A new genus of temperate woody bamboos (Poaceae, Bambusoideae, Arundinarieae) from a limestone montane area of China

Abstract Ampelocalamuscalcareus is a climbing and slender bamboo, known from south Guizhou, China. This species grows in broadleaved forests of limestone montane areas. Recent molecular phylogenetic analyses demonstrated that A.calcareus was sister to all other lineages of the tribe Arundinarieae rather than a member of Ampelocalamus. The morphological features and habitats of A.calcareus and related genera including Ampelocalamus, Drepanostachyum and Himalayacalamus were compared and discussed. The characteristics of the branch complements, nodes and foliage leaves distinguish A.calcareus from morphologically similar taxa. On the basis of molecular and morphological evidence, we propose to establish a new genus, Hsuehochloa, to accommodate A.calcareus and to honour the late Chinese bamboo taxonomist Chi-Ju Hsueh (Ji-Ru Xue). In addition, we describe the inflorescence of Hsuehochloa for the first time.


Introduction
Temperate woody bamboos or the tribe Arundinarieae (Bambusoideae, Poaceae) comprise approximately 550 species in 31 genera (BPG 2012, Clark et al. 2015. They are mainly distributed in temperate to subtropical montane areas of East Asia (Ohrnberger 1999) with China as the centre of species diversity (Li et al. 2006), but also in Southeast Asia, south India, Sri Lanka, North America and Africa.
The recent plastid molecular phylogenetic results indicated that there were 12 major lineages in temperate woody bamboos, i.e. I. Bergbambos, II. Oldeania, III. Chimonocalamus, IV. Shibataea clade, V. Phyllostachys clade, VI. Arundinaria clade, VII. Thamnocalamus, VIII. Indocalamus wilsonii, IX. Gaoligongshania, X. Indocalamus sinicus, XI. Ampelocalamus calcareus and XII. Kuruna. However, relationships amongst them remain largely uncertain (Triplett and Clark 2010, Zeng et al. 2010, Yang et al. 2013, Attigala et al. 2014, Ma et al. 2014, Zhang et al. 2016, Zhang et al. 2017. Those lineages are strongly inconsistent with the morphological classification at the generic and subtribal levels (Keng and Wang 1996, Li 1997, Ohrnberger 1999. Most species and genera were nested within lineages IV, V and VI, while some lineages included only one species (lineages I, VIII, IX, X, XI). Lineages I and IX consisted of Bergbambos tessellata (Nees) Stapleton and Gaoligongshania megalothyrsa (Handel-Mazzetti) D. Z. Li, Hsueh & N. H. Xia, respectively and Bergbambos Stapleton and Gaoligongshania D. Z. Li, Hsueh & N. H. Xia are both monotypic (Li et al. 1995, Stapleton 2013. Lineages VIII and X were formed by Indocalamus wilsonii (Rendle) C. S. Chao & C. D. Chu and I. sinicus (Hance) Nakai, respectively, with I. sinicus as the lectotype of the genus Indocalamus Nakai. Ampelocalamus calcareus C. D. Chu & C. S. Chao (lineage XI) was recovered as the sister taxon to all the other temperate woody bamboos (Yang et al. 2013, Ma et al. 2014. The phylogenetic positions of the abovementioned five monotypic lineages have also obtained some support from nuclear gene trees (Zhang et al. 2012, Yang et al. 2013. Molecular phylogenetic results provide fresh perspectives for taxonomy, especially for lineages VIII, X and XI with only one species. Continuing to include these bamboos in the present genera renders these genera polyphyletic and causes problems when describing or citing them. In this paper, we propose to establish a new genus for Ampelocalamus calcareus based on morphological characters and previous molecular results. For the other two monotypic lineages (VIII and X), taxonomic revisions will be made in a separate paper.

Materials and methods
Drepanostachyum P. C. Keng and Himalayacalamus P. C. Keng are morphologically close to Ampelocalamus S. L. Chen, T. H. Wen & G. Y. Sheng (Li et al. 1996). These three genera all have pachymorph rhizomes, prominent or conspicuous nodal sheath scars and pendulous culms. Sometimes it is difficult to see the difference when only the vegetative features are available. Some species of Ampelocalamus were transferred from the genus Drepanostachyum (Keng and Wang 1996, Stapleton et al. 2005, Li et al. 2006 and several taxa of Drepanostachyum were combined into Himalayacalamus (Stapleton 1994). It is necessary to compare characters of Ampelocalamus calcareus with those two genera in order to clarify their morphological similarities and differences.

Specimen examination
The type specimen of Ampelocalamus calcareus was examined at the herbarium of Nanjing Forestry University (NF). We also examined specimens of A. calcareus, other species of Ampelocalamus, Drepanostachyum and Himalayacalamus at herbaria of Kunming Institute of Botany, Chinese Academy of Sciences (KUN), Nanjing University (N), Institute of Botany, Chinese Academy of Sciences (PE) and Sichuan Agricultural University, Dujiangyan Campus (SIFS) (specimens of N and PE were checked through the website http://www.cvh.ac.cn/).

Living plant observation
In 2010, one clump of Ampelocalamus calcareus was introduced by P. F. Ma and Z. M. Cai from Libo, Guizhou and cultivated at the greenhouse of the Germplasm Bank of Wild Species (GBOWS), Kunming Institute of Botany, Chinese Academy of Sciences, Kunming. This introduced clump flowered between 2012 and 2013. Unfortunately, the clump did not grow well and only a small piece of inflorescence was collected before it died. After fieldwork in early 2015, it was confirmed that the wild populations also flowered and died during the same period. More seedlings were introduced into the greenhouse of GBOWS at Kunming by C. Guo and Y. Guo in March 2015. They grew well in the greenhouse and became mature individuals after three years. Vegetative features including culms, culm sheaths, branch complements and foliage leaves were observed based on those individuals.

Morphological comparison
Four reproductive characters and 15 vegetative characters were selected and compared across Ampelocalamus calcareus, Ampelocalamus, Drepanostachyum and Himalayacalamus. For A. calcareus, the morphological data were observed and obtained based on herbarium specimens, living plants and literature. We observed and measured the structure of the inflorescence of A. calcareus by hand lens (30×) and stereomicroscope (Leica M166FC) without dissecting the spikelet due to the scarcity of materials. For the other genera, the morphological data were obtained from specimens and literature. The habitats of Ampelocalamus calcareus, Ampelocalamus, Drepanostachyum and Himalayacalamus were also compared based on literature.

Results
Morphological characteristics and habitat data are summarised in Table 1. Ampelocalamus calcareus and species of Ampelocalamus, Drepanostachyum and Himalayacalamus are all unicaespitose. Culms of A. calcareus are procumbent or scrambling, while culms The inflorescence of Ampelocalamus calcareus is semelauctant and racemose. The spikelet has five florets and the floret possesses a purple-green lemma (ca. 1 cm long), palea shorter than the lemma (ca. 0.8 cm long), three purple stamens (4 mm long) and two plumose stigmas. The inflorescence of Ampelocalamus, Drepanostachyum and Himalayacalamus has been described in detail in other literature (e.g. Stapleton 1994, Li et al. 2006, therefore, we only list some key features in Table 1. Analysis of the habitat data demonstrates that Ampelocalamus calcareus mainly occurs under broadleaved forests of limestone areas below 1000 m; other Ampelocalamus species grow under broadleaved forests, on stony slopes (limestone, granite or basalt) and riverside slopes usually from 200 m to 1800 m alt.; taxa of Drepanostachyum are usually distributed under coniferous and broadleaved mixed forests from 1300 m to 3200 m alt.; species of Himalayacalamus occur under temperate forests from 1200 m to 3000 m alt. (Table 1).

Discussion
Ampelocalamus calcareus was described by Chao and Chu (1983) based on vegetative specimens. This species has pachymorph rhizomes with short necks and apically drooping culms (Fig. 1) that are similar to other species of the genus Ampelocalamus, especially to the type species A. actinotrichus (Merrill & Chun) S. L. Chen, T. H. Wen & G. Y. Sheng. Moreover, the conspicuous auricles and radiate oral setae on the culm sheath and leaf sheath are similar to A. actinotrichus as well. However, characteristics of nodes, branch complements and leaf blades are quite different from Ampelocalamus. Ampelocalamus calcareus has inconspicuous nodal sheath scars, a solitary branch at the base and 3-7 subequal branches at the middle and upper parts of the culm and leathery leaf blades. Other taxa in Ampelocalamus usually possess prominent nodal sheath scars with a corky collar, many branches with a central dominant one that may replace the culm and papery leaf blades. Branches at the nodes of A. calcareus are long (50-100 cm), pendulous and nearly as thick as the culm, which makes culms scrambling or procumbent. There are also some other vegetative features that can distinguish A. calcareus from typical Ampelocalamus species, as summarised in Table 1.
Culms of Drepanostachyum and Himalayacalamus are distally pendulous, but not scrambling, which is different from Ampelocalamus calcareus. Branches on mid-culms of Drepanostachyum and Himalayacalamus are usually more than 15 in number and subequal without a central dominant one, while A. calcareus has no more than 10 subequal branches. Culm sheaths of Drepanostachyum and Himalayacalamus are usually deciduous and glabrous abaxially, whereas culm sheaths of A. calcareus are persistent and densely white pubescent abaxially. Ampelocalamus calcareus has conspicuous auri- cles and oral setae on culm sheaths and leaf sheaths and ovate-lanceolate culm blades, while auricles and oral setae are often absent and culm blades are subulate or linear in Drepanostachyum and Himalayacalamus.
Due to the incomplete nature of the flowering material (Fig. 1), the description and comparison provided in Table 1 may not be fully accurate for healthy individuals flowering in the wild. The type of inflorescence of A. calcareus is similar to Himalayacalamus (racemose); the number of florets per spikelet is similar to Ampelocalamus and Drepanostachyum (5 vs. 2-7); they all have three stamens and two plumose stigmas, but the anther colour of A. calcareus is purple while anthers are yellow in Ampelocalamus, Drepanostachyum and Himalayacalamus.
Through comparison of morphological characters, we conclude that Ampelocalamus calcareus morphologically resembles species of Ampelocalamus, Drepanostachyum and Himalayacalamus in its pachymorph rhizomes and is especially similar to Ampelocalamus in its climbing habit. However, the branch complements and the characteristics of its nodes, culm sheaths and foliage leaves can distinguish this species from all taxa in these three genera. The inflorescence of A. calcareus is also similar to these three genera (on the basis of our incomplete material) in its semelauctant structure, the presence of three stamens and two stigmas.
Molecular phylogenetic studies indicated that Ampelocalamus, Drepanostachyum and Himalayacalamus had a close relationship in nuclear gene based phylogenies, although only limited taxa of those genera were sampled (Yang et al. 2013). Nonetheless, Ampelocalamus calcareus was sister to all the other taxa of the tribe Arundinarieae in plastid and nuclear gene trees (Yang et al. 2013, Ma et al. 2014, Attigala et al. 2016, Zhang et al. 2016. The morphological similarity between the distantly related A. calcareus and those three genera (Ampelocalamus, Drepanostachyum and Himalayacalamus) demonstrated that morphological characters had undergone complex evolutionary trajectories in those taxa and also in the whole tribe and some important features in bamboo taxonomy were homoplastic or convergent that was illustrated in other studies of Arundinarieae and some tropical woody bamboos (Yang et al. 2008, Tyrrell et al. 2012, Attigala et al. 2016. The habitat and altitude of A. calcareus are more similar to other typical species of Ampelocalamus than they are to Drepanostachyum and Himalayacalamus (Table 1).
Etymology for assistance in photographing. We also thank Dr Maria S. Vorontsova of Royal Botanic Gardens Kew and two anonymous reviewers for their constructive suggestions. This study was funded by the National Natural Science Foundation of China (Grants 31430011 and 31760049) and facilitated by the Germplasm Bank of Wild Species.