﻿Yushaniadezhui (Poaceae, Bambusoideae), a new bamboo species from Yunnan, China

﻿Abstract A new bamboo species, Yushaniadezhui, from Kunming, Yunnan, China is described and illustrated in this paper. The new species used to be misidentified as Y.polytricha. Based on careful comparison of morphological features and molecular phylogeny evidence, we confirmed its identity as a new member of the genus Yushania. Yushaniadezhui resembles Y.maculata, Y.polytricha and Y.weixiensis in several aspects, such as culm height and branch complement structure. However, the glabrous culm leaf sheaths and internodes, the absence of auricles and oral setae on most foliage leaves, except the one-year-old foliage leaves, the pubescence on the adaxial surface of the one-year-old foliage leaves and its limestone habitat preference can readily distinguish this new species from its related taxa. Moreover, we emphasise that individuals from various populations and molecular markers with different inheritance patterns for phylogeny reconstruction should be included in new species discovery, especially in plant groups with complex evolutionary histories.


Introduction
Yushania P.C. Keng is a genus diversified within the Hengduan Mountains region and followed by subsequent spreading outside this region (Ye et al. 2019).It belongs to the subtribe Thamnocalaminae of the tribe Arundinarieae (Poaceae, Bambusoideae) (Zhang et al. 2020) and consists of more than 90 species (Vorontsova et al. 2017;Shi et al. 2022).Taxa of Yushania are usually distributed in mountainous areas above an elevation of 1000 m and most of them are narrowly endemic to a certain region (Li et al. 2006).Due to the special habitat and distribution pattern, new species of Yushania are continuously being described recently from different mountains, such as Y. tongpeii D.Z.Li, Y.X.
There are two Yushania species recorded in Kunming, Yunnan, China, i.e.Y. maculata T.P. Yi and Y. polytricha T.P. Yi (Sun et al. 2003).The type specimen of Y. polytricha was collected at the Qiongzhu Temple in the west of Kunming (Yi 1986).During field investigations in recent years, we found a species of Yushania occurring in western and northern Kunming, sometimes sympatric with Y. maculata and Y. polytricha.This species has been misidentified as Y. polytricha for many years in floristic treatments and community ecology papers.The short and thin culms of this species resemble Y. polytricha, but the branches, auricles of culm leaves and foliage leaves and hairs on the culms, culm leaves and foliage leaves differ significantly from those of Y. polytricha.In order to clarify the identity of this species, we performed several field surveys at different times and localities during 2023.We finally confirm that it is new to science based on morphological and molecular evidence and describe it in this paper.

Field investigation, morphological feature observation and comparison
Several field investigations were carried out during late June to late August 2023 in Kunming, including Changchong Hill and Xiaoshao Village of Ciba Town, Haikou Forest Farm of West Hill District and Xianfeng of Xundian County.Specimens were collected in the field and deposited at the Herbarium of the Kunming Institute of Botany, Chinese Academy of Sciences.Young and healthy foliage leaves were sampled and dried in silica gel for the molecular experiment.Morphological features of the new species were observed and recorded in the field and the lab.Comparison of morphological characteristics amongst sympatric or morphologically similar taxa in Yushania (i.e.Y. maculata, Y. polytricha, Y. weixiensis T.P. Yi) was performed, based on field observation, specimens and literature (e.g.Li et al. (2006)).

DNA extraction, sequencing and assembly
Total genomic DNA was extracted from the silica gel-dried leaves using TIAN-GEN Magnetic Plant Genomic DNA Kit (TIANGEN, Beijing, China).All procedures were performed according to the manufacture's instruction.DNA concentration, integrity and purity were examined using the Agilent 5400 system (Agilent, USA).Subsequently, the DNA samples were fragmented by sonication to a size of 350 bp.Then DNA fragments were end-polished, A-tailed and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification.PCR products were purified by AMPure XP system (Beverly, USA).Library quality was assessed on the Agilent 5400 system (Agilent, USA)and quantified by QPCR (1.5 nM).The qualified libraries were pooled and sequenced on Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) with PE150 strategy.Finally, a total of 2 GB data per sample was generated.All those experiments were carried out at Novogene Bioinformatics Technology Co., Ltd (Beijing, China).
After quality control of the raw data by Fastp 0.23.2 (Chen et al. 2018) with default parameters, all paired reads were extracted for plastid assembly using Get-Organelle 1.7.2 (Jin et al. 2020) and the plastome sequence of Y. niitakayamensis (Hayata) P.C. Keng (MN310560.1)was used as a reference.Subsequently, the de novo assembled maps were conducted using Bandage 0.9.0 (Wick et al. 2015) to visualise the complete plastomes.The assembled plastome sequences were aligned and checked collinearly by Mauve 2.4.0 (Darling et al. 2004) with default settings in order to make sure the structure and direction were consistent with the reference plastome.The plastid genes were annotated using CPGAVAS2 online (Shi et al. 2019) based on the annotation of Y. niitakayamensis with manual adjustment in Geneious Prime 2022.0.1 (Kearse et al. 2012).In addition, the ribosomal DNA sequence of Oryza sativa cultivar TN1 (KM036285.1)(Guo et al. 2021) was used as a reference to assemble and annotate the entire nrDNA sequences in Geneious Prime 2022.0.1.

Phylogenetic analysis
To confirm the phylogenetic affinity of the new species, plastome and nrDNA sequences of representatives of the tribe Arundinarieae were used to reconstruct the phylogenetic trees, based on the results of Guo et al. (2021).A total of 30 sequences from 26 species were selected, including eight newly-sequenced plastome and nrDNA sequences, respectively (Table 1).The plastome and nrDNA sequences were aligned using MAFFT 7.520 (Katoh and Standley 2013).The TPM3+F+I+G4 model for plastomes and the TPM3+F+I+G4 model for nrDNA were selected using ModelFinder 2.2.5 (Kalyaanamoorthy et al. 2017), based on the Bayesian Information Criterion (BIC).Subsequently, Maximum Likelihood (ML) analyses were performed using IQ-TREE 2.2.5 (Nguyen et al. 2015) with 1000 ultrafast bootstrap replicates and SH-aLRT test (Hong et al. 2022).Bayesian Inference (BI) was conducted in MrBayes 3.2.7a(Ronquist et al. 2012) and the GTR+I+G model was selected by jModelTest 2.1.7(Darriba et al. 2012) using BIC.Markov Chain Monte Carlo (MCMC) simulations were run for 1,000,000 generations, with a sampling every 1000 generations.The initial 25% of generations were discarded as burn-in.A 50% majority-rule consensus tree was constructed when the average standard deviation of split frequencies was below 0.01.

Results
The new species (Yushania dezhui), Y. maculata and Y. polytricha all have solid rhizome necks, while Y. weixiensis possesses hollow ones.The culm height of Y. dezhui, Y. polytricha and Y. weixiensis is usually less than 2 m, whereas Y. maculata is more than 2 m tall.The internode and sheath scar of Y. dezhui are glabrous, which is different from the other three related species, which have at least some hairs on the internode and sheath scar.Dark purple-brown spotted culm leaf sheaths and purple oral setae differentiate Y. maculata from Y. dezhui.Auricles and oral setae of culm leaves and foliage leaves of Y. dezhui are usually absent, while Y. polytricha has conspicuous auricles and oral setae.Moreover, a different branch number per node and setae on culm leaves and foliage leaves can also distinguish Y. dezhui from Y. polytricha (see Table 2 for details).Although Y. dezhui and Y. weixiensis both have relatively short culms, usually glabrous culm leaves without auricles and oral setae and similar branch numbers, characters of rhizome neck, internode and foliage leaf can differentiate these two species (see Table 2 for details).The length of the eight newly-sequenced plastomes ranged from 139599 bp (Y.shuichengensis T.P. Yi & L. Yang LPS15) to 139653 bp (the new species Y. dezhui XS01), with a consistent GC content of 38.90%.They showed a typical quadripartite circular structure, which consisted of a pair of inverted repeat regions (IR), one large single copy region (LSC) and one small single copy region (SSC).The plastome length of the new species varied from 139616 bp (ZSC03) to 139653 bp (XS01).The newly-assembled eight nrDNA sequences had a length from 8337 bp in Y. shuichengensis (LPS22) to 9010 bp in Y. polytricha (QZS001) and comprised of 18S (1811 bp), ITS1 (215-216 bp), 5.8S (165 Foliage leaf blade 2.5-12.5 × 0.5-1.3cm, pubescent adaxially for the one-year-old blades, glabrous abaxially, secondary veins 2-4 paired 9-15 × 0.9-1.1 cm, glabrous, secondary veins 4-paired 9-21 × 1.2-2.5 cm, abaxially grey pubescent, secondary veins 4-6-paired 3.4-7 × 0.3-0.6 cm, glabrous, secondary veins 2-or 3-paired bp), ITS2 (215-217bp), 26S (3392 bp) and an intergenic spacer (IGS, 2539-3212 bp).For the new species, individuals ZSC03 and CCS03 had the same length (8695 bp) of nrDNA sequences, while the length of the individual XS01 was four bp longer than the individual DSTQ02 (216 bp vs. 217 bp in the ITS2 region and 2898 bp vs. 2893 bp in the IGS region).
Phenology.New shoots June to August.2021).The relationships amongst the five subtribes in the nrDNA tree were not well resolved, which was also consistent with Guo et al. (2021).The four individuals of Yushania dezhui were grouped into two subclades in the plastome tree (Fig. 1), whereas they formed a clade with high support (100%) in the nrDNA tree (Fig. 2).A similar situation applied to Y. shuichengensis.Discordance between plastome and nuclear gene trees has been recovered in the genus Yushania and other genera, even in the tribe Arundinarieae (e.g.Zhang et al. (2012); Yang et al. (2013); Guo et al. (2019); Guo et al. (2021); Ye et al. (2021a)).The cytonuclear incongruence was mostly caused by the complex evolutionary history of those taxa, including hybridisation, introgression, incomplete lineage sorting and so on.Ye et al. (2021a) revealed the reticulate evolutionary history of the genera Fargesia Franchet and Yushania, based on genome skimming and double digest restriction-site-associated DNA sequencing data.In their study, only several species with multiple individuals were clustered as monophyletic and most species with multiple individuals were resolved as polyphyletic in the plastome trees.Our results referring to Y. dezhui and Y. shuichengensis were consistent with Ye et al. (2021a).In the DNA barcoding study of Fargesia, the nrDNA sequences showed better discriminatory power than the plastomes and some species recovered as polyphyletic in the plastome trees were resolved as monophyletic in the nrDNA phylogenies (Lv et al. 2023).The cases of Y. dezhui and Y. shuichengensis were also congruent with that of Fargesia.However, the specific reasons for the cytonuclear incongruence of Y. dezhui and Y. shuichengensis need to be tested within a more comprehensive sampling background in the future.On the whole, all the aforementioned studies, including our own, have demonstrated that it was better to exploit molecular markers with different inheritance patterns in discovering new species, especially those with complicated evolutionary histories.

Figure 1 .
Figure 1.Maximum Likelihood phylogenetic tree reconstructed from plastome sequences of the tribe Arundinarieae.Numbers along branches indicate the Maximum Likelihood bootstrap values (MLBP) and Bayesian posterior probabilities (BI).* means MLBP/BI=100/1.00.The Roman numbers on the right of this tree correspond to those lineages recovered in previous studies(Yang et al. 2013).The branch in red (MLBP/BI = 88/1.00)denotes the clade containing Yushania dezhui and its relatives.

Figure 2 .
Figure 2. Phylogenetic tree reconstructed from nrDNA sequences of the tribe Arundinarieae by using the Maximum Likelihood method.Numbers along branches indicate the Maximum Likelihood bootstrap values (MLBP) and Bayesian posterior probabilities (BI).* means MLBP/BI = 100/1.00.

Figure 3 .
Figure 3. Yushania dezhui Y.X.Zhang & R.L.Zhang A clump B foliage leafy branch C branches D internode and culm leaf.

Figure 5 .
Figure 5. Yushania dezhui Y.X.Zhang & R.L.Zhang A foliage leaves with green sheath B foliage leaves with purple sheath C, D branches E internode with culm leaf F the one-year-old foliage leaf blade, showing the pubescent adaxial epidermis, ab = abaxial epidermis, ad = adaxial epidermis G auricles, oral setae and ligule of the one-year-old foliage leaf, a = auricles, l = ligule, s = oral setae.Photos A-E by Yu-Xiao Zhang F, G by Shun-Shun He.

Table 1 .
Voucher information and GenBank accession numbers for plant materials used in this study.

Table 2 .
Morphological comparison of Yushania dezhui and related species.