Khoonmengia honbaensis, a new genus and species of temperate bamboo (Poaceae, Bambusoideae) from central-southern Vietnam

Abstract A new genus of Arundinarieae, Khoonmengia, is established to accommodate a unique new bamboo species, K. honbaensis, from central-southern Vietnam. The morphological features, habitats and distribution of Khoonmengia and related genera, i.e. Ampelocalamus and Hsuehochloa, are compared. The characters of its scrambling habit, internodes with brownish green dots, conspicuous nodes swollen at one side, elliptic buds wholly sunken into culm, extravaginal branching pattern, mid-culm branch complement with one central dominant branch elongating to reiterate the culm accompanied by several lateral slender branches, swollen culm sheath base with a distinctive zone of transverse wrinkles, synflorescence composed of only one spikelet, single or several to many synflorescences arranged into a raceme or panicle terminal on leafy branches, purple anthers and nut-like caryopsis with hardened pericarp and loosely adherent lemma and palea distinguish K. honbaensis from morphologically similar taxa. In order to investigate the phylogenetic position of this unknown bamboo, molecular phylogenetic analyses based on the nuclear gene GBSSI were also conducted, and the results proved that K. honbaensis is definitely a member of Arundinarieae with an isolated position, which also indicated that this species could not be assigned to any of the already described genera and supported the establishment of the new genus.


Introduction
Bamboos, including a single evolutionary radiation of 1,642 species in the grass family Poaceae, subfamily Bambusoideae, are important components in tropical to warm temperate forests (Vorontsova et al. 2016). Bambusoideae is classified into two tribes of woody bamboos (the tropical Bambuseae and the temperate Arundinarieae) and one tribe of herbaceous bamboos (the Olyreae) (Soreng et al. 2015). The Arundinarieae are the temperate woody bamboos, a diverse clade of 31 genera and ca. 546 species, with the center of diversity in East Asia (ca. 430 species), distributed primarily in forests of the northern temperate zone, but also in some high elevation tropical regions (BPG 2012;Clark et al. 2015). Arundinarieae is not only a taxonomically difficult group of bamboos, but also a troublesome one in molecular phylogenetics . Previous phylogenetic studies mainly based on plastid DNA divided Arundinarieae into twelve lineages, but the phylogenetic relationships among many clades were not well resolved (Zeng et al. 2010;Zhang et al. 2012;Ma et al. 2014;Attigala et al. 2016). Some analyses also revealed many inconsistencies between the plastid and the nuclear gene trees (Zhang et al. 2012;Yang et al. 2013). For example, Ampelocalamus actinotrichus had an affinity with individuals of Chimonocalamus in the plastid phylogeny (Zeng et al. 2010), rather than other taxa of Ampelocalamus. However, in the nuclear gene phylogenies, this species formed a clade with the congeneric taxa . These results implied that the nuclear genome and the plastid genome may have different evolutionary trajectories (Zhang et al. 2012;Yang et al. 2013). The most recent study based on phylogenetic analyses with RAD-seq data identified eight major lineages in Arundinarieae with strong support, which conflicts with earlier studies (Wang et al. 2017).
During an investigation of the bamboos in Hon Ba Nature Reserve, Khanh Hoa Province of central-southern Vietnam in October 2017, an unusual bamboo with unicaespitose habit, scandent stems, pachymorph rhizomes and semelauctant inflorescences on leafy flowering branches caught our attention. This species was misidentified as Bambusa tulda Roxb. during the background survey of Hon Ba Nature Reserve (Lee et al. 2014). The semelauctant inflorescence is a relatively rare condition in the Bambuseae. After closer examination, we found that it has three stamens and two stigmas. By its habit and floral characters, it should be a member of the tribe Arundinarieae. In Vietnam, only five clambering genera, i.e. Melocalamus Benth., T. Tran, are currently recognized, but all of these genera belong to Bambuseae (Wong 1993;Tran et al. 2013). In Southeast Asia, many other genera also have climbing or clambering culm habits, such as Holttumochloa K. M. Wong, Kinabaluchloa K. M. Wong, Dinochloa Büse, Racemobambos Holttum, Chloothamnus Büse, etc. However, none of these genera belong to Arundinarieae either. There are only two genera belonging to Arundinarieae in subtropical Asia, i.e. Ampelocalamus S. L. Chen, T. H. Wen & G. Y. Sheng and Hsuehochloa D. Z. Li & Y. X. Zhang, which have a combination of morphological characters including scandent stems, semelauctant inflorescences, three stamens and two stigmas (Chen et al. 1981;Zhang et al. 2018). However, this unknown bamboo has some both reproductive and vegetative characters that are different from these two subtropical genera (see Table 1).
The nuclear gene GBSSI (granule-bound starch synthase I) occurs as a single copy in Poaceae and was often used in recent phylogenetic studies on woody bamboos (Zhang et al. 2012;Goh et al. 2013;Yang et al. 2013). Zhang et al. (2012) showed that the phylogeny based on GBSSI was better resolved at the generic level than the plastid phylogeny. Therefore, in order to investigate the phylogenetic position of this unknown bamboo, we conducted molecular phylogenetic analyses of Asian Arundinarieae based on GBSSI.

Sampling and morphological study
Samples of this putative new species were collected for morphological and molecular phylogenetic studies from the only known population in Hon Ba Nature Reserve, Khanh Hoa Province, central-southern Vietnam during our field investigation in Oct. 2017. Photographs were taken with a CANON EOS 60D camera and dried flowers were dissected and examined under an Olympus SZX16 Microscope; line drawings and descriptions were made by reference to dried specimens.

DNA amplification and sequencing
Total genomic DNA was isolated from silica gel-dried leaf material using the Plant Genomic DNA Extraction Kit (Tiangen, Beijing, China), following the manufacturer's instructions. The nuclear GBSSI sequence was amplified following the protocol used in Zeng et al. (2010). All PCR were performed in 25 μL volumes with a Senso-Quest Labcycler 48 Gradient. A fragment from this unknown species was successfully sequenced by the DNA sequencing facility at Sangon Biotech (China). Automated sequencing output was checked visually for correct automated base-calling. Sequences were aligned using Bioedit v7.2.0 (Hall 1999) and adjusted manually where necessary. The newly obtained sequence has been deposited in Genbank.
In addition, sequences from the other 42 taxon representing nearly all known genera of Arundinarieae and outgroups, mainly following prior studies (Zeng et al. 2010;

Phylogenetic analyses
Gaps were coded as present or absent using the simple indel coding method (Simmons and Ochoterena 2000). The best-fitting models were selected using jModeltest v2.1.4 under the Akaike Information Criterion (AIC) (Darriba et al. 2012). The model used for the GBSSI in this study was TrNef+G.  (Zwickl 2006). MP analyses were conducted using PAUP*v.4.0b10 (Swofford 2003). Heuristic searches were performed with 1000 homogeneity replicates, tree-bisection-reconnection (TBR) branch swapping, MUL-TREES option off, and random addition of sequences with 1000 replicates.
BI analyses were conducted using MrBayes 3.2.5 (Ronquist et al. 2012). The runs were conducted starting with random trees, consisting of a single cold chain and three heated chains, with the temperature set to 0.1. The Markov chain Monte Carlo (MCMC) chains were run for 10 million generations and sampled trees every 1000 generations for the GBSSI gene data set. A 50% majority-rule consensus tree was constructed from the remaining trees, yielding the posterior probability (PP) values for each clade.

Results
A total of 1414 characters were included in the maximum parsimony (MP) analyses matrix, of which 133 characters were parsimony-informative, 238 variable characters were parsimony-uninformative and 843 characters were constant. The strict consensus tree for the 234 most parsimonious trees (tree length = 531; CI = 0.787; RI = 0.675; RC = 0.532) is shown in Fig. 1. The results of the MP, BI and ML analyses were almost identical except for slight position changes of some species (not shown). PP (posterior probabilities), MPBS (maximum parsimony bootstrap support) and MLBS (maximum likelihood bootstrap support) were included on the strict consensus tree from MP analyses. PP< 0.95 and MPBS/MLBS < 70% were considered as lacking support for a clade.
In the current study, the monophyly of the temperate woody bamboo clade was strongly supported, with 100% MPBS, 100% MLBS and 1.00 PP. Our putative new species was nested in the monophyletic clade of Arundinarieae. However, the phylogenetic relationships among groups of temperate woody bamboos were not resolved in this study. Diagnosis. Khoonmengia resembles Hsuehochloa and Ampelocalamus in having pachymorph and short-necked rhizomes, florets with 3 stamens and 2 stigmas, but differs from the former by its scrambling habit, nodes swollen at one side, mid-culm branch complement with one central dominant branch elongating to reiterate the culm accompanied by 1-4 lateral slender branches, swollen culm sheath base with a distinctive zone of transverse wrinkles, culm and leaf sheaths without auricle or oral setae, and single or several to many synflorescences arranged into a raceme or panicle, and can be distinguished from the latter by extravaginal branching pattern, buds wholly sunken into culm, culm sheath base with a distinctive zone of transverse wrinkles, synflorescence composed of only one spikelet, purple anthers and nut-like caryopsis with hardened pericarp and loosely adherent lemma and palea.

Khoonmengia honbaensis
Etymology. Khoonmengia is named in honor of Dr. Khoon Meng Wong, a renowned botanist who has studied the bamboos and other plant groups of Southeast Asia for more than 35 years. The specific epithet is named after Hon Ba Nature Reserve, the type locality of this species.
Distribution and habitat. This species was only found in the type locality, i.e. Hon Ba Nature Reserve, Khanh Hoa Province of Vietnam. It occurs in high mountain broadleaved forests at an elevation of ca. 1500 m.

Discussion
Morphological analysis (Table 1) revealed that this unknown bamboo owns several unique vegetative and reproductive characters that are different from the two closely related genera, i.e. Ampelocalamus and Hsuehochloa, such as culm with brownish green dots (Figs 2F, 5A), swollen culm sheath base with a distinctive zone of transverse wrinkles (Fig. 5A), synflorescence composed of solitary spikelet, single (Figs 2J-K, 3B, 4H) or several to many synflorescences arranged into a raceme or panicle (Figs 3A, 4G) terminal on leafy flowering branches, and nut-like caryopsis with loosely adherent lemma and palea (Fig. 3J). The nut-like bamboo caryopsis is different from the usual grain-like one by the hardened pericarp, and is reported only in some species of Bambuseae such as Cephalostachyum pallidum Munro, Dendrocalamus membranaceus Munro and D. strictus Nees before (Yu et al. 1993). Thus, the nut-like caryopsis type seems very rare in Arundinarieae. Our unknown bamboo species is also different from Ampelocalamus in the extravaginal branching pattern (vs. transferring), elliptic buds wholly sunken into culm (vs. ovate to broad ovate, not sunken or only base sunken into culm) (Fig. 6), and purple anthers (vs. yellow). For these three important generic characters, Hsuehochloa is the same as our unknown bamboo, which makes us infer that the closest genus to Khoonmengia may be Hsuehochloa. Although some important characters of Hsuehochloa such as number of glume, caryopsis type, are still unknown, besides the differences mentioned above, our unknown bamboo species can be further distinguished from Hsuehochloa by its scrambling habit (vs. pendulous or procumbent, not scrambling, according to the third author's field observation), nodes swollen at one side (vs. flat, Fig. 5B), mid-culm branch complement with one central dominant branch elongating to reiterate the culm accompanied by 1-4 lateral slender branches (vs. subequal 3-7 branches), and culm and leaf sheath auricle or oral setae absent (vs. present). Moreover, Hsuehochloa grows on the limestone mountain, while the unknown species grows in granitic montane broadleaved forests. More detailed comparisons of  these genera are summarized in Table 1. The GBSSI phylogeny revealed that our putative new species is definitely a member of Arundinarieae with an isolated position, which indicated that this species could not be assigned to any of the already   described genera. Based on the above analysis of morphology, molecular phylogenetic relationships and habitat, we propose to establish a new genus to accommodate this unknown bamboo.