ISSN 1346-7565 Acta Phytotax. Geobot. 67 (2): 83–96 (2016) doi: 10.18942/apg.201518

Flora of Bokor National Park, Cambodia IV: A New Section and Species of Euphorbia Subgenus Euphorbia

1,* 1 2 Hironori Toyama , Shuichiro Tagane , Phourin Chhang ,

3 1 Hidetoshi Nagamasu and Tetsukazu Yahara

1Center for Asian Conservation Ecology, Kyushu University, 744 Motooka, Fukuoka 819-0395, Japan. * [email protected] (author for correspondence); 2 Institute of Forest and Wildlife Research and Development, Forestry Administration, 40 Preah Norodom Blvd, Phnom Penh, Cambodia; 3 The Kyoto University Museum, Kyoto University, Yoshida-Honmachi, Sakyo-ku, Kyoto 606-8501, Japan

A new section, Euphorbia sect. Bokorenses under subgenus Euphorbia is established for Euphorbia bo- korensis H. Toyama & Tagane, sp. nov., endemic to Bokor National Park, Cambodia. Euphorbia sect. Bokorenses is distinguished from related sections Denisophorbia and Goniostema by unbranched stems and smooth seeds, respectively. Bayesian phylogeny using matK, ndhF, and ITS regions supports its monophyly from the currently recognized 21 sections. It has a sister relationship with the Malagasy clade, including sections Denisophorbia, Deuterocalli and Goniostema, and to the northeastern African clade, including sect. Rubellae. A description, illustration, photographs and preliminary conservation status of the new species, and an updated identification key to the sections ofEuphoria subg. Euphorbia is provided.

Key words: Bokor National Park, Cambodia, Euphorbia, new section, new species

Euphorbia subg. Euphorbia () in E. subg. Euphorbia. Euphorbia sect. Euphor- exhibits the greatest diversity among subgenera bia is the largest clade in E. subg. Euphorbia hav- in both species richness and growth forms (Horn ing over 340 species extending from Africa to et al. 2012). The last revision of E. subg. Euphor- Asia, and is a lineage sister to E. sect. Monadeni- bia based on comprehensive taxonomic, geo- um of eastern, central, and southeastern Africa. graphic and phylogenetic studies using 661 spe- In Indochina, 10 species of Euphorbia. subg. cies (Dorsey et al. 2013) showed revealed four Euphorbia are known, of which six are cultivated major clades, a New World clade, a Pacific clade, and the other four, E. antiquorum L., E. lacei an Old World clade I, and an Old World clade II. Craib., E. ridleyi Croizat, and E. sessiliflora Twenty one sections were recognized, including Roxb., are native (Gagnepain 1931, Dy Phon 9 newly described (Fig. 1). According to the clas- 2000, Hô 2003, Esser 2005, Newman et al. 2007). sification of Dorsey et al. (2013), all of the Asian All species native to Indochina included in E. species are included in E. sect. Pacificae and E. sect. Euphorbia generally have succulent stems sect. Euphorbia. Euphorbia sect. Pacificae was with a spine shield that is a horny pad of tissue established by Dorsey et al. (2013) and occurs in subtending or surrounding each leaf base (Dors- Australia, Indonesia, New Guinea, and the Pacif- ey et al. 2013). ic islands. It has a unique distribution in the In 2011–2013, we investigated the flora in Bo- southern Pacific, and is one of the basal lineages kor National Park, Cambodia, and collected ca. Figure 1 Toyama et al.

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100 69 sect. Euphorbia_Asia/Africa/Arabia 100 sect. Monadenium_E./C./SE. Africa 100 65 sect. Deuterocalli_Madagascar 68 70 100 sect. Denisophorbia_Madagascar 89 Old World sect. Goniostema_Madagascar clade II 99 100 100 sect. Goniostema_Madagascar sect. Goniostema_Madagascar

85 sect. Goniostema_Madagascar

100 sect. Rubellae_NE. Africa 56 100 sect. Pervilleanae_Madagascar 97 Old World sect. Tirucalli_Madagascar clade I sect. Pachysanthae_Madagascar

100 sect. Crepidaria_N. America 100 100 sect. Brasilienses_Brazil 57 100 100 sect. Stachydium_S. America

99 sect. Nummulariopsis_N./C./S. America 97 sect. Portulacastrum_C./S. America New World 99 sect. Calyculatae_N. America 99 clade 93 sect. Euphorbiastrum_C./S. America 75 66 sect. Mesophyllae_C./S. America

sect. Lactifluae_C./S. America 100 51 sect. Cubanthus_Caribbean sect. Tanquahuete_N. America 100 sect. Pacificae_Pacfic Pacific clade Other subgenerasubg.

Fig. 1. Schematic phylogeny of Euphorbia subg. Euphorbia in Dorsey et al. (2013) at section level. Branches are labeled with bootstrap support greater than 50. Distribution follows Dorsey et al. (2013).

3,100 specimens, including ca. 770 identified sect. Euphorbia. Comparative morphological and woody species, and 22 new species (Tagane et al. phylogenetic studies provided evidence to elimi- 2015a,b, Naiki et al. 2015, Tanaka et al. 2015, nate its placement within any of the 21 sections rec- Toyama et al. 2016, Yahara et al. in press). Addi- ognized by Dorsey et al. (2013). Here, we describe a tionally, we discovered a spurge with subsuccu- new section, E. sect. Bokorenses H. Toyama & lent stems, gland-like stipules and conspicuous Tagane, to accommodate it and a new species, Eu- cyathophylls that did not belong in Euphorbia phorbia bokorensis H. Toyama & Tagane, based on Figure 2. Toyama et al.

June 2016 Toyama & al.— New Section and Species of Euphorbia from Cambodia 85

1000

15°N 200 10°40′N 7 11

1000 5 400

600 800 400 Cambodia

13°N 1 10 1000 1000 1000 Bokor 3 9 National Park 800 1000 800 2 600

600 11°N 400 400 6 4 8 200 10°36′N

200

9°N 1 km

103°E 105°E 107°E 104°02′E 104°06′E

Fig. 2. Location of surveyed sites in Bokor National Park, Cambodia. Surveyed sites are shown by black dots; number above black dot indicates plot ID. Gray line indicates road. Dashed gray lines are contours. Arrows indicate locality where Eu- phorbia was collected. morphological and phylogenetic evidence. baria BKF, FU, KAG, TI, and VNM, and digi- tized specimen images on the web (e.g., JSTOR Global ). The collections of spurge were Materials and methods also compared with the original descriptions of closely related species and with the taxonomic Field surveys keys of Dorsey et al. (2013). We conducted seven field surveys (December 3–23 in 2011; May 8–16, July 14–20, and October Molecular phylogenetic analysis 15–26 in 2012; February 16–17, August 6–13, and Leaf pieces of the spurge were dried using sil- December 7–12 in 2013) on the southern slope ica-gel in the field (voucher specimen: Toyama et and the top plateau of Bokor National Park, Kam- al. 1912, FU). DNA isolation was performed by pot Province, Cambodia (Fig. 2). We recorded the the CTAB method (Doyle & Doyle 1987) with flora by using plot-based surveys and general col- minor modifications. Before DNA extraction, lections. We placed 21 rectangular plots of 100 × dried leaf material was milled by QUIAGEN Tis- 5 m2 along an altitudinal gradient from 266 m to sueLyser to obtain fine powder. The powder was 1,043 m. Data from 11 plots were used in this washed up to five times in a 1 mL buffer (0.1 M study; in the other 10 plots we recorded only HEPES, pH 8.0; 2% Mercaptoetanol; 1% PVP; above 4 m tall (Fig. 2, black dots with the number 0.05 M Ascorbic acid). We sequenced the partial of plot ID). Each plot was divided into 10 subplots genes for the large subunit ribulose-1,5-bisphos- of 10 × 5 m2, in which we recorded all the species phate carboxylase oxygenase (rbcL) and mat- of vascular plants we encountered. In addition, urase K (matK) according to published protocols we collected specimens at 94 locations along the (Kress et al. 2009, Dunning & Savolainen 2010) whole altitudinal gradient of the park (from 15 to for DNA barcoding (CBOL Working Group 1,047 m) (Fig. 2, black dots). 2009). Each sequence was BLAST searched against GenBank to find species having similar Morphological comparison sequences. We also examined dried specimens in the her- In addition to the barcoding regions, we se- 86 Acta Phytotax. Geobot. Vol. 67

Table 1. Summary statistics of datasets used for phylogenetic inference. Chloroplast DNA data set consisted of concatenated matK and ndhF sequences and indel data. Three-gene dataset consisted of concatenated cpDNA and ITS sequences. *In Dorsey et al. (2013), E. bongensis is not included in the supplementary Data of “ITS Alignment”, but in the “3-gene Align- ment”. Here, we used the ITS sequence of E. bongensis in the file of “3-gene Alignment”.

Regions matK ndhF ITS* cpDNA 3-gene

Number of accessions 178 163 283 233 315 Aligned sequence length 2082 1449 669 3531 4200 Variable DNA sites (%) 829 (40) 548 (38) 449 (67) 1377 (39) 1826 (43) Parsimony-informative sites (%) 490 (24) 357 (25) 404 (60) 847 (24) 1251 (30) Indels 95 40 125 137 262 Specific variable DNA sites to Toyama et al. 1912 (FU)(%) 11 (0.53) 3 (0.21) 2 (0.30) 14 (0.40) 16 (0.38) Specific indels to Toyama et al. 1912 (FU) 0 2 1 2 3 Sequence length of Toyama et al. 1912 (FU) 1979 916 623 2895 3518

quenced matK including the partial trnK intron, phyly of each species for faster calculations. We NADH dehydrogenase subunit F (ndhF) gene, ran five independent chains of 100 million gen- and the internal transcribed spacer region of the erations each, sampling every 10,000 genera- nuclear ribosomal DNA (ITS) according to pub- tions. The first 1,000 trees were discarded as lished protocols (Dorsey et al. 2013). All three at- burn-in from each run, and then 9,000 trees were tempts of PCR for ndhF region using the primer resampled from all runs using LogCombiner v sets of 536F/1318R, 5F/972R, 536F/1318R, 1.6.1 (Drummond & Rambaut 2007). Among the 5F/1318R, and 972F/3R (Lohmann 2006) failed, posterior distribution of 9,000 trees, the maxi- so we used the primer sets of 972F (Lohmann mum clade credibility was identified using 2006) and 2110Ri (Steinmann & Porter 2002). We TreeAnnotator v 1.6.1 (Drummond & Rambaut downloaded available sequences from the supple- 2007) with a posterior probability limit of 0.5 and mentary data of Dorsey et al. (2013) and aligned median node heights. with our sequences in which we deleted the same regions of the partial trnK intron flanking the matK following Dorsey et al. (2013). The se- Results quence alignment was performed by MAFFT v7.220 (Katoh & Standley 2013), in which the “-- Field surveys auto” option that automatically selects the opti- Among 11 plots, we found fewer than ten in- mal options was used. MEGA v 6.06 (Tamura et dividuals of Euphorbia bokorensis in one plot al. 2013) was used to check electropherograms (No. 2) placed in an evergreen forest at 888 m alt. and to edit aligned sequences. We used a Bayes- (Fig. 2) where Castanopsis accuminatissima ian methods implemented in the program BEAST (Blume) A. DC., Olea salicifolia Wall. ex G. Don, v 1.6.1 (Drummond & Rambaut 2007). We set the hypoleucum Kurz, Dracaena gracilis GTR + γ model of molecular evolution following Wall. Illicium cambodianum Hance, Schima cre- a previous study (Dorsey et al. 2013) and used an nata Korth., and Xanthophyllum schizocarpon uncorrelated lognormal (UCLN) relaxed-clock Chodat dominated. In general collections from model to infer relative divergence times. A ran- 94 locations, we found E. bokorensis at only two dom starting tree was used, and a Yule process locations, at 602 m and 912 m alt. (Fig. 2). In each was set for the tree prior that was useful for spe- location, we found less than ten individuals. The cies-level analyses (Drummond & Rambaut evergreen forest at 602 m alt. contained Mallotus 2007). Topological constraints include the mono- paniculatus (Lam.) Müll. Arg., Alphitonia incana FigureJune 3A 2016 Toyama etT oyamaal. & al.— New Section and Species of Euphorbia from Cambodia 87

Fig. 3B 1 1 E. bracteata 1 E. lomelii 0.88 0.68 E. cymbifera 1 1 E. calcarata sect. Crepidaria_N. America 1 E. colligata E. finkii 1 1 E. tithymaloides E. personata 1 E. attastoma 1 E. phosphorea sect. Brasilienses_Brazil 1 E. sipolisii 1 1 1 E. lagunillarum 1 E. heterodoxa sect. Stachydium_S. America 0.93 1 E. comosa 1 0.61 E. portulacoides 0.96 1 E. caespitosa 1 E. sp. 1 0.84 E. elquiensis 1 E. peperomioides E. elodes sect. Nummulariopsis_N./C./S. America 1 0.98 E. papillosa 1 E. thinophila 1 0.77 E. rosescens E. telephioides E. germainii sect. Portulacastrum_C./S. America 1 1 E. hoffmanniana E. pteroneura 1 E. dussii sect. Euphorbiastrum_C./S. America 1 E. weberbaueri 0.93 E. cestrifolia 0.87 E. laurifolia 1 1 E. sinclairiana sect. Mesophyllae_C./S. America 1 E. lactiflua 1 sect. Lactifluae_C./S. America 1 E. calyculata 0.69 E. xylopoda sect. Calyculatae_N. America 1 E. tanquahuete sect. Tanquahuete_N. America 1 1 E. gymnonota 0.99 E. umbelliformis 1 E. punicea 1 1 E. cubensis sect. Cubanthus_Caribbean 1 E. helenae 1 E. podocarpifolia E. munizii 1 1 E. tirucalli 0.95 1 0.93 E. kamponii 0.97 1 1 E. decorsei 0.99 1 E. arahaka 1 1 E. enterophora 1 1 E. fiherenensis sect. Tirucalli_Madagascar 1 E. stenoclada 1 1 E. xylophylloides 0.5 E. dhofarensis 1 E. arbuscula E. damarana 0.91 E. gummifera 1 E. sp. 6 1 1 E. sp. 12 0.81 E. sp. 5 1 E. sp. 4 0.93 E. sp. 3 E. sp. 2 E. randrianjohanyi sect. Pervilleanae_Madagascar 0.99 E. tetraptera 0.96 1 E. pervilleana 1 E. sp. 7 1 E. rauhii 1 E. intisy 0.81 E. haeleeleana 0.69 E. boophthona 1 E. plumerioides sect. Pacificae_Pacific 1 0.61 E. sarcostemmoides E. stevenii 1 1 E. planiticola E. tannensis 0.77 1 E. spinea E. aequoris subg. Chamaesyce 1 E. plagiantha 1 E. guiengola E. cotinifolia 0.67 E. gariepina 0.79 E. horrida 0.93 E. hadramautica subg. Athymalus 1 E. bongensis 1 E. aphylla 1 E. orthoclada 1 E. helioscopia subg. Esula E. hirsuta

0.03 Fig. 3A Fig. 3. Bayesian phylogeny of 315 taxa in E. subgen. Euphorbia based on matK, ndhF, and ITS sequences. Multiple accessions of same species are summarized as empty triangles. Clades represented by filled triangles in Fig. 3A are expanded to Figs. 3B and 3C. Branches are labeled with posterior probabilities greater than 0.5. Figure 3B Toyama et al.

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Fig. 3C

0.92 E. tulearensis 0.73 E. rossii 0.66 1 E. capsaintemariensis 1 E. beharensis 0.9 E. decaryi 0.85 E. ambovombensis E. cylindrifolia E. croizatii E. francoisii sect. Goniostema_Madagascar 1 E. aff. retrospina 0.84 E. capuronii 0.85 E. horombensis E. sp. 17 0.6 0.5 E. aff. mahafalensis E. didiereoides E. pedilanthoides 0.52 1 E. milii 0.92 E. lophogona 1 1 E. alluaudii 1 E. sp. 19 1 E. famatamboay sect. Deuterocalli_Madagascar E. sp. 18 0.57 E. cedrorum 0.95 E. sp. 14 0.73 E. sp. 11 1 1 E. hedyotoides sect. Denisophorbia_Madagascar 0.54 E. sp. 10 0.86 E. sp. 9 0.76 E. sp. 8 0.55 E. viguieri 1 0.51 E. neohumbertii 0.86 E. iharanae sect. Goniostema_Madagascar E. geroldii 0.82 0.93 E. alfredii 0.93 E. bokorensis sect. Bokorenses_Cambodia 1 E. rubella E. brunellii sect. Rubellae_NE. Africa 1 0.78 E. lugardiae 1 E. kimberleyana 1 E. schubei 1 1 E. heteropoda 1 E. guentheri 1 E. succulenta 1 1 0.95 E. rhizophora 1 E. neostolonifera 0.94 E. neomontana 1 E. neorubella 1 0.9 0.81 E. invenusta E. pseudolaevis 1 1 E. bisellenbeckii 1 E. pseudostellata 1 E. neoreflexa E. major sect. Monadenium_E./C./SE. Africa 0.5 1 E. neovirgata E. lindenii 1 E. pseudotrinervis E. sp. 13 1 1 E. neospinescens 1 E. torrei 1 0.66 E. bicompacta 1 E. umbellata 1 E. pseudomollis 1 E. neogossweileri 1 1 E. neoarborescens 0.89 1 E. magnifica 1 E. spectabilis E. neococcinea E. neogracilis E. mandravioky sect. Pachysanthae_Madagascar

0.03 Fig. 3A

Fig. 3B Figure 3C Toyama et al.

June 2016 Toyama & al.— New Section and Species of Euphorbia from Cambodia 89

1 1 E. grandicornis 0.98 1 E. pseudocactus 0.88 E. breviarticulata 0.67 E. pseudoburuana 0.69 1 E. bussei E. bougheyi E. brevitorta 1 0.99 E. lividiflora 1 1 E. cooperi 1 1 E. enormis E. groenewaldii 1 0.82 E. caerulescens E. curvirama 0.79 E. ledienii 0.72 0.88 E. triangularis E. keithii 0.52 1 E. perangusta 1 E. limpopoana E. avasmontana E. ammak E. fractiflexa 0.99 E. cactus E. longispina E. robecchii 1 0.64 0.75 E. heterospina 1 1 E. heterochroma 0.62 E. elegantissima 0.53 0.95 E. cuprispina 0.91 E. parciramulosa E. ambroseae 1 E. clivicola E. lenewtonii 1 1 E. sudanica 1 0.52 1 E. venenifica 1 E. unispina sect. Euphorbia_Asia/Africa/Arabia E. sapinii 0.97 E. resinifera 1 E. seibanica E. fruticosa E. classenii 0.85 1 0.86 E. griseola E. persistentifolia 1 E. fanshawei 0.65 E. decidua 1 E. micracantha E. stellata 1 1 E. drupifera E. desmondii 1 1 E. abyssinica 0.9 0.75 E. ingens 1 1 E. nivulia 1 1 E. neriifolia E. teke 1 E. caducifolia 1 1 E. antiquorum 0.92 E. lactea E. sp. 16 0.96 0.89 E. sp. 15 1 1 E. vajravelui 1 E. evansii 1 E. grandidens 1 E. ramipressa 1 1 E. confinalis 1 E. tanaensis E. sekukuniensis 0.93 E. tetragona 1 0.99 E. lacei E. abdelkuri

0.03 Fig. 3B

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(Roxb.) Teijsm. & Binn. ex Kurz, Archidendron five similar glands and ecarunculate seeds (Figs. quocense (Pierre) I. C. Nielsen, Phoebe lanceo- 4 & 5). Euphorbia bokorensis is morphologically lata (Nees) Nees, and Gironniera subaequalis similar to E. boivinii Boiss. (E. sect. Denisophor- Planch.; the site at 912 m contained Macaranga bia) and E. boissieri Baill. (E. sect. Goniostema) andamanica Kurz, Beilschmiedia penangiana in its monoecious sexuality, shrub-like habit and Gamble, Illicium tenuifolium (Rindl.) A. C. Sm., well-developed oblanceolate leaves. Euphorbia Ardisia sanguinolenta Blume, and Antidesma boivinii has sympodial branching (vs. un- laurifolium Airy Shaw. branched), smaller leaves (to 10 by 4 cm vs. 13– 24 by 3–6 cm), attenuate leaf base (vs. cuneate Sequence variation and molecular phylogeny base), and reduced (vs. conspicuous) cyatho- Summary statistics for each dataset are given phylls. Euphorbia boissieri has larger leaves (34 in Table 1. The aligned sequences of matK, ndhF, by 6 cm), attenuate leaf base, fewer cyathia (to 4) and ITS were 2082, 1449, and 669 base pairs per cyme (vs. to 8), reduced cyathophylls, and long, respectively, of which 490, 357, and 404 verrucose (vs. smooth) seeds. base pairs were parsimony-informative. Three Euphorbia bokorensis is morphologically and specific indels and 16 specific variable sites were phylogenetically (Fig. 3) distinct from the species in Euphorbia bokorensis. DNA sequences of the of all known sections. We therefore propose a barcoding regions of E. bokorensis are the most new section, Bokorenses, to accommodate E. bo- similar to Euphorbia abyssinica (accession no. korensis. AY794824, rbcL identity: 566/567) and E. cactus (accession no. KF408914, rbcL identity: 565/566) Molecular phylogeny of Euphorbia subg. Euphor- of sect. Euphorbia, and E. ambarivatoensis (ac- bia cession no. JQ951981, matK identity: 763/775) of Our molecular phylogenetic analysis recog- sect. Goniostema. nized three major clades and one unplaced sec- The Bayesian phylogeny supports the mono- tion in Euphorbia subg. Euphorbia (Fig. 3). The phyly of Euphorbia subg. Euphorbia (with 93% first clade with 100% posterior probability in- posterior probability) and comprising three major cludes 11 sections, Brasilienses, Calyculatae, clades and one unplaced section (Fig. 3). Euphor- Crepidaria, Cubanthus, Euphorbiastrum, Lacti- bia bokorensis was in one of the major clades, but fluae, Mesophyllae, Nummulariopsis, Portulac- clearly separate from the other sections and astrum, Stachydium, and Tanquahuete, and cor- showed a sister relationship with the Malagasy responds to the New World clade of Dorsey et al. clade, including Denisophorbia, Deuterocalli, (2013). The second clade with 93% posterior and Goniostema; this group is sister to the north- probability includes six sections, Denisophorbia, eastern African section, Rubellae (Fig. 3). Deuterocalli, Euphorbia, Goniostema, Monade- nium, and Rubellae, and corresponds to the Old World clade II of Dorsey et al. (2013). The third Discussion clade with 99% posterior probability includes three sections, Pacificae, Pervilleanae, and Tiru- Morphology, phylogeny, and of Eu- calli, which correspond to the Pacific Clade and phorbia bokorensis part of the Old World clade I of Dorsey et al. Euphorbia bokorensis was not assignable to (2013). One unplaced section is Pachysanthae, any of the sections recognized by Dorsey et al. which corresponds to part of the Old World clade (2013). Based on the key of Dorsey et al. (2013), I in Dorsey et al. (2013). Each section recognized E. bokorensis is included in E. sect. Euphorbia or by Dorsey et al. (2013) was monophyletic except in E. sect. Monadenium, but is distinguished for Goniostema (Fig. 3). from the former by its spineless stems and con- Our phylogeny (Fig. 3) had two topological spicuous cyathophylls, and from the latter by the conflicts with the phylogeny previously presented June 2016 Toyama & al.— New Section and Species of Euphorbia from Cambodia 91

Fig. 4. Euphorbia bokorensis H. Toyama & Tagane, sp. nov. A & B, cyathium; C, glands and lobes of involucre; D, staminate flower; E, filamentous structure; F, pistillate flower; G, immature ; H, seeds; I, (A & G from Fuse 6192, FU; B, C, D, E & F from Toyama et al. 1912, FU; H & I from Toyama et al.4583, KYO. Drawn by H. Toyama). 92 Acta Phytotax. Geobot. Vol. 67

(Dorsey et al. 2013). First, sect. Monadenium is the presumed ancestral character states of the the basal lineage of the Old World clade II, in- clade including sect. Denisophorbia, Deuterocal- cluding sections Bokorenses, Denisophorbia, li, and Goniostema in having alternate leaves, Deuterocalli, Euphorbia, Goniostema, and Ru- five glands lacking appendages and ecarunculate bellae, but is treated as a monophyletic clade with seeds, but shows differences in having a shrub- sect. Euphorbia in Dorsey et al. (2013). Second, like habit and terminal and axillary synflores- sect. Tanquahuete was sister to sect. Calyculatae, cences (Horn et al. 2012). Phylogeographically, but was sister to sect. Cubanthus in Dorsey et al. E. bokorensis may link Southeast Asia to Mada- (2013). These conflicts might be due to our use of gascar. Further phylogeographic studies may MAFFT v 7.220 (Katoh & Standley 2013), which help us to understand the evolutionary history of is known as one of the best performers for align- subgenus Euphorbia. ments (Pervez et al. 2014).

Phytogeography of E. bokorensis and perspec- Taxonomic Treatment tives for future research The phylogenetic relationship among sections Euphorbia sect. Bokorenses H. Toyama & close to Bokorenses (Fig. 3) suggests long-dis- Tagane, sect. nov. tance dispersal from Africa or Madagascar to Southeast Asia. The sister relationship of African This section is the most similar to Sect. Denisophorbia in and Southeast Asian clades has been observed in having spineless stems with gland-like stipules, cyathia with usually 5 glands, and ecarunculate smooth seeds. It 15 taxonomic groups and is explained by the out differs in having unbranched (not sympodially branch- of India hypothesis (Datta-Roy & Karanth 2009), ing) stems. which proposed that rafting of India carried Typus. Euphorbia bokorensis H. Toyama & Tagane Gondwanan forms to Asia after the break-up of the Gondwana super continent. However, conti- Description. Shrubs, monoecious. Stems sub- nental drift occurred 160–40 Ma (Datta-Roy & succulent, smooth, usually unbranched. Stipules Karanth 2009), while the oldest fossil of Euphor- gland-like. Leaves alternate, spirally arranged. bia are 37.2–33.9 Ma (Behrensmeyer & Turner Synflorescences terminal and axillary, peduncu- 2013). It is therefore more likely that the relation- late, simple or compound cymes. Cyathia bisexu- ships among the sections close to Bokorenses are al; cyathophylls conspicuous, fused basally, sur- the result of a more recent trans-oceanic dispersal rounding the involucre; involucre lobes triangu- rather than continental drift. Alternatively, E. bo- lar, with feathery tips; glands 5, entire, typically korensis may be a relic species that was distrib- elliptic, without appendages. Capsules smooth. uted more extensively in the past (but after 37.2– Seeds smooth, ecarunculate. 33.9 Ma), and thereafter retreated to Bokor Na- tional Park. In our floristic survey of this area, we Species included. Euphorbia bokorensis H. found 34 endemic species and 11 widely distrib- Toyama & Tagane uted species from Africa to Asia (Tagane et al. in prep.). Comparative phylogeography and popula- tion genetics among these species, and further Euphorbia bokorensis H. Toyama & Tagane, sp. botanical surveys in Indochina, will help in un- nov. —Figs. 4 & 5. derstanding the phytogeography and history of E. bokorensis. Euphorbia bokorensis resembles E. boivinii Boiss. (E. Euphorbia bokorensis provides an interesting sect. Denisophorbia) and E. boissieri Baill. (E. sect. Go- niostema) in its monoecious cyathia, shrub-like habit, and opportunity for studying the evolution of mor- well-developed oblanceolate leaves, but differs from the phological character and phylogeography of E. former in having unbranched (vs. sympodially branching) subg. Euphorbia (Fig. 3). In morphology, it shows stems, larger leaves (13–24 by 3–6 cm vs. to 10 by 4 cm), June 2016 Toyama & al.— New Section and Species of Euphorbia from Cambodia 93

Fig. 5. Euphorbia bokorensis H. Toyama & Tagane, sp. nov. A, branch with cyathia; B, abaxial surface of leaf; C, cyathium; D, synfl orescence; E, [Photographs by S. Tagane & K. Fuse on 10 December 2011 (A) or 9 December 2013 (B–E)] 94 Acta Phytotax. Geobot. Vol. 67 cuneate leaf base (vs. attenuate base), and conspicuous runculate. (vs. reduced) cyathophylls; from the latter in having smaller leaves (vs. 34 by 6 cm), cuneate (vs. attenuate) leaf base, to 8 cyathia per cyme (vs. to 4 cyathia per Distribution. Cambodia (known only from cyme), conspicuous (vs. reduced) cyathophylls, and smooth Bokor National Park). (vs. verrucose) seeds. Typus. CAMBODIA, Kampot Province: Bokor Na- Other specimens examined. CAMBODIA, Kampot tional Park, evergreen forest near Popokvik waterfall, 917 Province: Bokor National Park, evergreen forest, 888 m m alt., 10°39′30.82″N and 104°03′05.14″E, 25 October alt., 10°37′15.48″N and 104°05′10.71″E, 10 December 2012, Toyama H., Tagane S., Ide T., Chhang P., Nagamasu 2011, Toyama H., Tagane S., Ide T., Chhang P., Nagamasu H. & Yahara T. 4583 [fl. & fr.] (holo- KYO. iso- FU, her- H. & Yahara T. 1912 [fl. & fr.] (FU, Forest administration barium of the Forest Administration in Cambodia). of Cambodia); 602 m alt., 10°36′44.87″N and 104°06′0.79″E, 9 December 2013, Fuse K. 6192 [fl. & fr.] (BKF, FU, Forest Administration of Cambodia). Shrubs, monoecious, ca. 0.7 m tall; stems sub- succulent, terete, smooth, usually unbranched Phenology. Flowers and fruits were observed (branched when cut), ca. 0.8 cm in diam. bark in October and December. green when young, greenish-gray when old, gla- Preliminary conservation status. Euphorbia brous. Stipules ca. 0.2 mm long, gland-like. Leaves bokorensis is known from three localities be- alternate, spirally arranged; petioles 2–6 cm long, tween 600–900 m alt. Less than 10 mature indi- glabrous; leaf blades carnose, oblanceolate, (4.5– viduals were observed at each site in moist ever- )13–24 by (1.5–)3–6 cm, base cuneate, margin green forests. It is qualified as critically endan- entire, apex acuminate, membranous when dry, gered (CR) using the criterion D of the Red List glabrous; venation brochidodromous, secondary Categories (IUCN 2012), although additional in- veins 10–14 pairs, abaxially slightly prominent dividuals may be discovered by more through when dry, tertiary veins obscure on both surfac- surveys. es, but becoming faintly visible when dry. Synflo- Mt. Bokor is considered to be one of the biodi- rescences terminal and axillary, pedunculate, versity hotspots in Southeast Asia. In our field simple and compound cymes. Cyathia to ca. 8 per survey, we collected ca. 3,100 specimens includ- cyme, bisexual, 7–10 mm long; peduncle gla- ing ca. 770 identified species and 22 new species brous, 1–4 mm long; cyathophylls 2, conspicu- (Tagane et al. 2015a,b, Naiki et al. 2015, Tanaka ous, orbicular, ca. 4 by 5 mm, fused basally, sur- et al. 2015, Toyama et al. 2016, Yahara et al. in rounding the involucre, white or with pink grada- press). However, recent development for tourism tion; two axial cyathium buds between cyatho- (e.g., construction of a casino and hotels) has phylls and involucre; involucre lobes triangular, threatened biodiversity of the area (Kosterin ca. 1 mm long, with feathery tips; glands 5, en- 2012). Euphorbia bokorensis occurs in the under- tire, typically elliptic, cream, ca. 0.5 by 1.5 mm, story of moist evergreen forests along and close without appendages. Staminate flowers 10, gla- to the main road from the bottom to the resort to brous; pedicels 1–2.5 mm long, filaments ca. 1 the plateau on the summit. To conserve E. boko- mm long, anther lobes yellow, ca. 0.4 mm long. rensis, as well as other endemic and rare species Filamentous structures 5, liner-cuneate, at base in Bokor National Park, eliminating habitat de- of staminate flowers, 2–3 mm long. Pistillate struction by logging and land use change is need- flowers glabrous; pedicel 1.4–2 mm long; reduced ed. perianth forming an annular protuberance at base Vernacular name. Champa Phnom. of , 0.2–0.3 mm long; ovary 3-locular, glabrous; 6-lobed, ca. 0.3 mm long, joint- GenBank accession Nos. Toyama et al. 1912: ed at base. Capsules obtusely 3-lobed, smooth, AB936028 (rbcL), AB936029 (matK), LC054296 (ndhF), ca. 8 mm in diam., greenish red. Seeds 3 per fruit, LC054297 (ITS). ca. 5 by 4 mm, ovoid to subglobose, smooth, eca- June 2016 Toyama & al.— New Section and Species of Euphorbia from Cambodia 95

Key to the sections of E. subg. Euphorbia sis is distinguished from those two sections by its Based on the key to the sections of Euphorbia spineless stem, 5 similar glands and ecarunclu- subg. Euphorbia in Dorsey et al. (2013), Euphor- late seeds (Figs. 4 & 5). The revised key is as fol- bia bokorensis is included in E. sect. Euphorbia lows. The numbers shown in the key are same as and E. sect. Monadenium. However, E. bokoren- in Dorsey et al. (2013).

Key to the sections of Euphorbia subg. Euphorbia

11. Plants varied in habit, but if geophytic then with glands fused into a horseshoe shape, or with 5 similar glands; widespread (Africa, southern/eastern Asia) ...... 12a 12a. Stems usually succulent, angled or tuberculate, generally with distinctive spine shields composed of a horny pad and associated spines; cyathophylls reduced, scale-like...... E. sect. Euphorbia 12a. Stems spineless or with spines of stipular origin, but without spine shields; cyathophylls conspicuous...... 12b 12b. Cyathia with one large horseshoe-shaped or circular gland; seeds carunculate ...... E. sect. Monadenium 12b. Cyathia with 5 similar glands; seeds ecarunculate...... E. sect. Bokorenses

11 is revised from the lower key of 11 in Dorsey et al. (2013) p. 304. 12a & 12b are revised from the upper and lower keys of 12 in Dorsey et al. (2013) p. 304.

Euphorbia sect. Bokorenses is closely related E. sect. Goniostema, and E. sect. Rubellae (Fig. to E. sect. Denisophorbia, E. sect. Deuterocalli, 3). The following is the key to sections.

Key to sections of Euphorbia sect. Bokorenses, E. sect. Denisophorbia, E. sect. Deuterocalli, E. sect. Goniostema, and E. sect. Rubellae

1a. Cyathia with 4 equal-sized glands and 2 smaller glands produced by division of a single gland; seeds carunculate ...... E. sect. Rubellae 1b. Cyathia usually with 5 glands (or 4 or 6); seeds ecarunculate ...... 2 2a. Stipules spine-like, papyraceous or comb-like, more or less branched, diversely colored; seeds verrucose ...... E. sect. Goniostema 2b. Stipules glandular or punctiform, never spiny or papyraceous; seeds smooth ...... 3 3a. Stem unbranched; cyathophylls conspicuous; cyathia bisexual ...... E. sect. Bokorenses 3b. Stem branched; cyathophylls inconspicuous to well developed; cyathia unisexual (rarely bisexual in E. sect. Denisophorbia) ...... 4 4a. Plants essentially leafless (leaves very small, soon caducous); stipules modified as a waxy or glandular ring around leaf insertion ...... E. sect. Deuterocalli 4b. Plants with leaves spirally arranged; stipules reduced to glandular dots ...... E. sect. Denisophorbia

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Received September 17, 2014; accepted December 28, 2015