﻿Chimonobambusasangzhiensis (Poaceae, Bambusoideae), a new combination supported by morphological and molecular evidence

﻿Abstract This study elucidates the taxonomic position of Indosasasangzhiensis in considering whether it belongs to Indosasa or Chimonobambusa. Based on morphological and molecular phylogenetic evidence, our results explicitly indicated that I.sangzhiensis should be a member of Chimonobambusa, rather than Indosasa, and is a distinct species closely related to C.communis, C.opienensis and C.puberula. Thus, the new combination Chimonobambusasangzhiensis (B.M.Yang) N.H.Xia & Z.Y.Niu is made. A detailed description as well as two color plates of this species are also provided.


Introduction
Chimonobambusa Makino (1914), belonging to the subtribe Arundinariinae of the tribe Arundinarieae (Poaceae, Bambusoideae) (Zhang et al 2020), is characterized by leptomorph rhizomes, diffuse culms, basal internodes that are 4-angled and frequently with a ring of root thorns, internodes with 2 longitudinal ridges and 3 grooves just above branching points at the node, three branches at each mid-culm node, small, triangular or subulate culm leaf blades (usually less than 1 cm long), pseudospikelets, and 3 stamens in each floret (Makino 1914;Xue and Zhang 1996;Li and Stapleton 2006). It contains about 42 species mainly distributed in China, Japan, Myanmar and Vietnam (Vorontsova et al. 2016). There are 38 species known, mainly distributed in Central, South and Southeast China (Li and Stapleton 2006;Vorontsova et al. 2016). Recent phylogenetic studies that focused on Arundinariinae have suggested that Chimonobambusa should be defined in a broad sense, including Qiongzhuea Hsueh & T.P.Yi (Hsueh and Yi 1980;Xue and Yi 1996), and thus it can be accepted as a monophyletic group, except for C. sichuanensis (T.P.Yi) T.H.Wen (Li and Stapleton 2006;Peng et al. 2008;Zhang et al. 2012;Guo et al. 2021).
Indosasa sangzhiensis B.M.Yang was described based on a collection (Vegetation Survey Group 00549) from Badagongshan, Sangzhi Xian, Hunan Province, China. In the protologue, Yang (1989) stated that this species resembled I. glabrata C.D.Chu & C.S.Chao (Chao and Chu 1983), but can be distinguished by its densely pubescent culms, solid distal internodes but hollow basal ones, few foliage leaf oral setae, as well as small and adaxially pubescent foliage leaf blades. However, after examination of the type specimen and protologue of I. sangzhiensis, we found that this species is characterized by small, erect and triangular culm leaf blades, which falls within the circumscription of Chimonobambusa rather than Indosasa.
During fieldwork at the type locality of I. sangzhiensis (Badagongshan, Sangzhi), we collected a bamboo with leptomorph rhizomes, diffuse culms and three branches at mid-culm nodes. After comparison of the specimens we collected and possibly related species, we found that it matches the type of I. sangzhiensis in both having culm internodes with densely white pubescence, solid distal internodes but hollow basal ones, ovate culm buds, small, erect and triangular culm leaf blades, glabrous culm leaf sheaths, 2-3 leaves per ultimate branches, developed foliage leaf oral setae and leaf blades with length of 9-19 cm and width of 1.2-2 cm. Therefore, we concluded that the specimens we collected are I. sangzhiensis. In addition, we found that this species is characterized by internodes with two longitudinal ridges and three grooves above the branching point, slightly 4-angled basal internodes, flat or only slightly prominent nodes, small, erect and narrowly triangular or subulate culm leaf blades, which is not typical of Indosasa species as far as we know, but conforms well with the known morphology of Chimonobambusa. Therefore, we conclude that I. sangzhiensis should be a member of Chimonobambusa, rather than Indosasa. After checking records of Chimonobambusa species from the Flora of China, we found that I. sangzhiensis is closely related to three species, viz.,  Yi 1980, 1983;Hsueh et al. 1996). In order to elucidate the relationship between I. sangzhiensis and related species, a detailed comparison of vegetative morphological characters and phylogenetic analyses were conducted in this study.

Morphological observation
The main morphological characters of I. sangzhiensis and three species of Chimonobambusa, viz., C. communis, C. opienensis and C. puberula, were compared based on protologues and descriptions from floras. Some detailed characters, such as indumentum of the culm leaf ligule, were observed with a stereomicroscope (Mshot MZ101). Measurements were taken using a ruler or micrometer.

Taxon sampling for phylogenetic analyses
Two different molecular regions including complete chloroplast genome (cpDNA) and nuclear ribosomal DNA (nrDNA) were utilized to reconstruct the phylogenetic relationships of I. sangzhiensis. A total of 23 species representing 7 genera from Arundinarieae were utilized to reconstruct the plastid tree, for which Bambusa emeiensis L.C.Chia & H.L.Fung and B. sinospinosa McClure were set as the outgroup taxa. For nrDNA, 16 species representing 5 genera from Arundinarieae were utilized to reconstruct the nrDNA tree, for which B. sinospinosa and B. multiplex (Lour.) Raeusch. ex Schult.f. were set as the outgroup taxa. The generic type of Indosasa McClure, I. crassiflora Mc-Clure, was added here to clarify the systematic position of I. sangzhiensis. All voucher information and accession number of cp genomes are listed in Table 1. Our sample information for nrDNA and its sequence matrix can be found in the supplementary material (Suppl. material 1: Table S1).

DNA extraction, sequencing, assembly and annotation
Total genomic DNA was isolated from silica-dried leaves following manufacturer specifications TIANGEN Genomic DNA Extraction Kit (TIANGEN, Beijing, China). DNA samples of concentration up to standard (≥1 μg) were randomly sheared into fragments using Covaris M220 (Covaris, Woburn, MA). Insert size of 350 bp fragments were enriched by PCR, and the paired-end (2 × 150 bp) libraries were constructed on NovaSeq 6000 platform. As a result, about 20 G genome skimming data were generated.
To improve assembly accuracy and efficiency, Trimmomatic v 0.39 were utilized to filter out unpaired and low-depth reads from clean data using default parameters (Bolger et al. 2014). The whole cpDNA and nrDNA were assembled using the software GetOrganelle v 1.7.4 pipeline (Jin et al. 2018), with five k-mer sets of 45, 65, 85, 105, 125 bp. The filtered reads were transferred to Bandage (Wick et al. 2015) for plastid and ribosomal DNA scaffolds connection. Two opposite plastid scaffolds exported from Bandage were aligned with the reference I. shibataeoides (MF066251), and the one matching the reference was annotated using the PGA software (Qu et al. 2019) based on the annotation of I. shibataeoides. The sequences were finally checked manually in Geneious 9.1.4 (Kearse et al. 2012). The assembled nrDNA sequences were annotated directly using Geneious. Illustration of the newly sequenced plastome of I. sangzhiensis was drawn by OGDRAW with default settings (Greiner et al. 2019).

Phylogenetic analysis
To determine the systematic position of I. sangzhiensis, maximum likelihood (ML) and Bayesian inference (BI) analyses were conducted. A total of 22 complete cp and 16 nrDNA genomes were aligned with MAFFT v 7.450 (Katoh and Standley 2013). Maximum likelihood (ML) analysis was generated by RAxML v 8.0.0 (Stamatakis 2014). Rapid bootstrap analysis and GTRGAMMAI were set as the best-fit algorithm and model. The number 12345 was specified as the random seed of parsimony tree inference with 1000 replicates performed. Bayesian inference (BI) was conducted using MrBayes v 3.2.6 (Ronquist et al. 2012). The model of SYM+G was defined by MrModeltest v 2.4 (Nylander 2004). The rates of variations across sites were trimmed as gamma. At least 6,000,000 generations were run to ensure average standard deviation of split frequencies (ASDFs) < 0.01 with sampling frequency set as 100 generations. After rejecting the first 25% burn-in samples, the optimized topology was printed.

Basic features of plastome and nrDNA
The plastid genome of I. sangzhiensis exhibited a typical quadripartite structure. Its genome size is 139,595 bp including a large single copy region (LSC) of 83,190 bp, a small single copy region (SSC) of 12,811 bp and a pair of inverted repeat regions (IRs) of 21,797 bp (Fig. 1A). The overall GC content is 38.9%. The whole plastid genome contains 108 unique genes, including 76 protein-coding genes, 28 transfer RNAs and 4 ribosomal RNAs. Among them, 43 genes are associated with photosynthesis and 59 genes are relevant to gene expression. For the tandemly repeated nrDNA, our de novo assembly obtained 5,799 bp sequences comprising 18S (1,811 bp), 5.8S (164 bp), and 26S (3,391 bp) ribosomal RNA gene along with two internal transcribed spacer I (ITS1) (215 bp) and ITS2 (217 bp) in the middle (Fig. 1B).

Phylogenetic analysis
The topologies based on ML and BI methods did not indicate any conflict between the cpDNA and nrDNA phylogenetic analyses, thus only ML cladograms are shown here (Figs 5, 6), with BP and PP values noted at each node. The plastid and nrDNA phylogenetic trees both strongly supported the case that I. sangzhiensis is distantly related to I. crassiflora (the type of Indosasa) (BS = 100% & PP = 1.00), but forms a sister clade with members of Chimonobambusa (BS = 95% & PP = 1.00) (Fig. 5). Our results also indicate that all samples of Chimonobambusa formed a monophyletic clade while Indosasa was found to be polyphyletic, as the samples of Indosasa and those of two other genera, viz. Acidosasa and Oligostachym, were intermixed within a clade.

Discussion
Indosasa was published by McClure (1940) based on a collection of flowering material in Vietnam. It is conventionally defined by having leptomorph rhizomes, diffuse culms, three branches per mid-culm node, prominent nodes, pseudospikelets and six stamens per floret (McClure 1940;Zhu and Stapleton 2006). Until now, it was thought that there were 18 species of Indosasa mainly distributed in South, Southwest and East China, Vietnam and Laos (Zhu and Stapleton 2006;Vorontsova et al. 2016;Niu et al. 2021). However, recent molecular evidence based on chloroplast genome and ddRAD data indicated that Indosasa is a highly polyphyletic group placed in the subtribe Arundinariinae of the tribe Arundinarieae (Ma et al. 2017;Zhang et al. 2020), and the phylogenetic relationships among Indosasa and several closely related genera with similar vegetative characters, such as Acidosasa, Oligostachyum, Sinobambusa, etc., have not been resolved (Guo et al. 2021;Niu et al. 2021). However, a broad concept was proposed for Chimobambusa based on recent molecular evidence (Li and Stapleton 2006;Peng et al. 2008;Zhang et al. 2012;Guo et al. 2021), and hence this genus has been assumed to be monophyletic so far. Although Chimonobambusa resembles Indosasa in having leptomorph rhizomes, diffuse culms, three branches at each mid-culm node and pseudospikelets, it can still be distinguished from Indosasa in having slightly 4-angled basal internodes, internodes with two longitudinal ridges and three grooves above the branching point, basal nodes often with a ring of root thorns, small, triangular or subulate culm leaf blades (usually less than 1 cm long) and each floret with 3 stamens. In this study, based on the results of morphological and phylogenetic analyses, we consider that I. sangzhiensis should be a distinct species of Chimonobambusa, rather than Indosasa, and thus, a new combination in Chimonobambusa is proposed. Note. After examining the type specimen and other specimens collected from the type locality, we are able to provide a revised description of the morphology of this species below.
Phenology. New shoots produced during April to June. Vernacular names. Lěng Zhú (Chinese pronunciation), 冷竹 (Chinese name).     Distribution and habitat. The species has only been found at its type locality so far and is rather common on mountains between elevations of 1600 m to 1900 m. It prefers a cold and moist environment and often grows under forest cover.