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: 211 – 217 211 hila and scolecosporous, mostly acicular, pluriseptate, hyaline conidia, are typical for the genus Cercospora (Crous & Braun, 2003). Recent studies of cercosporoid fungi from Thailand have revealed several new species and new records of Cercospora on wild plants and economically important cultivated plants (Meeboon et al., 2007a, b, c; Nakashima et al., 2010; To–anun et al., 2009, 2011; Phengsintham et al., 2012, 2013). During the study of diversity of Cercospora and allied genera in Thailand (2008–2010), we found Cercospora leaf spot on an ornamental plant identified as Brunfelsia uniflora [syn. B. hopeana] at Royal Flora Garden, Chiang Mai. This plant belongs to family Solanaceae, originally from South America and now widely cultivated as ornamental plant. Brunfelsia uniflora is also recognized as INTRODUCTION Cercospora Fresen. is an anamorph–typified ascomycete genus, now considered and used as holomorph genus covering species only forming Cercospora–like asexual morphs as well as species with such asexual and Mycosphaerella–like sexual morphs (Braun et al., 2013). More than 3000 names have been described worldwide (Crous et al., 2000; Crous & Braun, 2003) [see also http:// www.mycobank.com/MycoTaxo.aspx]. Members of this genus are commonly found as saprobes, secondary invaders and plant pathogens causing leaf spot or leaf blight (Crous & Braun, 2003). The morphological characteristics of pigmented conidiophores, conspicuously thickened and darkened conidiogenous loci (scars) and conidial CERCOSPORA BRUNFELSIICOLA (FUNGI, MYCOSPHAERELLACEAE), A NEW TROPICAL CERCOSPOROID FUNGUS ON BRUNFELSIA UNIFLORA Received January 17, 2014; accepted September 26, 2014 IMAN HIDAYAT Microbiology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong, Indonesia. E-mail: imanhidayat@yahoo.com JAMJAN MEEBOON Department of Agriculture, 50 Phaholyothin Rd., Ladyao, Chatuchack, Bangkok, Thailand. ABSTRACT HIDAYAT, I. & MEEBOON, J. 2014. Cercospora brunfelsiicola (fungi, Mycosphaerellaceae), a new tropical cerco- sporoid fungus on Brunfelsia uniflora. Reinwardtia 14(1): 211 – 217. — Cercospora brunfelsiicola on Brunfelsia uniflora is proposed as a new species based on a combination of molecular phylogenetic and morphological data analyses. The molecular phylogenetic analysis based on combined multilocus analyses of the Internal Transcribed Spacer (ITS), part of the elongation factor 1–α gene (EF1–α), and part of the calmodulin (CAL) gene regions showed that C. brunfelsiicola is phylogenetically distinguishable from other Cercospora species, including members of the C. apii s. l. complex. Morphologically, C. brunfelsiicola differs from other closely related Cercospora species, in particular C. acaciae-mangii, by forming lesions with indistinct margin, larger stromata [(32) 48.5 ± 10.6 (68) μm diam.], and filiform to narrowly obclavate conidia [(45) 59 ± 9.1 (72) × (2.5) 2.5 ± 0.2 (3) μm]. Key words: Fungi, Hyphomycetes, leaf spot, phylogeny, taxonomy. ABSTRAK HIDAYAT, I. & MEEBOON, J. 2014. Cercospora brunfelsiicola (jamur, Mycosphaerellaceae), jenis jamur cercospor- oid daerah tropis pada Brunfelsia uniflora. Reinwardtia 14(1): 211 – 217. — Cercospora brunfelsiicola Hidayat & Meeboon yang ditemukan pada bercak daun Brunfelsia uniflora diusulkan sebagai spesies baru berdasarkan kombinasi hasil analisis data morfologi dan analisis filogenetik sekuen DNA gabungan dari daerah Internal Transcribed Spacer (ITS), sebagian daerah gen elongation factor 1–α gene (EF1–α) dan gen calmodulin (CAL). Hasil analisis menunjukkan bahwa C. brunfelsiicola secara filogenetik terpisah dari sekuen DNA Cercospora terdekat, termasuk anggota C. apii s.l. Secara morfologi, C. brunfelsiicola dapat dibedakan dari spesies Cercospora terdekat, khususnya C. acaciae-mangii, karena memiliki warna gejala pada daun kecoklatan dengan garis batas yang tidak jelas, stromata lebih besar [diameter (32) 48,5 ± 10,6 (68) µm] dan memiliki bentuk konidia obclavate [(45) 59 ± 9,1 (72) × (2,5) 2,5 ± 0,2 (3) m]. Kata kunci: Bercak daun, filogeni, Hifomisetes, jamur, taksonomi. mailto:imanhidayat@yahoo.com REINWARDTIA 212 [VOL.14 medicinal plants for diuretic, antirheumatic and anti –inflammatory (Plowman, 1977). In this report, Cercospora specimen on B. uniflora from Thailand is proposed as a new species based on a combination of morphology and molecular phylogenetic analyses involving ITS rDNA, part of the elongation factor 1–α (EF1–α) gene and part of the calmodulin (CAL) gene region. MATERIALS AND METHODS Collection and Observation Specimens of Cercospora leaf spot on B. uniflora were collected from Royal Flora, Chiang Mai Province, Thailand. Magnifying lenses of 10× and 20× magnifications were used during the observation of symptoms in the field. Specimens showing the presence of Cercospora caespituli were placed in plastic bags for further examination. The collecting bags were sealed and labeled as follows: name of host plant, collection site, collector/s and collection date. Macroscopic characteristics were observed using a stereo microscope (Olympus SZX7) to check the fungal caespituli on the leaf spots in detail. The examination of microscopic characters was carried out by means of an Olympus BX53 light microscope using oil immersion (1000×). Specimens for observation were prepared by hand sectioning. Water and Shear’s solution were used as mounting media. Thirty conidia, hila, conidiophores, conidiogenous loci and 10 stromata Fig. 1. Phylogenetic tree based on combination of ITS, partial EF1–α, and CAL genes region representing placement of members of C. brunfelsiicola within Cercospora s.str.. Bootstrap support ≥ 50% from Maximum Parsimony analysis are shown on the nodes. 2014] 213 HIDAYAT et al. : A new tropical cercosporoid fungus on Brunfelsia uniflora were measured for each specimen. Line drawings were prepared at a magnification of 400× or 1000×. Single spore isolation was carried out following the method outlined by Choi et al. (1999) with a modification. Dried herbarium specimens were deposited in the BIOTEC Herbarium (BBH), Thailand. Cultures were deposited at BIOTEC culture collection (BCC), Thailand and LIPI microbial culture collection (LIPIMC), Indonesia. DNA Extraction, Polymerase Chain Reaction (PCR), and Sequencing A culture of Cercospora isolated from B. uniflora was grown on potato dextrose broth (PDB, Difco) and incubated on waterbath shaker (100 rpm) (Taitec Personal–11) for 10 days at 28°C prior to DNA extraction. Fungal mycelia were harvested in a 1.5 ml tube with 500 µl miliQ water. Mycelial cell–walls were homogenized mechanically using Table 1. GenBank accession number of cercosporoid fungi used in this study. No. Species Strain Code GenBank Accession Number ITS EF CAL 1 Cercospora brunfelsiicola RF5 AB859638 AB863025 AB863026 2 Cercospora acaciae-mangii CPC 10526T AY752141 AY752176 AY752235 3 Cercospora acaciae-mangii CPC 10550 AY752139 AY752172 AY752231 4 Cercospora senecionis-walkeri CBS 132636 JX143649 JX143408 JX142916 5 Cercospora apii CBS 116455 AY840519 AY840486 AY840417 6 Cercospora apiicola CBS 116457 AY840536 AY840503 AY840434 7 Cercospora armoraciae MUCC 768 JX143554 JX143308 JX142816 8 Cercospora apii CBS 152.52 AY840515 AY840482 AY840413 9 Cercospora apiicola CPC 10248 AY840539 AY840506 AY840437 10 Cercospora beticola CBS 116456 AY840527 AY840494 AY840425 11 Cercospora cf. flagellaris CBS 132674 JX143606 JX143364 JX142872 12 Cercospora capsici MUCC 574 JX143569 JX143325 JX142833 13 Cercospora chenopodii CBS 132620 JX143571 JX143327 JX142835 14 Cercospora cf. citrulina MUCC 588 JX143582 JX143340 JX142848 15 Cercospora tezpurensis CS 2012T KC351743 KC513746 KC513745 16 Cercospora zeae-maydis CBS 117758 DQ185075 DQ185087 DQ185111 17 Cercospora zeina CPC 11998 DQ185082 DQ185094 DQ185118 18 Cercospora beticola CPC 5123 DQ233327 DQ185094 DQ233405 19 Cercospora cf. helianthicola MUCC 716 JX143615 JX143374 JX142882 20 Cercospora cf. ipomoeae MUCC 442 JX143618 JX143377 JX142885 21 Cercospora kikuchii CBS 135.28 DQ835071 DQ835089 DQ835135 22 Cercospora lactucae-sativae MUCC 570 JX143623 JX143382 JX142890 23 Cercospora cf. nicotianae CBS 132632 JX143631 JX143390 JX142898 24 Cercospora punctiformis CBS 132626 JX143638 JX143397 JX142905 25 Cercospora cf. richardiicola MUCC 138 JX143643 JX143402 JX142910 26 Cercospora rodmanii 15-GTOX GQ884185 GQ884191 GQ884195 27 Cercospora sojina CPC 17977 JX143674 JX143434 JX142942 28 Cercospora zeae-maydis CBS 117757 DQ185074 DQ185086 DQ185110 29 Cercospora zebrina CBS 118790 GU214657 KF253248 KF253963 30 Cercospora zeina CPC 11995T DQ185081 DQ185093 DQ185117 31 Cercospora cf. zinniae CBS 132676 JX143757 JX143519 JX143027 32 Pseudocercospora thailandica CPC 10621 AY752159 AY752189 AY752251 33 Pseudocercospora thailandica CPC 10547 AY752156 DQ835102 AY752248 REINWARDTIA 214 [VOL.14 plastic pestle. The mycelia were then centrifuged using a Centrifuge MiniSpin (Eppendorf, Germany) at 14.500 rpm for 10 minutes. DNA was extracted from the mycelia with a PHYTOPURETM DNA extraction kit (GE Healthcare, UK) according to the manufacturer’s instruction. DNA amplification was performed by polymerase chain reaction (PCR) using primer pairs of ITS5 (5’– GGAAGTAAAAGTCGTAACAAGG–3’) and ITS4 (5’–TCCTCCGCTTATTGATATGC–3’) for ITS (Internal Transcribed Spacer) region (White et al., 1990), EF1–728F (5’– CATCGAGAAGTTCGAGAAGG–3’) and EF1– 986R (5’–TACTTGAAGGAACCCTTACC–3’) for part of Elongation Factor 1–α region, and CAL– 228F (5’–GAGTTCAAGGAGGCCTTCTCCC–3’) and CAL–737R (5’– CATCTTTCTGGCCATCATGG–3’) for Calmodulin region (Carbone & Kohn, 1999). For the ITS region, PCR was performed in a 25 ml volume as follows: nuclease free water 10 µl, Go Taq® Green Mastermix (Promega, Madison, USA) 12.5 µl, ITS5 dan ITS4 primer 0.5 µl for each primer, DMSO 0.5 µl, and DNA template 1 µl. The PCR reaction was done using TaKaRa thermocycler (TaKaRa, Japan) as follows: initial denaturation at 95ºC for 90 s, followed by 35 cycles of 95ºC for 30 s, 55ºC for 30 s, 72ºC for 90 s, and final extension of 72ºC for 5 min. PCR reactions for EF and CAL genes were performed in 25 mL reaction volumes as follow: each reaction containing nuclease free water 8.75 µl, Go Taq® Green Mastermix (Promega, Madison, USA) 12.5 µl, forward and reverse primer 0.625 µl for each primer, DMSO 0.5 µl, and DNA template 2 µl. PCR was performed in a TaKaRa thermocycler (TaKaRa, Japan) with the following program: 94ºC for 5 min, 35 cycles {94ºC for 30 s, 52ºC for 30 s, 72ºC for 30 s} and followed by a final extension of 7 min at 72ºC (Groenewald et al., 2005). PCR results were visualized using electrophoresis method in a 1% agarose gel at 100 V for 30 min. Agarose gel was soaked in an ethidium bromide for 60 mins and visualized under UV light (Printgraph). PCR products were sent to 1stBase (Malaysia) for DNA sequencing. Phylogenetic Analysis Nucleotide sequences obtained from the respective primer pairs (ITS5 and ITS4, EF1–728F and EF1–986R, CAL–228F and CAL–737R) were examined and refined by direct examination using Chromas Pro 1.41 software (Technelysium Pty Ltd., Australia). Sequences generated from the respective ITS, EF, and CAL regions were aligned with sequences retrieved from DNA databases (DDBJ, NCBI) using MUSCLE (Edgar, 2004) implemented in MEGA 6 (Tamura et al., 2013). Pseudocercospora thailandica strain CPC 10621 and P. thailandica strain CPC 10547 were used as outgroups in the analysis. Regions designated as ambiguously aligned were excluded from the analyses. GeneBank accession number, strain code, and taxon names used in this study are given in Table 1. The phylogenetic analysis was conducted using the maximum parsimony (MP) method in PAUP* 4.0b10 (Swofford, 2002). The MP analysis was performed with the heuristic search option using the ‘tree–bisection-reconstruction’ (TBR) algorithm with 1000 random sequence additions to find the optimum tree. The stepwise addition option set as random and maximum tree number was set at 5000. Tree length (TL), consistency index (CI), retention index (RI), related consistency index (RC), and homoplasy index (HI) were also calculated. The Kishino–Hasegawa (KH) likelihood test (Kishino & Hasegawa, 1989) was carried out to compare the best tree topology obtained by the nucleotide sequence data with a constrained tree. The strength of the internal branches of the phylogenetic tree in MP analysis was tested with bootstrap (BS) analysis (Felsenstein, 1985) using 1000 replications. BS values of 50 % or higher are shown. Random sequence addition was used in the bootstrap analysis. All sites were treated as unordered and unweighted, and gaps treated as missing data. TreeGraph 2 (Stöver & Müller, 2010) was used to refine the phylogenetic tree. The partition homogeneity test (Farris et al., 1995) was carried out by using PAUP* to determine whether ITS, EF1 –α, and CAL datasets were in conflict with 1000 replicates. RESULTS Phylogenetic Analysis The alignment of the combined sequences from ITS, EF and CAL regions contained 33 sequences and 1120 total characters, of which 835 characters were constant, 40 characters were variable and parsimony-uninformative, 245 characters were parsimony–informative. The most parsimonius tree was generated in 515 steps (CI = 0.695, RI = 0.791, RC = 0.550, HI = 0.305). The phylogenetic tree generated from MP analysis showed that C. brunfelsiicola is phylogenetically distinguishable from other Cercospora sequences (Fig. 1). Sequences of C. brunfelsiicola formed an independent lineage and showed a close relationship to sequences of C. acaciae–mangii Crous, Pongpan. & M. J. Wingf. on Acacia mangium Willd. (Leguminosae) with 62% BS (bootstrap support). 2014] 215 HIDAYAT et al. : A new tropical cercosporoid fungus on Brunfelsia uniflora This clade is sister to a clade containing sequences of C. tezpurensis M. K. Meghvansi & Md. Haneef Khan on Capsicum assamicum Purkayastha & Singh, C. rodmanii on Eichhornia crassipes (Mart.) Solms, and C. cf. richardiicola on Fuchsia×hybrida with 94% BS. Taxonomy Cercospora brunfelsiicola Hidayat & Meeboon, spec. nov. – Fig. 2. MYCOBANK MB805994 Cercospora brunfelsiicola differs from C. acaciae- mangii and other closely related Cercospora species by forming lesions with indistinct margin, larger stromata [(32) 48.5 ± 10.6 (68) μm diam.], and filiform to narrowly obclavate conidia [(45) 59 ± 9.1 (72) × (2.5) 2.5 ± 0.2 (3) μm]. ― Type: Thailand, Chiang Mai Province, Royal Flora Garden, on leaves of Brunfelsia uniflora (Pohl) D. Don [= B. hopeana (Hook.) Benth.] (Solanaceae), 27 July 2008, Jamjan Meeboon, RF5 (BBH 23764: Holotype). Ex-type culture: BCC32756, other culture: LIPIMC 774. GenBank accession number (ITS: AB859638, EF: AB863025, CAL: AB863026). Leaf spots amphigenous, distinct, circular to angular, 2–7 mm diam., brown, sometimes forming larger lesions, margin indistinct. Caespituli mainly epiphyllous, dark or blackish, punctiform, scattered within the lesions. Stromata (32) 48.5 ± 10.6 (68) μm diam. (n = 10), intraepidermal, well-developed, composed of globular to angular, brown to blackish brown cells. Conidiophores numerous, in dense fascicles aris- ing from stromata, (34) 98.5 ± 28.8 (151) × (2.5) 4 ± 0.6 (5.5) μm (n = 30), rarely branched, subcylindrical, strongly geniculate, 2–5–septate, simple, straight, erect to decumbent, smooth, pale Fig. 2. Cercospora brunfelsiicola sp. nov. (from holotype) a. Conidia. b. Stromata and conidiophores. (Scale bar = 50µm). REINWARDTIA 216 [VOL.14 yellow to pale brown. Conidiogenous cells integrated, terminal, (10) 21.5 ± 8.2 (47.2) × (2.5) 4.1 ± 1.1 (4.8) µm long (n = 30), holoblastic, monoblastic to polyblastic, sympodially proliferating. Conidiogenous loci 2.5–3 μm diam. (n = 30), conspicuous, thickened and darkened. Conidia (45) 59 ± 9.1 (72) × (2.5) 2.5 ± 0.2 (3) μm (n = 30), solitary, filiform to narrowly obclavate, 4 –8–septate, straight, hyaline, smooth, base obconically truncate, with subacute apex, hila 2– 2.5 μm diam. (n = 30), thickened and darkened. Distribution. Only known from its type locality. Etymology. The new species is named after its host generic name. DISCUSSION The current study is the first report of Cercospora found on B. uniflora. About 12 species of Cercospora have been recognized on hosts of the plant family Solanaceae, viz, C. canescens Ellis & G. Martin (C. apii s. l.), C. lanugiflori Chupp & A.S. Mull. [on Solanum velutinum Dunal (= S. lanugiflorum Pittier)], C. nigri Tharp var. microsporae L. N. Bhardwaj & Y. S. Paul (on S. nigrum L.), C. nicandrae Chupp [on Nicandra physalodes (L.) Gaertn.)], C. nicotianicola J. M. Yen (on Nicotiana tabacum L.), C. physalidis Ellis (C. apii s. lat.), C. physalidis–angulatae J.M. Yen (on Physalis angulata L.), C. puyana Syd. (on S. trachycyphum Bitter), C. sciadophila (Speg.) Chupp (on S. violifolium Schott. ex Spreng), C. solanacea Sacc. & Berl. (on Solanum spp.), C. solani Thüm. (on Solanum spp.), and C. Solani- nigri Chidd. (on S. nigrum) [Chupp, 1954; Crous & Braun, 2003]. Cercospora venezuelae var. indica Govindu & Thirum. and C. solanigena Bhartiya, R. Dubey & S. K. Singh are noted as uncertain species by Crous & Braun (2003) as type material could not be traced and due to the as- sumption that the original description was based on young conidia, respectively. Based on the morphological examination, C. brunfelsiicola is distinguishable from other Cercospora species found on hosts of the plant family Solanaceae by having strongly geniculate, densely fasciculate conidiophores and filiform to narrowly obclavate conidia with a few septa (Fig. 2). Cercospora brunfelsiicola differs from the plurivorous C. apii s. l. on Solanaceae (C. canescens, C. physalidis) by having well–developed stromata, numerous conidiophores in dense fascicles and strongly geniculate conidiophores. The filiform to narrowly obclavate conidia of C. brunfelsiicola (Fig. 2) are apparently distinct from the long acicular conidia of C. apii s.l. The molecular phylogenetic analyses based on combined sequence of ITS, EF1–α and CAL gene regions apparently showed that C. brunfelsiicola has a close phylogenetic relationship to C. acaciae –mangii (Groenewald et al., 2013). Cercospora brunfelsiicola is distinguishable from C. acaciae- mangii on A. mangium, which was originally de- scribed from Thailand as well (Crous et al., 2004), by having lesions with indistinct margin, larger stromata [(32) 48.5 ± 10.6 (68) μm diam.] and filiform to narrowly obclavate conidia [(45) 59 ± 9.1 (72) × (2.5) 2.5 ± 0.2 (3) μm]. Cercospora acaciae-mangii was morphologically described by Crous et al. (2004) based on distinct symptom (lesions medium brown, surrounded by a raised, dark brown border), stromata lacking to well– developed (up to 30 μm diam.) and conidia acicular (50–350 × 3.5–5 μm). In conclusion, our morphological data showed that the lesions with indistinct margin, densely fasciculate, strongly geniculate conidiophores and filiform to narrowly obclavate conidia are distinct characters that dis- tinguish C. brunfelsiicola from other closely related Cercospora species and justify the intro- duction of a new species for this fungus. 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L. 2002. PAUP*: phylogenetic analysis using parsimony (*and other methods), version 4. Sinauer Associates, Sunderland, Massachusetts. TAMURA, K., STECHER, G., PETERSON, D., FILIPSKI, A. & KUMAR, S. 2013 – MEGA6: Molecular Evolutionary Genetics Analysis Version 6.0. Molecular Biology and Evolution 30: 2725– 2729. http://dx.doi.org/10.1093/molbev/mst197. TO–ANUN, C., HIDAYAT, I. & MEEBOON, J. 2011. Genus Cercospora in Thailand: Taxonomy and phylogeny (with a dichotomous key to species). Plant Pathology & Quarantine 1: 11–87. WHITE, T. J., BRUNS, T., LEE, S. & TAYLOR, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. 315–322 pp. In: INNIS, M. A., et al. (Eds.), PCR Protocols: a guide to methods and applications. San Diego, Academic Press. http://dx.doi.org/10.1093/molbev/mst197 REINWARDTIA 218 [VOL.14 INSTRUCTION TO AUTHORS Scope. Reinwardtia is a scientific irregular journal on plant taxonomy, plant ecology and ethnobotany published in December. Manuscript intended for a publication should be written in English. Titles. Titles should be brief, informative and followed by author's name and mailing address in one- paragraphed. Abstract. English abstract followed by Indonesian abstract of not more than 250 words. Keywords should be given below each abstract. Manuscript. Manuscript is original paper and represent an article which has not been published in any other journal or proceedings. The manuscript of no more than 200 pages by using Times New Roman 11, MS Word for Windows of A4 with double spacing, submitted to the editor through . New paragraph should be indented in by 5 characters. For the style of presentation, authors should follow the latest issue of Reinwardtia very closely. Author(s) should send the preferred running title of the article submitted. Every manuscript will be sent to two blind reviewers. 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Legends or illustration must be submitted separately at the end of the manuscript. References. Bibliography, list of literature cited or references follow the Harvard system as the following examples. Journal : KRAENZLIN, F. 1913. Cyrtandraceae novae Philippinenses I. Philipp. J. Sci. 8: 163-179. MAYER, V., MOLLER, ML, PERRET, M. & WEBER, A. 2003. Phylogenetic position and generic differentiation of Epithemateae (Gesneriaceae) inferred from plastid DNA sequence data. American J. Bot. 90: 321-329. Proceedings :TEMU, S. T. 1995. Peranan tumbuhan dan ternak dalam upacara adat "Djoka Dju" pada suku Lio, Ende, Flores, Nusa Tenggara Timur. In: NASUTION, E. (Ed.). Presiding Seminar dan Lokakarya Nasional Etnobotani II. LIP1 & Perpustakaan Nasional: 263-268. (In Indonesian). SIMBOLON, H. & MIRMANTO, E. 2000. Checklist of plant species in the peat swamp forests of Central Kalimantan, Indonesia. In: IWAKUMA et al. (Eds.) Proceedings of the International Symposium on: Tropical Peatlands. Pp. 179-190. Book : RIDLEY, H. N. 1923. Flora of the Malay Peninsula 2. L. Reeve & Co. Ltd, London. Part of Book : BENTHAM, G. 1876. Gesneriaceae. In: BENTHAM, G. & HOOKER, J. D. Genera plantarum 2. Lovell Reeve & Co., London. Pp. 990-1025. Thesis : BAIRD, L. 2002. A Grammar of Keo: An Austronesian language of East Nusantara. Australian National University, Canberra. [PhD. Thesis]. Website : http://www.nationaalherbarium.n1/fmcollectors/k/Kostermans AJGH.htm). Accessed 15 February 2012. Reinwardtia Published by Herbarium Bogoriense, Botany Division, Research Center for Biology, Indonesian Institute of Sciences Address: Jin. Raya Jakarta-Bogor Km. 46 Cibinong 16911, P.O. Box 25 Cibinong Telp. (+ 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. Molecular phylogeny of maidenhair fern genus Adiantum (Pteridaceae) from Lesser Sunda Islands, Indonesia based on Rbcl and Trnl-f 143 ELIZABETH A. WIDJAJA & DANIEL POTTER. Floristic study of Mekongga Protected Forest: towards establishment of the Mekongga National Park 157 YESSI SANTIKA, EKA FATMAWATI TIHURUA & TEGUH TRIONO. Comparative leaves anatomy of Pandanus, Freycinetia and Sararanga (Pandanaceae) and their diagnostic value 163 SUHARDJONO PRAWIROATMODJO & KUSWATA KARTAWINATA. Floristic diversity and structural characteristics of mangrove forest of Raj a Ampat, West Papua, Indonesia 171 IAN M. TURNER. A new combination in Orophea (Annonaceae) for Uvaria nitida Roxb. ex G. Don 181 IVAN S AVINOV. Taxonomic revision of Asian genus Glyptopetalum Thwaites (Celastraceae R. Br.) 183 YUSI ROSALINA, NISYAWATL ERWIN NURDIN, JATNA SUPRIATNA & KUSWATA KARTAWINATA. 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 417-601-2-PB belakangbaru img577_Page_1 img577_Page_2 img577_Page_3 img577_Page_4