vs. imbricate, not forming rings) and larger nut size (1.7-2.0 cm long, 2.1-2.4 cm in diam. vs. 0.6-0.8 cm long, 1.2-1.5 cm in diam.). The new species differs from L. vinhensis in having fewer secondary veins (7-10 pairs vs. 11-12 pairs), shorter infructescences (4-7 cm long vs. 10 cm long) and larger nut size (1.7-2.0 cm In the molecular phylogenetic tree (Fig. 2), L. vuquangensis is sister to L. hongiaoensis with the posterior probability of 0.94. One nucleotide substitution in rbcL, six in matK and six in ITS were found between these two species. On the other hand, L. vinhensis, another Vietnamese species most similar to L. vuquangensis, is placed in a separated clade which includes L. longipedicellatus, L. ombrophilus, L. gigantophyllus, L. licentii, L. pseudomagneinii and L. lemeeanus, with a posterior probability 0.93.
Both Lithocarpus vuquangensis and L. vinhensis were collected in Vu Quang National Park, but these two species occur at different altitudes: L. vuquangensis was found between 1,500 m and 1,700 m altitude, while L. vinhensis was found at a lower elevation, below 1,100 m.

Discussion
Phylogenetically, L. vuquangensis is sister to L. hongiaoensis in ined. collected from Lam Dong Province located in southern Vietnam. These two species are morphologically Table 2. Morphological comparison of Lithocarpus vuquangensis with three related species: The measurements of L. nantoensis is derived from Hayata (1911), Liao (1996), Huang et al. (1999) and from digitised type specimen image (Kawakami & Mori 1157, TI); The measurements of L. vinhensis and L. hongiaoensis are derived from Camus (1948) and Ngoc et al. (in review), respectively. Concave, 0.5-0.8 cm in diam.
Slightly concave, 1.2-1.4 cm in diam. Nearly flat distinguished in their length of infructescences and fruiting stalk, the arrangement of cupule bracts, nut size and other characteristics as summarised in Table 2. Further molecular phylogenetic studies, using additional DNA markers, are needed to clarify the relationship between L. vuquangensis and L. hongiaoensis. However, morphological differences are sufficiently distinct to distinguish them as different species. Lithocarpus vuquangensis is also morphologically similar to L. vinhensis in having an entire leaf margin, solitary cupules not completely enclosing nut, scales arranged into concentric rings and glabrous nut, but these two species are not closely located in the phylogeny. This morphological similarity may have evolved in the similar habitat of the montane evergreen forest in Vu Quang National Park. Whereas L. vuquangensis and L. vinhensis were collected at 1,518 m and 1,062 m, respectively, altitudinal distributions of the two species may overlap in the montane evergreen forest.
The morphological comparison provided evidence to distinguish L. vuquangensis from a Taiwanese species, L. nantoensis, although the relationship between them remains to be clarified by further molecular phylogenetic studies. Diagnosis. Similar to Lithocarpus nantoensis, L. hongiaoensis and L. vinhensis, but distinguished from L. nantoensis mainly by its fewer secondary veins, shorter infructescences, longer fruiting stalk, larger nut size and larger scar size of the nut, from L. hongiaoensis by its much shorter petioles and infructescences, longer fruiting stalk, scales united into concentric rings and much larger nut size and from L. vinhensis by having fewer secondary veins, shorter infructescences and much larger nut size (Table 2).
Phenology. Mature fruits were collected in June. Distribution. Vietnam (so far known only from Vu Quang National Park, Ha Tinh Province) (Figure 1).
Etymology. The specific epithet is derived from its type locality, Vu Quang National Park.
Preliminary conservation status. Critically Endangered (CR). In the field observation, less than 10 individuals were found along the trail to the summit of Mt. Rào Cô, in lower montane forest. The habitat is inside the protected areas of Vu Quang National Park, but based on criterion D of the IUCN Red List criteria (IUCN 2012), this species is qualified as CR. Further intensive inventories are needed to find additional populations in Vu Quang National Park and its surrounding areas.