Research Article |
Corresponding author: Bing-Hua Chen ( bhchen@fjnu.edu.cn ) Academic editor: Alexander Sennikov
© 2022 Bing-Hua Chen, Miao Zhang, Kai Zhao, Xiao-Hui Zhang, Chang-Li Ge.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Chen B-H, Zhang M, Zhao K, Zhang X-H, Ge C-L (2022) Polypleurum chinense (Podostemaceae), a new species from Fujian, China, based on morphological and genomic evidence. PhytoKeys 199: 167-186. https://doi.org/10.3897/phytokeys.199.85679
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We describe Polypleurum chinense, 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. Polypleurum chinense 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.
Fujian Province, molecular identification, morphology, Podostemaceae, Polypleurum chinense
Podostemaceae, widely known as “river-weeds”, are a vast family of unique haptophtic angiosperms that grow in a variety of wetlands in the tropics and subtropics around the world (
Phylogenetically, Podostemaceae are placed in the eudicot order Malpighiales and are sister to Hypericaceae (
Podostemoideae, the largest subfamily, is divided into various clades, including Ceratolacis, Cipoia, Diamantina & Podostemum, American genera (Apinagia, Castelnavia, Jenmaniella, Marathrum, Monostylis, Mourera, Noveloa, Rhyncholacis, Wettsteiniola), Aulea (Saxicolella pro parte), African genera (Dicraeanthus, Djinga, Inversodicraea, Ledermanniella, Leiothylax, Letestuella, Macropodiella, Monandriella, Saxicolellas.s., Stonesia, Winklerella), Madagascan genera (Endocaulos, Thelethylax) and Asian/Australian genera (Cladopus, Farmeria, Griffithella, Hanseniella, Hydrobryum, Hydrodiscus, Paracladopus, Polypleurum, Thawatchaia, Terniopsis,Willisia, Zeylanidium) (
Podostemoideae are the largest subfamily in the family and are widely distributed in Asia, especially in Thailand (42 species with 4 varieties in 10 genera) (
Polypleurum (Taylor ex Tul.) Warm. is a genus previously known from Sri Lanka, India, Thailand and Laos with 17 species (
In January 2021, during a field investigation in Yunxiao County, within the Wushan Mountains in Zhangzhou City, Fujian, China, we unexpectedly discovered a little-known 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.
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.
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 (
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 GetOrganelle 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 (
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
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
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
Morphological differences between Polypleurum chinense, P. longistylosum and P. schmidtianum.
Characteristics | P. chinense | P. longistylosum | P. schmidtianum |
---|---|---|---|
Root width/mm | 0.6–0.8(−1.0) | 1–1.5 | 2–4 |
Tufts of leaves position | On both flanks, alternate, subopposite or opposite | On both flanks | Near both sides |
The number of leaves | 6–12, usually 8–12 | 4–8 | 2–4 |
Leaves length/mm | 12.4–23.1 | 5 | 1.5–3(−6) |
The number and morphology of bracts | 4, needle-like | 6, needle-like | 3–4(−6), sheathed |
Bracts length(mm) | 5–6 | 4 | 2–3 |
Spathella length/mm | 3 | - | 1.5–2 |
Spathella coat | Short spiny or glandular hairs | Papillate | Not papillate |
Peduncle length/mm | 0.7 | 1 | 6–7 |
Tepals length/mm | 0.3 | 0.2 | 0.5–0.7 |
Stamen length (mm) | 2.9 | 1.7 | 1.2 |
Ovary locular | 1 | 1 | 2 |
Ovary length (mm) | 1.8 | 1 | 1.2–1.5 |
Stigmas quantity | 2, unequal | - | 2 or 3 |
Stigmas length (mm) | 0.6–1.2 | 1–1.2 | 0.2–0.4 |
Ovules locule | 25–35 | 10–15 | 25–35 |
Capsule stalk length (mm) | 1.1–1.6 | - | 6–12 |
Capsule ribs | 12–14, conspicuous | 10–12, inconspicuous | 8 |
China. Fujian Province: Yunxiao County, Wushan Mountains, elevation 430 m, 117°14'E, 23°53'N, 4 January 2021, Bing-Hua Chen CBH 04407 (Holotype FNU barcode FNU0041131; isotype FNU barcode FNU0041132).
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.
Polypleurum chinense A plants in bud adhering to rock surface B ribbon-like root with young floriferous shoots on flanks C flower bud covered by spathella(s) above bracts (b) D shoots along the flanks of the ribbon-like root between the root branches E, F flower at anthesis on peduncle with ruptured spathella G spathella H flower with spathella removed, stamen, and ovary, arrow shows a tepal on side of stamen I gynoecium without bracts J ovules on ovary septum K, L, M stamen, arrows show two tepals on sides of stamen. Scale bar: 1 mm (C, F, J); 2 mm (B, E, M); 500 μm (G, H, I, K, L).
Polypleurum chinense A habitat in the dry season when the river level is reduced B habitat showing mature fruits C elliptic pale bodies along the flank of the root D stalked fruit, showing ribs E dehisced capsule, showing seven ribs on the half of capsule, conspicuous F dehisced capsule, showing persistent valve and seeds G seeds. Scale bars: 20 mm (B); 5 mm (C); 500 μm (D); 400 μm (E); 1 mm (F); 200 μm (G).
Polypleurum chinense is only known from Fujian, China (Fig.
Illustration of Polypleurum chinense A ribbon-like root with tufts of leaves B flower bud covered by spathella above bracts C floriferous shoots along the flanks of the root between the root branches D flower at anthesis on peduncle with ruptured spathella E flower with spathella removed, stamen and ovary, a tepal on side of stamen F stamen G ovules on ovary septum H stalked fruit, showing ribs I seeds.
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.
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.
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 (
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
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.
Category, Group of Genes | Gene Names |
---|---|
Photosynthesis: | |
Subunits of ATP synthase | atpA, atpB, atpE, atpF*, atpH, atpI |
Subunits of NADH dehydrogenase | ndhA*, ndhB*(x2), ndhC, ndhD, ndhE, ndhF, ndhG, ndhH, ndhI, ndhJ, ndhK |
Cytochrome b/f complex | petA, petB*, petD*, petG, petL, petN |
Subunits of photosystem I | psaA, psaB, psaC, psaI, psaJ |
Subunits of photosystem II | psbA, psbB, psbC, psbD, psbE, psbF, psbH, psbI, psbK, psbJ, psbL, psbM, psbN, psbT, psbZ |
Large subunit of rubisco | rbcL |
Other genes: | |
Subunit of Acetyl-CoA-carboxylase | accD |
c-type cytochrome synthesis gene | ccsA |
Envelope membrane protein | cemA |
Protease | clpP |
Maturase | matK |
Self-replication: | |
Large subunit of ribosome | rpl2*(x2), rpl14, rpl16*, rpl20, rpl22, rpl32, rpl33, rpl36 |
DNA-dependent RNA polymerase | rpoA, rpoB, rpoC1*, rpoC2 |
Small subunit of ribosome | rps2, rps3, rps4, rps7 (x2), rps8, rps11, rps12*a (x2), rps14, rps15 (x2), rps18, rps19 |
rRNA Genes | rrn4.5S (x2), rrn5S (x2), rrn16S (x2), rrn23S*(x2) |
tRNA Genes | trnA-UGC*(x2), trnC-GCA, trnD-GUC, trnE-UUC, trnF-GAA, trnfM-CAU, trnG-GCC, trnH-GUG, trnI-GAU*(x2), trnI-CAU (x2), trnK-UUU*, trnL-CAA (x2), trnL-UAA*, trnL-UAG, trnM-CAU, trnN-GUU (x2), trnP-UGG, trnQ-UUG, trnR-ACG (x2), trnR-UCU, trnS-UGA*, trnS-GCU, trnS-GGA, trnT-CGU, trnT-GGU, trnT-UGU, trnV-GAC (x2), trnV-UAC*, trnW-CCA, trnY-GUA |
Unknown function: | |
Conserved open reading frames | ycf3*, ycf4 |
Circular gene map of the plastid genome of Polypleurum chinense. Genes inside the circle are transcribed clockwise, while those drawn outside are transcribed counterclockwise. Genes are colour-coded according to their functional groups. The circle inside the GC content graph marks the 50% threshold.
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.
Phylogenetic tree of Asian Podostemaceae, based on Bayesian Inference of matK sequences. Numbers above and below branches indicate RAxML (left) bootstrap probabilities (BP) and Bayesian (right) posterior probabilities (PP), respectively. Triangles indicate clades containing multiple species (samples) of one genus examined and the vertical lengths of triangles reflect the number of species (samples) examined.
Amongst the 17 known species of Polypleurum, only two species, i.e. P. longistylosum and P. schmidtianum, have a single stamen and the shoots or tufts of leaves borne on both sides of the root between the root branches. Although P. chinense has an overall morphology similar to P. longistylosum, there are some obvious differences, such as narrower roots (0.6–0.8 mm vs.1.0–1.5 mm), leaves appearing in tufts of 8–12 (vs. 4–8) and being as long as 23.1 mm (vs. 5 mm) long; fewer bracts (4 vs. 6); spathella with short spiny or glandular hairs (vs. papillate) on its coat; capsule with 12–14 conspicuous ribs (vs. 10–12 inconspicuous ribs) (
A comparison of the plastome of Polypleurum chinense is made with six other species of Podostemaceae with available data (Table
Statistics on the basic features of the plastid genomes of Polypleurum chinense and related taxa.
Species | Voucher | Accession no. | Length (bp) | LSC (bp) | SSC (bp) | IR(bp) | GC content (%) | No. of PCGs | No. of tRNA | No. of rRNA |
---|---|---|---|---|---|---|---|---|---|---|
Polypleurum chinense | CBH 04407 | OL944404 | 132,110 | 79,022 (~ 59.8%) | 12,310 (~ 9.3%) | 20,389 × 2(~ 30.9%) | 34.85 | 74 | 30 | 4 |
Apinagia riedelii | C.P. Bove 2513 (R) | MN165812 | 134,912 | 85,377 (~ 61.0%) | 12,437 (~ 8.9%) | 21,049 × 2 (~ 30.1%) | 34.90 | 74 | 30 | 4 |
Marathrum utile | AMB 497 (ANDES) | MN165814 | 131,951 | 79,778 (~ 60.5%) | 12,283 (~ 9.3%) | 19,945 × 2 (~ 30.2%) | 35.10 | 73 | 29 | 4 |
Marathrum capillaceum | C.P. Bove 2493 (R) | MN165813 | 134,374 | 79,990 (~ 59.5%) | 12,302 (~ 9.2%) | 21,041 × 2 (~ 31.3%) | 35.00 | 75 | 30 | 4 |
Marathrum foeniculaceum | W. D. Stevens - 32072 | MK995178 | 131,600 | 79,506 (~ 60.4%) | 12,262(~ 9.3%) | 19,916×2 (~ 30.3%) | 35.10 | 76 | 30 | 4 |
Tristicha trifaria | A. Mesterhazy MLI 128(Z) | MN165816 | 130,285 | 78,925 (~ 60.6%) | 12,662 (~ 9.7%) | 19,349 × 2 (~ 29.7%) | 36.40 | 74 | 30 | 4 |
Terniopsis yongtaiensis | CBH 04587 | OM717943 | 129,074 | 79,000 (~ 61.2%) | 13,066 (~ 10.1%) | 18,504 × 2 (~ 28.7%) | 36.20 | 72 | 30 | 4 |
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.
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 (
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.
1 | Flowers axillary; spathella absent; tepals 3; stamens 2 or 3 | Terniopsis |
– | Flowers terminal; spathella present; tepals 2; stamens 1 or 2 | 2 |
2 | Leaves of fertile stems digitate, 3–9-segmented, scattered, imbricate; capsule smooth | Cladopus |
– | Leaves of fertile stems scale-like or needle-like, distichous; capsule ribbed | 3 |
3 | Stamens 2, forked; leaves of fertile stems scale-like; capsules appressed to roots; bracts boat-shaped, entire; roots foliose, with tufts of linear leaves scattered over dorsal surface | Hydrobryum |
– | Stamen 1, simple; leaves of fertile stems needle-like, capsules erect; bracts ribbon-like; roots, ribbon-like, with tufts of leaves along both sides of root | Polypleurum |
We are grateful to Ms. D.L. Cai for the illustration and Mr. T. M. Fang and J.M. Li for his kind help during our fieldwork. D.L. Lin assisted in the construction of the phylogenetic tree. This work was financially supported by Special Project of Orchid Survey of National Forestry and Grassland Administration(contract no. 2020-07), the Sub-project VI of National Program on Key Basic Research Project (Grant No. 2015FY110200), the National Special Fund for Chinese Medicine Resources Research in the Public Interest of China (Grant No.2019-39), the Natural Science Foundation of Fujian Province (2020J05037 to MZ), the Foundation of Fujian Educational Committee (JAT190089 to MZ) and the scientific research innovation program “Xiyuanjiang River Scholarship” of College of Life Sciences, Fujian Normal University.
Appendix
Data type: Primers, Voucher information (docx. file)
Explanation note: Table S1. Primers used in this study. Table S2. Voucher information for the taxa used in the present study (matK). Table S3. Voucher information for the taxa used in the present study (ITS).