﻿Kengiochloa, a new bamboo genus to accommodate the morphologically unique species, Pseudosasapubiflora (Poaceae)

﻿Abstract Pseudosasa was confirmed as polyphyletic by recent phylogenetic analyses, with Chinese species of Pseudosasa distantly related to those from Japan. Among the Chinese species of Pseudosasa, Pseudosasapubiflora is a morphologically unique as well as taxonomically problematic species endemic to South China, of which the generic designation is still uncertain. Molecular analyses based on both plastid and nuclear genomic data demonstrated that this species is closest to the recently published genus Sinosasa. Morphologically, the two are somewhat similar to each other in flowering branches developing at the nodes of every order of branches, raceme-like units of inflorescence with 3–5 short spikelets, each spikelet with few florets including a rudimentary one at the apex, and each floret with 3 stamens and 2 stigmas. However, P.pubiflora is very different from Sinosasa species in many reproductive and vegetative characters, such as the morphology of paracladia (lateral spikelet “pedicels”), the absence or existence of pulvinus at the base of paracladia, the relative length of the upper glume and the lowest lemma, the shape of lodicules and primary culm buds, the branch complement, the morphology of nodes, culm leaves and dried foliage leaf blades, and the number of foliage leaves per ultimate branchlet. The morphological and molecular evidence warrants recognition of a new genus to accommodate this unique species, which is here named Kengiochloa. After consulting related literature and examination of herbarium specimens or specimen photos, a taxonomic revision of K.pubiflora and its synonyms was made, and it was confirmed that four names, viz. P.gracilis, Yushanialanshanensis, Arundinariatenuivagina and P.parilis, should be merged with K.pubiflora, while Indocalamuspallidiflorus and Acidosasapaucifolia are distinct species.

Arundinaria teuivagina W. T. Lin and P. parilis T. P. Yi & D. H. Hu, were further synonymized with P. pubiflora. Consequently, the distribution of this species was extended to include three provinces, i.e., Guangdong, Hunan and Jiangxi. Moreover, Zhu et al. (2006) argued that, for P. pubiflora, "the woolly internode apex is unusual in Pseudosasa and rather suggestive of Indocalamus Nakai". Thus, the relationships between P. pubiflora and its synonyms and the generic designation of this species need a further study.

Materials and methods
Field trips to the type locality of P. pubiflora as well as its several synonyms including I. pallidiflorus, Arundinaria teuivagina and P. parilis were conducted from 2018 to 2022. The complete specimens including rhizomes, culms with culm leaves, branches and foliage leaves as well as fresh young foliage leaves used for molecular analyses were collected. The types and other specimens of the pertinent species housed at CANT, IBSC, N, SYS and ZJFI, as well as photographs of specimens housed at K, US and W, were examined. Herbaria acronyms follow Thiers (2022). The morphological description is based on specimens and referred to related literature Wen 1985Wen , 1986Lin and Wu 1990;Yi 1995;Chen et al. 1996;Zhu et al. 2006). General morphological terms follow Beentje (2016). The terms applied to the flowering structure in this study mainly follow the synflorescence concept applied to grasses (Vegetti and Anton 1996;Tivano et al. 2009;Cai and Xia 2021).
To study the phylogenetic position of P. pubiflora within the tribe Arundinarieae, the whole chloroplast genomes were used for building the phylogenetic tree. Five Pseudosasa species including one from Japan, also the type species of the genus, viz. P. japonica, and four from China were sampled. In total, there were 29 species  represented by 30 samples of all the five subtribes in the tribe Arundinarieae (two samples for P. pubiflora) and one sample of Bambusa bambos (L.) Voss from the tribe Bambuseae was the outgroup. Species names, voucher information and GenBank accession numbers are provided in Table 1.
Total genomic DNA was isolated from silica gel-dried leaves using TIANGEN Genomic DNA Extraction Kit (TIANGEN, Beijing, China). The extracted genomic DNA was fragmented randomly by Covaris M220 (Covaris, Woburn, MA), and the fragments with insert size of 350 bp were selected by using AxyPrep Mag PCR Clean Up Kit. The selected fragments were enriched by PCR after undergoing end repair, the addition of polyA tail and adaptor ligation. The paired-end (2 × 150 bp) libraries were constructed on NovaSeq 6000 platform. Finally, 20 Gb genome skimming data were generated for each sample. After quality control of 20 Gb clean data by Trimmomatic v 0.39 (Bolger et al. 2014), the whole chloroplast (cp) genomes were assembled using the software GetOrganelle v 1.7.4 pipeline (Jin et al. 2018), with the reference cp genome sequence of Ampelocalamus sinovietnamensis (MW525255) and K-mer sizes of 45, 65, 85, 105 and 125. The generated whole cp genome was annotated using the program Plastid Genome Annotator (Qu et al. 2019) based on the annotation of A. sinovietnamensis. The transferred RNAs (tRNAs) were adjusted using the software tRNAscan-SE service (Schattner et al. 2005). The initiation and termination codons of all the coding genes were manually verified in Geneious Prime v 9.1.4 (Kearse et al. 2012).
All the cp genomes were aligned using MAFFT v. 7.450 (Katoh and Standley 2013). Maximum Likelihood (ML) analysis was conducted by RAxML v 8.2.12 (Stamatakis 2014) with the rapid bootstrap algorithm. GTRGAMMAI was selected as the best-fit model recommended by jModelTest v2.1.6 (Darriba et al. 2012). The number of 12345 was specified as the random seed of parsimony tree inference with 1000 replicates performed. For Bayesian Inference (BI), the data matrix was calculated using MrBayes v3.2.2 software (Ronquist et al. 2012). The best-fit model was selected as SYM+G under the Akaike information criterion (AIC) using MrModeltest v 2.3 (Nylander 2004). Rates of variations across sites were trimmed as gamma. For each analysis, two simultaneous runs of four Monte Carlo Markov Chains (three heated and one cold) were run for 20 million generations with a random tree as the starting point and saving trees every 1000 generations. After discarding the first 25% samples as burn-ins, the optimized topology was generated. The final results were visualized with Figtree 1.4.3 (http://tree.bio. ed.ac.uk/software/figtree/).
The reconstructed phylogenetic tree is shown in Fig. 2. Similar to some previous analyses on plastid sequence data (Triplett and Clark 2010;Zhang et al. 2012

Taxonomic revision of Pseudosasa pubiflora and its synonyms
After consulting related literature and examining the types and other herbarium specimens or specimen photographs, we agree with Yang and Chao (1994) that P. gracilis, Yushania lanshanensis should be merged into P. pubiflora, and with Zhu et al. (2006) that Arundinaria teuivagina and P. parilis are also synonyms of P. pubiflora, since all these species share the same key diagnostic characters, mainly including the leptomorph rhizome, the pluricaespitose, short (< 2 m) and thin (< 0.8 mm) culm (Fig. 3C), the flat node, the narrowly ovate culm buds (Fig. 4B), the branch complement with two to four branches at each mid-culm node, the persistent papery culm leaf sheath with fragile oral setae that are adnate at base (Fig. 3E), the erect and amplexicaul culm leaf blades that are usually longer than sheath (Fig. 3D), and the ultimate branchlet with one to four lanceolate or narrowly lanceolate foliage leaves (Fig. 3B). Among these characters, its oral setae and culm leaf blade are very special in Arundinarieae.   However, we do not agree that I. pallidiflorus and Acidosasa paucifolia are identical to P. pubiflora. Indocalamus pallidiflorus (≡ Pseudosasa pallidiflora (McClure) S. L. Chen & G. Y. Sheng) markedly differs from P. pubiflora in the woolly (vs. glabrous) infranodal region, the culm leaf sheath oral setae present (vs. absent) and the culm leaf blade much shorter (vs. usually longer) than sheath. Actually, the description of P. pubiflora in Flora of China is mainly based on that of I. pallidiflorus made by McClure (1940), which conflicts a lot with the protologue of P. pubiflora . We cannot find the type specimens of Acidosasa paucifolia despite an exhaustive search in CANT where they should be preserved, as indicated by the author (Lin 1992). According to the description and illustration in the protologue (Lin 1992), this species significantly differs from P. pubiflora by the deciduous culm leaf with abaxially more or less hispid (vs. usually glabrous) sheaths and much longer ligules (2 mm vs less than 0.5 mm) and without (vs. with) oral setae, the absence (vs. existence) of foliage leaf oral setae and much longer foliage leaf inner ligules (3 mm vs. 0.3-1.5 mm). Thus, this species should not be synonymized with P. pubiflora either. The identities of these two taxa need further study.

Generic designation of Pseudosasa pubiflora
Recent research has proved that the formerly recognized Arundinaria in a broad sense should not be adopted any more, which at present is thought to be a small genus with only three species restricted to North America (Zhang et al. 2012(Zhang et al. , 2016(Zhang et al. , 2020Guo et al. 2021;Soreng et al. 2022). Thus, Pseudosasa pubiflora should not be placed into Arundinaria. This species has many characteristics that are very unusual for Pseudosasa, such as the lanceolate (vs. narrowly terete or linear in other Pseudosasa species, the same below) and rather short (1.6-2 cm long vs. usually much longer and up to 20 cm) spikelets with robust and erect (vs. slender and porrect) pedicels and very few florets (2-4 vs. 3-30), each floret with two (vs. three) stigmas, the papery (vs. leathery) culm leaf sheath and the glabrous and nonpowdery (vs. conspicuously powdery) infranodal regions ( Table 2). The phylogenetic analyses of both our study based on the chloroplast genomes and Guo et al. (2021) based on nuclear markers from ddRAD data demonstrated that this species had a distant relationship with other Pseudosasa. Thus, P. pubiflora should not be a member of Pseudosasa.
Interestingly, both of the two phylogenetic studies suggested a close relationship between Pseudosasa pubiflora and Sinosasa. In the analysis of Guo et al. (2021), Indocalamus sinicus (Hance) Nakai is closest to the Pseudosasa pubiflora + Sinosasa clade (called "Pseudosasa gracilis + Sasa subg. Sasa" clade ( Fig. 1) in that paper) with a strong support. Morphologically, this small clade is characterized by having persistent papery culm leaf sheath, short (less than 2.5 cm) and lanceolate spikelet with few florets (2-4) including a terminate rudimentary one and each floret with two stigmas in the subtribe Arundinariinae. However, the flowering branch of Indocalamus sinicus is terminal on culm or foliage leaf branches with a large and panicle-like unit of inflorescence composed of many spikelets, while the flowering branches of the other two develop at the nodes of every order of branches with a raceme-like unit of inflorescence composed of only 3-5 spikelets. The culm buds of Indocalamus sinicus are also very special; they are triangular with lower half completely adnate to the culm (Fig. 4A). Last, there are so many different vegetative and productive characters between Pseudosasa pubiflora and Sinosasa that they cannot belong to the same genus. Specifically, Pseudosasa pubiflora differs from Sinosasa in having narrowly ovate culm buds (vs. trullate) (Fig. 4), branch complement with 2-4 branches at mid-culm nodes (vs. solitary), flat nodes (vs. prominent with a strongly raised supranodal ridge), infranodal region being glabrous and nonpowdery (vs. with a sericeous or villous band), culm leaf with oral setae adnate at base (vs. distinct) and amplexicaul blade being usually longer than sheath (vs. not amplexicaul and much shorter than sheath), 1-4 foliage leaves per ultimate branchlet (vs. 5-17) with blades flat when dry (vs. wavy) and much shorter inner ligule (0.3-1.5 mm vs. (8-)10-20 mm), robust and erect (vs. slender and porrect to reflexed) paracladia being appressed to the axis and without (vs. with) pulvinus at base, upper glumes typically shorter than (vs. nearly equal to) the lowest lemma and lodicules with a rounded (vs. acute) apex (Detailed comparison is provided in Table 2). In conclusion, both the morphological and molecular phylogenetic evidence strongly supports the recognition of a new genus to accommodate Pseudosasa pubiflora. Diagnosis. This new genus is close to Sinosasa, but differs in having narrowly ovate culm buds, branch complement with 2-4 branches at mid-culm nodes, flat nodes, glabrous and nonpowdery infranodal region, culm leaf oral setae adnate at base, amplexicaul culm leaf blades that are usually longer than sheath, 1-4 foliage leaves per ultimate branchlet with blades flat when dry and short inner ligule, robust and erect paracladia (lateral spikelet pedicels) appressed to the axis and without pulvinus at base, upper lemma typically shorter than the lowest lemma and lodicules with a rounded apex.
Description. Shrubby bamboo. Rhizomes leptomorph. Culms pluricaespitose, short and thin, less than 2 m tall and 8 mm in diam.; nodes flat. Culm buds narrowly ovate. Branches intravaginal, 2-4 at each mid-culm node. Culm leaf sheath persistent, papery; auricles absent; oral setae fragile, adnate at base; blade erect and amplexicaul, usually longer than sheath; ligule convex, short. Foliage leaves 1-4 per ultimate branchlet; auricles obscure; inner ligule short. Flowering branches developing at the nodes of every order of branches; the unit of inflorescence of synflorescence raceme-like with 3-5 spikelets; paracladia robust and erect, appressed to the axis and without pulvinus at base; florets 2-4 per spikelet including a rudimentary one at the apex; glumes 2, upper one shorter than the lowest lemma; lemma longer than palea; palea 2-keeled; lodicules 3, apex rounded; stamens 3, anthers pale yellow; styles 2, base slightly connate; stigmas 2, plumose. Caryopsis unknown. Etymology. Kengiochloa is named in honor of Professor Yi-Li Keng (1897-1975, a renowned botanist and the first Chinese who studied the bamboo taxonomy in China. The type species of this genus was first described by him, too. Its Chinese name is given as 以礼竹属 (pinyin: yĭ lĭ zhú shŭ).
Phenology. Culm shoots produced in April to July, flowering in May. Distribution and habitat. This species is distributed in Hunan, Jiangxi and Guangdong, China. It grows under evergreen broadleaved forests at an elevation from 1100-1600 m.