﻿Terniopsisyongtaiensis (Podostemaceae), a new species from South East China based on morphological and genomic data

﻿Abstract The new species Terniopsisyongtaiensis X.X. Su, Miao Zhang & Bing-Hua Chen, from Fujian Province, China, is described and illustrated. It is similar to T.heterostaminata from Thailand, but differs in its two fertile stamens, fewer but longer vegetative ramuli, fewer but shorter flowering ramuli, shorter pedicels, capsule-stalk and stamens. The complete chroloplast genome of the new species is 129,074 bp long and has a typical quadripartite structure, including two inverted repeat regions (IRs) of 18,504 bp in length, separated by a large single-copy (LSC) and a small single-copy (SSC) regions of 79,000 bp and 13,066 bp, respectively. The ycf1 and ycf2 genes were lost compared to most higher plants, leading to a substantial reduction in the IR. The phylogenetic analysis using both matK and nrITS revealed that T.yongtaiensis is sister to T.heterostaminata with moderate support, and formed a clade with other Terniopsis species, suggesting that the new species belongs to Tristichoideae.


Introduction
The Podostemaceae (river-weeds) are unique aquatic angiosperms that exist in various wetlands across the world's tropics and subtropics (Philbrick and Novelo 1995;Cook Kato, T. savannaketensis Koi & Kato, T. sesadensis Koi & Kato, T. sessilis, T. ubonensis Kato, T. vapyensis Koi & Kato and T. daoyinensis Q.W.Lin, G. Lu & Z.Y.Li. A Terniopsis species that resembles T. heterostaminata from Thailand was discovered during our field investigation in Yongtai County, Fujian Province. As a result of comprehensive research, we observed that the species has considerable variation in plant morphology, flower and fruit characteristics, and that its phylogenetic position is supported by molecular-level data. As a result, we conclude that it is a new species, Terniopsis yongtaiensis, based on morphological distinctions, geographical isolation, and molecular evidence.

Morphological description
The morphological description of the new species was based on the specimens collected in a variety of localities in 2022. 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 the sizes of the morphological characters. Field observations provided habitats and phenology for the new species.
The leaf sample from Yongtai County, Fujian, China, was collected for DNA extraction.
DNA extraction, Genome sequencing, assembly, annotation and analysis In this study, total DNA was extracted from freeze-dried material using DNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA). Purified total DNA of the new species 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 Berry Genomics Co. Ltd. (Beijing, China), and 13.98 GB of reads was obtained. The paired-end reads were filtered and assembled into complete plastome using GetOrganelle v.1.7.5 with appropriate parameters, with K-merset "21,45,65,85,105" (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 visualized by Bandage to assess their completeness (Wick et al. 2015). Gene annotation was performed using CPGAVAS2 and PGA. Geneious v.2021.2.2 was used to manually calibrate the start and finish points for disputed positions (Jin et al. 2020a). 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
Phylogenetic analyses were conducted using Maximum likelihood (ML) and Bayesian Inference (BI) analyses, based on the matK and nrITS sequences. To construct the phylogenetic tree using matK sequence, 27 species (Suppl. material 1: Table S1) of Terniopsis, Tristicha, Dalzellia, Weddellina, Polypleurum, Zeylanidium and Tristellateia were included in our analysis. A species of Tristellateia was selected as 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 to other positions. The best nucleotide substitution model according to Bayesian Information Criterion (BIC) was TVM+F+G4, 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, 2000000 generations), in which the initial 25% of sampled data were discarded as burn-in. Phylograms were visualized in iTOL v.5.

Taxonomic treatment
The variations in morphology between T. yongtaiensis and the other two Terniopsis species from China, T. sessilis and T. daoyinensis, are more obvious. T. yongtaiensis shows clear differentiation between vegetative and reproductive stems, the erectness of the ramuli, and the characteristics of flower and fruit are distinctive from those of T. sessilis from Changting County, Fujian Province (Table 1, Suppl. material 1: Figs S2-S4). However, T. daoyinensis from Hainan differs significantly from other species of the genus by its long (up to 1 mm) and distinctly multi-furcated stigmas (Table 1).
Florescence December to January, fruiting season January to February. Distribution, habitat and conservation status. Terniopsis yongtaiensis is only known from Yongtai, Fujian, China (Suppl. material 1: Fig. S1), where it    also be included on the national secondary protection list during the upcoming revision process.
Etymology. The epithet yongtaiensis (永泰) refers to Yongtai County, Fujian Province where this new species was found.

Characteristics of the Terniopsis yongtaiensis plastome
The plastome of Terniopsis yongtaiensis (Fig. 5) is 129,074 bp in length, and exhibits a typical quadripartite structure, consisting of a large single copy (LCS) region of 79,000 bp and a small single copy (SSC) region of 13,066 bp, which were separated by a pair of 18,504 bp inverted repeat regions (IRs). The gene map of T. yongtaiensis is presented in Fig. 5. The gene composition in plastome of T. yongtaiensis would be divided into four categories: gene related to photosynthesis, genes related to self-replication, proteincoding genes with unknown functions, and other genes. A total of 106 unique genes were identified in the plastome; it contains 72 protein-coding genes, 30 tRNAs, and 4rRNAs. A total of 16 genes were duplicated in the IR regions, including ndhB, rpl2, rpl23, rps7,rps12, rrn4.5S, rrn5S, rrn16S, rrn23S, trnA-UGC, trnl-GAU, trnl-CAU, trnL-CAA, trnN-GUU, trnR-ACG, trnV-GAC. A total of six genes were lost, including psbZ, clpP, rpl 22, rpl32, and uncommon losses of ycf1 and ycf2. The annotated plastome was documented in GenBank (accession number OM717943).

Comparative analysis of the plastomes
A comparison of the plastome of Terniopsis yongtaiensis is made to five other species of Podostemaceae with available data (Table 3). The plastome lengths of the six species varied from 129,074 bp (T. 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. In all compared species, the ycf1 and ycf2 genes, which are two giant open reading frames found in most higher plants, are lost. In T. yongtaiensis and Tristicha trifaria, the rps15 gene is found at the SSC/IR border, but it is shifted to IRs in Apinagia riedelii, Marathrum utile, M. capillaceum and M. foeniculaceum due to the expansion at the IR/SSC boundary. In T. yongtaiensis, the trnG-UCC gene mutates to trnT-CGU, and in M. capillaceum, it is lost. Further, all the compared species have a gene inversion from trnK-UUU to rbcL in the LSC region, and the gene inversions are of similar size (ranging from 50.4 kb for T. yongtaiensis to 52 kb for A. riedelii). It represents an essential mechanism for plastome rearrangements (Mower and Vickrey 2018).

Phylogenetic analysis
Phylogenies were reconstructed by Maximum likelihood (ML) and Bayesian Inference (BI) analyses using the matK and nrITS sequences. The phylogenetic analysis based on matK sequences suggested that Terniopsis yongtaiensis is sister to T. heterostaminata with moderate support, and nested in a clade formed by T. brevis, T. minor, T. malayana with strong support (Fig. 6). Similar results showed by the phylogenetic analysis based on nrITS, suggested T. yongtaiensis is closely related to T. heterostaminata with moderate support, and sister to a clade comprising T. chanthaburiensis, T. filiformis, T. vapyensis, T. microstigma, T. ubonensis, T. savannaketensis, and T. malayana (Suppl. material 1: Fig. S5).

Discussion
The Terniopsis sessilis Chao was first discovered in 1948 in the Tingjiang River basin of Changting County in northwest Fujian Province (Chao 1948(Chao , 1980. The literatures indicated that this species has a wide distribution, but to date, 80 years after its report, it has not been found elsewhere after a long and continuous investigation, such as around the Min River, under the Wanshou Bridge (i.e. Jiefang Bridge) in Cangshan District, Fuzhou City, Fujian Province, where a distribution has been noted. This is possibly due to environmental changes and urbanization. Fortunately, some botanical enthusiasts discovered plants that were morphologically similar in Guilin, Guangxi Zhuang Autonomous Region, which our team analyzed and determined were consistent with T. sessilis based on matK sequences (data not published). While looking for other distribution sites of T. sessils in Fujian Province, the new species T. yongtaiensis was discovered in Yongtai county; it differs greatly in appearance from T. sessilis (Suppl. material 1: Figs S2-S4), especially in the ramuli, flower and fruit. Roots of T. yongtaiensis are often dark green in water, and the vegetative and flowering ramuli can be clearly distinguished. There are more leaves on vegetative ramuli (up to 55), the leaves are spatulate, and they wither during flowering. The ramuli of T. sessilis, on the other hand, are often attached to rock surfaces, and are obviously shorter (7-9 mm long), and have fewer leaves (< 12). The number of flowering ramuli branches varies between Terniopsis species. The flowering ramuli of T. yongtaiensis are usually two-branched, with one flower. The flowering ramuli are shorter and single branched with one or two flowers, but the leaf shape is similar. And the flowering ramuli of T. heterostaminata are often single to four-branched, with one flower (Chao 1980;Fujinami and Imaichi 2009; The plastome of T. yongtaiensis was compared with the plastome of 5 other species within the Podostemaceae family. All of the studied species lack the ycf1 and ycf2 genes, which are giant open reading frames found in most higher plants, resulting in a significant reduction of IR regions, thus reducing the size of their plastomes. Based on the available data, we believe that the absence of ycf1 and ycf 2 genes is typical for Podostemaceae. The ycf1 and ycf2 genes were also lost in the plastome of Poaceae (Guisinger et al. 2010), Geraniaceae (Weng et al. 2014) and Ericaceae (Braukmann et al. 2017). There is still debate over the functions of the ycf1 and ycf2 genes, and they have yet to be classified as genes involved in genetic or photosynthetic systems (Drescher et al. 2000).
According to molecular data on matK comparison, the new species from Yongtai was closely related to T. heterostaminata from Thailand, and was in the sister group of the same cluster in the phylogenetic tree. Additionally, due to its geographical distance and the unique river habitat, this species was identified as a new species and named T. yongtaiensis. Investigations of other rivers in Yongtai and surrounding counties have revealed that the species was only found in the upper reaches of the first discovery site, indicating that the species has a very limited distribution area. Meanwhile, a whole-genome analysis will be carried out to ascertain its phylogenetic and evolutional position among angiosperms.

Conclusion
Terniopsis yongtaiensis should be classified as a new species of Tristichoideae, based on the facts presented in the current study. The plastome of species of genus Terniopsis was studied for the first time, and the discovery of T. yongtaiensis provides new supporting materials for the phylogeny and evolution for the Podostemaceae family.
Key to the species of Terniopsis H. C. Chao