﻿Gelidocalamuszixingensis (Poaceae, Bambusoideae, Arundinarieae), a new species from southern China revealed by morphological and molecular evidence

﻿Abstract The genus Gelidocalamus T. H. Wen, endemic to southern China, is a small but taxonomically problematic genus of Arundinarieae (Poaceae, Bambusoideae). During field work, a population of Gelidocalamus from Zixing, Hunan, was discovered, appearing to be distinct from our previously identified collection. Comparisons of the population of Zixing were performed by using scanning electron microscopy (SEM) and a plastid genome-based phylogeny. Morphologically, it was mostly similar to G.multifolius, but differed by culm leaf erect with densely white pubescence, apical branch sheath much longer than the internodes and foliage leaf larger. Phylogenetically, the new species was well-supported as a sister to the clade of G.multifolius + G.tessellatus, and the above three taxa were clustered in the Shibataea clade (IV) of Arundinarieae. Thus, the new species, formally named as Gelidocalamuszixingensis W.G.Zhang, G.Y.Yang & C.K.Wang, was described and illustrated herein.


Introduction
Arundinarieae (Poaceae: Bambusoideae), i.e., accommodating the temperate woody bamboos, including ca. 581 species in 31 genera (Clark and de Oliveira 2018), is widely accepted as a monophyletic tribe (Guo et al. 2021;Gallaher et al. 2022;Huang et al. 2022), and has been one of the main focuses of botanical research due to its significant ecological and economic value (Triplett 2008). It is mainly distributed in the tropical and subtropical mountains of East Asia, central and southern Africa, Madagascar and eastern North America (Keng and Wang 1996;Soreng et al. 2022). Due to complex allopolyploid history and adaptive radiation events, Arundinarieae has evolved complex and diverse morphological characters, e.g., semelauctant and iterauctant inflorescences, pachymorph and leptomorph rhizomes, and growth habits from solitary to multiple branches, which made it a taxonomically complicated group (Li et al. 2006;Vorontsova et al. 2016;Guo et al. 2021).
As a small but taxonomically problematic genus of Arundinarieae, Gelidocalamus T. H. Wen, 1982, containing ca. 11 species (Li et al. 2006;Zhang et al. 2017;Cai et al. 2021), is endemic to southwestern China, and characterized by a set of morphological features including several branches per node, a single foliage leaf on each ultimate branch typically (except G. multifolius B. M. Yang, 1986) (Yang 1986), and semelauctant inflorescence. In addition, the new shoots occurring in autumn to winter are also a key feature of Gelidocalamus (Keng and Wang 1996;Liu et al. 2017;Nie et al. 2018). Members of Gelidocalamus have a relatively narrow distribution in the southern provinces of China and usually occur along ravines under broad-leaved evergreen forest below 1,000 m elevation, except G. monophyllus (Yi et B. M. Yang) B. M. Yang, 1989, distributed at 1250 m (Li et al. 2016;Nie et al. 2018). However, some "spring-shoot" species (as opposed to some others that produce shoots in the autumn-winter period), e.g., G. rutilans Wen, 1983, G. subsolidus W. T. Lin & Z. J. Feng, 1990, G. solidus C. D. Chu & C. S. Chao, 1984, and G. longiinternodus W. T. Wen & S. C. Chen, 1986, complicate the delimitation of the genus.
During field work in August 2014, a population of Gelidocalamus sp. in Zixing City of Hunan Province, China (25°54'1.75"N, 113°34'9.18"E), was found, and mistakenly identified as G. multifolius due to a somewhat similar morphology. In this study, a detailed comparison among the new species, G. multifolius and G. tessellatus, including characters obtained with scanning electron microscope (SEM) of the foliage leaf epidermis, was made. Moreover, the phylogenetic relationships of the new species including above taxa and allied genera were reassessed based on complete chloroplast genomes.

Field investigation and sample collection
Mature bamboo leaves were collected from the individuals of the type localities: G. sp from Zixing, Lianping Township of Zixing City in Hunan; G. stellatus, Xiazhuang of Jinggang Mountain in Jiangxi; G. tessellatus, Maolan of Libo County in Guizhou; G. multifolius, Jiuyi Mountain of Ningyuan County in Hunan. Foliage leaves were fixed with the FAA (acetic acid: formalin: ultrapure water: ethanol = 1:2:3:14), and some dried in silica-gel for storage. All voucher specimens were deposited in the herbarium of the College of Forestry, Jiangxi Agricultural University, China (JXAU).

Micromorphological observations of foliage leaf epidermis
After cleaning in the ultrasonic cleaner CPX2800H-C (Branson, USA), the middle portion of foliage leaf (5×5 mm) was dried at room temperature, mounted on stubs, and coated with gold sputtering. Using a scanning electron microscope S-4800 (Hitachi, Japan), leaf epidermal characters were observed and photographed. Terminology for epidermal appendages and leaf blades follows previous studies (Ellis 1979;Ellis et al. 2009;Zhang et al. 2014;Leandro et al. 2019).

Sequencing, assembly and annotation
Total genomic DNA was isolated from foliage leaves dried over silica-gel by a modified CTAB method (Murray and Thompson 1980). Illumina paired-end (2×150 bp) libraries were constructed and sequenced at Novogene Bioinformatics Technology Co. Ltd. (Beijing, China), and ca. 6 GB raw data for each sample was acquired. To improve assembly accuracy, FastQC 0.11.9 (Andrews 2016) and Fastp 0.12.4 (Chen et al. 2018) were used to filter out unpaired and low-depth reads by using default parameters. Complete chloroplast genomes were assembled using the software GetOrganelle 1.7.4 (Jin et al. 2018) with a range of k-mers of 45, 65, 85, 105 and 121, and the filtered reads were transferred to Bandage (Wick et al. 2015) for chloroplast genome scaffolds connection. Then, chloroplast genome sequences were annotated by using CPGAVAS2 (Shi et al. 2019) and manually checked in Geneious 9.1.4 (Kearse et al. 2012), and illustration of the newly sequenced plastome was drawn in the software Chloroplot with default settings (Zheng et al. 2020).

Phylogenetic analysis
To determine the position of the new species, phylogenetic analyses using maximum likelihood (ML) and Bayesian inference (BI) were performed. Besides G. sp. from Zixing (OP920758) and G. multifolius (OP920759), another 18 complete chloroplast genomes of the tribe Arundinarieae were obtained from the National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/). Hsuehochloa Zhang, 2018 was selected as outgroup (Genbank accession numbers see the Table 1 for details).
After alignment with MAFFT 7.450 (Katoh and Standley 2013), Maximum likelihood (ML) analysis was generated by IQ-TREE (Nguyen et al. 2015), bootstrap analyses were performed with 1,000 replications, and the best-fit BIC model GTR + F + I + G4 was defined by ModelFinder (Kalyaanamoorthy et al. 2017). Bayesian inference (BI) was conducted using MrBayes 3.2.6 (Ronquist et al. 2012) with the same model. 20,000,000 generations were run to ensure average standard deviation of split frequencies (ASDFs) < 0.01 with sampling frequency set as 2,000 generations. Discarding the first 25% burn-in samples, the optimized topology was printed.

Morphological comparison
Compared to G. tessellatus, G. zixingensis was mostly similar to G. multifolius in the habit, the morphological characters and new shoots sprouting season, but can be distinguished by the following morphological characters: (a) a ring of white-gray (vs. yellow-brown) appressed pubescence below each culm node; (b) culm leaf sheaths densely white pubescent (vs. glabrous), with brown patches (vs. pale brown), and erect (vs. reflexed) culm sheath blades; (d) branch leaf sheaths setose (vs. glabrous) and much longer, (>3cm) (vs. slightly longer, < 1cm or as long) as the internodes; foliage leaf blades mesophyll (vs. notophyll). (see Table 2, Fig. 1 for details).

Micromorphological comparison of abaxial foliage leaf epidermis
Epidermal traits of the foliage leaf, e.g., short papillae, microhairs, silica bodies and prickles, can be clearly identified under the scanning electron microscope (Fig. 2). Main characters shared by the four selected taxa were: (a) exposed stomatal apparatus, densely covered with white wax and surrounded by 8-10 short papillae; (b) bicellular microhairs, of which the apical one was withered; (c) saddle-shaped silica body, mainly distributed between veins (Table 3). Prickles were sparsely distributed between the veins in G. zixingensis and G. multifolius, and more densely distributed in the G. tessellatus, while no prickles were observed in G. stellatus. Besides, the number of stomatal rows was different, e.g., 3 in G. tessellatus, 3 or 4 in G. stellatus, 4 in G. multifolius, but 5 in G. zixingensis.

Phylogenetic analyses based on complete chloroplast genomes
The complete chloroplast genome of Gelidocalamus zixingensis was 139,500 bp in length, comprising a large single copy (LSC) region of 83,007 bp, a small single copy (SSC) region of 12,809 bp and two inverted repeat (IR) regions of 21,842 bp, and its GC content was 39%. The chloroplast genome contained 132 genes, including 85 protein-coding genes, 39 transfer RNAs and 8 ribosomal RNAs (Fig. 3), and the total length of the aligned plastid matrix data was 143,738 bp. Compared to that in G. zixingensis, the total length of chloroplast genome of G. multifolius and G. tessellatus was longer (>200bp), and the differences were mainly in the LSC region (Table 4)    The majority-rule consensus tree with both maximum likelihood (ML) and Bayesian inference (BI) analyses was shown in Fig. 4. Arundinarieae is well-supported as a monophyletic entity, finely divided into 12 lineages (I-XII). There is high support for the G. zixingensis being a sister to the G. multifolius + G. tessellatus clade (bootstrap value of 100% in ML analysis and posterior probability of 1.0 in BI analysis), and the above 3 species were clustered with members of Shibataea e.g., S. chiangshanensis and S. kumasaca, member of Sinosasa, e.g., S. longiligulata, to form the IV clade (bootstrap value of 100% in ML analysis and posterior probability of 1.0 in BI analysis).   Description. Rhizomes leptomorph. Culms 1.7-4.2 m, 3.5-10 mm in diameter; erect, apically slightly nodding; internodes initially covered with white pubescence, ca.  14-35 cm long, wall 0.6-1.9 mm thick; each node with a ring of white-gray appressed pubescence below sheath scar; branching intravaginal, arising from 5 th node above ground, ca. 4-11 (16) branches per node; branches equal or subequal, ca. 5-30 cm long. Culm leaves sheaths persistent, 12-19 cm, culm leaf sheath abaxially with brown patches, densely white pubescent and sparsely setose near the base; culm leaf blade erect, linearlanceolate, 0.5-2 cm long, 2 mm wide, apex acuminate, base blunt or truncate, ca.1/3 as wide as sheath apex, oral setae 2-4 on each side of the sheath apex, ca. 4 mm long; auricles absent; ligule truncate, ca. 0.5 mm high, scabrous. Branch sheath papery, white setose, without black spots, margins ciliate; sub-apical branch sheath ca. 3 cm beyond the internode. Foliage leaves usually solitary on ultimate branches; ligule truncate, ca. 1 mm, scabrous; auricles absent; oral setae 1-3 pairs straight or curved; leaf blade broadly lanceolate, usually 23.4-32.5×3.2-4.9 cm, lateral veins 6-8 pairs, abaxial surface basally pubescent, base cuneate and asymmetrical, margins serrulate and slightly revolute near base.
Phenology. New shoots in October. Etymology. The species epithet refers to the locality of the type specimen: Zixing County, Hunan, China.
Morphological key of the species of Gelidocaldums of China

Discussion
Morphologically, although its inflorescence is not seen so far, G. zixingensis is undoubtedly a member of the genus Gelidocalamus, because it possesses all key characters of the genus, i.e., leptomorph rhizomes, several branches per node, typically a single foliage leaf on each ultimate branch, semelauctant inflorescence (Wen 1982;Li et al. 2006;Yi et al. 2008). However, it is obviously different from other species of the genus, e.g., conspicuously longer than the internodes and culm leaf densely white pubescent. At first glance, G. zixingensis is similar to G. multifolius in appearance, but can be distinguished by a ring of white-gray appressed pubescence below each node, culm sheaths densely pubescent with brown patches, sub-apical branch sheath much longer than the internode, and a single foliage leaf on each ultimate branch. Previous studies indicated that leaf epidermal features were of taxonomic significance in Bambusoideae (Soderstrom and Ellis 1988;Yang et al. 2008;Zhang et al. 2014;Leandro et al. 2019). According to papilla form and distribution patterns around the stomatal apparatus of the abaxial leaf epidermis, Gelidocalamus can be classified into at least three types: (a) short papillae, none on the stomatal apparatus, e.g., G. stellatus, G. multifolius, G. tessellatus; (b) elongate or short papillae overarching the stomata, e.g., G. subsolidus, G. solidus; (c) many short papillae, completely covering stomatal apparatus, e.g., G. monophyllus (Wu et al. 2014). Compared to those of the "spring-shoot" taxa, leaf epidermal characters in the "gelido-" members of Gelidocalamus are relatively stable, and can be used as a diagnostic feature. In the present study, epidermal traits of foliage leaf in G. zixingensis were found to be consistent with these of six "gelido-" members in Gelidocalamus (except G. monophyllus, Liu et al. 2017;Nie et al. 2018), and the difference mainly lay in the fact that G. zixingensis had 5 rows of stomatal apparatus.
The tribe Arundinarieae was known for its complex phylogenetic relationships. Despite many previous attempts based on different datasets having been made, intractable problems, such as low resolution or heavily conflicting topologies, still arose (Zhang et al. 2012;Yang et al. 2013;Zeng et al. 2014;Ma et al. 2017). Recently, with the wider sampling of Arundinarieae, Guo et al. (2021) provided a robust phylogenetic tree of the tribe, referred from the ddRAD dataset, which was mostly consistent with the morphological data. In the phylogenetic analysis, only six "gelido-" members formed a monophyletic lineage, although all members of Gelidocalamus belonged to the leptomorph lineage. Together with our present molecular phylogenetic analysis, we confirm that G. zixingensis belongs to the genus Gelidocalamus, and it is closely related to G. multifolius.