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Boehmeria nivea (L.) Gaudich (Urticaceae) , commonly known as ramie, is widely distributed in tropical and temperate regions of the old world; in China, it mainly occurs in the middle and lower reaches of the Yangtze River (Fig.1) (Wang &Chen, 1995; Chen et al., 2003) . Ramie is an economically valuable crop due to the remarkable properties of its fibers (Roy &Lutfar, 2012; Wang et al., 2019) , and it shows promise for use in the biomedical and phytochemistry industries (Lee et al., 2009; Kandimalla et al., 2016; Chew et al., 2022; Choi et al., 2022; Fauzani et al., 2022) . B. nivea is first described by Gaudichaud-Beaupré (1830) , and two varieties are currently recognised: B. nivea var. nivea has an unbranched and patently hirsute stem, free stipules, and mostly white or grey abaxial surfaces of the leaf blades, whereas B. nivea var. tenacissima has branched and appressed-strigose stems, partly connate stipules, and mostly green abaxial surfaces of the leaf blades (Chen et al., 2003) .
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During lengthy field investigations, we encountered specimens from Southwest China that did not match either variety. These specimens are characterized by green abaxial leaf blades, dense patently strigose hairs on upper stems and petioles, and partly connate stipules (Fig.2) . Therefore, we employed morphologic and phylogenomic methods to test the hypothesis that these individuals might present a new variety of B. nivea.
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1 Materials and Methods
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In this study, 24 individuals of the putative new variety were collected from different locations in Yunnan Province and Guangxi Zhuang Autonomous Region during field surveys from 2020 to 2021 (Fig.1) . Comparisons with its relatives were made by consulting specimens stored in KUN, PE, and some virtual specimen databases (BM, E and K) . Morphological observations and measurements were made from live collections and herbarium specimens, and leaf material from each accession was desiccated with silica gel for DNA analysis. Total DNA was extracted from 0.2 g of gel-dried leaves using a modified 4×CTAB method (Doyle &Doyle, 1987) . The quality of DNA was assessed using1% agarose gel electrophoresis and a NanoDrop® ND-1000 spectrophotometer.Both PCR amplification and sequencing of rbcL and nrDNA followed the method of Wu et al. (2013) . Collection location, i.e. GPS and altitude were recorded, and all sequences were uploaded to NCBI (Table S1) .
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We selected two species of Boehmeria and four species of different genera of Urticaceae as outgroups (Table S1) . We constructed maximum likelihood (ML) and Bayesian inference (BI) trees based on each of three datasets: (a) rbcL, (b) nrDNA and (c) rbcL + nrDNA. The sequences were aligned using MAFFT implemented in Geneious v.9.0.2 (Kearse et al., 2012) . The ML tree was constructed using RAxML v.8.2.11 (Stamatakis, 2014) using the GTRGAMMAI model with 1 000 rapid bootstrap replicates. MrBayes v.3.2.7 (Ronquist et al., 2012) was employed to do Bayesian inference (BI) analysis, and the best-fit model was determined by Akaike Information Criterion (AIC) in jModeltest v.2.1.7 (David, 2008) . Phylogenetic trees were then visualized in iTOL (Letunic &Bork, 2021) .
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2 Results
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2.1 Taxonomic treatment
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Boehmeria nivea var. strigosa Zeng Y. Wu &Y. Zhao, var. nov.
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糙毛苎麻 新变种 Figs.2,3
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2.2 Diagnosis
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Boehmeria nivea var. strigosa Zeng Y. Wu &Y. Zhao is similar to B. nivea var. nivea in having patently strigose stem and petiole hairs. However, B. nivea var. strigosa differs from B. nivea var. nivea by its branched stem, partly connate stipules, and primarily green abaxial surfaces of the leaf blades. Conversely, B. nivea var. strigosa shares these three characteristics with B. nivea var. tenacissima, but differs from that variety in its patently strigose hairs, whereas those of B. nivea var. tenacissima are appressed.
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图1 苎麻在中国的分布
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Fig.1 Distribution of Boehmeria nivea in China
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2.3 Type
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China. Yunnan Province: Honghe City, Hekou County, Xiaonanxi Village, sunny limestone hillside.103°56′13.2″ E, 22°39′25.2″ N, elev.134 m, 11 December 2020, WuZY20201096 (Holotype, KUN!)
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2.4 Description
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Subshrubs or shrubs, dioecious. 0.5-4.0 m tall. Stem branched, the upper part with dense, patently strigose hairs ca. 0.5-1.0 mm long. Leaves alternate; petiole6 mm long; stipules lanceolate, partly connate, 8.0-10.0 mm; leaf blade ovate, elliptic-ovate or deltoid, (6.0-13.0) cm × (3.5-7.5) cm, herbaceous, secondary veins ca.3 each side of midvein, abaxial surface greenish, with strigose hairs on veins ca. 0.3 mm long, base subtruncate, cuneate, or aequilateral, margin dentate from the base, apex acuminate. Glomerules unisexual, on specialized, unisexual flowering branches in the axils of current or recently fallen leaves, these paired and much branched, usually longer than petioles and often congested in fruit; stigma filiform, ca. 0.5 mm. Achenes rhomboid-obovoid, compressed, ca.1.5 mm, strigose-hairy on the shoulder, base constricted and stalk-like, apex almost without neck, 2-3-toothed.
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图2 糙毛苎麻的生境和形态
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Fig.2 Habitat and morphology of Boehmeria nivea var. strigosa Zeng Y. Wu &Y. Zhao
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2.5 Phenology
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Flowering from May to August and fruiting from September to January.
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2.6 Phylogenetic relationships
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There were some discrepancies between topologies from ML and BI analyses based on rbcL dataset (Figs. S1, S2) . However, consistent topologies were observed when using nrDNA dataset and the combined rbcL+nrDNA dataset (Figs. S3, S4; Fig.4) . In the ML and BI trees constructed using the three datasets, all samples of B. nivea formed a well-supported monophyletic clade (Fig.4; Figs. S1-S4) . Within this, all samples of B. nivea var. strigosa clustered into a monophyletic group (Fig.4; Figs. S1-S4) , whereas B. nivea var. nivea and B. nivea tenacissima were intermixed and could not be separated by these datasets (Fig.4; Figs. S1-S4) . In this tree, and all trees except for the BI tree based on the rbcL dataset (Fig. S2) , B. nivea var. strigosa clade was sister to the rest of B. nivea (Fig.4; Figs. S1, S3, S4) . The rbcL dataset-based trees showed the highest support rates for B. nivea var. strigosa clade (BS = 94%, PP = 0.93; Figs. S1, S2) , followed by the rbcL+nrDNA dataset (BS = 73%, PP = 0.98; Fig.4) , while the lowest support rate was observed in the nrDNA dataset (BS = 41%, PP = 0.63; Figs. S3, S4) .
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2.7 Distribution and habitat
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B. nivea var. strigosa is known from Southeast Yunnan and Southwest Guangxi in China (Fig.1) and often grows on limestone areas at elevations ca.130-800 m. All known populations appear to be naturally wild, whereas the other two varieties tend to exist in cultivation or as feralized populations.
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图3 糙毛苎麻
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Fig.3 Boehmeria nivea var. strigosa Zeng Y. Wu &Y. Zhao
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图4 基于rbcL+nrDNA序列,使用最大似然法(ML)和贝叶斯推论(BI)构建的苎麻系统发育关系
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Fig.4 Phylogenetic tree produced by maximum likelihood (ML) and Bayesian inference (BI) methods, based on the rbcL+nrDNA of Boehmeria nivea
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2.8 Etymology
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The epithet “strigosa” refers to densely patent strigose hairs on upper stems, branchlets, and petioles in this new variety.
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2.9 Conservation status
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Based on field investigations, B. nivea var. strigosa grows on limestone forest edges or by roadsides, which are moderately disturbed by human activities. So far, it is only known from Southeast Yunnan and Southwest Guangxi in China. According to the red list standards, categories and criteria of the IUCN (2022) , B. nivea var. strigosa should fall under the “Near Threatened” (NT) category. However, more field studies are needed to define and determine the correct conservation category of this variety.
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2.10 Paratypes
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China. Guangxi: Napo County, Baisheng Town, Guilin New Village, roadside with sunshine, elev.326 m, 22 October 2020, WuZY2020071 (KUN) .
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China. Guangxi: Longzhou County, Shanglong Town, Sanlian Village, roadside with sunshine, elev.250 m, 23 October 2020, WuZY2020114, WuZY2020115 (KUN, PE) .
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China. Yunnan: Hekou County, Nanxi Town, Nanxi Village, roadside, elev.134 m, 11 December 2020, WuZY20201087, WuZY20201103 (KUN) .
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China. Yunnan: Pingbian County, Baihe Town, Aka Village, roadside, elev.339 m, 13 December 2020, WuZY20201186 (KUN) .
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China. Yunnan: Jinping County, Manhua Town, Lushuihe Tropical Rainforest Resort, next to the plank road, elev.571 m, 14 December 2020, WuZY20201224, WuZY20201233 (KUN) ; WuZY20201231, WuZY20201232, WuZY20201235 (PE) .
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China. Yunnan: Hekou County, Nanxi Town, Huayudong National Forest Park, roadside, elev.153 m, 30 January 2021, Liuj2021010 (PE) .
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China. Yunnan: Hekou County, Nanxi Town, Maduoyi Village, roadside, elev.136 m, 11 December 2020, WuZY20201082 (PE) .
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China. Yunnan: Mengla County, Menglun Town, Mengxing Village, roadside, elev.560 m, 3 August 2021, WuZY2021352 (PE) .
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China. Yunnan: Yuanyang County, Nansha Town, Shitouzhai Village, beside the banana grove, elev.717 m, 16 December 2020, WuZY2021356 (PE) .
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2.11 Key to varieties of Boehmeria nivea (L.) Gaudich
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1 Stipules partly connate, stem normally branched······2
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-Stipules free, stem unbranched······B. nivea var. nivea
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2 Upper stems, branchlets, and petioles densely and patently strigose······B. nivea var. strigosa
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-Upper stems, branchlets, and petioles assurgent or appressed strigose······B. nivea var. tenacissima
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3 Discussion
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Our phylogenetic result strongly supports that B. nivea var. strigosa is a monophyletic clade within B. nivea, sister to B. nivea var. nivea and B. nivea tenacissima. In morphology, B. nivea var. strigosa shares with B. nivea var. tenacissima a branched stem, a partly connate stipule, and mostly green abaxial surfaces of the leaf blades. Therefore, the similarities between B. nivea var. strigosa and B. nivea var. tenacissima might be due to the latter reverting to wild traits when artificial selection was relaxed. Other features, for example, the patently strigose stem hairs, link B. nivea var. strigosa to B. nivea var. nivea, suggesting that the trait was apomorphically changed in B. nivea var. tenacissima. Furthermore, the distribution range of B. nivea var. strigosa overlaps considerably with that of B. nivea var. nivea. Therefore, we consider that B. nivea var. strigosa is a new variety rather than a new species, and more extensive sampling and molecular markers should be used to reveal the evolutionary relationships among these three varieties within B. nivea in the future.
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4 Acknowledgments
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We thank the herbarium of Kunming Institute of Botany (KUN) for providing us with a safe and complete specimen preservation environment, so that all specimens of this species can be properly placed for future research. Thanks to Mr. HAN Zhoudong for drawing pictures of this variety. Thanks to Dr. LIU Jie for comments on the manuscript. Special thanks to Dr. YI Tingshuang, Ms. LI Yinlei, Ms. FU Xiaogang, and Dr. LI Dezhu for their available suggestions on this study.
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摘要
糙毛苎麻(Boehmeria nivea var. strigosa Zeng Y. Wu & Y. Zhao)是一个苎麻新变种,原产于中国西南地区。该文主要基于形态学和分子系统学证据对其进行了描述。该变种的主要特征包括叶片背面绿色、托叶部分合生以及茎和叶柄上浓密的糙毛。基于rbcL、nrDNA和rbcL+nrDNA 3个数据集构建的系统发育关系结果均表明strigosa糙毛苎麻的所有个体形成一个单系分支。根据IUCN的评估标准,糙毛苎麻被评估为“近危”( NT)。该新变种的发现对苎麻的分类至关重要,并且为苎麻资源的开发和利用提供了科学依据。
Abstract
Boehmeria nivea var. strigosa Zeng Y. Wu & Y. Zhao, a new variety of B. nivea (Urticaceae) from Southwest China, is here described based on evidence from morphology and molecular phylogeny. This new variety is mainly characterized by its green abaxial leaf blade, partly connate stipules, and densely patent strigose hairs on stems and potioles. The phylogenetic analysis based on rbcL, nrDNA and rbcL+nrDNA datasets, revealed that all individuals of B. nivea var. strigosa formed a monophyletic group. The conservation status of B. nivea var. strigosa is assessed as “Near Threatened” (NT) according to IUCN evaluation criteria. The discovery of this new variety is not only crucial for the taxonomy of ramie, but also provides reference for the exploration and utilization of ramie.
Keywords
Boehmeria nivea ; morphology ; molecular phylogeny ; new variety ; Southwest China ; Yunnan