﻿The identity of Sasaoblongula C.H.Hu (Poaceae, Bambusoideae, Arundinarieae): evidence from morphology and molecular data

﻿Abstract Sasaoblongula was described in 1987 based on a cultivated plant at the bamboo garden of Sun Yat-sen University. This species has two or three branches at the upper nodes, which differ from the rest of Sasa species that have a single branch per node. During the field trip to Baishi Town, Yunfu City, Guangdong Province in July 2021, one bamboo species with oblong foliage leaves was collected and matches the isotype. Then, our question was to test the identity of S.oblongula concerning other Sasa species based on morphology and molecular data. To do that, we sequenced the whole chloroplast genome of S.oblongula and did a phylogenetic analysis. Our morphological results indicate that the new collection is S.oblongula. The phylogenetic tree showed that S.oblongula is close to Pseudosasa, instead of Sasa species. Therefore, we transferred it to the genus Pseudosasa, and a revised description of P.oblongula is provided here.


Introduction
Sasa oblongula C.H. Hu (1987) was described based on two collections, i.e., Y. L. Yang & C. H. Hu 198001 (Type) and T. H. Wen & G. Y. Sheng 79413, from the bamboo garden of Sun Yat-sen University, Guangdong Province. According to its protologue, it was transplanted from somewhere in Guangdong with lack of a detailed address and could be distinguished by having small-medium-sized and oblong foliage leaves. It was well recognized and accepted as a distinctive species in the floras (Hu 1996;Wang and Stapleton 2006;Yi et al. 2008;Xia and Lin 2009;Vorontsova et al. 2016;Shi et al. 2022) and websites like GrassBase-The Online World Grass Flora (Clayton et al. 2016), Tropicos (www.tropicos.org), IPNI (www.ipni.org), POWO (powo.science. kew.org), The Plant List (www.theplantlist.org), GBIF (www.gbif.org). After examining paratype specimen T. H. Wen & G. Y. Sheng 79413 in N and a failed attempt of searching for it in the bamboo garden of Sun Yat-sen University during the revision of Sasinae Keng f. (Keng 1982), Li (2009) treated it as a suspicious species. Significantly, the type specimens and protologue all demonstrated that this bamboo species possessed two or three branches at upper culm nodes, which conflicted with the strictly solitary branch of Sasa at each node (Makino and Shibata 1901;Suzuki 1978;Kobayashi 2017;Qin 2019). Thus, S. oblongula should not belong to the genus Sasa and may be a member of Pseudosasa based on the evidence available.
However, the previous molecular phylogenetic analysis (Zeng et al. 2010) showed a surprising result, namely that S. oblongula, eight Japanese Sasa species (including generic type), and one Sasaella Makino (1929) (Keng 1957;Qin 2019;Qin et al. 2021;Li et al. 2023). We think that the voucher specimen Zeng & Zhang 06055 of S. oblongula used by Zeng et al. for the molecular analysis is probably misidentified. Thus, the phylogenetic position of S. oblongula needs to be further studied with correct samples.
During the field trip to Baishi Town, Yunfu City, Guangdong Province in July 2021, one bamboo species with oblong foliage leaves was found. It matched the isotype very well and shares the same key characters, such as the slightly prominent culm supranodal ridge, the white powdery infranodal region, the glabrous internodes with three branches at an upper node, the solitary secondary branch, three to six foliage leaves clustered at the top of ultimate branches, the small-medium-sized and oblong foliage leaves with glabrous blades and conspicuous transverse veins. Therefore, we are certain that the specimens we collected are S. oblongula. Then, our question was to test the identity of Sasa oblongula concerning other Sasa species based on morphology and molecular data.

Morphology
The sample of Sasa oblongula was collected from Hengjing Villiage, Baishi Town, Yunfu City, Guangdong, China during a field trip in July 2021. Observations and measurements were taken using a magnifier (SZ-6) and a ruler with a scale of 0.5 mm.
Some minor characters such as indumentum on ligules of both culm leaves and foliage leaves were observed with a stereomicroscope (Mshot MZ101). The description was made based on both living and dried material as well as relevant literature (e.g. Hu 1987Hu , 1996Wang and Stapleton 2006;Xia and Lin 2009). Comparisons between S. oblongula and Pseudosasa cantorii were conducted based on protologue and type specimens, and relevant specimens involved in the protologue of Arundinaria cantorii (≡Pseudosasa cantorii). The descriptive terms follow Beentje (2016) and herbaria acronyms follow Thiers (2021).

Sampling
For obtaining reliable results, a reasonable proof strategy with two steps was designed to identify the systematic position of S. oblongula. The first step is to test whether S. oblongula belongs to Sasa based on our plastid tree. The second step is to identify which genus S. oblongula belongs to based on SNP tree, mainly due to low discrimination rates for those 'three-branched' genera in plastid results (Guo et al. 2021). For the plastid tree, a total of 24 species from 11 genera were sampled. Bambusa multiplex and Dendrocalamus strictus were set as the outgroups. All accession numbers and voucher information are listed in Table 1. For the SNP tree, a total of 14 species from seven genera belonging to subtribe Arundinarieae were included. Chimonobambusa sangzhiensis was set as outgroup. Particular emphasis in our taxon sampling was placed on the inclusion that several key generic types were all involved in this study, including Acidosasa, Indosasa, Oligostachyum, Pseudosasa, and Sasa.

DNA extraction and sequencing
Young leaves at the vegetative growth stage were collected in the field. Total genomic DNA was isolated from silica-dried leaves following the manufacturer's specifications TIANGEN Genomic DNA Extraction Kit (TIANGEN, Beijing, China). DNA samples of concentration up to standard (≥1 μg) were 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. About 20G deep genome skimming (DGS) data were generated. Finally, adapters and lowquality reads were filtered from raw data using Fastp v 0.23.1  software.

Plastome assembly and chloroplast DNA regions mapping
The filtered clean reads were utilized to de novo assemble complete chloroplast (cp) genomes using GetOrganelle v 1.6.2 pipeline (Jin et al. 2018). Six k-mer values, including 21, 45, 65, 85, 105,125, were set for plastid contigs connection. Subsequently, the filtered plastid reads were transferred to Bandage (Wick et al. 2015) software for visualization processing. Two opposite plastid sequences exported from Bandage were aligned with the reference sequence Phyllostachys edulis (GenBank accession No. HQ337796), Table 1.
List of 24 species with species names, voucher information and GenBank accession numbers for the plastid tree based on eight combined plastid sequences extracted from whole chloroplast genomes (WCG). and one that matched the genomic direction of the reference was retained. The final cp genomes were manually corrected in Geneious 9.1.4 (Kearse et al. 2012). After referring to previous plastid phylogeny studies of Arundinarieae (Zeng et al. 2010;Zhang et al. 2012), eight plastid DNA regions  were selected to reconstruct plastid phylogenetic tree. Our cp genomes were annotated from eight DNA regions of Acidosasa purpurea with ≥ 70% sequence similarity in Geneious. Then, all the annotated plastid DNA regions were extracted from whole cp genomes. Sequence directions were visualized and adjusted using Mauve v 2.4.0 (Darling et al. 2004).

SNP calling
The latest high-quality genome sequence of moso bamboo (Phyllostachys edulis) (Zhao et al. 2018) was selected as the chromosome-level reference genome to build an index using the software SAMtools v 1.9 (Danecek et al. 2021) and Picard v 2.27.3 (Broad Institute 2019). After filtration of low-quality data, our clean reads were processed in removal of duplicates using Fastuniq v 1.1 (Xu et al. 2012). New filtered paired reads were aligned to the reference genome by Bowtie2 v 2.4.4 (Langmead and Salzberg 2012) with the parameter of minimum acceptable alignment score for L, 0.3, 0.3. After that, SAMtools was further employed to sort alignment (BAM files). Picard was utilized to remove duplicates again with the parameter "MarkDuplicates". GATK v 4.2.2.0 (Van der Auwera and O'Connor 2020) was performed to anchor variant calling including SNP and InDel using the joint calling method "HaplotypeCaller" in the genomic variant call format (GVCF). Each sample based on reads with mapping quality was set as at least 10 and the kmer size was set as 10 to 25. After completion of variants calling, the tool "CombineGVCFs" in GATK was carried out to combine all the GVCF files. The tool "GenotypeGVCFs" was then utilized to identify jointcalled variants. Subsequently, the tool "SelectVariants" was implemented to select single nucleotide polymorphic sites (SNPs). Filtration of SNPs of low quality was then conducted in the tool "VariantFiltration" with the parameter "QD < 2.0, MQ < 40.0, FS > 60.0, SOR > 3.0, MQRankSum < -12.5 and ReadPosRankSum < -8.0". Finally, the tool "SelectVariants" was run to extract filtered SNPs.
For a reliable phylogenetic tree based on SNP dataset, we considered that filtered raw SNPs with high missing genotype rates and low minor allele frequency will affect the accuracy of the phylogenetic trees and thus should be removed. Therefore, plink v 1.90b4.6 (Purcell et al. 2007) was operated to filter those low-quality SNPs with parameter "geno" set as 0.1 and "maf " set as 0.01. Filtered variants were then pruned with the parameter "indep-pairwise" set as 50, 10, 0.2, representing its window size, a variant count to shift the window and pairwise r2 threshold for SNPs, respectively. Finally, new clean SNP dataset was generated, and the GVCF format was transferred to PHYLIP format for phylogenetic analysis using the python script "vcf2phylip.py" (Ortiz 2019).

Alignments construction and phylogenetic trees inference
Chloroplast DNA regions and SNP dataset were utilized to reconstruct the phylogenetic tree, respectively. Eight plastid matrices were aligned using MAFFT v 7.450 (Katoh and Standley 2013) and concatenated as a super matrix. Maximum likelihood (ML) tree was inferred for plastid and SNP datasets using IQTREE v 1.6 in SH-aLRT test and ultrafast bootstrap (UFBoot) value (Nguyen et al. 2015). Node supports rates with SH-aLRT ≥ 80% and UFboot ≥ 95% were reliable and shown on each node. The final results were visualized with Figtree 1.4.4 (Rambaut 2018).

Morphological comparison
Ssasa oblongula has leptomorph rhizome, glabrous culm internodes, white powdery infranodal region, flat or slightly prominent nodes and culm supranodal ridge, mostly solitary branch at lower culm nodes and two to three (Fig. 3E, if three branches, central slightly dominant than lateral) branches at mid and upper culm nodes, glabrous culm leaf sheath (Fig. 3G) with erect and lanceolate blades, falcate auricles and ligules with ciliolate margin, glabrous foliage leaves blades and conspicuous transverse veins. These vegetative characters mentioned above make it fit well with the circumscription of Pseudosasa Makino ex Nakai (1925), rather Sasa. After examining the specimens of similar species and referring to the related literature (Munro 1868;Chia et al. 1983), we found that S. oblongula is most similar to P. cantorii (Munro) P. C. Keng ex S. L. Chen et al. (Zhu et al. 2006) by sharing one to three branches per nodes, glabrous internodes, the white powdery infranodal region, slightly prominent supranodal ridge, culm leaf sheath with falcate auricles, erect and lanceolate blades with serrulate margin, foliage leaf sheath with ciliate margin and truncate ligules, glabrous foliage leaf blades with conspicuous transverse veins, but differs by having nearly solid (vs. hollow) culm internode with appressed (vs. patent) branches, intravaginal (vs. transferred) and glabrous (vs. setose) abaxially culm leaf sheath with ciliate upper (vs. wholly) margin and arched (vs. truncate) ligules with ciliolate (vs. glabrous) margin, 3-6 foliage leaves with irregular (vs. coplanar) arrangement clustered at the top ultimate branch, glabrous (vs. hirsute) abaxially foliage leaf sheath with 1-4 mm (vs. 5-13 mm) long length per adjacent sheath apex, small-medium-sized (7-10 × 1.5-2.6 cm vs. 12.5-25 × 2.5-3.2 cm) foliage leaf blades with 6-7-paired (vs. 7-9-paired) secondary veins. A more detailed comparison between the two species is provided in Table 2.

Discussion
Sasa oblongula, mainly characterized by its oblong foliage leaves, was published based on sterile materials introduced in the bamboo garden of Sun Yat-sen University. It differed from Japanese Sasa species by having 1-3 branches per node (vs. 1 branch) and remote geographic distribution, indicating that it was not obviously the member of Sasa. After the examination of the voucher specimen Zeng & Zhang 06055 from Zeng et al. (2010), we were certain that this specimen does not represent S. oblongula since it possesses solitary branch at upper culm nodes, undeveloped or absent culm leaf auricles, and longlanceolate foliage leaf blades. Our phylogenetic study revealed that the actual S. oblongula and those Japanese Sasa species are dispersed in two different clades (Fig. 4, Clade A &  B). Furthermore, it and P. cantorii form a well-supported clade with two different branch lengths based on SNP phylogenetic tree (Fig. 5), supporting the result of morphology.
However, previous studies (Zhang et al. 2012;Guo et al. 2021) showed that Pseudosasa is polyphyletic, and the phylogenetic relationships between Pseudosasa and several other genera of subtribe Arundinariinae (Zhang et al. 2020), such as Pleioblastus, Oligostachyum, Indosasa, etc., have not been resolved. Sasa oblongula was closely related to Chinese Pseudosasa species in morphology and phylogeny, and thus was congruently assigned to the genus Pseudosasa here. Accordingly, a new combination Pseudosasa oblongula (C. H. Hu) N. H. Xia & X. Li was proposed.