Camellia sinensis var. madoensis is described and illustrated as a new variety of Camellia sinensis (section Thea, Theaceae) from Xuan Loc Commune, Song Cau District, Phu Yen Province. The new variety is easily distinguishable from C. sinensis var. sinensis by style free ½ to the base. The ITS sequence of this variety is also different from that of Camellia sinensis and its other varieties, while the matK gene sequences are nearly identical among Camellia taxa

Ba Nam Nguyen

Ngoc Trieu Le

Thi Huy Tra Trinh

Thi Phuong Nhu Van

Tran Vu Nguyen

Van Dung Luong

Van Ngoc Nguyen

Xuan Son Le

Tạp chí Khoa học Đại học Đà Lạt

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DALAT UNIVERSITY JOURNAL OF SCIENCE    Volume 14, Issue 1, 2024    5–12 5 CAMELLIA SINENSIS VAR. MADOENSIS (SECT. THEA, THEACEAE), A NEW TAXON FROM VIETNAM Nguyen Tran Vua, b, Van Thi Phuong Nhua, b, Le Xuan Sonc, Nguyen Van Ngocd, Luong Van Dungd, Trinh Thi Huy Trad, Nguyen Ba Namd, Le Ngoc Trieud* aLa Hieng Science and Technology Center, La Hieng Company Limited, Phu Yen, Vietnam bPhu Yen University, Phu Yen, Vietnam cPhu Yen Vocational College, Phu Yen, Vietnam dThe Faculty of Biology, Dalat University, Lam Dong, Vietnam *Corresponding author: Email: trieuln@dlu.edu.vn Article history Received: May 17th, 2023 Received in revised form: June 21st, 2023 | Accepted: June 29th, 2023 Available online: September 22nd, 2023 Abstract Camellia sinensis var. madoensisis is described and illustrated as a new variety of Camellia sinensis (section Thea, Theaceae) from Xuan Loc Commune, Song Cau District, Phu Yen Province. The new variety is easily distinguishable from C. sinensis var. sinensis by style free ½ to the base. The ITS sequence of this variety is also different from that of Camellia sinensis and its other varieties, while the matK gene sequences are nearly identical among Camellia taxa. Keywords: Camellia sinensis; ITS; Ma Do; matK; Section Thea; Theaceae; Vietnam.        DOI: http://doi.org/10.37569/DalatUniversity.14.1.1145(2024) Article type: (peer-reviewed) Full-length research article Copyright © 2023 The author(s).  Licensing: This article is published under a CC BY-NC 4.0 license. NATURAL SCIENCES AND TECHNOLOGY [ANNUAL ISSUE ON BIOLOGY - CAMELLIAS OF VIETNAM] 6 1. INTRODUCTION The scientific name of tea is Camellia sinensis (L.) O. Kuntze. It is a shrub or small tree with broad evergreen leaves belonging to Camellia section Thea in the Theaceae (Pierre, 1887; Sealy, 1958). Several varieties of tea have been described and illustrated, including C. sinensis var. sinensis, C. sinensis var. assamica (Masters) Kitamura, C. sinensis var. pubilimba Hung T. Chang, and C. sinensis var. dehungensis (Hung T. Chang & B. H. Chen) T. L. Ming. It is distributed over countries and/or territories Taiwan, China, northeast India, southern Japan, South Korea, Laos, Myanmar, Thailand, and Vietnam (Chang & Bartholomew, 1984; Ho, 1999; Ming & Bartholomew, 2007; Nguyen, 2017; Sealy, 1958). All varieties of C. sinensis are used as tea. In recent surveys in July 2019 and October 2020, we collected specimens of tea, and after analyzing and comparing morphological characteristics and ITS sequences with other varieties of tea, we assert that this is a new taxon. The scientific name of this new taxon is Camellia sinensis var. madoensis, and its local name is Ma Do tea. The recognition of Ma Do tea as a new taxon helps to better understand the diversity of tea taxa and contributes to the scientific basis for conservation, development, and sustainable exploitation of this endemic plant resource. 2. MATERIALS AND METHODS 2.1.  Materials Materials for taxonomic treatment were individuals belonging to the natural Ma Do tea population and specimens collected from them. Three leaf samples from different individuals were taken as materials for phylogenetic analysis. 2.2. Methods Taxonomic treatment was performed according to routine methods of comparing anatomical morphology. Phylogenetic analysis was performed on the basis of the nuclear ITS1-5.8S rRNA-ITS2 sequence region and the chloroplast matK gene sequence. DNA extraction from leaf samples was performed on the basis of the CTAB I protocol (Weising et al., 2005). The ITS1-5.8S rRNA-ITS2 sequence region was amplified using the primer pairs of ITS 5P (5'-GGA AGG AGA AGT CGT AAC AAG G-3') and ITS 8P (5'-CAC GCT TCT CCA GAC TAC A3') according to the method described in Möller and Cronk (1997). The matK gene sequence was amplified using the primer pairs matK472F (5'-CCC RTY CAT CTG GAA ATC TTG GTT C-3') and matK1248R (5'-GCT RTR ATA ATG AGA AAG ATT TCT GC-3') according to the method described in Yu et al. (2011). The primers used for amplification were also used for sequencing by the Sanger method. Sequences obtained after sequencing were compared to find homologous sequences from GenBank for phylogenetic analysis using the BLAST tool. MEGA 5.1 software (Tamura et al., 2011) was used to align sequences, determine variable locations, Nguyen Tran Vu, Van Thi Phuong Nhu, Le Xuan Son, Nguyen Van Ngoc, Luong Van Dung, Trinh Thi Huy Tra, Nguyen Ba Nam,  and Le Ngoc Trieu 7 calculate genetic distances, and construct a dendrogram for phylogenetic relationships among the surveyed taxa. 3. RESULTS 3.1. Taxonomic treatment Camellia sinensis var. madoensis T. V. Nguyen, V. D. Luong & N. T. Le var.nov. (Figures 1, 2) Type:—VIETNAM. Phu Yen Province: Song Cau District, Xuan Loc Commune, 400–500 m elevation, 22 July 2019, Nguyen Tran Vu, Luong Van Dung DL190702 (holotype, isotype, DLU); 10 October 2020, Nguyen Tran Vu, Luong Van Dung DL201001 (DLU) Description: Small or medium trees, 4–8 (10) m tall, evergreen; young branches sparsely pubescent to glabrous, purple; older branches gray. Leaves stalked, broad elliptic, 7.0–10.5 cm long, 3.0–4.7 cm wide, lamina thin, coriaceous; apex acute to attenuate, base obtuse to broadly obtuse, margins regularly serrate; above dark green, shiny and glabrous, below light green and sparsely villous, generally on the midrib; midribs and lateral veins sunken above, protruding below; secondary venation pinnate with 7–10 pairs of veins; petiole stout, 0.5–0.7 cm long, slightly curved, round, purple in color, sparsely villous. Flowers solitary or 2–(3), axillary, nodding, 2.7–3.3 cm diameter; pedicel 1.2–1.5 mm long, thickened toward the apex, glabrous, pale blue-purple in color. Bracteoles 2–3, ovate, 1.8–2.0 mm long, 1.0–1.5 mm wide, pale blue, glabrous, ciliolate, deciduous. Sepals 5–6, broadly ovate to suborbicular, 3.0–4.5 mm long, 3.0–4.0 mm wide, pale green, glabrous, ciliolate, persistent. Petals 6, outer whorl 2, subglobose, 1.1–1.3 cm long, 0.8–1.0 cm wide, green-white, glabrous; inner whorl 4, broadly obovate, 1.4–1.7 cm long, 1.1–1.4 cm wide, white, glabrous; inner whorl petals united with outermost filaments 1.5–2.0 mm at the base. Androecium numerous stamens in 3–4 circles, filaments 0.8–2.2 cm long, light yellow, glabrous, outer filaments united 3.0–4.0 mm from the base and form a short cup; anthers yellow, about 1.5 mm. Gynoecium (2)–3 loculi, ovary ovate, 2.0–3.0 mm long, 2.0–2.5 mm wide, densely white tomentose; style 3, free ½ to the base, 1.0–1.2 cm long, glabrous. Capsule globose to depressed globose, 1–3 locular, 2.0–3.5 cm in diameter, 2.0–2.5 cm high; seeds 1–2 per loculus, globose or hemispherical 1.4–1.8 cm long, 1.2–1.5 cm wide, glabrous; pericarp 2.0–2.5 mm thick, sparsely pubescent. Phenology: Flowers from May to July and fruits from October to December. Distribution and ecology: The distribution of this species is known from Ma Do Mountain, Xuan Loc Commune, Song Cau District, Phu Yen Province. It is located in secondary forests on rocky mountain slopes or in valleys. Etymology: The specific epithet refers to the type location, Ma Do Mountain, Xuan Loc Commune, Song Cau District, Phu Yen Province, Vietnam. Use: Leaves and buds are harvested and used as tea. NATURAL SCIENCES AND TECHNOLOGY [ANNUAL ISSUE ON BIOLOGY - CAMELLIAS OF VIETNAM] 8 Note: The new taxon has many characteristics of section Thea (Linnaeus) Dyer in J. D. Hooker, such as pedicel thick, thickened toward the apex; bracteoles 2(or 3), caducous; sepals persistent; petals white, basally connate; stamens numerous, in 4 or 5 whorls, glabrous, outer filament whorl basally connate into a tube. Ovary 3–5-loculed; style apically 3–5-parted or lobed (Ming & Bartholomew, 2007; Sealy, 1958). The new taxon is easily distinguishable from C. sinensis var. assamica and C. sinensis var. dehungensis by ovary tomentose (vs. ovary glabrous at least apically); from C. sinensis var. pubilimba by sepals glabrous (vs. sepals outside pubescent); and from C. sinensis var. sinensis by style free ½ to the base (vs. style fused, apically 3-lobed). The known variants of Camellia sinensis can be distinguished using the following key. 1a. Ovary densely tomentose..................................................................................2 1b. Ovary glabrous at least apically........................................................................4 2a. Sepals outside white pubescent ..................................................var. pubilimba 2b. Sepals outside glabrous....................................................................................3 3a. Style free ½ to the base..............................................................var. madoensis 3b. Style fused, apically 3-lobed..........................................................var. sinensis 4a. Leaf blade abaxially densely spreading villous along midvein......var. assamica 4b. Leaf blade abaxially appressed pubescent..............................var. dehungensis  Figure 1. Camellia sinensis var. madoensis T. V. Nguyen, V. D. Luong & N. T. Le Notes: (a) Leaf (adaxial view); (b) Venation detail of leaf (abaxial view); (c) Flower (top view);  (d) Flower (back view); (e) Sepals; (f) Petals; (g) Androecium; (h) Stamens; (i) Gynoecium; (j) Fruit;  (k) Capsule after dehiscence; (l) Seeds.  Source: Photos by Luong Van Dung. Nguyen Tran Vu, Van Thi Phuong Nhu, Le Xuan Son, Nguyen Van Ngoc, Luong Van Dung, Trinh Thi Huy Tra, Nguyen Ba Nam,  and Le Ngoc Trieu 9  Figure 2. Camellia sinensis var. madoensis T. V. Nguyen, V. D. Luong & N. T. Le Notes: (a) Natural plant; (b) Leader of field trip that recorded Ma Do tea; (c) Shoots and leaves;  (d) Flower buds; (e) Flowers; (f), (g) Fruit.  Source: Photos by Luong Van Dung. 3.2.  Phylogenetic analysis The sequencing results showed that the ITS1-5.8S rRNA-ITS2 region sequences of three Ma Do tea samples were identical, and the length of the clear region was 669 bp. NATURAL SCIENCES AND TECHNOLOGY [ANNUAL ISSUE ON BIOLOGY - CAMELLIAS OF VIETNAM] 10 The results of a sequence comparison on GenBank using the BLAST tool showed that the 100 sequences most nearly identical (94.84–99.19%) to ITS1-5.8S rRNA-ITS2 sequences of Ma Do tea were homologous sequences of the taxa belonging to the Camellia genus, including C. sinensis, C. sinensis var. sinensis, C. sinensis var. assamica, C. chekiangoleosa, C. tachangensis var. remotiserrata, and C. liberistamina. The genetic distances between Ma Do tea and Camellia sinensis varieties, including C. sinensis var. sinensis, C. sinensis var. assamica, and other Camellia taxa, range from 0.0081 to 0.0195 based on ITS region. The genetic distances were calculated using MEGA 5.1 software. The phylogenetic relationship dendrogram was constructed on the basis of the genetic distances using the maximum likelihood method, with Stewartia rostrata as the outgroup (Figure 3).  Figure 3. Phylogenetic relationship between Ma Do tea and other Camellia taxa based on the ITS sequence using the maximum likelihood method The sequencing results showed that the matK gene sequences of three Ma Do tea samples were identical, and the length of the clear region was 860 bp. A sequence comparison on GenBank using the BLAST tool showed that the 100 sequences most nearly identical (99.88–100.00%) with the matK gene sequences of Ma Do tea were homologous sequences of the taxa belonging to the Camellia genus, including Camellia sinensis, C. sinensis var. sinensis, C. sinensis var. assamica, C. sinensis var. lasiocalyx, C. atrothea, and C. ptilophyla,  The genetic distances between Ma Do tea and Camellia sinensis and its varieties, including C. sinensis var. sinensis, C. sinensis var. assamica, and C. sinensis var. lasiocalyx, range from 0.0000 to 0.0012 on the basis of the matK gene. Ma Do tea cannot be identified from several sources belonging to Camellia sinensis and C. sinensis var.  FJ004881.1 Cameliia sinensis var. sinensis cultivar Keemon EF544723.1 Cameliia sinensis var. assamica clone Taq 3 EF649692.1 Camellia liberistamina FJ432098.1 Cameliia sinensis var. assamica isolate S455 EF544730.1 Cameliia sinensis var. assamica clone Pfu 5 MH270493.1 Cameliia sinensis var. assamica FJ004872.1 Camellia sinensis var. sinensis cultivar Guey Hua FJ004861.1 Camellia sinensis var. sinensis cultivar TTES FJ004878.1 Cameliia sinensis var. sinensis cultivar Dah Nan FJ004870.1 Cameliia sinensis var. sinensis cultivar Horng Shoin Wuu EF637042.1 Camellia chekiangoleosa EU579779.1 Camellia taliensis AY096014.1 Camellia sinensis EU579725.1 Camellia tachangensis var. remotiserrata MA DO TEA MF171093.1 Stewartia rostrata95243638Nguyen Tran Vu, Van Thi Phuong Nhu, Le Xuan Son, Nguyen Van Ngoc, Luong Van Dung, Trinh Thi Huy Tra, Nguyen Ba Nam,  and Le Ngoc Trieu 11 sinensis. Based on these genetic distances, a phylogenetic relationship dendrogram was constructed using the maximum likelihood method with Stewartia rostrata as the outgroup (Figure 4).  Figure 4. Phylogenetic relationship between Ma Do tea and other Camellia taxa based on the matK gene sequence using the maximum likelihood method The results of phylogenetic analysis show that the ITS region and matK gene sequences of Ma Do tea are closely grouped with Camellia taxa, particularly C. sinensis and its varieties. However, based on the sequence of the ITS region, Ma Do tea has certain differences with the investigated Camellia taxa, and this supports the identification of Ma Do tea as a separate taxon that does not overlap with published varieties of Camellia sinensis, such as C. sinensis var. sinensis or C. sinensis var. assamica. Based on the matK gene sequence, Ma Do tea is identical to several sources of C. sinensis var. sinensis released on GenBank. Combining the morphological data (especially the style characteristics) and the molecular data (especially the sequence of the ITS region), it is possible to identify Ma Do tea as a new variety of C. sinensis. 4. CONCLUSIONS Ma Do tea possesses its own morphological and molecular characteristics that are different from those of other varieties of Camellia sinensis varieties. This is the scientific basis to identify this tea as a new taxon from Vietnam with the scientific name of Camellia sinensis var. madoensis.  MN473197.1 Camellia sinensis var. sinensis cultivar TRF 2 OM677602.1 Camellia sinensis var. assamica OM677573.1 Camellia sinensis var. assamica isolate TCM219K-2020 OM677600.1 Camellia sinensis var. assamica isolate TMS295K-2020 OM677609.1 Camellia sinensis var. assamica OM677602 Camellia sinensis var. assamica isolate TMS297K-2020 MN138448.1 Camellia sinensis var. lasiocalyx cultivar UPASI 24 MA DO TEA AJ429305.1 Camellia sinensis MN138457.1 Camellia sinensis var. sinensis cultivar OSD 6 MN473198.1 Camellia sinensis var. sinensis cultivar TRF 4 MH753081.1 Stewartia rostrata matK63NATURAL SCIENCES AND TECHNOLOGY [ANNUAL ISSUE ON BIOLOGY - CAMELLIAS OF VIETNAM] 12 REFERENCES Chang, H. T., & Bartholomew, B. (1984). Camellias. Timber Press. Ho, P. H. (1999). Theaceae. In P. H. Ho (Ed.), An Illustrated flora of Vietnam (Vol. 1) (pp. 424–432). Youth Publishing House. Ming, T. L., & Bartholomew, B. (2007). Theaceae. In Z.-Y. Wu, P. H. Raven, & D. Y. Hong (Eds.), Flora of China (Vol. 12) Hippocastanaceae through Theaceae (pp. 366–478). Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis. Möller, M., & Cronk, Q. (1997). Origin and relationships of Saintpaulia (Gesneriaceae) based on ribosomal DNA internal transcribed spacer (ITS) sequences. American Journal of Botany, 84(7), 956–965. https://doi.org/10.2307/2446286 Nguyen, H. H. (2017). Thuc vat chi Viet Nam (Flora of Vietnam), ho Che-Theaceae D. Don (pp. 160–273). Publishing House for Science and Technology. [In Vietnamese]. Pierre, L. (1887). Flore forestière de la Cochinchine (Vol. 2). Octave Doin, Paris. Sealy, J. R. (1958). A revision of the genus Camellia. Royal Horticultural Society. Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., & Kumar, S. (2011). MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. 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CAMELLIA SINENSIS VAR. MADOENSIS (SECT. THEA, THEACEAE), A NEW TAXON FROM VIETNAM

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CAMELLIA SINENSIS VAR. MADOENSIS (SECT. THEA, THEACEAE), A NEW TAXON FROM VIETNAM

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