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Research Article
Wikstroemia fragrans (Thymelaeaceae, Daphneae), a new species from Mount Danxia, China based on morphological and molecular evidence
expand article infoJing-Rui Chen‡, Shiou Yih Lee§‡, Jian-Qiang Guo|, Jie-Hao Jin‡, Qiang Fan‡, Wen-Bo Liao‡
‡ Sun Yat-Sen University, Guangzhou, China
§ INTI International University, Nilai, Malaysia
| Administrative Commission of Danxiashan National Park, Shaoguan, China

Corresponding author: Wen-Bo Liao ( lsslwb@mail.sysu.edu.cn )

Academic editor: Laurence J. Dorr
© 2022 Jing-Rui Chen, Shiou Yih Lee, Jian-Qiang Guo, Jie-Hao Jin, Qiang Fan, Wen-Bo Liao.
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 J-R, Lee SY, Guo J-Q, Jin J-H, Fan Q, Liao W-B (2022) Wikstroemia fragrans (Thymelaeaceae, Daphneae), a new species from Mount Danxia, China based on morphological and molecular evidence. PhytoKeys 213: 67-78. https://doi.org/10.3897/phytokeys.213.91116
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Abstract

A new species, Wikstroemia fragrans (Thymelaeaceae, Daphneae), from Danxiashan National Park, Shaoguan, Guangdong of China is described and illustrated. It is similar to the sympatric W. trichotoma, but can be differentiated easily from the latter by its shorter racemose inflorescences, yellowish green calyx tube, and smaller leaves. It also resembles the allopatric W. fargesii, but differs from it by its strigose-pubescent ovary and disk scale that is 2- or 3-dentate apically. Phylogenetic analysis using the nuclear DNA internal transcribed spacer (ITS) region revealed that W. fragrans falls within the Wikstroemia clade; based on current sampling, W. fragrans is closely-related to W. capitata. It is also the first species of Wikstroemia known to be endemic to the Danxia landform and is classified provisionally as Critically Endangered according to the IUCN Red List Categories and Criteria.

Keywords

Danxia landform, flora, internal transcribed spacer (ITS), IUCN Redlist, phylogenetics, taxonomy, Wikstroemia

Introduction

Thymelaeaceae comprise 50 genera and about 900 species widely distributed in both temperate and tropical regions (Herber 2002, 2003). It is circumscribed to include three subfamilies: Thymelaeaoideae (ca. 42 genera), Octolepidoideae (8 genera), and a yet to be validly published “Tepuianthoideae” (1 genus). Wikstroemia Engl. is a member of the Daphne group (Daphneae) of the Thymelaeaoideae, comprising approximately 70 species. Wikstroemia is widely distributed in Asian and Oceanic regions, with some populations scattered around the Hawaiian Islands. Among these species, 54 were reportedly naturally distributed in China (Rogers 2009-onwards).

Since 2004, we have conducted a series of biological surveys on Mount Danxia to elucidate biodiversity patterns in the Danxia landform. Mount Danxia in Guangdong, China, from which the name Danxia landform is derived, is characterized by steep slopes derived from sandstones and conglomerates (Peng et al. 2018). Owing to its unique geological and geomorphic structure, the ecological succession of biota is strongly differentiated and a variety of ecosystems appear in a small scale. Mount Danxia harbors a handful of endemic plant species, of which more than ten have been discovered in the last decade, including the recently published Selaginella orientali-chinensis Ching & C.F. Zhang ex H.W. Wang & W.B. Liao (Selaginellaceae) (Wang et al. 2022) and Lespedeza danxiaensis Q. Fan, W.Y. Zhao & K.W. Jiang (Fabaceae) (Zhao et al. 2021). The discovery of these species serves to reveal the biodiversity richness of Mount Danxia.

During one of our floristic inventories in Danxiashan National Park in 2022, a plant species that most closely resembled Wikstroemia was found. It was previously overlooked and misidentified as W. nutans Champion ex Benth, which is a common species widely distributed in Guangdong. The 5-lobed calyx obviously differed from the 4-lobed calyx of W. nutans. After careful morphological comparison using herbarium specimens, digital images, and relevant literature of other similar species, we confirmed that our specimens represented an undescribed species, which we here describe as W. fragrans. Taxonomic information, including the distribution, habitat, phenology, etymology, and the International Union for Conservation of Natures (IUCN) preliminary conservation status also are provided. The epithet, fragrans, refers to the attractive scent of the flowers, which is like a mixed aroma of orchid and jasmine. A molecular phylogenetic analysis based on the nuclear DNA internal transcribed spacer (ITS) was conducted to evaluate the phylogenetic position and relationship of W. fragrans within Wikstroemia.

Materials and methods

Morphological study

The morphological characters of Wikstroemia fragrans were compared with similar species, using living plants, relevant literature, and herbarium specimens, including the Chinese Virtual Herbarium (https://www.cvh.ac.cn/) and the China Field Herbarium (https://www.cfh.ac.cn/). Morphological examination was conducted in the Herbarium of Sun Yat-Sen University (SYS). Herbarium acronyms are based on those reported by Thiers (2022).

Taxon sampling and molecular analyses

Three individuals of Wikstroemia fragrans were collected in Danxiashan National Park, Guangdong, China, during the March to April flowering season in 2022. Voucher specimens were deposited in SYS. The ITS region was used for phylogenetic reconstruction of Wikstroemia. Despite insufficiency in delimiting plants at the species level, the ITS regions of most Wikstroemia species are publicly available and partly provide an insight into phylogenetic relationships between closely related species (He et al. 2021; Lee et al. 2022). We downloaded all species of Daphneae (Thymelaeaceae) with ITS gene sequences available in NCBI GenBank for analysis. In total, 24 taxa were selected, including 17 species of Wikstroemia. Two closely-related species, Aquilaria sinensis (Lour.) Spreng. (Thymelaeaceae, Aquilarieae) and Edgeworthia chrysantha Lindl. (Thymelaeaceae, Daphneae) were included as outgroups. The GenBank accession numbers for each species used in this study are listed in Suppl. material 1.

Total genomic DNA was extracted from silica-gel-dried leaves using the modified cetyltrimethyl ammonium bromide (CTAB) protocol (Doyle and Doyle 1987). The quality and quantity of the DNA extract were determined using Nanodrop spectrophotometer (Thermo Fisher, USA). Polymerase chain reaction was carried out based on the program setting as proposed by Lee et al. (2022) using the universal primers, ITS1 and ITS4 (White et al. 1990). The amplicon was verified under UV and the PCR product was Sanger sequenced both forward and reverse directions. The ITS sequence was assembled and multiple sequence alignment was performed using ClustalW which is embedded in MEGA-X (Kumar et al. 2018). The sequences in the alignment were manually trimmed, in which the nucleotides that correspond to the primer regions were removed from the sequences to obtain a clean sequence read. Phylogenetic trees were reconstructed using the maximum likelihood (ML) and Bayesian inference (BI) methods via MEGA-X (Kumar et al. 2018) and MrBayes v3.2.7a (Ronquist et al. 2012), respectively. For ML, the optimum DNA substitution model calculated using the “Find best DNA/Protein Models (ML) function embedded in MEGA-X was Kimura two-parameter model (K2P) with gamma incorporated (+G) (=K2P+G). All branch nodes were calculated with 1000 bootstrap replicates. For BI, a mixed substitution type and a four by four nucleotide substitution model were selected for the likelihood model, and the Markov chain Monte Carlo simulations were run twice independently for 2000000 generations. Four chains were selected and sampling of data was conducted every 100 generations.

Results

Morphological comparison

Wikstroemia fragrans is similar to the sympatric W. trichotoma (Thunb.) Makino. The two species share identical features such as papery leaves, a glabrous calyx tube, 5-lobed calyx, and a lobed disk scale. However, W. fragrans differs from the latter by its smaller leaves (1.2–1.6×0.5–0.9 vs. 1.2–3.5(-8) × (0.5-)1–2.2(-4) cm), densely racemose to capitate inflorescence (vs. loose panicle), yellowish green calyx (vs. white), and strigose-pubescent ovary (vs. apically strigose ovary) (Table 1).

Table 1.
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Comparison of morphological features and distribution between Wikstroemia fragrans, W. capitata, W. fargesii, and W. trichotoma.

Characters W. fragrans W. capitata* W. fargesii* W. trichotoma*
Leaf shape ovate to ovate-lanceolate elliptic or obovate-elliptic, rarely obovate-oblong elliptic, suborbicular, or oblong-lanceolate ovate to ovate-lanceolate
Leaf size (cm) 1.2–1.6 × 0.5–0.9 1–2 × 0.4–1.0 1–2.2 × 0.8–2.0 1.2–3.5(-8) × (0.5–)1–2.2(-4.0)
Inflorescences densely racemose to nearly capitate, 4–8-flowered capitate, 3–7-flowered capitate, 7–10-flowered a panicle of spikes, few to 10(-26)-flowered
Indumentum of tube abaxially Glabrous sericeous-strigose glabrous glabrous
Number of calyx lobes Five four five five
Color of calyx yellowish green yellowish green yellowish green white, rarely yellowish green
Indumentum of ovary strigose-pubescent strigose-pubescent apically strigose apically strigose
Shape of subgynoecial disk scale linear or linear-oblong, apex 2- or 3-dentate linear, apex 2- or 3-dentate linear, apex entire or slightly retuse linear or linear-oblong, membranous, lobed or truncate
Distribution (Province) Guangdong Guizhou, Hubei, Shanxi, Sichuan Chongqing, Hunan** Anhui, Guangdong, Guangxi, Hunan, Jiangxi, Zhejiang

*Characters of Wikstroemia capitata, W. fargesii, and W. trichotoma are from the original descriptions in Flora of China (2007). **We checked the herbarium specimens of Wikstroemia fargesii collected from Mount Tianmen, Hunan (CSFI026054, CSFI034280) and confirmed their identifications.

Among the Wikstroemia species known from China, W. fragrans also resembles the allopatric and stenochoric W. fargesii (Lecomte) Domke: both species have capitate inflorescences, yellowish green calyces, glabrous tubes, 5-lobed calyces, and a linear disk scale. However, W. fragrans differs from W. fargesii by its strigose-pubescent ovary (vs. apically strigose ovary) and disk scale 2- or 3-dentate apically (vs. entire or slightly retuse apically) (Table 1).

Molecular analysis

The final sequence alignment based on the ITS dataset was 687 bp. Both the ML and BI trees revealed identical topologies; thus, the trees were merged and only the ML tree is displayed (Fig. 1). The backbone of the phylogenetic tree was not well-supported when using both the ML and BI methods, in which the bootstrap support (BS) value was less than 75% and the posterior probability was less than 0.90. Based on current sampling, species of Wikstroemia are monophyletic; W. fragrans was placed close to W. capitata and the divergence between the two species was strongly supported (BS = 75%, PP = 0.98). Wikstroemia trichotoma was placed distant from W. fragrans (Fig. 1).

Figure 1. 

Phylogenetic inference of Wikstroemia fragrans and related species based on ITS sequences. Values of Bootstrap support (BS) and posterior probability (PP) are labeled at each branch node, in which BS ≥ 75% and PP ≥ 0.90 are indicated with an asterisk (*). Wikstroemia fragrans, described in this study, is shown in bold.

Taxonomic treatment

Wikstroemia fragrans W.B.Liao, Q.Fan & J.R.Chen, sp. nov.

urn:lsid:ipni.org:names:77307946-1
Figs 2, 3, 4

Type

China. Guangdong Province, Danxia National Park, 25.0°N, 113.7°E, 74 m alt., 16 March 2022 [fl.], Qiang Fan, DNPC 1597 (Holotype SYS! Barcode SYS00236854, Isotypes SYS! Barcode SYS00236855, CSFI!).

Diagnosis

Wikstroemia fragrans differs from W. trichotoma, by its smaller leaves (1.2–1.6×0.5–0.9 vs. 1.2–3.5(-8) × (0.5-)1–2.2(-4) cm), densely racemose to nearly capitate inflorescence (vs. loose panicle), yellowish green calyx (vs. white), and strigose-pubescent ovary (vs. apically strigose ovary). Wikstroemia fragrans differs from W. fargesii by its strigose-pubescent ovary (vs. apically strigose) and a disk scale 2- or 3-dentate apically (vs. entire or slightly retuse apically).

Description

Shrub, 0.6–1.6 m tall; perennial branches reddish brown, rugose, annual branches yellowish green, glabrous. Leaves opposite or subopposite, ovate to ovate-lanceolate, 1.2 – 1.6×0.5 – 0.9 cm, thinly papery, grayish green adaxially, light yellowish green abaxially, glabrous on both surfaces, apex acuminate or obtuse, margin entire, base cuneate or subrounded, midrib flat adaxially, prominent abaxially, secondary veins 4–8 pairs per side, slightly prominent abaxially; petiole ca. 1 mm long. Inflorescence 4–8-flowered, densely racemose to capitate; peduncle 5 – 15 mm long, glabrous; pedicels absent or ca 0.4–0.7 mm long, glabrous. Calyx tube yellowish green; 9–11 mm long, exterior glabrous, lobes 5 (sometimes abnormally 6), elliptic, 2.5–3.3×1.2–1.7 mm, margin undulate, glabrous on both surfaces. Stamens 10 (sometimes abnormally 11), lower whorls of 5 anthers inserted 2–4 mm above middle of hypanthium, upper whorl of 5 anthers at throat; free portion of filaments ca 0.3 mm long; anther linear-oblong, ca 0.8 mm long; subgynoecial disk scale 1, linear or linear-oblong, apex 2- or 3-dentate, 0.8–1.0×0.3–0.7 mm, membranous, glabrous. Ovary obovoid, 3–4 mm long, ca 0.6 mm in diam., subsessile, strigose pubescent; style ca 0.2 mm long, glabrous; stigma yellow, globose, ca 0.5 mm in diam., surface papillate. Drupe ca. 6 mm long, yellowish green, ovoid-globose, glabrous, 1-seeded, enclosed by persistent calyx; fruiting pedicel ca 8 mm long. Seed ovoid, ca. 4 mm long, black, glabrous.

Distribution and habitat

Wikstroemia fragrans is currently known only from the type locality, Danxiashan National Nature Reserve (Ba Zhai, Mount Shaoshi, Shuang He Zhai, Yu Nv Lan Jiang), Guangdong, China. It occurs in xerophytic hillside thickets on sandstone and conglomerate based soil at 100–300 m elevation. In this habitat, the most common shrubby and herbaceous species are Lagerstroemia indica Linn. (Lythraceae), Symplocos tanakana Nakai (Symplocaceae), Decaspermum gracilentum (Hance) Merr. et Perry (Myrtaceae), Viola hybanthoides W. B. Liao & Q. Fan (Violaceae) and Salvia scapiformis Hance (Lamiaceae).

Phenology

Wikstroemia fragrans was observed flowering from March to April, fruiting from April to June.

Etymology

Latin fragrans, smell or odor, alluding to sweet-scented flowers. The Chinese name is given as 香花荛花 (xiāng huā ráo huā).

Conservation status

During our intensive floristic inventories in Danxiashan National Nature Reserve from September 2021 to May 2022, only 5 populations of Wikstroemia fragrans comprising 5–20 individuals each were found. Due to the limited extent of occurrence (ca. 40 km2) and area of occupancy (ca. 5 km2) and small population sizes (<100 individuals totally), W. fragrans is proposed to be classified as Critically Endangered (CR B1ac(i)+2ac(i)) according to the IUCN Categories (IUCN 2012).

Figures 2. 

Wikstroemia fragrans W.B.Liao, Q.Fan & J.R.Chen, sp. nov. A flowering branch B flower C subgynoecial disk scale opened out D dissected flower showing the normal condition of 10 stamens E strigose ovary and glabrous disk scale A–E DNPC 1597 deposited in SYS. Drawn by Rong-En Wu.

Figure 3. 

Habitat and morphological features of Wikstroemia fragrans W.B.Liao, Q.Fan & J.R.Chen, sp. nov. A, B habitat, hillsides thickets of Danxiashan National Park C flowering branches D immature fruits E densely racemose inflorescence F, G flowers, showing 5-lobed calyx. (A–C, E–G photographs by Jing-Rui Chen of unvouchered plants in the original habitat in April 2022 D photograph by Jian-Qiang Guo taken in April 2021).

Figure 4. 

Wikstroemia fragrans W.B.Liao, Q.Fan & J.R.Chen, sp. nov. A hypanthium dissected showing 10 stamens B hypanthium dissected showing abnormal 11 stamens C subgynoecial scale disk, linear-oblong, apex 2–3 dentate D pistil with strigose-pubescent ovary, glabrous style, and light green stigma. A–D DNPC 1597 deposited in SYS.

Additional specimens examined (paratypes)

China, Guangdong Province, Danxia National Park, 25.0°N, 113.7°E, 384 m alt., 14 August 2022, Wan-yi Zhao & Jing-rui Chen, DNPC 2966 (SYS); China, Guangdong Province, Danxia National Park, 24.9°N, 113.7°E, 162 m alt., 17 August 2022, Wan-yi Zhao & Jing-rui Chen, DNPC 3029 (SYS).

Discussion

The taxonomic status of Wikstroemia has long been debated; species exhibit continuous morphological variation, which has complicated efforts to distinguish between species and has created problems in the classification of the genus and its sister genera (Skottsberg 1972; Halda 2001; Wang et al. 2007; Zhang et al. 2007). However, it is generally accepted that Wikstroemia can be distinguished from related genera through its inflorescence and scale type (distinct or annular) (Domke 1934; Herber 2002; 2003; Wang et al. 2007).

Wikstroemia fragrans was previously confused with W. nutans, but can be distinguished easily by the length of its inflorescence axis and number of calyx lobes. After checking all species of Wikstroemia distributed in Guangdong, we turned our eyes to the sympatric W. trichotoma. These two species share identical features such as papery leaves, a glabrous calyx tube, five calyx lobes, and a lobed disk scale, but differ in leaf size, inflorescence, and calyx color. To support these morphological findings, molecular methods to distinguish closely similar species were also utilized. However, DNA studies of W. trichotoma are limited; there is no publicly available record of the ITS sequence of W. trichotoma. Thus, we sequenced two individuals of W. trichotoma collected from Mount Babao, which is adjacent to Mount Danxia, to be included in our phylogenetic analysis. The phylogenetic tree placed W. trichotoma distant from W. fragrans, indicating that W. trichotoma is at least not considered to be the most immediately genetically affiliated species to W. fragrans. Additionally, we noticed that among the Chinese mainland species the allopatric W. fargesii is also morphologically similar to W. fragrans. We faced enormous difficulties in collecting samples of W. fargesii, which had not been collected since the 1890s until it was allegedly rediscovered on limestone in Mount Tianmen, Hunan, but with the help of CSFI, we acquired pieces of flowers and leaves from herbarium specimens of W. fargesii from Mount Tianmen. This enabled us to make further comparisons. Dissection of these herbarium materials confirmed the identification of W. fargesii and showed differences from W. fragrans in the ovary indumentum and shape of the disk scale (Table 1). As the two species grow on different types of substrates (W. fargesii on limestone and W. fragrans on sandstone and conglomerate from red beds), we are convinced that they represent two independent species.

It is worth mentioning the fragrant flowers of Wikstroemia fragrans, which are quite rare in Wikstroemia. In general, members of Thymelaeaceae with fragrant flowers are usually found in the sister genus Daphne. Based on its fragrance, W. fragrans has the potential to be domesticated and cultivated for horticulture purposes. Based on the morphological and molecular evidence obtained through this study, we confirmed that the newly described W. fragrans is a distinct species.

Acknowledgements

We are deeply grateful to Ms. Rong-En Wu for her excellent illustration. We appreciate the staff of Danxiashan National Nature Reserve and Dr. Xun-Lin Yu of Central South University of Forestry and Technology for their generous help in collecting samples. We also thank Dr. Cui-Ying Huang and Dr. Wan-Yi Zhao for suggestions in revising this article. This study was supported by the Guangdong Provincial Special Research Grant for the Creation of National Parks (2021GJGY034).

References

  • Domke W (1934) Untersuchungenüber die systematische und geographische Gliederung der Thymelaeaceen nebst einer Neubeschreibung ihrer Gattung. Bibliotheca Botanica 27(111): 1–151.
  • Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small amounts of fresh leaf tissue. Phytochemical Bulletin 19: 11–15.
  • Halda JJ (2001) Subgenus Wikstroemia. In: Halda JJ (Ed.) The genus Daphne. SEN, Dobré, 102–153.
  • He L, Zhang Y, Lee SY (2021) Complete plastomes of six species of Wikstroemia (Thymelaeaceae) reveal paraphyly with the monotypic genus Stellera. Scientific Reports 11(1): 1–14. https://doi.org/10.1038/s41598-021-93057-3
  • IUCN (2012) IUCN Red List Categories and Criteria: Version 3.1 (2nd Edn). Gland, Switzerland and Cambridge, UK: IUCN, [iv +] 32 pp.
  • Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: Molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution 35(6): 1547–1549. https://doi.org/10.1093/molbev/msy096
  • Lee SY, Xu KW, Huang CY, Lee JH, Liao WB, Zhang YH, Fan Q (2022) Molecular phylogenetic analyses based on the complete plastid genomes and nuclear sequences reveal Daphne (Thymelaeaceae) to be non-monophyletic as current circumscription. Plant Diversity 44(3): 279–289. https://doi.org/10.1016/j.pld.2021.11.001
  • Peng H, Liu P, Zhang GH (2018) Small scale vegetation differentiation structure in Danxia Landforms, Southeast China. Dili Kexue 38(6): 944–953.
  • Rogers Z (2009 onwards) A World Checklist of Thymelaeaceae. Missouri Botanical Garden, St. Louis.
  • Ronquist F, Teslenko M, Van Der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Skottsberg C (1972) The genus Wikstroemia Endl. in the Hawaiian Islands. Acta Regiae Societatis Scientiarum et Litterarum Gothoburgensis. Botanica 1: 1–166.
  • Thiers B (2022) [continuously updated] Index Herbariorum: A global directory of public herbariaand associated staff. New York Botanical Garden’s Virtual Herbarium. http://sweetgum.nybg.org/science/ih/ [accessed 19.07.2022]
  • Wang Y, Gilbert MG, Mathew B, Brickell CD, Nevling LI (2007) Thymelaeaceae. In: Wu ZY, Raven PH, Hong DY (Eds) Flora of China, vol. 13. Science Press & Missouri Botanical Garden Press, Beijing & St. Louis, 213–250.
  • Wang HW, Dai JM, Chen ZX, Fan Q, Liao WB (2022) Selaginella orientali-chinensis, a new resurrection spikemoss species from southeastern China based on morphological and molecular evidences. Acta Scientiarum Naturalium Universitatis Sunyatseni 61(2): 57–64.
  • White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fugal ribosomal RNA genes for phylogenetics. In: Innis M, Gelfand D, Sninsky J, White T (Eds) PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, 315–322. https://doi.org/10.1016/B978-0-12-372180-8.50042-1
  • Zhao WY, Jiang KW, Chen ZX, Tian B, Fan Q (2021) Lespedeza danxiaensis (Fabaceae), a new species from Guangdong, China, based on molecular and morphological data. PhytoKeys 185: 43–53. https://doi.org/10.3897/phytokeys.185.72788

Supplementary material

Supplementary material 1 

List of the GenBank accession numbers of the ITS sequences of sampled species in this study

Jing-Rui Chen, Shiou Yih Lee, Jian-Qiang Guo, Jie-Hao Jin, Qiang Fan, Wen-Bo Liao

Data type: Phylogenetic.

Explanation note: The appendix shows the GenBank accession numbers of the ITS sequences of sampled species in this study.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
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