REINWARDTIA A JOURNAL ON TAXONOMIC BOTANY, PLANT SOCIOLOGY AND ECOLOGY Vol. 14(1): 1 - 2 4 8 , December 23, 2014 Chief Editor Kartini Kramadibrata (Mycologist, Herbarium Bogoriense, Indonesia) Editors Dedy Darnaedi (Taxonomist, Herbarium Bogoriense, Indonesia) Tukirin Partomihardjo (Ecologist, Herbarium Bogoriense, Indonesia) Joeni Setijo Rahajoe (Ecologist, Herbarium Bogoriense, Indonesia) Marlina Ardiyani (Taxonomist, Herbarium Bogoriense, Indonesia) Topik Hidayat (Taxonomist, Indonesia University of Education, Indonesia) Eizi Suzuki (Ecologist, Kagoshima University, Japan) Jun Wen (Taxonomist, Smithsonian Natural History Museum, USA) Managing Editor Himmah Rustiami (Taxonomist, Herbarium Bogoriense, Indonesia) Lulut Dwi Sulistyaningsih (Taxonomist, Herbarium Bogoriense, Indonesia) Secretary Endang Tri Utami Layout Editor Deden Sumirat Hidayat Medi Sutiyatno Illustrators Subari Wahyudi Santoso Anne Kusumawaty Correspondence on editorial matters and subscriptions for Reinwardtia should be addressed to: HERBARIUM BOGORIENSE, BOTANY DIVISION, RESEARCH CENTER FOR BIOLOGY- INDONESIAN INSTITUTE OF SCIENCES CIBINONG SCIENCE CENTER, JLN. RAYA JAKARTA - BOGOR KM 46, CIBINONG 16911, P.O. Box 25 Cibinong INDONESIA PHONE (+62) 21 8765066; Fax (+62) 21 8765062 E-MAIL: reinwardtia@mail.lipi.go.id 1 2 3 4 1 3 4 4 Cover images: 1. Begonia holosericeoides (female flower and habit) (Begoniaceae; Ardi et al.); 2. Abaxial cuticles of Alseodaphne rhododendropsis (Lauraceae; Nishida & van der Werff); 3. Dipo- dium puspitae, Dipodium purpureum (Orchidaceae; O'Byrne); 4. Agalmyla exannulata, Cyrtandra coccinea var. celebica, Codonoboea kjellbergii (Gesneriaceae; Kartonegoro & Potter). The Editors would like to thanks all reviewers of volume 14(1): Abdulrokhman Kartonegoro - Herbarium Bogoriense, Bogor, Indonesia Altafhusain B. Nadaf - University of Pune, Pune, India Amy Y. Rossman - Systematic Mycology & Microbiology Laboratory USDA-ARS, Beltsville, USA Andre Schuiteman - Royal Botanic Gardens, Kew, UK Ary P. Keim - Herbarium Bogoriense, Bogor, Indonesia Barry Conn - Royal Botanic Gardens National Herbarium of New South Wales, Sydney, Australia Dato' Abdul Latiff Mohamad - Universiti Kebangsaan Malaysia, Bangi, Selangor, Malaysia Daniel Potter - Department of Plant Sciences, University of California, Davis, California, USA Deby Arifiani - Herbarium Bogoriense, Bogor, Indonesia Ferry J. W. Slik - University of Brunei Darussalam, Brunei Henti H. Rachmat - Conservation and Rehabilitation Research and Development Center, Bogor, Indonesia Ian M. Turner - Royal Botanic Gardens, Kew, UK Iskandar Z. Siregar - Bogor Agricultural University, Bogor, Indonesia Jay H. Bernstein - Kingsborough Community College, Brooklyn, New York, USA Jens G. Rohwer - University of Hamburg, Hamburg, Germany Joan Pereira - SAN Herbarium, Sabah Forestry Department, Sabah, Malaysia Kuswata Kartawinata - Herbarium Bogoriense, Bogor, Indonesia Lars H. Schmidt - University of Copenhagen, Copenhagen, Denmark Mark Hughes - Royal Botanic Gardens, Edinburgh, UK Masahiro Kato - Kyoto University, Kyoto, Japan Nuril Hidayati - Herbarium Bogoriense, Bogor, Indonesia Ong Poh Teck - Forest Research Institute Malaysia, Kepong, Malaysia Peter C. van Welzen - National Herbarium Netherlands, Leiden University Branch, Leiden, Netherlands Reuben Nilus - Sabah Forestry Department, Sabah, Malaysia Rugayah - Herbarium Bogoriense, Bogor, Indonesia Ruth Kiew - Forest Research Institute of Malaysia, Kepong, Malaysia Uwe Braun - Institut fur Biologie Bereich Geobotanik und Botanischer Garten, Halle (Saale), Germany Yasuaki Sato - Osaka-Sangyo University, Osaka, Japan REINWARDTIA Vol 14, No 1, pp: 53 − 66 53 species are placed in Persea (ca. 80 species), Apollonias consists of one species in the Canary Islands and one in India, while the remaining genera range from Pakistan to Japan and south to New Guinea. Machilus (ca. 100 species) and Phoebe (100 species fide Li et al., 2008, but ca. 50 species fide Kochummen, 1989) have their centers of diver- sity in southern China. Alseodaphne (50 sp. or more), Nothaphoebe (40 sp.) and Dehaasia (35 sp.) are most common in tropical Asia with only few species in southern China. Modern treatments for Asian members of these INTRODUCTION The Persea group as currently accepted consists of seven genera, Alseodaphne Nees, Apollonias Nees, Dehaasia Blume, Machilus Nees, Nothaphoebe Blume, Persea Mill. and Phoebe Nees and includes 400–450 species (Li et al., 2011). The group is well represented in subtropical and tropical America, is absent from Africa and Madagascar and has a large number of species in subtropical and tropical Asia. No members of this group are known from Australia nor the Pacific area. All Neotropical DO CUTICLE CHARACTERS SUPPORT THE RECOGNITION OF ALSEO- DAPHNE, NOTHAPHOEBE & DEHAASIA AS DISTINCT GENERA? Received January 17, 2014; accepted May 21, 2014 SACHIKO NISHIDA Nagoya University Museum, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-8601, Japan. E-mail: nishida@num.nagoya-u.ac.jp HENK VAN DER WERFF Missouri Botanical Garden, 4344 Shaw Blvd., St. Louis, MO 63110, U.S.A. E-mail: henk.vanderwerff@mobot.org ABSTRACT NISHIDA, S. & VAN DER WERFF, H. Do cuticle characters support the recognition of Alseodaphne, Nothaphoebe and Dehaasia as distinct genera? Reinwardtia 14(1): 53 – 66. ― The Asian members of the Persea group are divided among the genera Alseodaphne, Apollonias, Dehaasia, Machilus, Nothaphoebe and Phoebe. A recent phylogenetic analysis has shown that Machilus and Phoebe are supported as monophyletic genera but evidence that the closely related genera Alseodaphne, Dehaasia and Nothaphoebe are monophyletic or not was equivocal. In this study we analyzed cuticle characters of 95 collections belonging to the Asian members except for Apollonias. We anticipated two possible outcomes. If the genera were not monophyletic, we expected that the groups based on cuticle characters would consist of species belonging to different genera. If the genera were monophyletic, we expected that the groups based on cuticle characters would consist of species belonging to the same genus. We found 16 groups based on cuticles. Of these, 12 consisted of species of a single genus (one group included a single species and thus a single genus). The four mixed groups included mostly species of one genus with 1 or 2 species of a different genus. Our results support the recognition of Alseodaphne, Dehaasia, Machilus, Nothaphoebe and Phoebe as distinct genera. Key words: Alseodaphne, cuticle, Dehaasia, Lauraceae, Machilus, Nothaphoebe. ABSTRAK NISHIDA, S. & VAN DER WERFF, H. Apakah karakter kutikula mendukung pengenalan Alseodaphne, Nothaphoebe dan Dehaasia sebagai marga yang berbeda? Reinwardtia 14(1): 53 – 66. ― Kelompok Persea dari Asia dibedakan menjadi beberapa marga yaitu Alseodaphne, Apollonias, Dehaasia, Machilus, Nothaphoebe dan Phoebe. Hasil analisis kekerabatan menunjukkan bahwa Machilus dan Phoebe adalah marga yang monofili, namun tidak demikian halnya dengan tiga marga yang berkerabat dekat yaitu Alseodaphne, Dehaasia dan Nothaphoebe. Pada studi ini telah dianalisis karakter kutikula dari 95 koleksi yang termasuk dalam kelompok Asia kecuali Apollonias dengan dugaan dua hasil yang telah diantisipasi. Pertama, jika marga-marga tersebut tidak monofili, maka pengelompokan yang didasarkan pada karakter kutikula pada jenis-jenis tersebut berasal dari marga yang berbeda. Kedua, jika marga-marga tersebut monofili maka pengelompokan yang didasarkan pada karakter kutikula jenis-jenis tersebut berasal dari marga yang sama. Dari hasil studi ditemukan 16 kelompok berdasarkan karakter kutikulanya. Dua belas kelompok terdiri atas jenis-jenis yang berasal dari satu marga (satu kelompok terdiri atas satu jenis yang berarti satu marga juga). Sedangkan empat kelompok yang bercampur termasuk jenis-jenis yang berasal dari satu marga dengan satu atau dua jenis dengan marga yang ber- beda. Hasil studi ini mendukung pengenalan Alseodaphne, Dehaasia, Machilus, Nothaphoebe dan Phoebe sebagai marga yang berbeda. Kata kunci: Alseodaphne, kutikula, Dehaasia, Lauraceae, Machilus, Nothaphoebe. REINWARDTIA 54 [VOL.14 genera are few or lacking. Regional treatments of Machilus and Phoebe are published in the Flora of China (Li et al., 2008), covering the majority of the species of these genera. Kostermans published synopses of Alseodaphne (1973a) in which he recognized 50 species and of Dehaasia (1973b) with 35 species. These synopses consist of descrip- tions of new species, listing of accepted species with citation of specimens, but do not include keys to species. There is no recent treatment of Nothaphoebe (ca. 40 sp.). Morphologically, the Asian genera of the Persea group are poorly defined. No floral differences have been reported between Machilus and Phoebe; the sole difference is found in the condition of the per- sistent tepals in fruit and the shape of the fruit: spreading to recurved tepals and round fruits in Machilus vs. erect, clasping tepals and ovoid fruits in Phoebe. A few species, such as M. calcicola Qi or M. glabrophylla Zuo, have round fruits and deciduous tepals. A few species placed in Phoebe are described as having globose fruits and loose, lax or slightly clasping tepals in fruit (P. chinensis Chun, P. microphylla H.W. Li, P. faberi (Hemsley) Chun; see Li et al., 2008). Assigning flowering specimens to either Machilus or Phoebe remains problematic. The majority or possibly all species of Machilus and Phoebe have perulate vegetative buds, leaving clusters of scars at the base of the sea- sonal shoots; Alseodaphne, Nothaphoebe and Dehaasia do not have perulate buds (pers. obs.). There is no consensus on differences between Alseodaphne and Nothaphoebe. During the last sixty years a variety of opinions have been published. Kostermans (1957) placed Alseodaphne and Nothaphoebe in Persea, but later (Kostermans, 1973a) reconsidered and accepted both as good genera without indicating how they could be separated. In an unpublished treatment of the Lauraceae in Thailand (a copy owned by one of the authors, HvdW) Kostermans separated Alseodaphne from Nothaphoebe based on the fruiting pedicels: thick and fleshy in Alseodaphne, cylindrical and not fleshy in Nothaphoebe. This manuscript was probably written in the early 1970’s. Kostermans also identified many collections in L; he placed nearly all specimens with unequal tepals in Nothaphoebe and those with equal or subequal te- pals in Alseodaphne (pers. obs.). Kochummen (1989) treated species with unequal tepals in Nothaphoebe and species with subequal tepals in Alseodaphne. Rohwer (1993) separated the two by the size of the staminodes of whorl four: Alseo- daphne was said to have well-developed, heart- shaped staminodes and Nothaphoebe small stami- nodes. Van der Werff (2001) included Nothaphoebe in Alseodaphne. Most recently, Julia et al. (2009) studied Alseodaphne and Nothaphoebe for the Tree Flora of Sabah and Sarawak; they separated the two genera on a variety of characters (petioles canalicu- late vs. rounded, few-flowered vs. many-flowered inflorescences, distinct vs. very short or absent fila- ments of the fertile stamens and fleshy vs. woody fruiting pedicels). Dehaasia is generally considered to be closely related to Alseodaphne and Nothaphoebe, differing only in the number of pollen sacs per anther, Alseodaphne and Nothaphoebe being 4-locular and Dehaasia 2-locular (Kostermans, 1973b; Rohwer, 1993; van der Werff, 2001). Fruiting specimens therefore cannot be iden- tified to genus with any confidence and Kochum- men (1989) already deplored the description of new species based solely on fruiting specimens. The lack of recent revisions with keys to species makes even identification of flowering specimens difficult and this lack of reliable identifications poses large problems for studies of relationships in this group. Two recent studies have presented phylogenetic analyses of the Persea group (Rohwer et al., 2009; Li et al., 2011). The phylogenies found in these studies are not identical, however, these studies share a number of conclusions. The main ones referring to Asian members are the following: Machilus is a monophyletic group and separate from Persea; Persea is not monophyletic and consists of a large group (mostly of subg. Erio- daphne) and a small group (mainly subg. Persea), plus (sometimes) a few species currently placed in Alseodaphne; Phoebe is not monophyletic in Rohwer et al. (2009), but monophyletic in Li et al. (2011); Alseodaphne is not monophyletic, but consists of two groups; Dehaasia is nested in one of the Alseodaphne groups and Nothaphoebe was only represented by one species which was part of the Alseodaphne/Dehaasia group. In our study we focused on characters of the cu- ticles found in the Asian Persea group. Cuticular characters are features of the cutinized epidermal cells or stomatal complex. They have been long used in identifying fossil leaves (e.g. Upchurch, 1984a, 1984b; Carpenter et al., 2010) and investi- gating relationships among extant taxa (Baranova, 1972, 1987, 1992; Stace, 1984; Yang & Lin, 2005). Observation of cuticles requires relatively simple methodology. One can use fresh or dried specimens for cuticle studies, including material that is not suitable for molecular analyses. Sterile specimens might also provide useful information for classifica- tion, which cannot be expected for conventional Lauraceae systematics that usually requires some reproductive characters. We analyzed cuticle characters of 95 collections 2014] 55 NISHIDA et al. : Cuticular characters of the Asian Persea group belonging to the Asian members except for Apollonias. We anticipated two possible outcomes. If the genera were not monophyletic, we expected that the groups based on cuticle characters would consist of species belonging to different genera. If the genera were monophyletic, we expected that the groups based on cuticle characters would consist of species belonging to the same genus. MATERIALS AND METHODS Cuticles of 95 leaf samples of Persea complex were examined (Appendix 1). All were from Asian countries (Cambodia, China, Malaysia, Indonesia, Philippines, Thailand and Vietnam), and included species of Alseodaphne, Dehaasia, Machilus, Nota- phoebe, and Phoebe (one species of Persea, which should belong to Machilus was also included). Leaves were collected from herbarium specimens at MO and L, using one leaf sample per species. Iden- tities (genus names, species names, localities or col- lector’s names) of the plant samples were not known to the first author who examined the cuticles before her grouping them by the cuticular charac- ters. The examination procedure basically followed that of Christophel et al. (1996), Nishida and Chris- tophel (1999), and Nishida and van der Werff (2007). A 1 cm square sample was taken from left basal part of the leaf (with the adaxial surface up) for each species. The leaf samples were soaked in 90% ethanol overnight then placed in a test tube with ca. 2 mL 30% H2O2 and ca. 1 mL 90% etha- nol. The test tubes were heated around 100°C in a heated dry block bath for about 3 hr. When the sam- ples turned soft and yellow, they were placed in a Petri dish with tap water, tenderly cleaned with a fine artist’s brush to remove the cellular contents or leaf veins, then placed in bottles with 90% ethanol for more than one night. Each sample was then rinsed in 2% ammonia (to adjust the pH), trans- ferred to a Petri dish with tap water to clean with a fine artist’s brush once more. The cuticles were stained in 0.1% crystal violet for ca. 1 min., then mounted in phenol glycerin jelly on a slide, covered with an 18 mm square cover glass and observed under an optical microscope. Feature descriptions follow Christophel et al. (1996), Nishida and Chris- tophel (1999), or Nishida and van der Werff (2007, 2011). All the cuticles (except for three species, Dehaasia sp. 1, Machilus sp. 10, and Phoebe formo- sana, whose cuticles were fragile and broke apart during the preparation) were also examined using an SEM. Sample preparation was the same as described above. Samples were dehydrated in a t- butanol series (90% ethanol : t-butanol = 3:1; 1:3; 100% t-butanol twice), freeze-dried using a JFD- 310 (JEOL, Tokyo, Japan) at -5°C, then coated with platinum, and observed under a JSM-6060B micro- scope (15 kV; JEOL). Without being informed about the identity (the genus or species names, the locality, or the repro- ductive characters), one of the authors (Nishida) observed cuticles firstly only under optical microscope and grouped samples by their overall similarity of the cuticles. SEM was not available at the time. We later reexamined cuticles with SEM, and reconsidered the groups if the characters under SEM were largely different from the previous im- pression we had under optical microscope. Because generic concepts have varied from author to author, we give below the generic charac- ters we have used. Machilus and Phoebe are separated on their fruit characters, Machilus with reflexed or spreading tepals and round fruits, Phoebe with clasping tepals and ovoid fruits; Dehaasia is characterized by its 2-locular stamens Fig. 1. Optical micrographs of the abaxial cuticles (A1, B1, C1, D1, E1) and SEMs of the stomatal com- plex (A2, B2, C2, D2, E2). A. Alseodaphne sp. 7; B. Alseodaphne sp. 10; C. Alseodaphne sp. 15; D. Alseodaphne rhododendropsis; E. Phoebe lucida. Scale bars = 50 µm. REINWARDTIA 56 [VOL.14 and Alseodaphne and Nothaphoebe are separated on their tepals; unequal tepals in Nothaphoebe and equal tepals in Alseodaphne. RESULTS Among the 95 samples, two samples (Alseodaphne peduncularis and A. sp. 16) were ex- cluded from the results because cuticles were not well removed from leaf tissue and unobservable. Cuticular features were different between the adaxial and abaxial leaf surfaces; stomata were observed only on the abaxial leaf surfaces. Features consistent within each of the samples but varied among the species were observed mainly on the abaxial leaf surfaces. They are listed in Table 1, and samples with the representative features are shown in Figs. 1-4. Drawings of the cuticle parts or fea- tures we refer to (and may be difficult to understand from the sample pictures) are shown in Figs. 5-7. The following are brief descriptions of the features. In many species, periclinal walls of epidermal cells or stomatal complex (Fig. 5) were smooth (Figs. 2B2, 2C2, 2D2, 3B2, 3C2), or granular (e.g. Figs. 1A2, 1B2, 1D2, 2A2, 3D2, 3E2, 4A2). The struc- tures mentioned here as “granular” may be remains of epicuticular waxes, but we used the term because the appearance of them under microscope resem- bled the ones referred to as granular by Christophel & Rowett (1996). Epidermal cells were sometimes papillose or strongly protruding upward (Figs. 1D2, 1E2, 2E2, 3A2), although surface of the papillae or the protruding cells themselves could be smooth (Figs. 1E2, 2E2) or granulous (Fig. 1D2, 3A2). Peri- Table 1. Cuticle character states recognized for the Asian Persea genera. * Characters used for the grouping. Part of cuticle Characters Character states Epidermal anticlinal walls Straightness of walls straight to slightly curved / with loose U-shaped curves / with tight U-shaped curves Epidermal periclinal walls Surface texture* smooth / weakly granulous / granulous Surface appearance* flat / each cell protruding upward / papillose Stomatal complex Overall shape* narrowly rectangular / elliptic / broadly elliptic / (hidden under papillae or protrusion of epidermal cells) Stainability of subsidiary cells stained as much as epidermal cells / darkly stained / inner part scarcely stained but outer part darkly stained Lower ledges* lip-shaped / butterfly-shaped Surface appearance* almost flat / irregularly protruding / circular and protrud- ing / reniform and protruding / lip-shaped and protruding / dome-shaped and protruding / (hidden among protrusion or papillae of epidermal cells) Surface texture* smooth /granulous 2014] 57 NISHIDA et al. : Cuticular characters of the Asian Persea group clinal walls were usually homogeneous within each sample, but in some species they were different between the cells surrounding stomata and the other part of the epidermal cells (Fig.1B2): the former had granulous periclinal walls, whereas the other part had smooth ones. A few species had only their sub- sidiary cells with granulous periclinal walls (Fig. 1C2). Anticlinal walls of epidermal cells were usually straight to slightly curved, and sinuous anticlinal walls were rarely seen. Straightness of anticlinal walls of subsidiary cells made the stomatal overall shape (Fig. 6) different: usually the walls were curved outward and the stomatal complexes look elliptic to broadly elliptic (Fig. 6A; e.g. Fig. 2B1), but in a few cases they were straight and the stomatal complexes look narrowly rectangular (Fig. 6B; Figs.1A1, 1B1, 4A1). Stomatal ledges (the most inside part of the stomatal complex and along the stomatal opening slit; Fig. 5) are usually stained only weakly (e.g. “sl” of Figs. 2B1 and 2D1), but other part of the complex, which mainly consists of subsidiary cells, may have their periclinal walls stained by crystal violet. If the entire periclinal walls are stained only weakly (e.g., “su” of Fig. 2C1), darkly (e.g. “su” of Fig. 2D1), or only outer edge of the walls stained darkly (e.g. “su” of Fig. 2B1) is observable under the microscope, and we listed the states as dyed patterns of the subsidiary cells (Table 1). Surface appearance of the stomata recognized under SEM had some variation: almost flat and in- conspicuous (Fig. 7A; Figs.1A2, 1B2, 3D2, 3E2), irregularly protruding (Fig. 7B; Fig. 4A2), circular and protruding (Figs. 1C2, 3A2, 3B2, 3C2), reniform and protruding (Fig. 7C; Fig. 2A2), lip-shaped and protruding (Fig. 7D; Fig. 2B2), dome-shaped and protruding (Fig. 7E; Figs. 2C2, 2D2, 2E2). In the Fig. 2. Optical micrographs of the abaxial cuticles (A1, B1, C1, D1, E1) and SEMs of the stomatal com- plex (A2, B2, C2, D2, E2). A. Alseodaphne in- signis; B. Nothaphoebe sarawacensis; C. Phoebe neurantha; D. Alseodaphne andersonii (#Poilane 19847); E. Nothaphoebe cavalieriei. sl = stomatal ledges. su = subsidiary cells. Scale bars = 50 µm. Fig. 3. Optical microgaphs of the abaxial cuticles (A1, B1, C1, D1, E1) and SEMs of the stomatal com- plex (A2, B2, C2, D2, E2). A. Machilus kurzii. B. Alseodaphne sp. 8. C. Dehaasia sp. 4. D. Dehaasia cairocan. E. Dehaasia sp. 3. Scale bars = 50 µm. REINWARDTIA 58 [VOL.14 cuticle of circular and protruding stomatal surface, circles may appear perfect (Fig. 7F; Fig. 3B2) or broken at both ends of the stomatal slit (Fig. 7G; Fig. 3A2, 3C2). Differences among circular surface, lip-shaped surface and dome-shaped surface were relative width of the complex (length of the com- plex crossing the stomatal slit perpendicularly) and shape of the part near the stomatal slit: the width was usually as long as or longer than the slit in the circular shaped surface or dome shaped surface but shorter than the slit in the lip shaped surface; the part near the stomatal slit was slightly depressed in the circular shaped surface or lip shaped surface whereas it was protruding in the dome shaped surface. In some species, stomatal complexes were hidden under the papillae of the epidermal cells or protruding surface of the surrounding epidermal cells (Figs. 1D2, 1E2). As we mentioned in the description of the periclinal walls, some species had only the stomatal complex with granulous periclinal Fig. 4. Optical micrographs of the abaxial cuticles (A1) and SEMs of the stomatal complex (A2). A. Alseodaphne sp. 12. Scale bars = 50 µm. Fig. 5. Diagram of a typical stomatal complex of the Asian Persea group. Fig.6. Overall shape of the stomatal complex in the Asian Persea group. A. Elliptic to broadly ellip- tic. B. Narrowly rectangular. Fig. 7. Stomatal surface appearance patterns of the Asian Persea group. A. Almost flat. B. Irregu- larly protruding. C. Reniform and protruding. D. Lip-shaped and protruding. E. Dome- shaped and protruding. F. Circular and protrud- ing, with the circle perfect. G. Circular and protruding, with the circle broken at both ends of the stomatal slit. 2014] 59 NISHIDA et al. : Cuticular characters of the Asian Persea group walls (Fig. 1C2) or stomatal complex and epidermal cells surrounding the stomata with granulous walls (Fig. 1B2). DISCUSSION We found 16 groups based on cuticles (Table 2, 3). If groups share the same number (eg. 1a and 1b) this indicates that their cuticles appear more similar to each other than to the other groups. Names of species that have exceptional features and are only tentatively placed in one of the groups are placed in parentheses in the table. These species must be further investigated to examine their attribution. As we mentioned in Materials and Methods, we firstly observed the cuticles under optical microscope and grouped the species, then later reexamined with SEM and reconsidered the groups if the characters observed under SEM were largely different from the previous impression we had with optical microscope. This way, seven of our 93 samples (#Hyland 14931 of Alseodaphne andersonii, A. glaucina, A. sp. 14, Dehaasia sp. 3, Phoebe neuran- tha and P. formosana, P. tavoyana) were moved to a different group. The cuticular characteristics used for the grouping mainly belong to the stomatal complex, but the features of the periclinal walls were also used. As mentioned earlier (Nishida and van der Werff 2011), features of the stomatal complex might be better correlated with molecular phylogeny than features of epidermal cells, and we also considered the former ones more important. In the key to the groups (Table 3), however, we used features of the epidermal cells more often, because they are more easily recognized. Dyed patterns of the stomatal complex (whether subsidiary cells are stained darkly or not) might be a new character recognized for cuticular studies of Lauraceae. Twelve of the 16 groups consist of species of a single genus (including one group with just a single species). The four mixed groups included mostly species of one genus with 1 or 2 species of a different genus. Because the taxonomy of the genera is poorly known, it is very well possible that the mixed groups are a consequence of misidentifi- cations. For example, the species identified as Nothaphoebe cavaleriei is a species of Phoebe and the specimen identified as Alseodaphne sp. 13 has unequal tepals and is thus a species of Nothaphoebe. Our results, therefore, support the recognition of Alseodaphne, Dehaasia, Machilus, Nothaphoebe and Phoebe as distinct genera. Most of the species were grouped by the first author with- out their generic or distributional information, which indicates the groupings were not biased by such information. This suggests that the cuticular characters might be useful to recognize some natu- ral taxa, if we use the characters carefully. We, of course, still have a problem to rely on the cuticles. For instance, the groupings were based on the over- all similarity of the features and without any quanti- fication. It was hard to quantify features like the appearance of the stomatal complex, but we need some systems of the cuticular characters that are more accessible and easier to recognize even for non-specialists. More objective ways of evaluation of the features, including more comparison with the other morphologies or molecular phylogeny, are also needed. ACKNOWLEDGEMENTS We gratefully acknowledge Leiden Herbarium (L) and the herbarium of Missouri Botanical Garden (MO) for allowing us to sample leaves from the specimens. We also thank the Kyoto University Museum for use of the scanning electron microscope and Dr. H. Nagamasu for his guidance on the procedure. REFERENCES BARANOVA, M. 1972. Systematic anatomy of the leaf epidermis in the Magnoliaceae and some related fami- lies. Taxon 21: 447–469. BARANOVA, M. 1987. Historical development of the present classification of morphological types of sto- mata. Botanical Review (Lancaster) 53: 53–79. BARANOVA, M. 1992. 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Annals of the Missouri Botanical Garden 98: 2014] 61 NISHIDA et al. : Cuticular characters of the Asian Persea group G ro u p S p e c ie s n a m e S u rf a c e a p p e a ra n c e a n d te x tu re o f a b a x ia l le a f e p id e rm is a n d s to m a ta O v e ra ll s h a p e o f st o m a ta S ta in a b il it y o f su b si d ia ry c e ll s L o w e r st o m a ta l le d g e s S u rf a c e a p p e a ra n c e o f st o m a ta 1 a A ls e o d a p h n e e lo n g a ta , A . sp . 6 , A . sp . 7 , A . sp . 9 g ra n u lo u s n a rr o w ly r e c - ta n g u la r d a rk ly s ta in e d li p -s h a p e d a lm o st f la t 1 b (A ls e o d a p h n e h a in a n e n si s) , A . g la u c in a , A . sp . 1 0 , A . sp . 1 4 . c e ll s a ro u n d s to m a g ra n u lo u s, o th e r c e ll s sm o o th n a rr o w ly r e c - ta n g u la r d a rk ly s ta in e d li p -s h a p e d a lm o st f la t 2 (A ls e o d a p h n e o b o v a ta ), A . sp . 5 , A . sp . 1 5 . su b si d ia ry c e ll s g ra n u - lo u s, o th e r c e ll s sm o o th b ro a d ly e ll ip ti c d a rk ly s ta in e d li p -s h a p e d c ir c u la r a n d s li g h tl y p ro tr u d in g 3 A ls e o d a p h n e r h o d o d e n d ro p si s, A . sp . 1 , A . sp . 2 . g ra n u lo u s, e a c h c e ll p ro tr u d in g u p w a rd h id d e n u n d e r e p id e rm is sc a rc e ly s ta in e d li p -s h a p e d h id d e n u n d e r e p id e rm is 4 P h o e b e l u c id a , P . m a c ro p h y ll a , P . sp . 1 , P . sp . 2 . p a p il lo se ( p a p il la s u r- fa c e s m o o th ) h id d e n u n d e r p a p il la e sc a rc e ly s ta in e d li p -s h a p e d h id d e n u n d e r e p id e rm is 5 A ls e o d a p h n e i n si g n is , A . o b la n c e o la ta sl ig h tl y g ra n u lo u s b ro a d ly e ll ip ti c d a rk ly s ta in e d b u tt e rf ly - sh a p e d re n if o rm a n d p ro tr u d - in g 6 N o ta p h o e b e c u n e a ta , N . sa - ra w a c e n si s, N . sp . 1 , N . sp . 2 , A ls e o d a p h n e s p . 1 3 * 1 sm o o th b ro a d ly e ll ip ti c o n ly o u te r p a rt d a rk ly s ta in e d li p -s h a p e d li p -s h a p e d a n d p ro tr u d - in g 7 a (P h o e b e n e u ra n th a ), P . fo rm o - sa n a , P . la n c e o la ta , (M a c h il u s sp . 1 ) sm o o th b ro a d ly e ll ip ti c w e a k ly s ta in e d li p -s h a p e d d o m e -s h a p e d a n d p ro - tr u d in g 7 b A ls e o d a p h n e a n d e rs o n ii , A . sp . 3 a lm o st s m o o th b ro a d ly e ll ip ti c d a rk ly s ta in e d li p -s h a p e d d o m e -s h a p e d a n d p ro - tr u d in g 8 N o th a p h o e b e c a v a li e ri * 2 , P h o e b e b o u rn e ri , P . c a th ia , P . c h e k ia n - g e n si s, P . fo rr e st ii , P . h u n a n e n si s, P . sh e a re ri , P . ta v o y a n a , P . sp . 4 e a c h c e ll p a p il lo se o r st ro n g ly p ro tr u d in g u p w a rd ( p a p il la e s u r- fa c e s m o o th ) b ro a d ly e ll ip ti c w e a k ly s ta in e d li p -s h a p e d d o m e -s h a p e d a n d p ro - tr u d in g T a b le 2 . G ro u p in g s o f sp e c ie s o f th e A si a n P e rs e a g ro u p b y c u ti c u la r c h a ra c te rs . R E IN W A R D T IA 6 2 [V O L .1 4 Species temporally placed in the group in parentheses. *1 Alseodaphne sp. 13 should belong to Nothaphoebe based on floral morphology (van der Werff, unpublished). *2 Nothaphoebe cavalerieri should belong to Phoebe based on ITS-based phylogeny (Rohwer et al., 2009) and general morphology (van der Werff, p. 200 in Li et al, 2008). *3 The two samples, which were named as Alseodaphne sp. 8 and A. sp. 11, came from the same tree, one collection with flowers, the other with fruits. *4 This species should belong to Machilus based on floral morphology (van der Werff, unpublished). Table 2. Groupings of species of the Asian Persea group by cuticular characters (continued). 9 Alseodaphne sp. 4., Machilus bombycina, M. breviflora, M. cal- cicola, M. chekiangensis, M. de- cursinervis, M. gamblei, M. glabrophylla, M. grandibracteata, M. grijsii, M. kurzii, (M. minuti- loba), M. nakao, M. odoratissima, M. oreophila, M. parabreviflora, (M. phoenics), M. platycarpa, M. pomifera, M. velutina, M. sp. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, Persea rimosa, (Phoebe sp. 3) granulous, each cell usually protruding up- ward broadly elliptic scarcely stained lip-shaped circular and protruding, circles broken at the both ends of the stomatal slit 10a Alseodaphne sp. 8, 11 *3 smooth elliptic darkly stained lip-shaped circular and protruding, circles perfect 10b Dehaasia turfosa, D. sp. 1, 2, 4, (6), (Persea chatacea) *4 smooth elliptic scarcely stained lip-shaped circular and slightly protruding, circles al- most perfect 11 (Alseodaphne semecarpifolia), Dehaasia annamensis, D. cairo- can, (D. suborbicularis) granulous broadly elliptic only outer part darkly stained lip-shaped flat to slightly irregu- larly protruding 12 (Dehaasia tomentosa), D. sp. 3, 5. granulous broadly elliptic scarcely stained lip-shaped almost flat 13 Alseodaphne sp 12. slightly granulous narrowly rec- tangular scarcely stained lip-shaped irregularly protruding 2014] 63 NISHIDA et al. : Cuticular characters of the Asian Persea group Table 3. Key to the cuticular groupings. 1a. All or a part of abaxial leaf epidermis (including stomatal complex) with granulous walls, whether the cells protruding upward or not ...................................................................................................... 2 b. All of abaxial epidermis with smooth periclinal walls or papillose (pallila surface smooth) ........... 10 2a. Only abaxial epidermal cells surrounding stoma with granulous periclinal walls .............................. 3 b. All the abaxial epidermal cells with granulous walls ......................................................................... 4 3a. Subsidiary cells and epidermal cells surrounding the subsidiary cells with granulous periclinal walls; stomatal surface almost flat .................................................................................................. Group 1b b. Only subsidiary cells with granulous periclinal walls; stomatal surface circular and slightly protrud- ing (Fig. 7G) ........................................................................................................................... Group 2 4a. Stomatal surface reniform (Fig. 7C) and protruding; stomatal ledges butterfly-shaped ......... Group 5 b. Stomatal surface almost flat to slightly protruding, but not reniform; stomatal ledges lip-shaped .... 5 5a. Stomatal surface circular and protruding ................................................................................ Group 9 b. Stomatal surface almost flat, irregularly protruding or hidden under epidermis ............................... 6 6a. Epidermal cells protruding conspicuously upward, with stomatal surface (subsidiary cell surface) hid- den under the epidermal cells ................................................................................................. Group 3 b. Epidermal cells flat or only slightly protruding, with stomatal surface (subsidiary cell surface) visi- ble, whether it is flat or protruding ..................................................................................................... 7 7a. Subsidiary cells narrowly rectangular, with the outer anticlinal walls almost straight (Fig. 6B) ...... 8 b. Subsidiary cells broadly elliptic, with the outer anticlinal walls curved outward (Fig. 6A) ............... 9 8a. Stomatal surface almost flat ................................................................................................... Group 1a b. Stomatal surface irregularly protruding ................................................................................ Group 13 9a. Outer part of subsidiary cells darkly stained, making a contrast with the inner part of the subsidiary cells that are scarcely stained............................................................................................... Group 11 b. Outer part of subsidiary cells stained only weakly as the inner part ..................................... Group 12 10a. Stomatal surface (subsidiary cell surface) hidden under papilla ........................................... Group 4 b. Stomatal surface protruding ............................................................................................................ 11 11a. Stomatal surface lip-shaped and protruding (Fig. 7D), with width of the protrusion usually shorter than the stomatal slit ............................................................................................................. Group 6 b. Stomatal surface circular or dome-shaped and protruding, with width of protrusion usually as long as or longer than the stomatal slit ................................................................................................... 12 12a. Stomatal surface dome shaped and protruding, with the part near stomatal slit also protruding .... 13 b. Stomatal surface circular and protruding, with the part near stomatal slit slightly depressed ........ 15 13a. Epidermal cells papillose or strongly protruding upward ..................................................... Group 8 b. Epidermal cells scarcely or only weakly protruding ....................................................................... 14 14a. Outer part of subsidiary cells only weakly stained .............................................................. Group 7a b. Outer part of subsidiary cells stained darkly ....................................................................... Group 7b 15a. Rim of the circular stomatal surface darkly stained and conspicuous under optical microscope ....... ........................................................................................................................................... Group 10a b. Rim of the circular stomatal surface scarcely stained and inconspicuous under optical microscope .......................................................................................................................................... Group 10b REINWARDTIA 64 [VOL.14 Species Specimen no. Locality Alseodaphne andersonii (King) Kosterm. Poilane 19847 Vietnam Alseodaphne andersonii (King) Kosterm. Hyland 14931 China Alseodaphne elongate (Blume) Kosterm. de Wilde 18784 Indonesia Alseodaphne glaucina (A.Chev.) Kosterm. Chevalier 38873 Vietnam Alseodaphne hainanensis Merr. Yu 103147 China Alseodaphne insignisGamble Mohtar S 54836 Sarawak Alseodaphne oblanceolata (Merr.) Kosterm. SAN 35191 Indonesia Alseodaphne obovata Kosterm. Ashton 5823 Indonesia Alseodaphne peduncularis Hook.f. Rahmat 2980 Indonesia Alseodaphne rhododendropsis Kosterm. Poilane 3566 Vietnam Alseodaphne semecarpifolia Nees Bernardi 15385 Ceylon Alseodaphne sp. 1 van der Werff 23932 Vietnam Alseodaphne sp. 2 van der Werff 23889 Vietnam Alseodaphne sp. 3 van der Werff 23855 Vietnam Alseodaphne sp. 4 van der Werff 17084 Vietnam Alseodaphne sp. 5 Lee S 45516 Sarawak Alseodaphne sp. 6 Mohtar S 59461 Sarawak Alseodaphne sp. 7 Jamree S 73282 Sarawak Alseodaphne sp. 8 Maxwell 07-702 Thailand Alseodaphne sp. 9 Garcia 15942 Philippines Alseodaphne sp. 10 Gaerlan 26377 Philippines Alseodaphne sp. 11 Maxwell 06-515 Thailand Alseodaphne sp. 12 Julaihi S 83465 Sarawak Alseodaphne sp. 13 Julaihi S 83482 Sarawak Alseodaphne sp. 14 Wu WP 409 Vietnam Alseodaphne sp. 15 Enjah S 81836 Sarawak Alseodaphne sp. 16 de Wilde 20304 Indonesia Dehaasia annamensis Kosterm. Poilane 2786 Vietnam Dehaasia cairocan (S.Vidal) C.K.Allen Curran 10392 Philippines Dehaasia suborbicularis (Lecomte) Kosterm. Poilane s.n. Vietnam Dehaasia tomentosa (Blume) Kosterm. Kostermans 4896 Indonesia Dehaasia turfosa Kosterm. SAR 9268 Indonesia Dehaasia sp. 1 Arifiani 37 Indonesia Dehaasia sp. 2 de Wilde 15572 Indonesia Dehaasia sp. 3 Kessler 2130 Indonesia Dehaasia sp. 4 de Wilde 14420 Indonesia Dehaasia sp. 5 Adriansyah AA 2476 Indonesia Dehaasia sp. 6 Kessler 303 Indonesia Appendix 1. List of the samples examined. Specimens are deposited in MO or L. 2014] 65 NISHIDA et al. : Cuticular characters of the Asian Persea group Species Specimen no. Locality Machilus bombycina King Maxwell 02-155 Thailand Machilus breviflora Hemsl. Wang 37282 China Machilus calcicola S.Lee & C.J.Qi Guo 80136 China Machilus chekiangensis S.K.Lee Cheng 170507 China Machilus decursinervis Chun Chen 14184 China Machilus gamblei King Wang 39173 China Machilus glabrophylla J.F.Zuo China Germany T 618 China Machilus grandibracteata S.K.Lee & F.N.Wei Zhou 10355 China Machilus grijsii Hance Huang 161402 China Machilus kurzii King Maxwell 13971 Thailand Machilus minutiloba S.K.Lee Zhang 5346 China Machilus nakaoi S.K.Lee Hou 72073 China Machilus odoratissima Nees Middleton 643 Cambodia Machilus oreophila Hance Chen 23180 China Machilus parabreviflora H.T.Chang Tsang 23803 China Machilus phoenicis Dunn He 15100 China Machilus platycarpa Chun Wang 38678 China Machilus pomifera (Kosterm.) S.K.Lee Wang 34302 China Machilus velutina Champ. Guangdong 73 T 2980 China Machilus sp. 1 Harder 4179 Vietnam Machilus sp. 2 Harder 4775 Vietnam Machilus sp. 3 van der Werff 14068 Vietnam Machilus sp. 4 van der Werff 14104 Vietnam Machilus sp. 5 van der Werff 14255 Vietnam Machilus sp. 6 VH 5117 Vietnam Machilus sp. 7 HAL 111 Vietnam Machilus sp. 8 Lowry 4918 Vietnam Machilus sp. 9 Lowry 4921 Vietnam Machilus sp. 10 van Beusekom 4078 Thailand Machilus sp. 11 van Beusekom 4793 Thailand Machilus sp. 12 Poilane 24913 Vietnam Machilus sp. 13 Poilane 19114 Vietnam Machilus sp. 14 Chevalier 38790 Vietnam Machilus sp. 15 Poilane 23288 Cambodia Machilus sp. 16 Gao 50232 China Nothaphoebe cavalieriei (H.Lév.) Yang Xu 527 China Nothaphoebe cuneata Blume Kostermans 7103 Indonesia Nothaphoebe sarawacensis Gamble Chai S 35449 Sarawak Nothaphoebe sp. 1 James S 34453 Sarawak Nothaphoebe sp. 2 Lam 3564 Indonesia Persea chartacea Kosterm. Maxwell 00-44 Thailand Phoebe bournei (Hemsl.) Yang He 4270 China Appendix 1. List of the samples examined. Specimens are deposited in MO or L (continued). REINWARDTIA 66 [VOL.14 Species Specimen no. Locality Phoebe cathia (D.Don) Kosterm. Maxwell 98-664 Thailand Phoebe chekiangensis P.T.Li Ho 30223 China Phoebe formosana Hayata Liu 305 Taiwan Phoebe forrestii W.W.Sm. Hyland 14912 China Phoebe hunanensis Hand.-Mazz. Zuo 859 China Phoebe lanceolata (Nees) Nees Maxwell 05-387 Thailand Phoebe lucida Blume Beaman 9598 Sabah Phoebe macrophylla (Nees) Blume Jacobs 4572 Indonesia Phoebe neurantha Gamble Xu 1995119 China Phoebe sheareri Gamble Tan 58303 China Phoebe tavoyana Hook.f. Hou 70523 China Phoebe sp. 1 de Vogel 3625 Indonesia Phoebe sp. 2 Julaihi S 81357 Sarawak Phoebe sp. 3 Gaoligong SBS 23334 China Phoebe sp. 4 Gaoligong SBS 22844 China Appendix 1. List of the samples examined. Specimens are deposited in MO or L (continued). INSTRUCTION TO AUTHORS Scope. Reinwardtia is a scientific irregular journal on plant taxonomy, plant ecology and ethnobotany published in December. 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(+ 62) 21 8765066; Fax (+62) 21 8765062 E-mail: reinwardtia@mail.lipi.go.id REINWARDTIA Author Agreement Form Title of article Name of Author(s) : I/We hereby declare that: • My/Our manuscript was based on my/our original work. • It was not published or submitted to other journal for publication. • I/we agree to publish my/our manuscript and the copyright of this article is owned by Reinwardtia. • We have obtained written permission from copyright owners for any excerpts from copyrighted works that are included and have credited the sources in our article. Author signature (s) Date Name MUHAMMAD EFFENDI, TATIK CHIKMAWATI & DEDY DARNAEDI. New cytotypes of Pteris ensiformis var. victoria from Indonesia 133 SUZANA SABRAN, REUBEN NILUS, JOAN T. PEREIRA & JOHN BAPTIST SUGAU. Contribution of the heart of Borneo (HoB) initiative towards botanical exploration in Sabah, Malaysia 137 WENNI SETYO LESTARI, BAYU ADJIE, TASSANAI JARUWATANAPHAN, YASUYUKI WATANO & MADE PHAR- MAWATI. 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Floristic compo- sition and structure of a peat swamp forest in the conservation area of the PT National Sago Prima, Selat Panjang, Riau, Indone- sia 193 IMAN HID AY AT & JAMJAN MEEBOON. Cercospora brunfelsiicola (Fungi, Mycosphaerellaceae), a new tropical Cercosporoid fungus on Brunfelsia uniflora 211 MAX VAN BALGOOY & ELIZABETH A. WIDJAJA. Flora of Bali: a provisional checklist 219 EKA FATMAWATI TIHURUA & INA ERLINAWATI. Leaf anatomy of Pandanus spp. (Pandanceae) from Sebangau and Bukit Baka-Bukit Raya National Park, Kalimantan, Indonesia 223 JULIA SANG & RUTH KIEW. Diversity of Begonia (Begoniaceae) in Borneo - How many species are there? 23 3 DIAN LATIFAH, ROBERT A. CONGDON & JOSEPH A. HOLTUM. A Physiological approach to conservation of four palm species: Arenga australasica, Calamus australis, Hydriastele wendlandiana saALicuala ramsayi 237 REINWARDTIA Vol. 14. No. 1.2014 CONTENTS Page ABDULROKHMAN KARTONEGORO & DANIEL POTTER. The Gesneriaceae of Sulawesi VI: the species from Mekongga Mts. with a new species of Cyrtandra described 1 LIM CHUNG LU & RUTH KIEW. Codonoboea (Gesneriaceae) sections in Peninsular Malaysia 13 WISNU H. ARDI, YAYAN W. C. KUSUMA, CARL E. LEWIS, ROSNIATI A. RISNA, HARRY WIRIADINATA, MELISSA E. ABDO & DANIEL C. THOMAS. Studies on Begonia (Begoniaceae) of the Molucca Islands I: Two new species from Halmahera, Indonesia, and an updated description of Begonia holosericea 19 YUZAMMI, JOKO R. WITONO & WILBERT L. A. HETTERSCHEID. Conservation status of Amorphophallus discophorus Backer & Alderw. (Araceae) in Java, Indonesia 27 MOHAMMAD F. ROYYANI & JOENI S. RAHAJOE. Behind the sacred tree: local people and their natural resources sustainabil- ity 35 FIFI GUS DWIYANTI, KOICHI KAMIYA & KO HARADA. Phylogeographic structure of the commercially important tropical tree species, Dryobalanops aromatica Gaertn. F. (Dipterocarpaceae) revealed by microsatellite markers 43 SACHIKO NISHIDA & HENK VAN DER WERFF. Do cuticle characters support the recognition of Alseodaphne, Nothaphoebe and Dehaasia as distinct genera? 53 NURUL AMAL LATIFF, RAHAYU SUKMARIA SUKRI & FAIZAH METALI. Nepenthes diversity and abundance in five habi- tats in Brunei Damssalam 67 NURUL HAZLINA ZATNI & RAHAYU SUKMARIA SUKRI. The diversity and abundance of ground herbs in lowland mixed Dipterocarp forest and heath forest in Brunei Darussalam 73 MUHAMMAD AMIRUL AIMAN AHMAD JUHARI, NORATNI TALIP, CHE NURUL ATNI CHE AMRI & MOHAMAD RUZI ABDUL RAHMAN. Trichomes morphology of petals in some species of Acanthaceae 79 DIAN ROSLEINE, EIZI SUZUKI, ATIH SUNDAWIATI, WARDI SEPTIANA & DESY EKAWATI. The effect of land use history on natural forest rehabilitation at corridor area of Gunung Halimun Salak National Park, West Java, Indonesia 85 JULIUS KULIP. The Ethnobotany of the Dusun people in Tikolod village, Tambunan district, Sabah, Malaysia 101 PETER O'BYRNE. On the evolution of Dipodium R. Br 123 Reinwardtia is a LIPI accredited Journal (517/AU2/P2MI-LIPI/04/2013) Herbarium Bogoriense Botany Division Research Center for Biology - Indonesian Institute of Sciences Cibinong Science Center Jln. Raya Jakarta - Bogor, Km 46 Cibinong 16911, P.O. Box 25 Cibinong Indonesia barudepan 395-569-2-PB belakangbaru img577_Page_1 img577_Page_2 img577_Page_3 img577_Page_4