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Sulistyaningsih LD, Abinawanto, Ardiyani M, Salamah A. 2018. Short Communication: Phylogenetic analysis and molecular identification of Canar (Smilax spp.) in Java, Indonesia Based on DNA Barcoding Analysis. Biodiversitas 19: 364-368. Smilax spp. (Smilacaceae) has long been used as medicinal herbs especially in East Asia and North America as they were known to be rich in steroidal saponin. Pharmacological study has been carried out in Indonesia. This genus is widespread in Indonesia and fairly abundant in Java and has been known either as edible fruit or medicinal plants. Characteristics of Smilax as a dioecious plant with high morphological variations make it thorny in species identification. Various molecular approaches have been devised to overcome identification problems such as DNA barcoding. This study, therefore was conducted to analyze the DNA barcoding application for phylogenetic and identification of Smilax in Java. A total of 31 samples were used in this study including 19 accession numbers from NCBI GeneBank. The genus Ripogonum was used as the out-group in phylogenetic reconstruction. Samples were successfully extracted by CTAB method with some modifications. rbcL region was used as the DNA barcode showed sufficient variation and conserved flanks. Two unidentified specimens have high similarity with S. leucophyla and lies in the same clade. The phylogenetic tree constructed by Maximum Likelihood analysis. The result showed that the monophyletic of Smilacaceae consisted of four clades. The genus Heterosmilax nested with Smilax though with low bootstraps value. It supports the monogeneric status of Smilacaceae.

Phylogenetic study to determine the phylogenetic relationship of Smilacaeae of Java based on morphological data has been conducted. Smilacaceae has a well-deserved reputation for being taxonomically difficult since this family has wide phenotypic variation and lack of fertile collections. Smilacaceae species from Java consist of species which vary in both vegetative and reproductive characters. A total of ten species of Smilacaceae in Java with an addition of 78 taxa representing all genera and sections of the family from all over the world were used for phylogenetic analysis. Two species of Ripogonum (Ripogonum album and R. discolor) which were known as the sister of Smilacaceae were incorporated as an outgroup. A matrix consisted of 38 binary and ten multistate morphological characters were constructed. Strict consensus tree revealed from Maximum Parsimony (MP) as well as Neighbor-Joining (NJ) showed that Smilacaceae is a monophyletic family. Smilacaceae species of Java which belong to the section Macranthae are supposed to be closely related with Smilacaceae species from section Coilanthus and China based on MP analysis and supposed to be closely related with Smilacaceae species from section China based on NJ analysis. Sections of Smilacaceae are not monophyletic except section Coprosmanthus. Heterosmilax is considered to be derived from ancestral Smilax and occurs as a section of Smilax. This study supports previous studies that merged Heterosmilax to Smilax and made Smilacaceae as monogeneric taxa.

A new hopantenic acid derivative, (R)-methyl-4-(2,4-dihydroxy-3,3-dimethylbutanamido)butanoate (1), was isolated from the rhizomes of Heterosmilax japonica Kunth, together with three known compounds, (3S)-1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (2), daucosterol (3), and ethyl-β-D-xylobioside (4). The structure of 1 was determined by extensive spectroscopic analyses, including 1D and 2D NMR and HR-ESI-MS. Compounds 2 and 4 are new for the genus Heterosmilax.

Confusion in the species associated with the name of "Bixie" in Chinese Materia Medica began centuries ago. In recent decades, diverse medicinal plants from the genera Dioscorea and Smilax, and even minor species from the genus Heterosmilax, have been documented under the name Bixie or a very similar name as folk medicines in different areas of China. However, the traditional efficacies and chemical profiles of these herbs are not exactly the same and even vary wildly. Comprehensive authentication of multiple Bixie herbs has not yet been attempted. To differentiate and ensure the correct use of these Bixie-related herbs, in this study, seven sorts of representative Bixie herbs (Dioscorea collettii, D. zingiberensis, D. nipponica, D. septemloba, Smilax china, S. glabra, and Heterosmilax japonica) were characterized based on the microscopic examination of their powders and cryotomed transverse sections. This is not only the first attempt to distinguish Bixie herbs by a comprehensive microscopic techniques, including common light microscopy, fluorescence microscopy, and polarized light microscopy, but also it is the first research to observe characteristics of transections of crude drugs under polarized lighting for the purpose of authentication. Polarized light has been found to provide a number of unique characteristics. The results indicate that starch granules, stone cells, vascular bundles, and other significant tissue features can be used to authenticate "Bixie" herbs. The method was proven to be quick, handy, specific, and simple. It should be widely applicable to other herbal materials.

Smilacaceae, composed of Smilax and Heterosmilax, are a cosmopolitan family of > 200 species of mostly climbing monocots with alternate leaves characterized by reticulate venation, a pair of petiolar tendrils and usually prickly stems. Although there has been a long history of studying Smilax since Linnaeus named the genus in 1753, the phylogenetic history of this dioecious family remains unclear. Here we present results based on nuclear ribosomal internal transcribed spacer (nrITS) and plastid matK and rpl16 intron DNA sequence data from 125 taxa of Smilacaceae. Our taxon sampling covers all sections of Smilax and Heterosmilax and major distribution zones of the family; species from Ripogonaceae and Philesiaceae are used as outgroups. Our molecular analysis indicates that phylogenetic relationships largely contradict the traditional morphological classification of the family, instead showing a conspicuous geographical pattern among the species clades. The previously recognized genus Heterosmilax was found to be embedded in Smilax. Species in the family are separated into primarily New World and Old World clades, except for a single species lineage, Smilax aspera, that is sister to the remaining species of the family, but with poor statistical support. Ancestral character state reconstructions and examination of distribution patterns among the clades provide important information for future taxonomic revisions and historical biogeography of the group. © 2013 The Linnean Society of London, Botanical Journal of the Linnean Society, 2013, 173, 535–548.

Smilacaceae are a small family of dioecious, mostly climbing, net-veined monocotyledons with a cosmopolitan distribution. Relatively little is known about the variation of pollen morphology within the family. For this reason, and to investigate the systematic value of palynology in Smilacaceae, pollen from 125 species of Smilax, Heterosmilax, and Ripogonum was examined using light and scanning electron microscopy. Ten of these were examined further by transmission electron microscopy. Four distinct pollen types grouped into two major pollen classes were distinguished: Class 1, represented by the pollen of all Smilax and Heterosmilax species, is mostly spheroidal, inaperturate, and spinulate or microspinulate, with a thin, fragile exine of varied sculpturing; three pollen types are represented within this class. Class 2 is found only in Ripogonum and contains a single pollen type with prolate, monosulcate, reticulately-sculptured pollen. The unique pollen morphology of Ripogonum supports its removal from Smilacaceae. In contrast, the characteristics of Heterosmilax pollen intergrade with those seen in Smilax, suggesting that the former might be better reduced to synonymy with the latter. A key to the identification of these pollen types is presented along with a discussion of geographic and possible evolutionary trends among them.

Smilacaceae are a taxonomically confused, cosmopolitan family of Liliales characterized by climbing habit, reticulate leaf venation, paired petiolar tendrils, unisexual flowers, and superior ovaries. Deviations from this generalized morphology have led to the division of Smilacaceae into at least seven different genera and five sections within the large genus Smilax. In particular, taxa with connate tepals (Heterosmilax), more than six stamens (Pleiosmilax, Oligosmilax), or herbaceous habit (Nemexia) have been variously classified. Using DNA sequences of 96 taxa from the nuclear rDNA ITS gene region, parsimony analyses provide moderate resolution, but generally poor bootstrap support for phylogenetic relationships in the family. Rhipogonum and Lapageria are closely related to Smilax, but may be better classified in separate families. Within Smilax few of the sections are monophyletic, but three major lineages are evident. The first is a primarily Old World clade that contains species of Heterosmilax, the erect, woody species of Smilax from Asia, and the herbaceous Smilax species. Within this clade there is evidence that the S. herbacea complex represents a single eastward dispersal from Asia to North America. A primarily New World clade (or paraphyletic grade in some trees) is also present in the ITS cladogram. From within this group there is evidence of a single westward dispersal from North America to Asia. The third lineage is represented by S. aspera, which is the sole member of Smilax section Smilax, and is sister to the entire genus. Its position in the cladogram is intriguing and may help to shed light on the family's greater evolutionary and biogeographic history.

Petiole anatomy of 12 species, 11 species of Smilax L. and one species of Heterosmilax Kunth., of the family Smilacaceae have been studied. Anatomical features of the petiole provide supporting evidences for the infra-specific circumscription of the occurring in India.

A new species, Smilax hirtellicaulis (Smilacaceae), from southwestern China is described and illustrated. The new species differs from all known Smilax species in its unique tepals, i.e., basally connate in male flowers but separate in female flowers. Phylogenetic analysis supports its species delimitation, and suggests a sister position to the clade of Smilax sect. Heterosmilax which have flowers with connate tepals. Their closest phylogenetic relationship and resembled flower morphology may represent an evolutionary transition from separate to united tepals in Smilax. It thus provides us an ideal model for studying the evolution and development of such an innovative floral trait.

Smilax Linnaeus (1753: 1028) is the only genus of Smilacaceae, comprising ca. 210 species, which are lianas, shrubs, and herbs (Cameron & Fu, 2006; Qi et al. 2013a). The genus shows a near-cosmopolitan distribution (Qi et al. 2013a). In the large genus, Smilax sect. Heterosmilax (Kunth 1850: 270) Judd (1998: 158), formerly treated as an independent genus Heterosmilax Kunth in Smilacaceae, has 12 species and is distributed only in Asian and Malesian regions (Judd 1998, Qi et al. 2013b). All species of the section have slender stem without pricks, cordate leaves, and tendrils in bronze color. Especially, flowers with connate perigone are one of the most important characteristics to distinguish the section from the other sections (Koyama 1984, Qi et al. 2013a).

By various chromatographic methods, one new phenylpropanoid glycoside, heterosmilaside (1), two known phenylpropanoid glycosides, helonioside B (2), and 2',6'-diacetyl-3,6-diferuloyl sucrose (3), and three known flavonoids, isoquercetin (4), quercetin-3-O-beta-D-glucuronopyranoside (5), and quercetin-3-O-(2''-alpha-L-rhamnopyranosyl)-beta-D-glucuronopyranoside (6) were isolated from the methanolic extract of the aerial part of Heterosmilax erythrantha Baill. Their structures were elucidated on the basis of spectroscopic analyses. All the isolated compounds were tested for antioxidant activity in the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging assay. Among them, compounds 5 and 6 showed significant antioxidant activity with SC(50) values of 3.7 and 6.5 microg/mL, respectively.

Background Dioscorea is a genus of flowering plants, and some Dioscorea species are known and used as a source for the steroidal sapogenin diosgenin. To screen potential resource from Dioscorea species and related medicinal plants for diosgenin extraction, a rapid method to compare the contents of diosgenin in various plants is crucial. Results An ultra-performance liquid chromatography (UPLC) coupled with diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS) method was developed for identification and determination of diosgenin in various plants. A comprehensive validation of the developed method was conducted. Twenty-four batches of plant samples from four Dioscorea species, one Smilax species and two Heterosmilax species were analyzed by using the developed method. The present method presented good sensitivity, precision and accuracy. Diosgenin was found in three Dioscorea species and one Heterosmilax species, namely D. zingiberensis, D. septemloba, D. collettii and H. yunnanensis. Conclusion The method is suitable for the screening of diosgenin resources from plants. D. zingiberensis is an important resource for diosgenin harvesting.

Smilax Linnaeus (1753: 1028), the only genus of Smilacaceae, consists of over 200 species (with ca. 90 in China), and is widely distributed from tropical to temperate areas (Qi et al. 2012). Li (1992: 21) published Smilax jiankunii H.Li, based on specimens collected in the Dulongjiang Valley, Gongshan County, northwestern Yunnan Province, China. Smilax jiankunii was characterized by having a smooth stem, abaxially glaucous leaves, slender peduncles 9 to 10 cm long, densely flowered umbels (with 60–100 flowers), and the whole plant becoming blackish when dried (Li 1992), a feature shared with Smilax pottingeri Prain (1900: 174). Since Wang & Tang (1978) transferred S. pottingeri to the genus Heterosmilax Kunth (1850: 270) as H. pottingeri (Prain) Wang & Tang (1978: 245), based on the leaf shape and flattened peduncles, it is understandable that Li failed to compare her new species with it at that time. However, the name Smilax pottingeri was more recently accepted under Smilax again, due to more collections and knowledge of this plant becoming available (Chen & Koyama 2000), and the genus Heterosmilax having been merged with Smilax based on morphological and molecular studies (Judd 1998, Cameron & Fu 2006, Qi et al. 2012, 2013). After comparing the descriptions of both, we recognized that Smilax pottingeri appears to be similar to S. jiankunii, which has smooth stems, abaxially glaucous leaves, a slender peduncle 3 to 6 cm long and densely flowered umbels (with 40–70 flowers), but a more southern distribution in eastern Myanmar, southeastern Yunnan, Vietnam, northern Laos and northern Thailand (Koyama 1975, Chen & Koyama 2000, Nguyên 2007).

Judd (1998) formally transferred the species considered to be members of Heterosmilax Kunth (1850: 270; Koyama 1960, 1978, 1984) including Pesudosmilax Kunth (1850: 270) and Oligosmilax Seemann (1868: 258) into Smilax Linnaeus (1753: 1028) as a section, since “the segregation of these two genera would result in a paraphyletic Smilax sensu stricto because no apomorphies are known that would unite the remaining members of the group, which are characterized by flowers with free tepals and six more or less distinct stamens” (Judd 1998, p. 156). This proposal was further supported by recent molecular phylogenetic evidence (Cameron & Fu, 2006; Li et al. 2011, Qi et al. 2012), which shows that Heterosmilax distributed in Eastern and Southeast Asia is monophyletic and embedded within Smilax . The preparation of the treatment of Smilacaceae for the Flora of the Pan-Himalaya necessitates transfer of all species of Heterosmilax to Smilax . Nine new combinations are made, a new name is given and three names are resurrected.

Smilacaceae are a taxonomically confused,cosmopolitan family of Liliales characterized by their climbing habit,reticulate leaf venation,paired petiolar tendrils,unisexual flowers,and superior ovaries. Deviations from this generalized morphology have led the division of Smilacaceae into at least three different genera and five sections within the large genus Smilax. The karyotypes and chromosomes of 17 taxa of Smilax and Heterosmilax were examined and compared with previous studies of Smilacaceae. Phylogeny and karyotype evolution of the two genera are discussed in this paper along with evolutionary trends in inflorescence morphology,in the context of gene trees based on nrDNA ITS sequences. Karyotypes of these 17 taxa are characterized as follows:(1) “woody S. riparia”,2n=32=L(2m+2st+2t)+M(4sm+4st)+S(2m+8sm+8st); (2) S. scobinicaulis,from Guizhou,2n=32=L(2sm+4st+2t)+M(6sm(SAT)+2st)+S(2m+6sm+8st); (3) S. china in Shennongjia,Hubei is a diploid,2n=32=L(6st)+M(4sm+6st)+S(2m+6sm+8st); (4) S. china in Guangxi with 4x,2n=64=L(2sm+12st+2t)+M(4sm+8st)+S(6m+20sm+10st); (5) S. china from Japan with 4x chromosomes,2n=64=L(4sm+14st+2t)+M(6st)+S(4m+28sm+6st); (6) S. trinervula from Guiyang,2n=32=L(8st)+M(2sm+2st)+S(2m+6sm+12st); (7) S. chingii,2n=32=L(2sm +6st)+M(4sm+4st)+S(2m+10sm+4st); (8) S. ferox from Kunming,a hexaploid,2n=96= L(6sm+22st+6t)+M(2sm+10st)+S(6m+28sm+16st); (9) S. lebrunii from Sichuan,a hexaploid,2n=96=L(4sm+24st)+M(4sm+10st)+S(10m+26sm+18st); (10) S. stans from Chongqing,2n=32=L(4st+2t)+M(2sm+4st)+S(12sm+8st); (11) S. austro-zhejiangensis found in Zhejiang,2n=32=L(4st+2t)+M(2sm+6st)+S(12sm+6st); (12) S. umbrosa found in Mt. Emei,2n=32= L(2sm+6st)+M(2m+2sm+2st)+S(4m+8sm+6s); (13) S. bockii from Mt. Jingfo,2n=32= L(2sm+6st)+M(6st+2m)+S(8sm+8st); (14) S. guiyangensis from Guiyang,2n=32=L(6sm+ 2st)+M(4sm+2st)+S(2m+10sm+6st); (15) S. vanchingshanensis from Guizhou,2n=32 =L(2sm+6st+2t)+M(4st)+S(4m+6sm+8st); (16) S. lunglingensis from Kunming,2n=32= L(4st)+M(4sm+4st)+S(6m+6sm+6st); and (17) S. polycephala from Yunnan,2n=32= L(2sm+ 6st)+M(2sm+6st)+S(6m+6sm+4st). The results showed that the karyotypes of Smilax and Heterosmilax are bimodal,with evidence of polyploidization,micro-variation,and variance ofbasic number from 16 to 15 and 13. We suggest that the karyotypes of the herbaceous Smilax species show a trend from symmetry in East Asian taxa to asymmetry in North America,but no such trend is obvious among woody species. Regarding the inflorescence,we consider the prophyll to be a persistent bract. Therefore,the branches composed of single umbels borne in axils and with a prophyll at the base of the entire branch (i.e.,sect. China and sect. Coilanthus) are homologous with the inflorescences possessing a prophyll at their base,or with a node on the peduncle and only a single umbel (in sect. Macranthae and sect. Smilax). Based on this interpretation and previous published ITS sequences,it is evident that a spike of umbels lacking peduncles is the primitive type of inflorescence in Smilacaceae from which two types of inflorescences are derived:(1) a solitary umbel without a prophyll,including species within Heterosmilax and Smilax section China and Coilanthus; and (2) a panicle-like type with one to many umbels subtended by a prophyll,i.e.,the species of Smilax sect. Macranthae. Furthermore,our data suggest that Heterosmilax should not be recognized as a separate genus,but as a subgenus closely related to species of the herbaceous section of Smilax and Smilax sect. Coilanthus. The herbaceous species of Smilax,originating in East Asia and then expanding into North America,form a monophyletic group and a common ancestry with sect. Coilanthus. They are recognized as Smilax sect. Nemexia. Finally,studies of S. china show that it is a widespread species with varying ploidy levels. In this paper,a diploid population has been documented from Hubei,suggesting that tetraploid populations were widely distributed in East Asia when Japan and Taiwan separated from the Asian mainland during the Tertiary to Middle Pleistocene. Hexaploid populations,which are common on the mainland today,were probably generated by hybridization after their isolation. In conclusion,these results show that Southwest China is a center for the distribution and differentiation of Smilacaceae today.

In order to construct phylogenetic relationships of the family Smilacaceae, 79 species representing all genera and sections of the family from all over the world were used for cladistic analysis, with Philesia and Lapageria of Philasiaceae as outgroups. A matrix of 36 binary and 11 multistate morphological characters was constructed. The first parsimonious analysis yielded numerous most parsimonious trees of 275 steps, and then 82 most parsimonious trees with 40.729 steps length were produced by reweighting characters using maximum values of rescaled consistency (RC), which remained stable after the second reweighting. There is a moderate resolution but generally poor bootstrap support for the phylogenetic relationships in the family. From the strict MP tree and the NJ tree, Ripogonum is a sister to Smilax, but may be better treated as a separate family. Heterosmilax is considered to be derived from ancestral Smilax and occurs as the most derived clade of the Smilacaceae, and thus it is reasonable to retain it as a section in Smilax s.l. Within Smilax most currently recognized sections are polyphyletic, but some major lineages are distinct. The herbaceous Smilax species form a monophyletic clade, and it is also the case for the South American species. Their positions in the cladogram are intriguing and may help to shed light on evolutionary and biogeographic history of the family.

Puccinia kraussiana, a rust fungus of Africa, was found in Taiwan for the first time in eastern Asia. It was parasiting on leaves ofHeterosmilax gaudichaudiana, on which all four stages were characteristically found. The host species,H. gaudichaudiana, newly added to the host range ofP. kraussiana, constitutes also a new entry to the phanerogamic flora of Taiwan.

Heterosmilax, a small southeastern Asiatic genus in the Smilacaceae, comprises 11 species in two sections, includingH. micrandra, which is described as new from Hainan, China. These taxa are taxonomically discussed, described, illustrated, and a key is provided to differentiate them.