Preliminary morphological assessment of six new, yellow flowering Camellia (Theaceae) species from Viet Nam

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1 Preliminary morphological assessment of six new, yellow flowering Camellia (Theaceae) species from Viet Nam George Orel and Anthony S. Curry (Royal Botanic Gardens, Mrs Macquaries Road, Sydney, NSW 2000, Australia) Author for correspondence: honorary research associate; Abstract: Six new, yellow flowering species of Camellia (Theaceae), endemic to the southern provinces of Viet Nam, were compared to one another and to nine Camellia species from Viet Nam and China. Morphological evidence indicates the dissimilarity of the majority of the newly discovered Camellia species to other yellow flowering Camellia taxa. Available data supports the assertion that: 1. the morphological differences are reflected in the respective genomes; 2. the morphological dissimilarities indicate modified primitive traits; 3. southern Viet Nam is an important centre of genetic diversity and the possible area of origin for genus Camellia. 83 Key words: Camellia; Lam Dong; new species; Theaceae; Viet Nam. 1 Introduction Historically only a relatively small number of Camellia species were considered to be native to the southern mountain regions of Viet Nam. The northern parts of Viet Nam (e.g. the Tam Dao National Park and the adjacent geographical areas which are relatively close to the Chinese border) were traditionally considered to be the major centres of distribution of Vietnamese Camellia species (Sealy, 1958; Chang & Bartholomew, 1984; Gao et al., 2005). Recent systematic exploration carried out by a team of Australian and Vietnamese scientists on the Da Lat Plateau and the Lang Biang Massiff in the southern provinces of Viet Nam resulted in the discovery of a number of new Camellia taxa, some of which have already been published (Orel, 2006; Orel & Wilson, 2010a, Orel & Wilson 2010b, Orel et al, 2012). These discoveries not only confirm the floral richness of this geographical area but also establish it as an important centre of Theaceae genetic diversity and the possible northern boundary of the hypothesized area of origin for genus Camellia (Orel & Marchant, 2006). Table 1. General provenance and population data for 15 yellow flowering Camellia species from Viet Nam and China that were used in this paper axon* Provenance Population notes C. dongnaiensis* Viet Nam Less than 20 individual plants collected C. luteocerata Viet Nam Small scattered population C. inusitata Viet Nam Small relatively protected population C. sp. 0720** Viet Nam Relatively large established population C. sp. 698** Viet Nam Small scattered population C. sp. CT5** Viet Nam Population size unknown C. gilbertii Viet Nam Recently discovered new population C. petelotii Viet Nam Population relatively large C. flava Viet Nam Population relatively large C. rosmanii Viet Nam Small population some in cultivation C. aurea P.R.China Population size unknown C. nitidissima P.R.China In cultivation C. nitidissima var. microcarpa P.R.China Taxonomic status uncertain C. luteoflora P.R.China Population size not known C. tunghinensis P.R.China Population size not known * Bold print denotes newly discovered Camellia species ** Species in the process of publication, accession Nos. only

2 84 2 Materials and Methods Morphological characters of 15 yellow flowering species of Camellia from Viet Nam and China were observed to indicate the interspecific status of the six newly-discovered Camellia taxa from Viet Nam (Table 1.). All materials were collected in the wild and the exact provenance details for each were withheld for conservation reasons. General provenance and population data for all species used is presented in Table 1. Morphological data was derived from observations of the six newly discovered Camellia species and the nine yellow flowering Camellia from Viet Nam and China. The flower colour and the dominant morphological traits of the six newly discovered species are presented in Table 2. Some 30 multiple state characters were scored from in situ observations. The assessments of herbarium materials were performed under laboratory conditions. The morphological matrix, where the absence of a character was denoted 0 and the presence of a character 1, 2, 3, 4, 5 and 6 was constructed and analyzed (Fig. 1.). Phylip 3.69, a computational program for inferring evolutionary phylogenies was used to generate the taxonomic trees. ITS (Internal Transcribed Spacer) molecular procedures were also used to attempt to resolve the taxonomic status of the six newly discovered Camellia species. Genomic DNA was extracted from the fresh leaves using a method similar to that of Dellaporta et al (1983, in Wilkie, 1997). ITS PCR amplification was achieved using a number of primer pairs, e.g. ITS4 and ITSleu1 (White et al 1990). Horizontal Agarose electrophoresis procedures followed the modified protocol of Sambrook et al (1989). 3 Results The attempts to directly sequence the ITS PCR product of the six new Camellia species, without cloning, were not completely successful. Camellia dongnaiensis, C. inusitata and C. sp provided marginally viable ITS PCR products. The ITS PCR products of the other three new Camellia species were judged to be inferior. As such the ITS molecular results were not included in this work. 4 Discussion Historically only a relatively small number of yellow flowering Camellia species were considered native to the southern provinces of Viet Nam. The northern parts of Viet Nam, e.g. the Tam Dao National Park and its environs as well as the geographical areas relatively close to the Chinese border, were traditionally considered to be the major centres of yellow Camellia distribution. This fact can be amply demonstrated by even a cursory look at the pertinent literature. The total number of published, southern Vietnamese yellow flowering Camellia species fluctuates from author to author and is subject to issues surrounding the flower colour of herbarium specimens, e.g. of C. krempfii (Gagnep.) Sealy and C. dormoyana (Pierre ex Laness.), and inconsistencies present in published descriptors (C. gilbertii (A. Chev.) Sealy) (Sealy, 1958; Chang & Bartholomew, 1984; Ho, 1991; Tran, 2002; Gao et al., 2005). The relatively recent exploration of the southern provinces of Viet Nam carried out by the Australian-Vietnamese scientific team resulted in the discovery of at least six new yellow flowering Camellia species (Orel, 2006; Orel & Wilson, 2010a, Orel & Wilson 2010b, Orel et al, 2012). These and numerous other Camellia and Theaceae finds established southern Viet Nam as Table 2. Morphological details for six, newly discovered, yellow flowering Camellia species from Viet Nam Taxon Flower colour Morphology, notes C. dongnaiensis yellow-apricot, pink margins large, up to 60.0 cm long leaves C. luteocerata intensely yellow flowers laterally oblongoid C. inusitata light yellow lacks secondary branching C. sp light to mid yellow, pink-lilac margins oblate fruit, 5.5 cm diam. C. sp. 698 dark yellow petals in spiral arrangement C. sp. CT5 pale yellow inflorescence of flower buds

3 Interspecific relationships of 15 yellow flowering Camellia species based on respective morphological traits Figure 1. Interspecific relationships of 15 yellow flowering Camellia species based on respective morphological traits 85 C. dongnaiensis* C. gilbertii C. petelotii C. rosmanii C. aurea C. nitidissima C. nitidissima var. microcarpa C. inusitata C. flava C. tunginhensis tunghinensis C. sp. CT5 C. luteoflora C. sp C. sp. 698 C. luteocerata an * Bold important print denotes centre the of genetic newly discovered diversity species and the possible area of origin for genus Camellia (Orel & Marchant, 2006). The discovery of more than 30 new Camellia species (so far) in this region places the geographical area of southern Viet Nam on a par with other known global centres of diversity, e.g. the Western Ghat Mountains that hold about 30% of India s plant species (Bawa & Krishnaswami, 2007) and the state of Georgia in the USA, which contains some 30% of all tree species of the USA and Canada (Brown & Kirkman, 1990). It is important to note, that the six newly-discovered Camellia species from south Viet Nam (Table 1.) possess a number of morphological characteristics that are quite dissimilar to those of the other, already described yellow Camellia taxa (Table 2.). Some of these traits are not present in the morphologies of known yellow Camellia species. For example, to name only the most obvious dissimilarities, C. inusitata Orel, Curry & Luu lacks secondary branching, C. sp. 698 and C. luteocerata possess a spiral and not whorled petal arrangement and C.

4 86 sp. CT5 has a large inflorescence composed of 12 to14 flower buds (Table 2.). Figure 1. which shows the interspecific relationships that exist between the six newlydiscovered Camellia species and the nine yellow Camellia species from Viet Nam and China reflects the morphological differences that exist between the new and the old Camellia species. C. luteocerata Orel, C. sp. 698, C. sp and C. sp. CT5 nest separately within the dendrogram (Fig. 1.). The position of C. dongnaiensis Orel with the rest of the Vietnamese and Chinese yellow Camellia species is consistent with the published data (Orel, Marchant & Curry 2007) and that yet to be published. C. dongnaiensis Orel seems to possess fewer primitive morphological traits than the geographically allied yellow Camellia species. The position of C. inusitata Orel, Curry & Luu is considered anomalous and the reasons for this are not precisely known. It appears that the reproductive characters of C. inusitata Orel, Curry & Luu, i.e. its floral parts, may be akin to other yellow Camellia species, but dissimilar to the other species vegetative characters. It is hoped that further morphological and molecular studies will more adequately explain this inconsistency. Data contained in Fig. 1. may have implications in regard to the general taxonomy of genus Camellia and also for the taxonomy of the family Theaceae. The question: do the rather unorthodox morphological dissimilarities of the six newly discovered species translate into a general genetic dissimilarity? This question can be answered in the affirmative. The phenotypic unorthodoxy is reflected in the respective Camellia genotypes. The attempts to directly sequence the ITS PCR product of the new Camellia species were only partially successful, even when using a number of previously tested, positive result bearing primers. As this paper is only a preliminary study into the interspecific relationships of the south Vietnamese yellow flowering species of Camellia, a larger study, that will eventually encompass all of the newly-discovered Camellia species, is already in progress and will be published later. Literature cited [1] Bawa, K.S., Das, A. & Krishnaswami J Western Ghats and Sri Lanka Biodiversity Hotspot. Critical Ecosystem Partnership Fund Publication. [2] Brown, C.L. & Kirkman, L.K Trees of Georgia and adjacent states. Portland Oregon: Timber Press. [3] Chang HT and Bartolomew B Camellias. Timber Press, Portland, Oregon. Dellaporta, S.F, Wood, T. & Hicks, J.B A plant DNA minipreparation: Version 2 in: Plant Molecular Biology Report`1: [4] Gao J, Parks CR and Du Y Collected species of the genus Camellia: An illustrated outline. Zhejiang Science and Technology Press, P.R. China. [5] Ho, P-H Cayco Viet Nam. Vol. 1., Part 1., Santa Ana, California, Mekong Printing. [6] Ming, T. L. & Bartholomew, B Theaceae. In: Z.-Y. Wu & P. H. Raven (editors), Flora of China, Vol. 12. Science Press, Beijing, and Missouri Botanical Garden Press, St. Louis. [7] Orel, G Notes on Camellia dongnaiensis a new specie from Vietnam. International Camellia Journal 2006, No. 38: [8] Orel, G., Marchant, A. D Investigation into the evolutionary origins of Theaceae and genus Camellia (Adapted from Melbourne Congress lecture) International Camellia Journal 2006, No. 38: [9] Orel, G., Marchant A. & Curry, A.S Molecular investigation and assessment of C. azalea Wei 1986 (SYN. C changii Ye 1985) as potential breeding material. International Camellia Journal 2007, No. 39: [10] Orel, G., Wilson, P. 2010a. Camellia luteocerata sp. nov. and a new section of Camellia (Dalatia) from Vietnam. Nordic Journal of Botany 28: [11] Orel, G. & Wilson, P. G. 2010b. A New Species of Camellia Sect. Stereocarpus (Theaceae) from Vietnam. - Novon 20:

5 [12] Orel, G., Wilson, P. G., Curry, A. S. & Luu Hong Truong Camellia inusitata (Theaceae), a new species forming a new section (Bidoupia) from Vietnam. Edinburgh Journal of Botany 69: [13] Sambrook, J., Fritch, E.F. & et al Molecular cloning. Cold Spring Harbor: Laboratory Press. [14] Sealy, J. R. (1958). A revision of the genus Camellia. Royal Horticultural Society. [15] Tran, N Biodiversity of the genus Camellia of Viet Nam. Proceedings of the First National Symposium on yellow Camellias in Viet Nam. Tam Dao. [16] White, T.J., Bruns, S., Lee, S. and Taylor, J Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In Innis, M.A., Gelfand, D.H. Sninski, J.J. and White, T.J. (eds), PCR protocols: a Guide to Methods and Amplifications. Academic Press, London, p [17] Wilkie, S Isolation of total genomic DNA. Plant molecular biologya Laboratory Manual. London: Springer 87 Camellia Dilinhensis: A New Yellow Species from Viet Nam Tran Ninh 1 Luong Van Dung 2 Camellia dilinhensis Tran & Luong, sp. nov. (Fig. 1) Small tree, 3-4 m high; young branches glabrous. Leaves stalked, petioles cm; blades oblong-elliptic or elliptic, cm. long, cm. wide, deep green above, light green below; midribs sunken above and protruding below, glabrous on both sides: base cuneate, apex acuminate, leaf margin shallowly serrulate; lateral veins 8-14 pairs. Flowers light yellow, cm. in diameter, 1-3 in each group in axillary; pedicels 5-7 mm long, bearing two or three tiny bracteoles, glabrous. Sepals 5, scale shape nearly rounded, 4-8 mm. high, 8-11mm. wide, glabrous. Petals 8-9, nearly rounded or elliptic, cm. wide, cm. high, glabrous. Androecium over 350 stamens, 5-9 mm. long, glabrous, outer filaments united from the base and forms a short cup, united with petals mm. at the base. Gynoecium 3, ovaries cylinder, pubescent, 3-4 mm wide, 7-8 mm high, 3 loculi; styles 3, free to the base, 5 mm. long, pubescent. Fruits obovoid, immature fruits scarred white hairy; mature fruits glabrous, cm. wide, cm. high, 3 loculi, 5-6 seeds in each loculus. Seeds cuneate, 7-10 mm. high, 8-11 mm. wide, pubescent. Blooming season: winter This species was collected in evergreen forest of Dilinh district (Lamdong province), at altitudes of m. Typus: Vietnam, Lam Dong, Dilinh, evergreen forest, at altitudes of m, 22/10/2012, Dung, Ninh (Holotypus, Isotypus HNU). Taxonomic remarks: Camellia dilinhensis possesses some morphological characteristics common to the species belonging to Section Chrysantha Chang such as flowers axillary, medium large, yellow, pedicellate but there are some differences: pedicels bear two or three tiny bracteoles (Sect. Chrysantha have 5-7 bract.); ovary cylindrical (Sect. Chrysantha ovary globose); fruits obovoid (Sect. Chrysantha fruits globose). Based on these differences we affirm that this species belongs to a new Section and will be named Obvoidea Tran et Luong, Sect. nov.

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