﻿Polypleurumchinense (Podostemaceae), a new species from Fujian, China, based on morphological and genomic evidence

﻿Abstract We describe Polypleurumchinense, a new species of Podostemaceae from Yunxiao County, Fujian Province, China, based on morphological and molecular data and the genus Polypleurum is recorded here for the first time from China. Polypleurumchinense has a gross morphology similar to P.longistylosum, but it can be distinguished from the latter by its narrower roots, more numerous and longer leaves, shorter stigmas and more numerous ovules per locule. To distinguish the new Polypleurum species and study its phylogenetic position, its complete plastome was sequenced and characterised. The plastome is 132,110 bp in length, including a pair of inverted repeat regions (IRs) of 20,389 bp divided by the large single-copy (LSC) and small single-copy (SSC) regions of 79,022 bp and 12,310 bp, respectively. The plastome size of P.chinense is relatively smaller compared to most angiosperms due to the absence of the ycf1 and ycf2 genes in the IR regions. The phylogenetic analyses also strongly support the separation of the new species from other taxa.


Introduction
Polypleurum species differ in number of capsule ribs and stamens. Species in Sri Lanka and India, including P. wallichii (R. Br. ex Griffz) Warm., the type of the genus, have two stamens and eight capsule ribs, while species in Thailand and Laos have a single stamen and eight to fifteen ribs (Kato 2006).
In January 2021, during a field investigation in Yunxiao County, within the Wushan Mountains in Zhangzhou City, Fujian, China, we unexpectedly discovered a littleknown Podostemaceae species during anthesis in a stream. The species has a completely distinct morphology from the other three known genera identified in China. In March and August of 2021, we collected its fruits and vegetative parts for molecular study. Based on morphological analysis and molecular phylogeny, we established that our recently obtained specimen is a new species of Polypleurum, a hitherto not recorded genus of Podostemaceae in China.

Morphological description
The morphological description of the new species was based on the study of specimens collected in a variety of spots in 2021. Live material adhering to rock surfaces from a river in Wushan Mountains, Yunxiao County, Fujian, China, was collected for DNA extraction. A stereoscopic zoom microscope (Carl Zeiss,Axio zoom. v.16,Germany), equipped with an attached digital camera (Axiocam) and a digital caliper were used to record details of roots, leaves, bracts, spathella, tepals, stamen, pistil and seeds. Field observations provided habitats and phenology for the new species.

DNA extraction, amplification and sequencing
In this study, total DNA was extracted from freeze-dried material using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). The phylogenetic position of the new species was determined by nrITS and plastid matK sequences. The nrITS and plastid matK regions were amplified via polymerase chain reaction (PCR) using MiniAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA) and 1.1xT3 Super PCR Mix (Tsingke Biotechnology, Beijing, China) under the following conditions: 5 min at 94 °C; 30 cycles of 45 s at 94 °C, 45 s at 55 °C, 60 s at 72 °C; and 10 min at 72 °C (Zhou and Jin 2018) and 3 min at 94 °C; 30 cycles of 30 s at 94 °C, 30 s at 55 °C, 90 s at 72 °C; and 7 min at 72 °C , respectively. The PCR products were treated with Mag-MK 96 Well PCR Products Purification Kit (Sangon Biotech, Shanghai) to remove the extra primers. Sequencing was conducted using the BigDye Terminator v.3.1 Cycle Sequencing Kit (Applied Biosystems) and the ABI 3130xl Genetic Analyser (Applied Biosystems). The primers used for the DNA amplification and the cycle sequencing are listed in Suppl. material 1: Table S1.

Genome sequencing, assembly, annotation and analysis
Purified total DNA of Polypleurum chinense was fragmented, genome skimming was performed using next-generation sequencing technologies on the Illumina Novaseq 6000 platform with 150 bp paired-end reads and 350 bp insert size by Wuhan Onemore-tech Co. Ltd. (Wuhan, China) and 15.88 GB of reads was obtained.
The paired-end reads were filtered and assembled into complete plastome using Ge-tOrganelle v.1.7.5.0 with appropriate parameters, with K-merset "21,45,65,85,105", the word size being 0.6 (Jin et al. 2020a). Following previous studies, our workflow includes five key steps as well (Camacho et al. 2009;Bankevich et al. 2012;Langmead and Salzberg 2012;Jin et al. 2020a). Graphs of the final assembly were visualised by Bandage to assess their completeness (Wick et al. 2015). Gene annotation was performed using CPGAVAS2 and PGA. The different annotations of protein coding sequences were confirmed using BLASTx. The tRNAs were checked with tRNAscan-SE v.2.0.3. Final chloroplast genome maps were created using OGDRAW.

Phylogenetic analysis
In an attempt to reconstruct the evolutionary history of Polypleurum chinense, phylogenies were constructed using Maximum Likelihood (ML) and Bayesian Inference (BI) analyses of the nrITS and matK sequences. To construct a phylogenetic tree, based on matK sequence, 114 samples (Suppl. material 1: Table S2) of Terniopsis, Cladopus, Paracladopus, Hanseniella, Hydrobryum, Hydrodiscus, Thawatchaia, Hydrobryopsis, Zeylanidium, Griffithella, Polypleurum, Willisia and Cratoxylum were included in our analysis. Cratoxylum cochinchinense was selected as the outgroup. Each individual sequence was aligned using MAFFT 7.310 (Katoh and Standley 2013) with default settings. A concatenated supermatrix of the two sequences was generated using PhyloSuite v.1.1.15 (Zhang et al. 2019) for the phylogenetic analysis. All missing data were treated as gaps. Gblocks 0.91b (Castresana 2000) was applied to eliminate poorly-aligned regions of the concatenated supermatrix with gaps set as no different from the other positions. The best nucleotide substitution model according to Bayesian Information Criterion (BIC) was TVM+F+R3, which was selected by Model Finder (Kalyaanamoorthy et al. 2017) implemented in IQTREE v.1.6.8. Maximum Likelihood phylogenies were inferred using IQ-TREE (Nguyen et al. 2015) under the model automatically selected by IQ-TREE ('Auto' option in IQ-TREE) for 1000 ultrafast (Minh et al. 2013) bootstraps. Bayesian Inference phylogenies were inferred using MrBayes 3.2.6 (Ronquist et al. 2012) under GTR+F+G4 model (2 parallel runs, 2,000,000 generations), in which the initial 25% of sampled data were discarded as burn-in. Phylograms were visualised in iTOL v.5.
To construct a phylogenetic tree based on nrITS, 42 species of Cladopus, Hanseniella, Hydrobryum, Hydrobryopsis, Zeylanidium, Griffithella, Polypleurum, Willisia and Cratoxylum were included in the analysis (Suppl. material 1: Table S3). Cratoxylum cochinchinense was employed as the outgroup. The study was carried out as described above and, according to the Bayesian Information Criterion (BIC), the optimal nucleotide substitution model was GTR+F+I+G4.

Taxonomic treatment
Polypleurum chinense B.Hua Chen & Miao Zhang, sp. nov. urn:lsid:ipni.org:names:77299427-1 Figs 1-4 Diagnosis. The new species can be easily distinguished from most other species, except Polypleurum longistylosum, by tufts of leaves on both sides of the root between the root branches, a more or less flattened, ellipsoid, rough capsule with a greater number of longitudinal ribs (> 12), a spathella nearly completely enclosing the ovary and stamen at anthesis, a solitary stamen and a very short capsule stalk (< 2 mm). The narrower roots (0.6-0.8 mm vs.1.0-1.5 mm) with leaves 8-12 per tuft (vs. 4-8), up to 23.1 mm (vs. 5 mm) long, fewer (4 vs. 6) bracts, short spiny or glandular hairs on the spathella (vs. papillate) in the new species differentiate it from P. longistylosum (Table 1).
Root creeping, adhering to rock surfaces, ribbon-like, branched, 0.6-0.8 (−1.0) mm wide, with tufts of leaves on both flanks, not associated with root branching, 2-4 mm apart; leaves 6-12 per tuft, in two ranks, to 17.6 (12.4-23.1) mm long, 0.2-0.4 mm wide, needle-like ( Fig. 1). Flowering shoots on both flanks of root, very short; bracts 2-6, needle-like, to 5-6 mm long, caduceus. Flower just prior to anthesis, with only two remaining bracts, pale purplish-red. The anthesis begins when the water level is further reduced, the bracts disappeared, but the base remained. Flower1, bud covered by ellipsoid spathella, spathella stalk ca. 3 mm in length, coated with short spiny hairs or glandular hairs, with a papilla-like tip, the papilla ruptured near apex at anthesis, but persisting spathella base keeps the ovary and lower bottom of stigma enclosed. Pedicel ca. 0.7 mm long; tepals 2, one on each side of stamen, linear, ca. 0.3 mm long; stamen 1, up to 2.9 mm long, protruding from spathella; ovary dark green, ellipsoid, ca. 1.8 mm long, 0.9 mm wide, 1-locular, free central placenta; stigmas 2, forked near base, thin, needle-like, ca. 0.9 mm long, as long as, or slightly shorter than ovary, branched at the top, upper part exerted from spathella; ovules on marginal surface of septum, 25-35 per septum (Fig. 2). Post-pollination, the spathella and ovary developed into a pale ellipsoid, ca. 2 mm long, arranged on both sides of the root, the ovary stalk lengthened and developed into pedicels (ca. 0.7 mm). Mature capsule 12-14-ribbed, conspicuous under microscope, fissured longitudinally; seed yellowish-brown, with shallow groove, ca. 300 μm long (Fig. 3). Distribution and habitat. Polypleurum chinense is only known from Fujian, China (Fig. 5), where it grows on rocks in unpolluted streams. In addition, Cladopus austrosinensis M. Kato & Y. Kita from the same family was found on the rock surfaces in the lower reaches of the stream. Many other plants grow in the surrounding habitat, whose tree layer includes Pinus massoniana Lamb.   Phenology. Polypleurum chinense was observed flowering and fruiting in its habitat from December to February when the water level is reduced to partly expose the rocks.
Etymology. The Zhong Guo Cha Pu Chao (中国叉瀑草). The specific epithet "chinense" refers to China, as the distribution of this genus was first identified there and it was proven to be a new species of Polypleurum.
Conservation status. According to our investigation, Polypleurum chinense was found in patches attached to rock surfaces in rapid-flowing streams in the Wushan Mountains range in Zhangzhou City, Fujian Province, China. It is difficult to count the exact number of individuals in the population. Its habitat is vulnerable to anthropogenic destruction and projects like reservoir construction. To determine the exact distribution of this species, further fieldwork is required around the Wushan Mountains in Zhangzhou City and nearby mountainous areas. Therefore, we suggest the species be classified as category DD (Data Deficient), according to the International Union for Conservation of Nature (IUCN 2022). According to the Updated List of National Key Protected Wild Plants (Decree No. 15) by the country's State Forestry  and Grassland Administration and the Ministry of Agriculture and Rural Affairs, all of the known genera of Podostemaceae found in China are classified in the national secondary protection list. The new recorded genus should also be included on the national secondary protection list during the upcoming revision process.

Morphology
The new species is morphologically similar to most Polypleurum species in that it has ribbon-like roots, tufts of linear leaves on roots, a single flower, a bud covered by spathella and an ellipsoid and rough capsule with longitudinal ribs. However, the new species differs from P. longtistylosum and P. schmidtianum in the length of leaves, the number of leaves per tuft and capsule ribs, as well as the spathella coat appearance (Table 1).

Characteristics of the Polypleurum chinense plastome
The complete plastome of Polypleurum chinense was sequenced and characterised in this study. It is 132,110 bp in length and exhibits a typical quadripartite structure, consisting of a large single copy (LCS) region of 79,022 bp and a small single copy (SSC) region of 12,310 bp, which were separated by a pair of 20,389 bp inverted repeat regions (IRs). The gene map of P. chinense plastome is presented in Fig. 6. The gene composition in the plastome of P. chinense could be divided into four categories: genes related to photosynthesis, genes related to self-replication, protein-coding genes with unknown functions and other genes. A total of 108 unique genes were identified in the plastome and it contains 72 protein-coding genes, 30 tRNAs, and 4 rRNAs. A total of 16 genes were duplicated in the IR regions, including ndhB, rpl2, rps7, rps12, rps15, rrn4.5S, rrn5S, rrn16S, rrn23S, trnA-UGC, trnI-GAU, trnI-CAU, trnL-CAA, trnN-GUU, trnR-ACG and trnV-GAC (Table 2). There were four genes lost, including rpl23, infA and uncommon losses of ycf1 and ycf2. The annotated plastome was documented in GenBank (accession number OL944404).

Phylogenetic analysis
Phylogenies were reconstructed by the Maximum Likelihood (ML) and Bayesian Inference (BI) analyses using the matK and nrITS sequences. The phylogenetic study, based on matK sequences suggested the two subfamilies, Tristichoideae and Podostemoideae are sister groups. Polypleurum is a monophyletic group within Podostemoideae, which is divided into two subclades. Polypleurum is closer to Griffithella. Polypleurum chinense is sister to P. longistylosum with strong support (PP = 1, BS = 100) and nested in a clade formed by nine other species of Polypleurum. Polypleurum chinense 1 is extracted from the complete chloroplast genome and P. chinense 2 is a cloned matK sequence (Fig. 7). The phylogenetic analysis, based on the nrITS sequences, suggested that P. chinense is sister to a clade formed by Hydrobryopsis sessilis, P. stylosum, P. schmiditianum, P. wallichii, P. munnarense, Zeylanidium lichenoides and Z. olivaceum with strong support (PP = 0.99, BS = 86%) (Fig. 8).

Comparative analysis of the plastomes
A comparison of the plastome of Polypleurum chinense is made with six other species of Podostemaceae with available data (Table 3). The plastome lengths of the seven species varied from 129,074 bp (Terniopsis yongtaiensis) to 134,912 bp (Apinagia riedelii), with T. yongtaiensis being the shortest. For the LCS and SSC regions, the extent of length variation between these species is not evident. The number of PCGs in these species is similar to that of most angiosperms, according to a comparative analysis of gene content (Jin et al. 2020b). The numbers of tRNA and rRNA genes, as well as the GC content, are substantially conserved in all of these plastomes, as shown by our findings. The structure of the IR greatly influences the structural integrity of the entire genome.
In all compared species, the ycf1 and ycf2 genes, which are two giant open reading frames found in most higher plants, are lost, resulting in a significant reduction of IR regions, hence reducing the size of their plastomes. The loss of ycf1 and ycf2 genes were also found in the plastome of Poaceae (Guisinger et al. 2010), Geraniaceae (Weng et al. 2014) and Ericaceae (Braukmann et al. 2017). The functions of ycf1 and ycf2 genes are still controversial and they have not been classified as genes involved in the genetic and photosynthetic systems (Drescher et al. 2000). The size of the IR regions varied amongst compared species, largely due to the evolutionary transfer of complete genes from the SSC regions into the IR or vice versa (Chumley et al. 2006;Wicke et al. 2011).
In Polypleurum chinense, Tristicha trifaria and Terniopsis yongtaiensis, the rps15 gene is found at the SSC/IR border, but it is shifted to IRs in Apinagia riedelli, Marathrum utile, M. capillaceum and M. foeniculaceum due to the expansion at the IR/SSC boundary. Jin et al. (2020b) found that the relocation of rps15 gene in M. foeniculaceum did not accumulate significant mutations, either because it occurred recently or because the substitution rate was too low to detect. In P. chinense, the trnG-UCC gene mutates to trnT-CGU and, in M. capillaceum, it is lost. In addition, it is found that all the species compared have a gene inversion from trnK-UUU to rbcLin the LSC region and the size of the inversions for each species is similar (ranging from 51 kb for P. chinense to 52 kb for A. riedelli). It represents an essential mechanism for plastome rearrangements (Mower and Vickrey 2018). The rpl23 gene is lacking in plastomes of the other five compared species, except T. yongtaiensis (Zhang et al. 2022). However, it is present in plastomes of non-Podostemaceae species, such as Bonnetia paniculata and Cratoxylum cochinchinense (Bedoya et al. 2019;Jin et al. 2020b). We do not know the extent of gene loss amongst other Podostemaceae species in China and future sequencing projects will inevitably offer insights into rates and mechanisms of gene loss in plastid genomes.

Phylogenetic analysis
The present study confirmed Polypleurum chinense is a new species, based on the phylogenetic analysis of matK and nrITS sequences, which indicated that P. chinense is related to P. longistylosum. The phylogenetic study demonstrated that matK sequence performed better for the phylogenetic analysis of P. chinense, which was consistent with the previous studies Kato et al. 2017). The ability of discrimination between species based on nrITS was comparatively poor (Khanduri et al. 2015).
Despite the fact that nrITS performed quite well (79%) in angiosperms, lower discrimination success was reported for Ranunculales (6.7%) and Laurales (14.3%) (China Plant BOL 2011). The inconsistencies in species ascriptions between nrITS may result from hybridisation and introgression or incomplete lineage sorting (Alvarea and Wendel 2003; Rieseberg et al. 2006;Feliner and Rosselló 2007). As a result, utilising plastid DNA markers alone may not be sufficient to discriminate between closelyrelated species. Furthermore, using a single individual for each species, based on plastid DNA markers, might be deceptive (China Plant BOL 2011).

The establishment of the new species
The identification of the new Polypleurum brings the total number of genera of Podostemaceae to four in China. The discovery of Polypleurum in China not only enriches the angiosperm flora of China, but also provides strong evidence for a close connection between the subtropical flora of Fujian and the tropical flora of Southeast Asia.
Key to the genera of Podostemaceae of China