https://orcid.org/0009-0000-7521-2065
Figures
Abstract
Three new species of Polycarpaea from Vietnam, Polycarpaea vanphongensis V.T. Tran, H.T. Truong & N.V. Binh, Polycarpaea chungana V.T. Tran, H.T. Truong & N.V. Binh, Polycarpaea phuquocensis V.T. Tran, H.T. Truong & N.V. Binh are described and illustrated based on evidence of molecular sequence data from two markers (ITS1-5.8S-ITS2 and rps16) and combined morphological characteristics. Polycarpaea vanphongensis is closely related to Polycarpaea gaudichaudi Gagnep., Polycarpaea arenaria (Lour.) Gagnep., Polycarpaea phuquocensis V.T. Tran, H.T. Truong & N.V. Binh but differs by its stem glabrous, leaf ovate to elliptic, glabrous, ovary oblong ovoid, base obtuse, apex attenuate, capsule oblong void, 3.8 mm long. Polycarpaea phuquocensis V.T. Tran, H.T. Truong & N.V. Binh differs from the three species mentioned above by its stem being densely villous, leaf spathulate, ciliate, ovary ovoid, base acute, apex obtuse, capsule ovoid, 1.2 mm long. Polycarpaea chungana V.T. Tran, H.T. Truong & N.V. Binh is most similar to Polycarpaea lignosa Gagnep., but differs in having leaf oblong or linear, sparse ciliate, sepal and petal apex deeply concaved or slightly bifid, ovary ovoid, ovoid, 0.8–1.0 mm long. Furthermore, the achievements of analysis using molecular data on the systematic positions of 7 other species are results that have not been in previous molecular analyses.
Citation: Hoang TT, Le LB, Le NTK, Nguyen MTA, Truong ATL, Tran NT, et al. (2024) Molecular phylogeny and cryptic morphology: A combined approach to taxonomic novelties in Polycarpaea (Caryophyllaceae) from Vietnam. PLoS ONE 19(10): e0301407. https://doi.org/10.1371/journal.pone.0301407
Editor: Waqas Khan Kayani, University of Kotli, PAKISTAN
Received: March 16, 2024; Accepted: May 17, 2024; Published: October 16, 2024
Copyright: © 2024 Hoang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting information files.
Funding: This research was funded by grants from the Vingroup Innovation Foundation (VINIF.2020.DA04). The funders supported money for all the study and publishing but had no role in study design, data collection, and analysis, the decision to publish, or the preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The perennial xerophytic genus Polycarpaea Lamrck (1792:3) [1] (Caryophyllaceae Juss) comprises approximately 50 species in the world, and 6 species have been recorded in Vietnam [2, 3]. It’s widely distributed in the tropics and subtropics of the Old World, and a few occur in the New World tropics [3]. The systematics of Polycarpaea is still mainly based on morphological characters used for recognizing taxa and placed in the tribe Polycarpeae along with 15 other genera with capsular fruits [4, 5]. It is widely known, and aspects of its morphological characters are often considered to be diagnostic for an herbaceous annual or herbaceous perennial in habit. Notable plants belonging to Polycarpaea have very similar vegetative and reproductive characteristics, such as dichotomously branched herbs, sometimes woody at the base, leaves opposite and often with developed axillary buds, which give the presence of whorls, stipules scarious, inflorescences a cyme, terminal provided with scarious bracts, sepals scarious, petals structurally similar to the sepals, stamens 5, style simple, stigma punctate to globular, capsule 3-valved [6–12]. However, the characters used for the most common classification of Polycarpaea are based on characteristics of the stipules, petals, sepals, ovaries, and fruits.
During collecting trips along coastal and island foredunes in Vietnam from 2020–2022, the authors found several perennial populations of xerophytic Caryophyllaceae distributed on dunes along the coast. Specimens of branches and flowering branches were collected. Specimens of branches and flowering branches were collected and represented by the collection VinIF0312, VinIF0029, and VinIF0388 (Table 1). The ranges of the three collections fall into three regions. One of these is the population of collection VinIF0029 growing sparsely scattered in the grass vegetation on the backdune, which is the climatic type of semi-arid in the south center. The population of collection VinIF.0312 is growing sparsely scattered in the shrubby vegetation on the foredune, which is the climatic type of arid in the north-south. The population of collection VinIF.0388 is growing sparsely scattered in the casuarina plantation, which is the climatic type of semi-arid in Phu Quoc Island, South Vietnam. All collected specimens were dissected and studied. The analysis results show that vegetative and reproductive characteristics are similar to the abovementioned characteristics. However, there could not be keyed out to any named species from Vietnam and neighboring regions. Although it is often assumed that vegetative and reproductive characteristics should be considered when delimiting species. However, the interrelationships between species and their allies are too complex to resolve based on morphological characteristics alone. The evidence provided for achieving clear delimitation is the use of molecular data and morphological characters in investigating relationships within taxa. Recently, a phylogenetic reconstruction based on sequences of the nuclear ITS gene and chloroplast gene regions (rps16) has been extensively proven valuable for studies of Caryophyllaceae at low or high taxonomic levels [13–16]. Therefore, in this study, details of morphological characteristics were used for comparison and combined with phylogenetic analysis based on the nuclear ITS gene sequence and the chloroplast gene region (rps16) to clarify the identities certainty of currently collected specimens of the genus Polycarpaea in Vietnam.
Materials and methods
Ethics statement
Plants studied were collected along coastal and island foredunes in Phu Hai District, Ha Tinh Province; Trieu Phong District, Quang Tri Province; Phu Loc District, Thua Thien Hue Province; Ninh Hoa District, Khanh Hoa Province; Bac Binh District, Binh Thuan Province; Xuyen Moc District, Baria—Vung Tau Province; Phu Quoc Island, Kien Giang Province. All locations are not protected in any way. The voucher specimens are deposited in the Dalat University Herbarium (DLU).
Morphological observation
Vegetative parts and flowers were observed under an Olympus SZX2-ILLK light microscope and color photographs were taken with a Canon Power Shot SX10IS. Based on field investigation and specimen examination, all vegetative and reproductive characters of the plant material were checked taxonomic key and descriptions of Polycarpaea, which were provided from Vietnam [2, 6, 7]; from China [3]; from Malaysia [8]. The features of the new species were verified by consulting herbarium specimens at Herbaria (HN, VMN, IBSC, K, KUN, and P). Botanical terminology of the description was followed by Gagnepain [6, 7], Cowie [8], Ho [2], Bakker [9], and Arya [11, 12].
Molecular markers
Taxon sampling and DNA extraction.
The fresh leaves of the new species and their allies were collected and summarized in Table 1. They were used for DNA extraction using a modified cetyltrimethylammonium bromide (CTAB) method [17]. The quality and quantity of extracted genomic DNA were measured using a UV-spectrophotometer, and agarose-gel electrophoresis.
PCR, PCR product purification, and sequencing.
The polymerase chain reaction (PCR) was performed in a 50 μl reaction mixture containing 25 μL My Red HS Taq mix (Bioline), 2.5 μL 10 pmol/μL forward primer, 2.5 μL 10 pmol/μL reverse primer, and approximately 50 ng of DNA templates. The PCR reaction was performed in thermocyclers (Eppendorf, Germany) with the following cycling parameters: 94°C (5 min); 35 cycles of 94°C (30 s), 55–60°C (30 s); 72°C (30 s), then 72°C (7 min). The DNA sequence of the ITS1-5.8S rRNA-ITS2 region was isolated and amplified using the primer pairs ITS 5P (5’- GGA AGG AGA AGT CGT AAC AAG G -3’) and ITS 8P (5’- CAC GCT TCT CCA GAC TAC A -3’), respectively (Moller et al. 1997) [18]. Forward and reverse primers for rps16 intron isolation and amplification were rpsF (5’- GTG GTA GAA AGC AAC GTG CGA CTT—3’) and rpsR2 (5’- TCG GGA TCG AAC ATC AAT TGC AAC—3’), respectively [19]. PCR products were first visualized on agarose gels (1.0%) containing safe gel stain (Inclone, Korea), and then were purified using Isolate II PCR and Gel purification Kits (Bioline). DNA sequencing was performed using Sanger method using ABI 3730 sequencers (Singapore).
Sequence alignment and phylogenetic analysis.
All sequences were assembled using SeqMan software (DNASTAR Lasergene, DNASTAR, USA) and then aligned using the MAFFT program [20]. Phylogenetic tree construction and the reliability assessment of internal branches were conducted using the maximum likelihood (ML) method with 1,000 bootstrap replicates using MEGA 6.0 [21].
Nomenclature.
The electronic version of this article in Portable Document Format (PDF) in a work with ISSN or ISBN will represent a published work according to the International Code of Nomenclature of algae, fungi, and plants, and hence the new names contained in the electronic publication of a PLOS ONE article are effectively published under that Code from the electronic edition alone, so there is no longer any need to provide printed copies.
In addition, new names contained in this work have been submitted to IPNI, from where they will be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix http://ipni.org/. The online version of this work is archived and available from the following digital repositories: PubMed Central, and LOCKSS.
Results and discussions
Phylogenetic relationships within Polycarpaea in Vietnam
The length of ITS1-5.8S-ITS2 regions in investigated taxa was 809–811 bp with 63 variable sites, while the length of the partial rps 16 gene was 882–885 with 8 variable sites. The combined sequence data sets (ITS1-5.8S-ITS2 and rps16) with 1693–1695 bp lengths and 71 variable sites were analyzed. The achieved phylogenetic relationship, dendrogram based on ML possessed the topologies with bootstraps values (MLBS)≥ 65% mapped on the corresponding nodes (Fig 1).
Numbers at the nodes are ML bootstrap values from 1000 replicates. Red filled round bullets indicate new species in the current study, and blue filled round bullets indicate previously recorded species.
In the strict phylogenetic relationship, dendrograms were consistently divided into two groups. The group with a long style can be recognized as having two subgroups. The first group, separated from the others with high bootstrap value (100%), included Polycarpaea gaudichaudii, P. areanria, P. vanphongensis, and P. phuquocensis, and another comprising P. lignosa and P. chungana with high bootstrap support value (100%). The group with short style revealed that P. stylosa was a sister.
Taxonomic implication of comparative molecular studies
As in previous analyses of the reproductive morphological characteristics of the genus Polycarpaea in Vietnam, Gagnepain [6, 7] indicated that species of this genus are divided into two groups; one group is characterized by a short style (P. corymbosa and P. stylosa) and the other group has a long style (P. arenaria and P. gaudichaudii). Most species that were currently collected from Vietnam were recovered within the group, which is the long style. Therefore, these new species are closely related to P. arenaria. P. lignosa and P. gaudichaudii. However, based on reproductive and vegetative characteristics, these new species are completely different.
P. chungana collected from Bac Binh, Binh Thuan Province is closely related to, but distinct from P. lignosa by its leaf oblong or linear, ciliate, sepal, and petal apex deeply concaved or slightly bifid. Polycarpaea vanphongensis collected from Van Ninh, Khanh Hoa Province, possesses a stem glabrous, leaf ovate to elliptic, glabrous, ovary oblong ovoid, base obtuse, apex attenuate. These characteristics can be distinguished considerably from P. duongana, P. arenaria, and P. gaudichaudii. P. phuqocensis collected from Phu Quoc island, Kien Giang Province, is similar to the three species mentioned above, but differs by leaf spathulate, ciliate, sepal and petal small, capsule ca. 1.2 mm long, stigma 1 mm long.
The molecular evidence (Fig 1), as well as morphological characteristics (Tables 2 and 3; Figs 2–8), indicated that P. vanphongensis, P. chungana, P. phuquocensis are closely related to, but differ from P. arenaria, P. lignosa, and P. gaudichaudii.
Ovaries: A1. P. gaudichaudii; A2. P. arenaria. A3. P. vanphongensis; A4. P. phuquocensis; A5. P. chungana; A6. P. lignosa; A7. P. stylosa. Stipules: B1. P. gaudichaudii; B2. P. arenaria; B3. P. phuquocensis; B4. P. vanphongensis; B5. P. chungana; B6. P. lignosa; B7. P. stylosa. Photographs by V.T. Tran, T.T. Vinh.
A. Habit. B. Section of the stem with pseudoverticillate leaves. C. Internode with stipules. D. Leaves. E. Inflorescence. F, H. Bracts. G. Flower cluster. I. Sepal. J. Petal with stamen. K. Gynoecium. L. Capsule. Photo by V. T. Tran, H. T. Truong.
A. Habit; B. Section of the stem with pseudoverticillate leaves; C. Stipule; D. Inflorescence. E. Bracts; F. Opening Flower; G Sepal; H. Petal; I. Dry Gynoecium; L. Capsule. Photo by V. T. Tran, T. T. Vinh.
A. Habit; B. Section of the stem with pseudoverticillate leaves; C. Flowers; D-E. Bracts; F. Stipule; G. Sepal; H. Petal; I. Fresh gynoecium; J. Dry Gynoecium. Photo by V. T. Tran, T. T. Vinh.
Taxonomic treatment
Polycarpaea vanphongensis V.T. Tran, H.T. Truong, N.V. Binh sp. nov.
[urn:lsid:ipni.org:names: 77342739–1] (Figs 3 and 4).
Type.
VIETNAM, Khanh Hoa Province, Van Ninh district, Van Tho village, Van Phong Bay, Xuan Dung beach, coastal sand beach, alt. 5m, 109040’85”E, 12068’76”N, 9 September 2023, V. T. Tran, H. T. Truong, N. V. Binh VinIF.0029 (holotype: DLU), the same locally, 11 September 2023 V. T. Tran, H. T. Truong, N. V. Binh VinIF.1223 (isotypes: DLU).
Diagnosis.
Polycarpaea vanphongensis is characterized by having a stem glabrous, leaf ovate to elliptic, glabrous, ovary oblong ovoid, base obtuse, apex attenuate, capsule oblong void, 3.8 mm long.
Description.
Perennial herbs, sub-erect and branched at base, ca. 25 cm high. Stems terete, glabrous, nodes reddish swollen, internodes 2–4 cm long. Leaves at the base, pseudoverticillate, sessile, oval, plump, slightly revolute, glabrous, light green, 8–10 mm long, 4–5 mm wide, base rounded, margin recurved, apex apiculate, veins 2-paired, prominent veins on abaxial surface; leaves at the top ovate to elliptic, 4–7 mm long, 2–4 mm wide; stipules scarious, triangle shape, concave, 1–1.5 mm long, 1–1.5 mm wide, margin fimbriate, inner surface villous, outer surface prominently nerved, white. Inflorescence terminal or axils of terminal leaves, compound cyme sparsely ca. 2.5–5 cm long; bracts paired, ovate-lanceolate, fimbriate along the margin, apex deeply bifid, white, ca. 1.8–2.0 mm long. Flowers 4–4.5 mm long; bracteoles, triangle, fimbriate along the margin, bifid when old; pedicels 2–3 mm long, glabrous. Sepals 5, free, ovate 2.8–3 mm long, ca. 1 mm wide, margin entire, apex acute, a yellowish circle around a reddish pot at the base, dark red when old, midrib prominent. Petals 5, lanceolate 3.8–4 mm long, 1–1.2 mm wide, margin entire, acute at apex, embraced the ovary, 1/3 longer than sepals, dark red at the base and along the midrib to 3/4 petals. Stamens 5, villous filament at the base, ca. 3–3.5 mm long; anthers oblong. Ovary 1-loculed, long ovoid, apex obtuse, apex attenuate 0.8–1.0 mm long, 0.4–0.5 mm wide; style 2 mm, slender; stigma lobbed. Capsule ovoid, 3.6–3.8 mm long, 1.2–1.5 mm wide, shortly stipitate, 3-valved, breaks along the suture, brownish, thin along the margin.
Etymology.
The specific epithet refers to the type locality, Van Phong Bay, Van Ninh district, Khanh Hoa Province.
Additional specimens examined.
Vietnam. Polycarpaea arenaria (Polycapaea arenaria var. condorensis): Indochine, Iles de Poulo-Condor (Cochinchine française), 1876–8, F.J. Harmand 662 (P01902862!, P01902863!); Polycarpaea arenaria (Polycarpaea arenaria var. longifolia). Cambodia monts de Knang-Krepeuh, May 1870, Pierre (P04925647!, P04925648!); Indochine, Pro. Ba Ria, Xuyen-mot, Feb. 1866, sept. 1860, sept. 1864, Mar 1868, Pierre (P04925632!, P04925633!, P04925636! P04925645!); Pro. Bien Hoa, Bao Chanh, sept. 1867, Pierre (P04925636!); Polycarpaea arenaria Gagnep. (Polycarpaea arenaria var. parviflora). Vietnam. Indo-Chine, Cochinchine, La-Thien, Bao-Chiang, Sept. 1865 (1862-8/1866-8), C. Thorel 1966 (P01902866!, P01902867!, P01902868!,); Polycarpaea arenaria Annam: Ca Na, Pro: Phanrang, 24-12-23, Poilane 9297 (P04925641!); Polycarpaea arenaria Ca Na, Pro: Phanrang, 3-3-23, Poilane 5531 (P04925638!);
Polycarpaea phuquocensis V. T. Tran, H.T. Truong & N. V. Binh sp. nov.
Type.
VIETNAM, Kien Giang Province, Phu Quoc district, Cua Duong village, Ong Lang beach, coastal sand-beach, alt. 1 m, 103092’70”E, 10027’05”N, 21 March 2021, V. T. Tran, H. T. Truong, N. V. Binh VinIF.0388 (holotype: DLU).
Diagnosis.
Polycarpaea phuquocensis is characterized by having a stem densely villous, leaf spathulate, ciliate, ovary ovoid, base acute, apex obtuse, capsule ovoid, 1.2 mm long.
Description.
Perennial herbs, sub-creeping and branched at base, 20–25 cm long. Stems terete, densely villous, nodes greenish, internodes 2–3 cm long. Leaves spathulate, pseudoverticillate to opposite, ciliate, 1.6–1.8 long, 3.0 mm wide; leaves at top: rare lanceolate, light green, 1.5–1.8 mm long, 1.9–2.1 mm wide, base truncate, entire margins, apex apiculate, 1 midrib and prominent on abaxial surface; stipules scarious, triangle, 0.8–1.0 mm long, 0.4–0.6 mm wide, apex apiculate ca. 0.3 mm, margins fimbriate, villous, outer surface prominently nerved, white. Inflorescence terminal, compound cyme sparsely with a 3.0–5.0 cm long; bracts paired, scarious, triangle, apex apiculate to attenuate, fimbriate along the margins, bifid or slightly bifid, white, ca. 1.5–1.6 mm long, 0.8–1.5 mm wide. Flowers 2.8–3.0 mm long, 0.8–2.0 mm white; pedicels 5–13 mm long, densely villous. Sepals 5, free, oval ca. 2.2 mm long, 1 mm wide, sparse fimbricate at the base, apex obtuse. Petals 5, oval 2.9–3.1 mm long, 1.0–1.2 mm wide, obtuse, margins entire, apex acute, embraced ovary, ca. 3/4 longer than sepals, lemon along the midrib from base to 3/4 of petals. Stamens 5, glabrous, ca. 2.2 mm long; anthers yellow, oblong, dorsifixed. Ovary 1-loculed, shortly stipitate, ovoid, 0.8–1.0 mm long, 0.7–1.0 mm wide, finely lined, placentation free central; style 1 mm. Capsule ovoid 1.0–1.2 mm long, 0.5–0.7 mm wide, 3-valved, breaks along the suture, brownish, thin along the margin.
Additional specimens examined.
Polycarpaea gaudichaudii. Mallaca, Voyage de M. Gaudichaud sur la Bonite 1836–37, Feb. 1837, s.n. 184 (P01902871!, P01902872!); Polycarpaea gaudichaudii. Vietnam. Cochinchine-Tourane, Voyage de M. Gaudichaud sur la Bonite 1836–37, Jan. 1837, s.n. 184 (P01902869!, P01902870!); Polycarpaea gaudichaudii. Annam. Tourane, 17-2-39, Poilane 28844 (dupl. VNM, E), Poilane 28844 (P05139175!, P04925935!, P04925942!); Polycarpaea gaudichaudii. Cochinchine, Phú Quốc, Dương Đông, 13-6-1938, E. Poilane 27275 (P04925936!); Polycarpaea gaudichaudii. Indochine, Tourane, 1912, H. Lecomte and A. Finet 874 (P04925937!); Polycarpaea gaudichaudii. Cochinchine, Nha Trang, Bá-Ta, 23-4-23, Poilane 6045 (P04925939!); Polycarpaea gaudichaudii. Indochine, Tourane, 12-II-II, Lazaret 902 (P04925941!); Polycarpaea gaudichaudii. Indo-chine, Tourane, 19 Jan. 1903, D. Bois 695 (P04925943!); Polycarpaea gaudichaudii. Indo-chine, Thừa Thiên Huế, Plain de Nước-Ngọt, 28 Juin 1916, s.n. 3197 (P04925948!).
Polycarpaea chungana V. T. Tran, H.T. Truong & N. V. Binh, sp. nov.
Type.
VIETNAM, Binh Thuan Province, Bac Binh District, Bình Châu village, coastal sand-beach, alt. 4 m, 1080499’552”E, 110126’395”N, August 6, 2023, V. T. Tran, H. T. Truong, N. V. Binh VinIF.0312 (holotype: DLU), the same locally, September 26, 2023 V. T. Tran, H. T. Truong, N. V. Binh VinIF.1227 (isotypes: DLU).
Diagnosis.
Polycarpaea chungana is characterized by having leaf oblong or linear, sparse ciliate, sepal, and petal apex deeply concaved or slightly bifid, ovary ovoid, ovoid, 0.8–1.0 mm long.
Description.
Perennial herbs, sub-creeping and branched at base, 25–35 cm long. Stems terete, nodes greenish, internodes 1.2–1.5 cm long, densely villous. Leaves pseudoverticillate, sessile, oblong or linear, 0.6–0.8 cm long, ca. 0.1 cm wide; margins entire, apex acute or obtuse, sparse white ciliate, 1 midrib, prominent midrib on abaxial surface; stipules scarious, oblong triangle, 2–3 mm long, 1–1.2 mm wide, apex apiculate ca. 3 mm, margin fimbriate, outer surface prominently nerved, white. Inflorescence terminal, compound cyme, densely 1.5–2.0 cm long; bracts paired, scarious, oblong triangle, 2–2.5 mm long, 0.8–1.0 mm wide, apex apiculate, ca. 2 mm, margin fimbriate, outer surface prominently nerved, white. Flowers 2.8–3.0 mm long, 0.8–2.0 mm white; pedicels 2–3 mm long, densely long villous. Sepals 5, free, oblong-ovate ca. 2.2 mm long, 1 mm wide, sparse fimbricate at the base, apex concave deeply or slightly bifid. Petals 5, oblong-ovate 2.0–2.5 mm long, 1.0–1.2 mm wide, apex concave deeply or slightly bifid, margins entire, embraced ovary, orange along the midrib from base to 3/4 of petals. Stamens 5, villous, ca. 2.2 mm long; anthers yellow, oblong, dorsifixed. Ovary 1-loculed, shortly stipitate, ovoid, 0.8–1.0 mm long, 0.7–1.0 mm wide, finely lined, placentation free central; style 1 mm. Capsule ovoid, 1.0–1.2 mm long, 0.5–0.7 mm wide, 3-valved, breaks along the suture.
Conclusions
Based on the molecular data with ITS1-5.8S-ITS2 region and rps16 gene and morphology, a combined approach to taxonomic novelties in Polycarpaea (Carophyllaceae) from Vietnam. Three new species, P. vanphongensis and P. duongana, P. chungana, are sparsely distributed along the coast and Island of South VietNam. The primary habitat of new Polycarpaea is the coastal dunes have an arid climate and are associated with Hedyostis pinifolia, Fimbristylis lasiophylla, Glinus oppositifolius, Euphorbia atoto, Gisekia pharmacoides, Ipomaea stolonifera, Ipomoea pes-caprae, Launaea sarmentosa. The result also suggests that integrated morphological and molecular data should used to recognize and describe new species of Polycarpaea.
Supporting information
S1 Fig. The ML consensus tree of two molecular marker rps16.
https://doi.org/10.1371/journal.pone.0301407.s001
(TIF)
S2 Fig. The ML consensus tree of marker ITS1-5.8S-ITS2.
https://doi.org/10.1371/journal.pone.0301407.s002
(TIF)
S3 Fig. The quality of total extracted genomic DNA and PCR products.
a. total extracted genomic DNA. b. ITS1-5.8S rRNA-ITS2 PCR products. c. rps16 intron PCR products. M. DNA ladder (Bioline a. 1kb. b. c. 100bp).
https://doi.org/10.1371/journal.pone.0301407.s003
(TIF)
S4 Fig. The quality of DNA sequencing products.
A, ITS1-5.8S rRNA-ITS2 sequences; B, rps16 intron sequences.
https://doi.org/10.1371/journal.pone.0301407.s004
(TIF)
S1 Table. The quality and quantity of total extracted genomic DNA.
https://doi.org/10.1371/journal.pone.0301407.s005
(DOCX)
References
- 1. Lamarck JB. Sur le nouveau Polycarpaea. Journal d’Histoire Naturelle. 1792; (2):3,5,478.
- 2. Pham HH. An illustrated Flora of Vietnam 1. Montréal. 1993; pp. 935–936.
- 3.
Dequan L, Gilbert MG. Polycarpaea Lamarck. In: Wu Z., Raven PH. (eds) Flora of China (Caryophyllaceae-Lardizabalaceae) 6. Science Press and St Louis, Missouri, USA: Missouri Botanical Garden Press. 2001; pp. 1–113.
- 4.
Bittrich V. Caryophyllaceae. In: Kubitzki K., Rohwer J., Bittrich V. (eds.). The Families and Genera of Vascular Plants. Magnoliid, Hamamelid and Caryophyllid Families 2. Springer, Berlin. 1993; pp. 206–236.
- 5. Smissen RD, Clement JC, Garnock-Jones PJ, Chambers GK. Subfamilial relationships within Caryophyllaceae as inferred from 5! ndhF sequences. Amer. J. Bot. 2002; (89):1336–1341. pmid:21665736
- 6. Gagnepain F. Nouveautés Asiatiques de L’herbier du Museum. Bulletin de la Société Botanique de France. 1909; (56):37–39.
- 7. Gagnepain F. Un genre nouveau de Butomacées et quelques espèces nouvelles d’Indo-Chine. Bulletin de la Société Botanique de France. 1939; (86):300–303.
- 8. Cowie ID. Three new species, a new name and notes on Australian Polycarpaea (Caryophyllaceae). Nuytsia. 1994; 9(3):319–332.
- 9. Bakker K. Revision of the genus Polycarpaea (Caryoph.) in Malaysia. Acta Botanica Neerlandica. 1957; (6):48–53.
- 10. Venu P, Muthukumar SA, Daniel P. Polycarpaea majumdariana (Caryophyllaceae)—a new species from Tamil Nadu, India. Nordic Journal of Botany. 2001; 21(6):577–579.
- 11. Arya S, Kumar VNSA, Nathanpillai AV, Philip AA, Sojan J, Suresh V. Polycarpaea palakkadensis (Caryophyllaceae), a new species from Kerala, South-West India. Phytotaxa. 2021; 527(2):151–157.
- 12. Arya S, Kumar VNSA, Pillai AV, Alen AP, Sojan J, Suresh V. Three new species of Polycarpaea (Caryophyllaceae) from Kerala, South India. PhytoKeys. 2022; 213:95–110 (2022) pmid:36762247
- 13. Fior S, Karis P O, Casazza G, Minuto L, Sala F. Molecular Phylogeny of the Caryophyllaceae (Caryophyllales) Inferred from chloroplast MatK and nuclear rDNA ITS sequences. American Journal of Botany. 2006; 93(3):399–411. pmid:21646200
- 14. Kool A, Bengtson A, Thulin M. Polyphyly of Polycarpon (Caryophyllaceae) inferred from DNA sequence data. Taxon; 2007; 56(3):775–782.
- 15. Harbaugh DT, Nepokroeff M, Rabeler RK, McNeill J, Zimmer EA, Wagner WL. A new Lineage-based tribal Classification of the Family Caryophyllaceae. Intnational Journal of Plant Sciences. 2010; 171(2):185–198.
- 16. Kool A, Perrigo A, Thulin M. Bristly versus juicy: Phylogenetic position and taxonomy of Sphaerocoma (Caryophyllaceae). Taxon. 2012; 61 (1):67–75.
- 17. Allen G, Flores-Vergara M, Krasynanski S, Kumar S, Thompson W. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethylammonium bromide. Nature Protocols. 2006; 1:2320–2325. pmid:17406474
- 18. Moller M, Cronk Q. Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences. American Journal of Botany. 1997; 84(7):956. pmid:21708650.
- 19. Oxelman B, Lidén M, Berglund D. Chloroplastrps16 intron phylogeny of the tribe Sileneae (Caryophyllaceae). Pl. Syst. Evol. 1997; 206:393–410.
- 20. Katoh K, Misawa K, Kuma K, Miyata T. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 2002; 30(14):3059–66. pmid:12136088.
- 21. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution. 2013; 30:2725–2729. pmid:24132122