Article Rhizanthes

7/31/2021 Email Universitas Bengkulu - article Rhizanthes

Agus Susatya

article Rhizanthes 1 pesan

Agus Susatya 3 Juli 2020 17.22 Kepada: [email protected]

ekology saya sisipkan di intro.... tapi kalau di arrange... silahkan mas

Rhizanthes, the forgotten relative of Rafflesia Jaime edits 1 - Rev AW-AS.docx 1563K

https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-a%3Ar-702760702106332019&simpl=msg-a%3Ar-701108218… 1/1 1 Rhizanthes, the forgotten relative of Rafflesia in Rafflesiaceae: A brief review on Formatted: Numbering: Continuous

2 ethnomedicine and propagation technology prospects for educational and conservation

4 Adhityo Wicaksono1a, Ghea Putri Cristy1, Reza Raihandhany12,23, Sofi Mursidawati34b, Agus

5 4 5c6 5 Susatya , Jaime A. Teixeira da Silva , Agus Susatya Comment [AW1]: Jaime, please help me with the content and I need your help to make 6 1Division of Biotechnology, Generasi Biologi Indonesia (Genbinesia) Foundation, Jl. Swadaya sure the content will focus on the educational aspect and propagation studies/prospect instead of prospecting that could encourage 7 Barat no. 4, Gresik Regency 61171, Indonesia people from looting the plant in the wild (similar as in our 2016 Rafflesia review) 8 2School of Life Sciences and Technology (SITH), Institut Teknologi Bandung, Jl. Ganeca no. Sure. The paper is still very under-developed and so not much to comment on. I think it will be easier to create a “conservation-based” 9 10-12, Bandung 40132, Indonesia message once we get several of the sections filled in. Also, it might be best that Agus be the 10 3Division of Botany, Generasi Biologi Indonesia (Genbinesia) Foundation, Jl. Swadaya Barat no. last, senior author.

11 4, Gresik Regency 61171, Indonesia

12 43Center of Plant Conservation, Bogor Botanical Garden – Indonesian Institute of Science (LIPI),

13 Jl. Ir. H. Juanda no.13, Bogor 16003, Indonesia

14 5Department of Forestry, Faculty of Agriculture, Universitas Bengkulu, Jl. Raya Kandang Limun,

15 Bengkulu 38371, Indonesia

16 6Independent 5Department of Forestry, Faculty of Agriculture, Universitas Bengkulu, Jl. Raya

17 Kandang Limun, Bengkulu 38371, Indonesia

18 4Independent Researcher, P.O. Box 7, Miki-cho post office, Ikenobe 3011-2, Kagawa-ken, 761-

19 0799, Japan

20 55Department of Forestry, Faculty of Agriculture, Universitas Bengkulu, Jl. Raya Kandang

21 Limun, Bengkulu 38371, Indonesia

23 a b 24 Co-corresponding authors emails: [email protected], [email protected], Formatted: Superscript Formatted: Superscript 25 [email protected] Formatted: Superscript 26

27 Abstract

28 Rhizanthes is a holoparasitic plant of the Rafflesiaceae, and, just like Rafflesia, its host is

29 Tetrastigma (Vitaceae). Unlike Rafflesia, only very few studieslittle research has been done

30 conducted on Rhizanthes. Some previousA few studies are have focused on the its taxonomy and Comment [Jaime2]: only very few studies have been published on OR 31 anatomy. There are more to be done sinceStudies on the propagation study of Rhizanthes is are only very little research has been conducted on Comment [AW3]: 32 limited, despite some uses of it as ethnomedicineethnomedicinal uses in some regions in of The second one

33 Indonesia. In the wWild, the populations of Rhizanthes is are declining due to deforestation and

34 overharvesting by locals. Some studies highlight the aArtificial pollination and possible seed

35 spread, which has are similar procedure to Rafflesia and, may be useful for future propagation-

36 based study studies, which in are generally very difficult and challenging for members of the

37 Rafflesiaceae is considered very difficult and challenging. This short-reviewpaper emphasizes

38 the cultural and ethnomedicinal importance of Rhizanthes in and seeks to define a conservation

39 road-map that incorporates a scientifically-based approach to research while also seeking a four-

40 pronged approach to conservation of Rhizanthes: 1) conventional and biotechnology-based

41 conservation; 2) germplasm multiplication and preservation; 3) reintroduction into the wild and

42 conservation of wild populations; 4) policy-based protective measures.multiple aspects and to Comment [Jaime4]: Try to consider these 4 steps as an integrated conservation approach. Think locally, how is this possible in the 43 encourage future studies. different Indonesian islands? So, what are practical limitations? Is reintroduction a 44 Key words: parasitic plant, holoparasite, biotechnology; conservation; ethnobotany; holoparasite; fantasy? Is legislation impossible? If yes then what would be the best conservation strategies based on Indonesia’s financial and 45 in vitro propagation; parasitic plant structural limitations? Be realistic, no need to apply strategies used in France or the USA to 46 Indonesia, because it might simply not work… careful reflection is needed.

Formatted: Font: Italic 47 Introduction

48 Rhizanthes belongs to the family Rafflesiaceae, alongside with Rafflesia and Sapria (REFMeijer

49 1997). The plant is also holoparasitic holoparasitizes some species of to Tetrastigma

50 (REFBänziger and Hansen 2000). Unlike Rafflesia, Rhizanthes is has not much been studied

51 much (Google Scholar search [June 7th, 2020]: “Rafflesia” 5090 results, “Rhizanthes” 254 results,

52 even “Sapria” is 855 results). Not much information covers the progress in propagation study Comment [Jaime5]: This could be a separate section if there is work on in vitro conservation, 53 (REF) and very limited studies covers the prospect of the plant, while the other studies have been seed propagation, cryoconservation, etc. Comment [AW6]: I’ve put in on later section 54 performed on Rafflesia and Sapria i.e. artificial pollination study which resulted in production of part

55 fruit (REFBänziger 2004). Comment [Jaime7]: This could be the first part of the introduction. 56 Comment [AW8]: I think this info is already good to be here, and the rest are already 57 described in later section Formatted: Line spacing: Double 58

59 Research on Rhizanthes can be traced back as early as 19 th century, when young German botanist, A Formatted: Font: (Default) Times New Roman 60 Zippelius found a speciment on Gunung Salak, Bogor, Indonesia which was later desccribed by Blume as Formatted: Font: (Default) Times New Roman 61 a new genus within Rafflesiaceae (Mejieer 1997). Meijer and Veldkamp (1988) then provided a Formatted: Line spacing: Double 62 revision on Rhizanthes and Meijer (1997) then mensioned two species Rhizanthes zippelii (Blume) Formatted: Font: (Default) Times New Roman 63 Spach and Rhizanthes lowii (Beccari) Harms in a published revision the whole family of Rafflesiaceae. Formatted: Font: (Default) Times New Roman 64 Finally after reviewing Meijer and Veldkamp‟s article especially variation of ramenta structures, and Formatted: Font: (Default) Times New 65 considering color, size, and sexuality of the flower and observing more available herbarium specimens , Roman Formatted: Font: (Default) Times New 66 Banzinger and Hansen (2000) came up with two additional new species, Rhizanthes infanticida Banzinger Roman

67 et Hansen and Rhizanthes deceptor Banzinger et Hansen. Existing information on Rhizanthes mainly

68 focusses on morphology and taxonomy, and rarely on ecological aspects, eventhough the ecological

69 information are of importance for future conservation of the species. Ecological Studies on Rhizanthes

70 were limited to R. zippelii (Bazinger, 1995) and R. lowii (Susatya 2003). Study on ecology on Rhizanthes

71 was initially carried out by Bazinger (1995) who observed bud‟s growth and also estimated the life cycle 72 of R.zippelii. He estimated that the life history of R. zippelii reached up to 200 and 255 days after

73 emerging from its host plant. This figure were shorter than the life cycle of Rafflesia patma (Hidayati et

74 al. 2000) and R. arnoldii (Susatya 2020). Banzinger (1995) unfortanely did not provide the population

75 dynamics of the bud of R. zippelii which was important in term of conservation. Susatya (2003) futher

76 observed the mortality, growth, and the life history of R. lowii. He found that the population size of R.

77 lowii was 32 flower buds, which was higher than those of species of Rafflesia (Susatya 2020). Within 4

78 months observations, R. lowii suffered 25 % loss of its flower buds. All mortality occurred at very small

79 size diameter (0.50-0.94 cm). After running transition matrix analysis, he found that the intrinctive rate

80 of increase (alpha) of the species was 0.762. This indicated that the local population of R. loweii would

81 likely go extinct within 30 months (Susatya 2003).

83 In some region of Indonesia, Rhizanthes has been known for its ethnomedicinal properties,

84 especially by locals in Borneo island (Syaifuddin et al. 2018; Uluk et al. 2001). This will triger

85 the uncontrolled harvests the flower of the species in the wield. Furthermore Susatya (2003)

86 predicted that high mortality caused extinction its local population For these reasons, the

Comment [Jaime9]: Make a section on 87 population will be declining fast in the wild.Despite the widespread use in ethnomedicine, the ethnomedicine. However, check those papers carefully for fake information! Medical studies 88 number of Rhizanthes in the wild is keep declining (REF) but none of Very unfornate that none appear on PubPeer all the time and so we don’t want to just blindly cite anything and everything that has been published. If there 89 of the all 4 species Rhizanthes such as (Rhizanthes deceptor Bänziger & B. Hansen, R. are studies with bad scientific evidence we can cite, but offer a cautious approach. Do not be 90 infanticida Bänziger & B. Hansen, R. lowii (Beccari) Harms, and R. zippelii Blume (Spach); afraid to include studies published in Indonesian language or dialects, but the core information must be accurately translated. I 91 Bänziger and Hansen 2000) are enlisted in IUCN Red List or in CITES AppendixCITES. assume that there is quite a bit of information in local journals not in English! 92 Moreover, there is no available propagation techniques for Rhizanthes , eventhough advanced Comment [AW10]: The problem is, the information is very limited! Indonesian study 93 propagation research especially on the biotechnology and metabolite testing had been on metabolites is only found in 1 thesis, the rest of information is on blogs which describes nothing 94 progressively carried on Rafflesia since 2001 (Wicaksono et al. 2016). Considering all those Comment [Jaime11]: Define them. OK, link 95 aspects, this paper is built to raise awareness of the future biology and conservation of better with the list later… Comment [AW12]: I place more descriptions of them on the next part 96 Rhizanthes through revealing its detailed biology, and its bioprospects.On R. lowii study in 2001 Formatted: Font: Italic

97 at Kerinci-Seblat National Park, Sumatra, Indonesia, a population with 32 flower buds were

98 found (Susatya 2003). However, no details if the population is viable and stable, especially that

99 Rhizanthes is mostly dioecious.

100

101 This paper aim is to expose some details in biology and to understand the bioprospects of

102 Rhizanthes in order to raise awareness for the importance of future biological studies, especially

103 for conservation. In many aspects of Rhizanthes, especially in the referral to previous studies, Comment [Jaime13]: Let’s see first what we have, then we can advance… 104 will mostly be compared to any study to Rafflesia. This is due to the progresses have been made

105 since 2001 in Rafflesia including conventional to biotechnological approach to propagation and

106 attempts on metabolite testings (Wicaksono et al. 2016). Formatted: Font: (Default) +Body (Calibri), 11 pt, English (Indonesia) 107

108 Biology

109 Rhizanthes (name derived from Greek words: Rhiza or root and –anthes flowering, as it believed

110 to bloom at the root) is also known as the deceptive flower, because to its tendency to deceive its

111 other visiting organisms in many ways: It dissimulate a corpse leading the pollinating insects to

112 lay many eggs with doom-fated hatchlings, a suspect of place to steal a scarce to non-existing

113 nectar for bees, wasps, ants, and butterflies, a vantage ground for male flies to find a female, and

114 to human, especially scientists, is a specimen which can lead to many disagreements for its

115 characters (Bänziger and Hansen 2000). Initially, two species of Rhizanthes, R. lowii and R.

116 zippelii were called Brugmansia lowi (sic) Beccari in 1868 and Brugmansia zippelii Blume in

117 1841 (Bänziger and Hansen 2000). There are 4 species of Rhizanthes in the world and spread

118 across the Malesian region (REF), including Indonesiafrom Peninsular Malaysia, to Sumatra, 119 Borneo (Kalimantan), and Java (REFMeijer 1997). The species are Rhizanthes deceptor

120 BanzigerBänziger &, B. Hansen, Rh. infanticida BanzigerBänziger &, B. Hansen, Rh. lowii

121 (Beccariari) Harms, and Rh. zippelii Blume (Spach) (Bänziger and Hansen 2000REF). Some

122 species can be seen in Fig. 1. R. zippelii is distributed in Sumatra, Peninsular Malaysia, Java

123 (specifically on West Java; Bänziger and Hansen 2000), and Borneo (Meijer and Veldkamp

124 1988), R. lowii in Sumatra, Peninsular Malaysia, and Borneo (Meijer and Veldkamp 1988;

125 Bänziger and Hansen 2000), R. infanticida in Thailand, Peninsular Malaysia, and Sumatra

126 (Bänziger and Hansen 2000), and R. deceptor in west area of Sumatra (Bänziger and Hansen

127 2000).

Comment [Jaime14]: Similar to the Rafflesia paper, maybe if you have a sketch of the floral 128 structures, could be useful… 129 Fig.ure 1. Rhizanthes upon anthesis: Two Rhizanthes lowii flower buds emerged and grown in Formatted: Font: Bold Formatted: Font: Bold 130 same spot (a), enlarged view of Rh. lowii flower bud (b), the fully bloomed Rh. lowii (sex Formatted: Font: Bold Formatted: Font: Bold 131 unidentified) (c), and the post-anthetic flower of Rh. lowii with flower bud underneath (yellow Formatted: Font: Bold 132 arrow) (d). The fully bloomed Rh. deceptor (sex unidentified) (e ) and its zoomed view of a male Formatted: Font: Bold Formatted: Font: Bold 133 flower pseudo-brown colored (color actually from the hairs) central area with anther region Formatted: Font: Bold 134 exposed (blue arrow) (f). Scale bars = 5 cm. Photo taken by Arbi Wiguna (a-d), Sofian Rafflesia Formatted: Font: Bold Formatted: Font: Bold 135 (e), and Neka Afnidarti (f); placed with permission via personal communication. Formatted: Font: Bold 136 Formatted: Font: Bold

Formatted: Font: (Default) Times New Roman, 12 pt

137

138 Fig. 2. The anatomy of Rhizanthes (modelled using R. lowii), showing the proto bud (prior to Formatted: Font: Bold

139 emergence from the host periderm), early, and late bud (with halved bud on inset, showing the

140 inward-folded caudal appendage), full organ male flower (perigone lobe surface without

141 epidermal features) and inset of female flower column. Additionally, inset of bisexual flower

142 column is also shown (but only available in R. zippelii and R. lowii). Anatomic figures of the

143 flower is inspired from Bänziger and Hansen (2000), and Bänziger et al (2007). Note: The term

144 of tepal described by from Bänziger and Hansen (2000) is mentioned as perianth in Nikolov et al

145 (2013), which is synonymous to perigone. The fully anthesis R. lowii and the late bud of R. lowii 146 are the same flowers from Fig. 1c and 1b, respectively, and photos are taken by Arbi Wiguna

147 (placed with permission via personal communication). Photo of proto bud, early bud, and halved

148 late bud are taken by Sofi Mursidawati.

149

Formatted: Font: (Default) Times New Roman, 12 pt

150

151 Fig. 3. The comparison of epidermal hair features in four species of Rhizanthes. Picture is Formatted: Font: Bold

152 redrawn and inspired from Bänziger and Hansen (2000).

153

154 As the plant belongs to Rafflesiaceae, the terms of morphology of Rhizanthes (Fig. 2) is almost

155 similar to Rafflesia and Sapria. The petal-like organ of Rhizanthes is referred as tepal (Bänziger

156 and Hansen 2000), but sometimes also referred as perianth (Nikolov et al. 2013), which is

157 synonymous to perigone (referred by Meijer 1997). Comparatively, in Rafflesia, the petal-like

158 organ is referred and mostly known as perigone. The unique part of Rhizanthes, it has caudal

159 appendage in the distal region of the perigone, which folded together in the hole of the central

160 column prior to anthesis and extended upon and prior anthesis (Bänziger et al. 2007; Saleh, 161 2015). The outer organ which protects the young flower bud is bract (Meijer 1997; Bänziger and

162 Hansen 2000). The flower has a column in the center, with gonadal region in the lateral part of

163 surrounding the column, and a hole or „crater‟ in the distal region (Bänziger and Hansen 2000).

164 Rhizanthes possesses three types of “hairs” or epidermal modified features (Fig. 3), which serve

165 as an important identification features between the species (Bänziger and Hansen 2000): The

166 bristles, the brush-like tuft hairs, the smaller and densely packed furry Organ description for

167 identification: Perigone, gonad, ramentahairs, and the tiny ramentae. Three of known Rhizanthes Comment [AW15]: Adhit: Make a figure

168 species are dioecious plant with separate male and female flower, while two species, R. zippelii

169 (Bänziger and Hansen 2000) and R. lowii (Bänziger et al. 2007) are bisexual.

170 Another feature of Rhizanthes flower is its endothermy and its capability to regulate its own Formatted: Indent: First line: 0.5 cm

171 temperature (observed in R. lowii by Patiño et al. 2000). In term of coloration, unlike the other

172 which is brown to yellowish, R. deceptor is whiteish (Bänziger and Hansen 2000). R. deceptor

173 name refers to the genus tendency of deceiving its pollinators and scientists, due to its apparent

174 brown coloration of the proximal area of the perigone in opposite to the central column, which is

175 actually caused by the overshadowing effect of the increasingly dense bristles and the

176 convergence of the radial lines, while actually the perigone wall is white (see Fig. 1e and f;

177 Bänziger and Hansen 2000). According to Cammerloher (1920), R. zippelii (still as Brugmansia

178 zippelii) possesses stomata with two guard cells on its epidermis. However, this feature requires

179 further investigation. In term of cytology (recorded in Meijer and Veldkamp 1988, contains the

180 old reference to van der Pijl 1933 cit. Harms 1935), there were 11 haploid chromosomes detected

181 in the pollen of R. zippelii (hence 2n = 22).

182 Prior anthesis, Rhizanthes grows as unicellular strands inside the host (Nikolov et al. 2014). It

183 grows near the phloem and xylem vessels of its host plant as it is seen in transversal sectioning. 184 However, longitudinal sectioning is required for future studies to obtain a clearer picture of the

185 endophyte and its possible behavior upon vegetative or generative stage of development. Upon

186 the flower bud development, the perigone lobes is derived from sepal, petal, and specific ring

187 derivative compared to Rafflesia only sepal and Sapria from both sepal and petal, while the

188 perigone cup or ring (proximal area of perigone) is derived from specific ring derivative, as

189 Rafflesia has no perigone ring but it forms a perigone tube or chamber derived from petal whorls

190 and sepal with the ring derivative forms the inner annuli (as described in Nais, 2001), and Sapria

191 also has perigone ring derived from specific ring derivative as Rhizanthes (Nikolov et al. 2013).

192 After anthesis and pollination, Rhizanthes will produce a fruit with tiny microseeds. As

193 observed in R. lowii, the fruit has labyrinth like lines with thousands of brownish seeds inside

194 (Bänziger et al. 2007). The seed, similar as in Rafflesia (Bouman and Meijer 1994; Nais 2001;

195 Mursidawati et al. 2014), Rhizanthes seed is bilobed with irregular sized beehives-like holes or

196 pores in over its surface (tegmic part) (observed in R. lowii by Bänziger et al. 2007; and R.

197 zippelii by Bouman and Meijer 1994).

198

199 Interaction biology: Hosts, pollinators, seed dispersal, etc. Formatted: Font: (Default) +Body (Calibri), 11 pt, Italic, English (Indonesia) 200 Nikolov’s study on endophyte and flower development. Flower endothermy and temperature Formatted: Font: Italic Formatted: Indent: First line: 0.5 cm 201 self-regulation (Banziger and Patino papers)

202 Rhizanthes grows in a range of Tetrastigma species for host hosts. R. zippelii grows in Formatted: Font: Not Italic

203 Tetrastigma papillosum Planch. (Meijer 1997), T. pedunculare (Wallich ex Lawson) Planch.

204 (Bänziger and Hansen 2000), and T. tuberculatum Latiff (syn. T. coriaceum (DC.) Gagnep.)

205 (Meijer 1997). R. lowii grows in T. papillosum (Meijer 1997) and T. pedunculare (Patiño et al.

206 2000). R. infanticida grows exclusively in T. papillosum (Bänziger 1995; Bänziger and Hansen 207 2000). R. deceptor grows in T. pedunculare (Bänziger and Hansen 2000) and T. papillosum

208 (Bänziger and Hansen 2000; Saleh et al. 2015).

209 As Rhizanthes emits rotting flesh-like odor as in Rafflesia, the pollination relies on the carrion

210 insects i.e. flies, hence making it sapromyophilous. R. lowii is known to be pollinated by carrion

211 flies (Calliphoridae), specifically Hemipyrellia ligurriens (Wiedemann) (Bänziger et al. 2007).

212 Rh. infanticida acquired its name “infanticide” due to its extremely effective deception nature to

213 a mammalian carcass to its pollinator (carrion flies, 10 species of Calliphoridae) which lead it to

214 laying hundreds of eggs, but then die by starvation upon hatching (Bänziger and Hansen 2000).

215 The pollinating insects of R. deceptor are including carrion flies Chrysomya defixa (Walker),

216 Lucilia porphyrina (Walker), and Hypopygiopsis fumipennis (Walker) (Bänziger in press cit. Comment [AW16]: It was told that the paper is in press in Bänziger and Hansen 2000, 217 Bänziger and Hansen 2000). R. zippelii pollination as observed by Bänziger (1996) are often but never seems to be published or updated

218 done by some members of carrion flies as well, mostly observed by Lucilia porphyrina (Walker),

219 Chrysomya pinguis (Walker), Ch. chani Kurahashi, Ch. villeneuvei Patton, Ch. megacephala

220 (Fabricius), and Hemipyrellia ligurriens (Weidemann), while the rests (Hypopygiopsis infumata

221 (Bigot), Hy. fumipennis (Walker), Hy. tumrasvini Kurahashi, and Lu.papuensis Macquart) are

222 considered exceptional and potential pollinators.

223 After fruit is formed, the seed is assumed to be dispersed by small mammals similar to

224 Rafflesia and Sapria, i.e rats (Bänziger, 2004). In Bänziger et al. (2007), it was found that R.

225 lowii fruit was bitten by giant rat (Leopoldamys or Sundamys). It is unclear if Rhizanthes seed is

226 dispersed via exozoochorous (i.e. stick on fur, snout, etc.) or endozoochorous (via digestive tract)

227 (Bouman and Meijer 1994). The discovery of squirrel (Callosciurus notatus (Bodaert)) and tree

228 shrew (Tupaia tana Raffles) eating Ra. keithii fruit in Borneo could indicate the importance of

229 endozoochorous possibilities, which also possibly occurring in Rhizanthes. 230 It is unknown so far if any species of Rhizanthes harbors endophytic fungal species as in

231 Refaei et al. (2011) study on Ra. cantleyi Solms-Laubach which shows the existence of

232 Colletotrichum, Cytospora, and Gliocladiopsis isolated from bud parts cultured in potato

233 dextrose agar (PDA) medium, and the fungi produce bioactive metabolites which possibly

234 contributes to plant defense and ethnomedicinal properties in addition to its natural secondary

235 metabolite biosynthesis.

236

237 Ethnobotany, ethnomedicinal, and metabolites

238 R. lowii is believed as a folk medicine from Banjarmasin, South Borneo (South Kalimantan)

239 province of Indonesia, known as “ulur-ulur”. Despite referred as “ulur-ulur” fruit, the taken part

240 of the flower is actually the flower bud prior the anthesis. The flower bud is taken from its host

241 stem, left to dry, and then boiled for consumption (Fig. 4a). It is believed that the boiled water of

242 the flower bud is a cure for hemorrhage, coronary disease, cholesterol, and female reproductive

243 organLocal name: Ulur-ulur. Local packed medicine from Banjarmasin, to cure hemorrhoid,

244 coronary disease, and cholesterol. supplement. According to Syaifuddin et al. (2018), “ulur-ulur” Comment [Jaime17]: Please assess the medical evidence very carefully… 245 referred as Rhizanthes sp., is used to cure hemorrhage, back pain, stomachache (acid reflux), and

246 hematuria by the people from Murung B Village, South Kalimantan, Indonesia by boiling the Comment [AW18]: The name is actually mentioned as “Murung B” 247 dried flower bud with 2 glass of water (± 500 mL), reducing it to 1 glass of water (±250 mL),

248 and consumed periodically. An ethnobotanical study by Uluk et al. (2001) mentioned that

249 Rhizanthes sp. is locally also known by the people of Dayak tribe near Kayan Mentarang

250 National Park as “aka kepun” is used locally as rope, despite it could be mistakenly thought as

251 the same part of its liana host, Tetrastigma sp. From different region of Indonesia, in Sumatra

252 island (region is not specifically mentioned) the flower bud Rh. deceptor is used to treat diarrhea 253 and stomachache by charring the bud on open flame, grinded to powder, mixed into water, and

254 then consumed (Quattrocchi, 2012).

255 A study of phytochemical analysis was performed on R. deceptor flower bud and its host T. Formatted: Indent: First line: 0.5 cm

256 papillosum root and stem taken from a site near Universitas Andalas, West Sumatra, Indonesia

257 (Saleh, 2015; Saleh et al. 2015). The samples (20 g) were grinded to powder and extracted three

258 times with ethanol (3×1000 mL) in room temperature for 24 hours. The obtained filtrate was

259 reduced until 100 mL with a rotary evaporator with 50 rpm at 40-50°C. Then 5 mL of the

260 obtained extract

261 Metabolites study was dried in an oven at 103°C for extract yield and the rest stored for

262 chemical test (all methods were using 0.1 g of samples, except for phenolic test in

263 spectrophotometry): alkaloid (the extract dissolved in 10 mL CHCl3 and added with 4 drops of Formatted: Subscript

264 NH4OH and 10 drops of 2 M H2SO4, then tested with Mayer, Wagner, and Dragendorf reagents Formatted: Subscript Formatted: Subscript 265 and positive results of the reagents shows white, brown, and orange coloration respectively), Formatted: Subscript 266 phenolic (Folin-Ciocalteu method), triterpenoid and steroid (the extract dissolved in 25 mL 50°C

267 ethanol, and then tested with Lieberman-Buchard test: in a test plate with 3 drops of anhidric

268 acetic acid and 1 drop of concentrated H2SO4, red or purple coloration indicates positive

269 existence of triterpenoid, while green or blue for steroid), saponin – which also a terpenoid (the

270 extract was mixed with 10 mL of water, boiled for 5 minutes, filtered, and the filtrate was

271 vigorously mixed for 10 seconds and left for 10 minutes, and the existence of stable foam/bubble

272 indicates the existence of saponin) and flavonoid (the extract was mixed with 10 mL of water,

273 boiled for 5 minutes, filtered, added with 0.5 g magnesium powder, 1 mL of concentrated HCl

274 and 1 mL amyl alcohol, and then mixed vigorously, positive existence of flavonoid is shown by

275 red, yellow, or orange coloration in the amyl alcohol layer). As for the results, no saponin and 276 steroid found in the R. deceptor extract, while only no steroid in T. papillosum root extract, and

277 no triterpenoid in T. papillosum stem extract. As for the total phenolic measurement, R. deceptor

278 extract possesses the highest amount, 431.52 mg GAE/g, followed by the root and stem of T.

279 papillosum extracts (323.93 mg GAE/g and 271.38 mg GAE/g, respectively; Note: GAE = Gallic

280 Acid Equivalent). The R. deceptor extract in the test (at IC50) was also revealed high antioxidant Formatted: Subscript

281 level (on the ethanol extract), 31.97±4.79 µg/mL comparatively to the root of T. papillosum

282 ethanol extracts, 21.71±8.01 µg/mL, but lower than the stem of T. papillosum ethanol extracts

283 35.23±2.42 µg/mL. The extracts appear to possess a prospect as biopesticide, shown in the brine

284 shrimp lethality test (BSLT), R. deceptor ethanol extract showed LC50 at 283.21±9.68 µg/mL.

285 Comparatively, the root ethanol extract of T. papillosum has fewer impact as the LC50 is at Formatted: Subscript

286 719.50±13.1 µg/mL, and the stem ethanol extract of T. papillosum is higher impact at LC50, at Formatted: Subscript

287 277.65±10.71 µg/mL. Despite the mentioned property and prospect, the exact properties of Comment [AW19]: Properties as in which compound of it, i.e. alkaloid -> is it caffeine or 288 alkaloid, phenolic, flavonoid, and triterpenoid in the R. deceptor extract is not yet described. nicotine?

289 Plant secondary metabolites are known for its purpose for plant protector against herbivores

290 and diseases (microbes), but also known to possess medicinal properties on human. Alkaloid

291 compounds in plants serve as protective compound against herbivory, plant antibacterial and

292 antifungal, while for human uses, it is applicable as anticancer, antibacterial, anti-inflammatory,

293 and even antiviral (Adamski et al. 2020). Some of alkaloids harvested from plants are already

294 clinically tested as anticancer drugs, i.e. taxol from Taxus brevifolia Nutt. (Wani et al. 1971) and

295 camptotechin from Camptotheca acuminate Decne. (Cragg and Newman 1995), and these

296 compounds have been screened by National Cancer Institute (NCI). Phenolic compounds or

297 polyphenols comprise of 8000 compounds, including flavonoids, phenolic acids, tannins,

298 stillbenes, anthocyanins, xanthines, and lignans have been characterized and identified with 299 distinctive in vivo test effect on each compounds (Sommerville et al. 2017). Flavonoid

300 compounds in plant are useful as antioxidants and act as growth regulators, while

301 pharmacologically useful also as antioxidant which scavenge free radicals, preventing coronary

302 heart diseases, act as hepatoprotective components, anti-inflammatory, antimicrobial, and

303 anticancer (Kumar and Pandey 2013). Triterpenoid is a precursor compound of steroid (Davis

304 and Croteau 2000), composed of three terpene units or six isoprene units (Du et al. 2014).

305 Triterpenoid is useful as plant defense mechanism against herbivory and disease, and the

306 following biosynthesis into steroid, steroid is an important constituent of cell membrane and

307 precursor to growth regulators (Haralampidis et al. 2002). Triterpenoid glycoside compound

308 saponin has been described in PubMed as inhibitor of cancer formation and progression by

309 modulation of multiple signaling target related to cellular proliferation, apoptosis, autophagy,

310 metagenesis, angiogenesis, inflammation, oxidative stress, multidrug resistance, cancer stem

311 cells, and miRNA (Du et al. 2014). On Ra. hasseltii Suringar and T. leucostaphyllum (Dennst.)

312 Alston ex Mabb study by Sofiyanti et al. (2008) has detected two alkaloid compounds (nicotine

313 and caffeine) and three phenolic compounds (catechin, proanthocyanidin/leucoanthocyanidin,

314 and phenolic acid). These compounds could be similar in Rhizanthes, hence metabolite

315 identification study is encouraged in the future.

Formatted: Indent: First line: 0.5 cm, 316 However, due to the increased rarity of Rhizanthes, the metabolite bioprospecting should be Space Before: 12 pt

317 balanced with the advances of propagation technology for both the host (Tetrastigma) and the

318 parasite (Rhizanthes). Study of Saleh (2015; Saleh et al. 2015) has revealed that the host, T.

319 papillosum might also have the near similar compounds as R. deceptor, which lead to the

320 suggestion of “why not using the host, instead of using the parasite?” especially since

321 Tetrastigma can be propagated via cuttings (specimens are currently found in Bogor Botanical Comment [AW20]: Show the cuttings of Tetrastigma in BBG 322 Garden, Indonesia; Fig. 4b). The problem is, the other species, R. lowii, which already known for

323 its ethnomedicinal properties, will bring more challenge to inform the locals to use the

324 Tetrastigma instead of R. lowii. Hence, the importance of Rhizanthes propagation is becoming

325 equally significant to balance the exploitation.

326

Formatted: Font: (Default) Times New Roman, 12 pt Formatted: Space Before: 12 pt

327

328 Fig. 4. The commercially sold dried bud of R. lowii (also known locally as “ulur-ulur”) a, and

329 the clones of T. leucostaphyllum (Dennst.) Alston ex Mabb (the host of some species of

330 Rafflesia) propagated via stem cuttings b. Photo taken by: Adhityo Wicaksono (a), Sofi

331 Mursidawati (b). Scale bar in a = 5 cm.

332

333 Propagation technology Comment [AW21]: So far, not much has been done. Jaime, do you have any suggestion 334 Until the completion of this paper, there is very little to limited research information has been to add?

335 published about the propagation attempts of Rhizanthes. No report on seed germination (in vivo 336 and in vitro) and not even report on tissue culture attempts. Comparatively, Rafflesia has been

337 tested on host grafting (Mursidawati et al. 2015), seed germination (in vivo, both in situ and ex Formatted: Font: Italic

338 situ, and in vitro, Mursidawati et al. 2015; Wicaksono et al. 2016; Molina et al. 2016), and tissue

339 culture (Sukamto 2001; Wicaksono and Teixeira da Silva 2015; Mursidawati and Handini 2009;

340 Sukamto and Mujiono 2010).

341 The closest attempt on propagation is the artificial pollination procedure. As Rafflesia is known

342 to agamospermy (producing fruit without pollination) as seen in the observation at Rafflesia

343 tengku-adlinii and Ra. cantleyii (Nais 2001), Rafflesia artificial pollination to allow fertile seeds

344 production is encouraged (Bänziger 2004; Nais, personal communication in 2015). On the other

345 hand, it is still unknown that Rhizanthes could also being agamospermy. BanzigerBänziger

346 artificial pollination paper.mentioned in his paper (2004) that artificial pollination can be done

347 using aluminum strip to gather pollens from the concealed part of male flower anther and

348 transfer it into the stigmatic part of the female flower as he performed on Ra. Kerrii and Sapria

349 himalayana, and as this procedure was done, the fruiting chance of S. himalayana increased to

350 78% from 8-12% as occurring via natural pollination.

351 No report on seed germination. No report on tissue culture attempt.

352

353 Future prospects Comment [AW22]: All authors, any suggestions?

354 In the future, more research topics on Rhizanthes are encouraged. From histological study to see Formatted: Font: Italic Formatted: Font: Italic 355 how the endophyte interact with its host tissue, histochemical study to see the accumulation of Formatted: Font: Italic 356 metabolites in the specific region of tissues or cells, metabolite identifications and simulations

357 (i.e. using molecular docking), to the propagation study, which is important to keep the

358 population in the wild. The closest possible suggestion is to propagate Rhizanthes and its host 359 Tetrastigma by using grafting as in Rafflesia (Mursidawati et al. 2015) or in vivo, in situ or ex Formatted: Font: Not Italic

360 situ seed germination (Mursidawati et al. 2015; Wicaksono et al. 2016; Molina et al. 2016).

361 Following the progression of propagation studies, it is highly encouraged to transfer the

362 knowledge to the local people so every element in the society could contribute to the supervised

363 propagation attempts. Only by any chance of successful propagation, re-introduction can be

364 performed in later stage. Also in the future, local and governmental protection policy can be

365 build. Comment [AW23]: This part probably requires more details 366

367 Conclusion Formatted: Font: Italic

368 Rhizanthes is not much highlighted compared to its Rafflesiaceae cousin, Rafflesia, which some

369 of Rafflesia species has been placed in IUCN Red List and CITES Appendix as rare or

370 endangered species. Rhizanthes is still understudied, while its life cycle is similarly enigmatic to

371 other Rafflesiaceae, thus making the propagation procedure remains unknown while some locals

372 harvested the bud as ethnomedicine. Future study on propagation is encouraged, followed by re-

373 introduction and protection policy establishment to protect the flora. This conservation attempts

374 require contributions of multiple elements from local tribes, scientists, to the government to

375 prevent the overexploitation of Rhizanthes.

376

377 Acknowledgement

378 We are thankful to Arbi Wiguna (Universitas Negeri Padang, West Sumatra, Indonesia; and

379 Division of Botany, Genbinesia Foundation, Indonesia), Sofian Rafflesia and Neka Afnidarti

380 (Komunitas Peduli Puspa Langka, Bengkulu) for providing us the figures for this paper.

381 382 Author contributions

383 AW conceived the idea. AW consulted the early concept of the paper with SM, AS, and JATS.

384 RR provided insights about local articles for Rhizanthes for ethnobotanical perspectives from

385 information articles, journals, and books, both locally and international published data. GPC

386 provided overview on the metabolite and ethnomedicinal prospects. AW, GPC, RR, SM, and

387 JATS collaborated equally to finish the paper.

388

389 Conflict of interest

390 All authors declare no conflicts of interest

391

392 References Formatted: Font: Bold

393 Adamski Z, Blythe LL, Milella L, Bufo SA (2020) Biological activities of alkaloids: From Formatted: Indent: Left: 0 cm, Hanging: 0.5 cm 394 toxicology to pharmacology. Toxins 12: 210. https://doi.org/10.3390/toxins12040210

395 Bänziger H (1996) Pollination of a flowering oddity: Rhizanthes zippelii (Blume) Spach

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399 Bänziger H (2004) Studies on hitherto unknown fruits and seeds of some Rafflesiaceae, and a

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403 (Rafflesiaceae) from Borneo: First description of flowers, fruits, and seeds. Nat Hist Bull

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412 https://doi.org/10.1007/3-540-48146-X_2

413 Du J-R, Long F-Y, Chen C (2014) Chapter Six – Research progress on natural triterpenoid

414 saponins in the chemoprevention and chemotherapy of cancer. In: Bathaie SZ, Tamanoi F

415 (eds) The Enzymes, vol. 36, pp.95-130. https://doi.org/10.1016/b978-0-12-802215-3.00006-9

416 Haralampidis K, Trojanowska M, Osbourn AE (2002) Biosynthesis of triterpenoid saponins in

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418 Advances in Biochemical Engineering/Biotechnology, vol. 75, pp.31-49. Springer, Berlin,

419 Heidelberg. https://doi.org/10.1007/3-540-44604-4_2

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421 World J 2013: 162750. https://doi.org/10.1155/2013/162750

422 Meijer W, Veldkamp JF (1988) A revision of Rhizanthes (Rafflesiaceae). Blumea 33:329-344.

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424 Mursidawati S, Irawati, Ngatari (2014) Rafflesia patma (Rafflesiaceae): notes on its field study,

425 cultivation, seed germination, and anatomy. Buletin Kebun Raya 17:9-14.

426 Nais J (2001) Rafflesia of The World. Sabah Parks, Malaysia. Formatted: Font: Italic 427 Nikolov LA, Endress PK, Sugumaran M, Sasirat S, Vessabutr S, Kramer EM, Davis CC (2013)

428 Developmental origins of the world‟s largest flowers, Rafflesiaceae. Proc Nat Acad Sci USA

429 110:18578-18583. https://doi.org/10.1073/pnas.1310356110

430 Nikolov LA, Tomlinson PB, Manickam S, Endress PK, Kramer EM, Davis CC (2014)

431 Holoparasitic Rafflesiaceae possess the most reduced endophytes and yet give rise to the

432 world‟s largest flowers. Ann Bot 114:233-242. https://doi.org/10.1093/aob/mcu114

433 Patiño S, Grace J, Bänziger H (2000) Endothermy by flowers of Rhizanthes lowii (Rafflesiaceae).

434 Oecologia 124:149-155. https://doi.org/10.1007/s004420050001

435 Quattrocchi UFLS (2012) World Dictionary of Medicinal and Poisonous Plants: Common

436 Names, Scientific Names, Eponyms, Synonyms, and Etymology. CRC Press, Boca Raton,

437 Florida, US. pp.3197-3198.

438 Refaei J, Jones EBG, Sakayaroj J, Santhanam J (2011) Endophytic fungi from Rafflesia cantleyi:

439 species diversity and antimicrobial activity. Mycosphere 2:429-447

440 Saleh Z (2015) Bioekologi dan bioprospeksi Rhizanthes deceptor sebagai stimulus konservasinya

441 di HPPB Universitas Andalas Padang Sumatera Barat. Master Degree Thesis, Institut

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443 Saleh Z, Zuhud EAM, Sari RK (2015) Phytochemical screening and antioxidant activity of

444 ethanolic extract of Rhizanthes deceptor (Rafflesiaceae) and its host Tetrastigma papillosum.

445 Res J Med Plant 9:293-299. https://doi.org/10.3923/rjmp.2015.293.299 Formatted: Default Paragraph Font, Font: (Default) +Body (Calibri), 11 pt, English 446 Sofiyanti N, Wahibah NH, Purwanto D, Syahputra E, Mat-Salleh K (2008) Alkaloid and (Indonesia)

447 phenolic compounds of Rafflesia hasseltii Suringar and its host Tetrastigma leucostaphylum

448 (Dennst.) Alston ex Mabb. in Bukit Tigapuluh National Park, Riau: A preliminary study.

449 Biodivers 9: 17-20. https://doi.org/10.13057/biodiv/d090105 450 Sommerville V, Bringans C, Braakhuis A (2017) Polyphenols and performance: A systematic

451 review and meta-analysis. Sports Med 47: 1589-1599. https://doi.org/10.1007/s40279-017-

452 0675-5

453 Susatya A (2003) Populasi dan siklus hidup tumbuhan langka Rhizanthes loweii (Becc) Harm

454 (Rafflesiaceae) di Taman Nasional Kerinci-Seblat Resort Katenong. Jurnal Ilmu-Ilmu

455 Pertanian Indonesia 5:71-76 [in Indonesian, with English abstract]

456 Syaifuddin, Andriani S, Suryanto E, Nisa LS, Wardana AZ, Kurniawan NMA (2018) Kajian

457 pemanfaatan tumbuhan obat sebagai obat tradisional di Kabupaten Hulu Sungai Tengah

458 Propinsi Kalimantan Selatan. Prosiding Seminar Nasional Teknologi Hasil Hutan 2018,

459 pp.317-325 [in Indonesian].

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461 Sekitar Taman Nasional Kayan Mentarang. Center for International Forestry Research

462 (CIFOR), Bogor, Indonesia [in Indonesian].

463 Wani MC, Taylor HL, Wall ME, Coggon P, McPhail AT (1971) Plant antitumor agents. VI.

464 Isolation and structure of taxol, a novel antileukemic and antitumor agent from Taxus

465 brevifolia. J Am Chem Soc 93: 2325-2327. https://doi.org/10.1021/ja00738a045

466 Wicaksono A, Mursidawati S, Sukamto LA, Teixeira da Silva JA (2016) Rafflesia spp.:

467 propagation and conservation. Planta 244: 289-296. https://doi.org/10.1007/s00425-016-2512-

468 8 7/31/2021 Email Universitas Bengkulu - Rhizanthes Paper Update

Agus Susatya

Rhizanthes Paper Update 1 pesan

Adhityo Wicaksono 3 Juli 2020 17.53 Kepada: Jaime Silva Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Jaime,

We have finished the Rhizanthes review latest version. About the propagation and a bit regarding the ethnobotany, I discussed it with Bu Sofi. The ethnobotany section is mostly from Reza. The details on metabolite function is from Ghea, but the one about the extraction and analysis of Rhizanthes (specific) metabolites was written by me. The ecological part is from Pak Agus. And we have put Planta (Springer) format for the references.

Looking forward for your input!

Best wishes,

Adhit

Rhizanthes, the forgotten relative of Rafflesia Jaime edits 1 - Rev AW-GPC-RR-SM-AS.docx 1553K

https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1671192766678529840&simpl=msg-f%3A16711927666… 1/1 7/31/2021 Email Universitas Bengkulu - File received: Rhizanthes Paper Update

Agus Susatya

File received: Rhizanthes Paper Update 1 pesan

Jaime Silva 3 Juli 2020 18.46 Kepada: Adhityo Wicaksono Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Adhit and colleagues,

Thank you so much for the updated version of the review/perspective.

Please give me a few days to assess the text in detail, and attempt to improve it.

I'll be back soon, hopefully with a more perfected and fortified version.

Warm regards and a good weekend to all. Please stay safe (I notice that Covid-19 is increasing in Indonesia).

Jaime

https://www.researchgate.net/profile/Jaime_Teixeira_Da_Silva

On Friday, July 3, 2020, 7:53:41 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

Looking forward for your input!

Best wishes,

Adhit

https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1671196125793881798&simpl=msg-f%3A16711961257… 1/1 7/31/2021 Email Universitas Bengkulu - Version 3.1: Rhizanthes paper

Agus Susatya

Version 3.1: Rhizanthes paper 2 pesan

Jaime Silva 9 Juli 2020 14.43 Kepada: Adhityo Wicaksono Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Adhit and colleagues,

I decided to prioritize this paper and made good progress. You will see that I am suggesting major structural edits, and cutting quite a bit or irrelevant text and references which will make the content "sharper" and the mini-review shorter, thus making it more "attractive" to editors and maybe easier to publish.

Good luck with the next edits!

Regards,

Jaime

On Wednesday, July 8, 2020, 4:11:02 PM GMT+9, Jaime Silva wrote:

Dear Adhit,

I have received the files safely.

I will need a few days as I want to read some of the literature in detail.

I'll get back to you with the next version ASAP.

Regards,

Jaime

On Wednesday, July 8, 2020, 2:06:21 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

Good news! Pak Agus and I managed to do the revision faster than previous plan. Bu Sofi also instructed me to edit the part about the conservation policy at the end (future prospects) section. I also added the Main Conclusion part before the abstract since it's mandatory if we want to submit to Planta.

Along with the revised draft, I attached the figure permission. It's in Indonesian but it could serve as our archive. Also, here is the folder in Google Drive that I made for the references (except Nais - I can only put one picture from it, and also the book about metabolites).

Best regards, Adhit

Virus-free. www.avast.com https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1671724393445953839&simpl=msg-f%3A16717243934… 1/3 7/31/2021 Email Universitas Bengkulu - Version 3.1: Rhizanthes paper

On Sun, Jul 5, 2020 at 1:04 AM Jaime Silva wrote: Dear Adhit and colleagues,

I spent the past 2 days focusing on the Rhizanthes paper. I made some good advances, but still a lot of work is needed on the paper. It will still need a few revisions before it is ready for submission.

I suggest that all authors make improvements to the attached file, and once you have compiled all authors' input, Adhit, please send me the next version.

Good luck to all!

Regards,

Jaime

On Friday, July 3, 2020, 8:46:59 PM GMT+9, Jaime Silva wrote:

Dear Adhit and colleagues,

Thank you so much for the updated version of the review/perspective.

Please give me a few days to assess the text in detail, and attempt to improve it.

I'll be back soon, hopefully with a more perfected and fortified version.

Warm regards and a good weekend to all. Please stay safe (I notice that Covid-19 is increasing in Indonesia).

Jaime

https://www.researchgate.net/profile/Jaime_Teixeira_Da_Silva

On Friday, July 3, 2020, 7:53:41 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

Looking forward for your input!

Best wishes,

Adhit

Rhizanthes, the forgotten relative of Rafflesia version 3.1.docx https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1671724393445953839&simpl=msg-f%3A16717243934… 2/3 7/31/2021 Email Universitas Bengkulu - Version 3.1: Rhizanthes paper 1525K

Adhityo Wicaksono 9 Juli 2020 21.12 Kepada: Jaime Silva Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Jaime,

I have discussed with Pak Agus this afternoon and managed to revise the paper. I made some major updates:

1. Page 6, I attached Nikolov et al (2014b, new, I didn't send you this one last time) to compare with Nikolov et al. (2013), just want to make sure about the organ derivation. I want to recheck, but I am not really sure about it. 2. About Saleh et al (2015), I made a table in the end. Should it be okay or we still need permission? 3. About the metabolite details, I turned it into a table rather than a paragraph. I compare each metabolite and each medical property. I hope the table is better than the long paragraph. 4. I have the R. lowii (ulur-ulur) extract, should I boil and taste it? Maybe we can put the boiled water in our last figure and describe the taste? I'm just asking by the way.

What do you think?

Anyway, let's try to submit in the Botanical Review (Springer). I think that is also good.

Best regards,

Adhit [Kutipan teks disembunyikan]

2 lampiran Rhizanthes, the forgotten relative of Rafflesia version 3.1 (Rev).docx 1518K 225.full.pdf 10738K

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Rhizanthes paper preFinal 3 pesan

Jaime Silva 13 Juli 2020 00.51 Kepada: Adhityo Wicaksono Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Adhit and colleagues,

I believe that the review is close to submission-ready.

Attached please find the prefinal files with just a few issues left to resolve.

I look forward to revising the final edits before submission.

Regards,

Jaime

On Sunday, July 12, 2020, 1:36:55 AM GMT+9, Adhityo Wicaksono wrote:

Dear all,

Since we are close to finish, I suggest that we do a recheck on figure 1 and 2. Since the picture of Rhizanthes are not as much as Rafflesia, we need to make sure that all the species are the correct species (except for R. deceptor, which is easily distinguishable since the perigone is white). And if you have a picture of Rhizanthes from other species blooming, please share with me so I can add to the figure plate.

Thank you.

Adhit

On 9 Jul 2020, at 11.23 PM, Jaime Silva wrote:

Dear Adhit,

Good work. We're getting there...

Jaime

On Thursday, July 9, 2020, 11:12:50 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

I have discussed with Pak Agus this afternoon and managed to revise the paper. I made some major updates:

1. Page 6, I attached Nikolov et al (2014b, new, I didn't send you this one last time) to compare with Nikolov et al. (2013), just want to make sure about the organ derivation. I want to recheck, but I am https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1672034440969950350&simpl=msg-f%3A16720344409… 1/4 7/31/2021 Email Universitas Bengkulu - Rhizanthes paper preFinal not really sure about it. 2. About Saleh et al (2015), I made a table in the end. Should it be okay or we still need permission? 3. About the metabolite details, I turned it into a table rather than a paragraph. I compare each metabolite and each medical property. I hope the table is better than the long paragraph. 4. I have the R. lowii (ulur-ulur) extract, should I boil and taste it? Maybe we can put the boiled water in our last figure and describe the taste? I'm just asking by the way.

What do you think?

Anyway, let's try to submit in the Botanical Review (Springer). I think that is also good.

Best regards,

Adhit

On Thu, Jul 9, 2020 at 2:43 PM Jaime Silva wrote: Dear Adhit and colleagues,

Good luck with the next edits!

Regards,

Jaime

On Wednesday, July 8, 2020, 4:11:02 PM GMT+9, Jaime Silva wrote:

Dear Adhit,

I have received the files safely.

I will need a few days as I want to read some of the literature in detail.

I'll get back to you with the next version ASAP.

Regards,

Jaime

On Wednesday, July 8, 2020, 2:06:21 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

Best regards, Adhit https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1672034440969950350&simpl=msg-f%3A16720344409… 2/4 7/31/2021 Email Universitas Bengkulu - Rhizanthes paper preFinal

Virus-free. www.avast.com

On Sun, Jul 5, 2020 at 1:04 AM Jaime Silva wrote: Dear Adhit and colleagues,

I suggest that all authors make improvements to the attached file, and once you have compiled all authors' input, Adhit, please send me the next version.

Good luck to all!

Regards,

Jaime

On Friday, July 3, 2020, 8:46:59 PM GMT+9, Jaime Silva wrote:

Dear Adhit and colleagues,

Thank you so much for the updated version of the review/perspective.

Please give me a few days to assess the text in detail, and attempt to improve it.

I'll be back soon, hopefully with a more perfected and fortified version.

Warm regards and a good weekend to all. Please stay safe (I notice that Covid-19 is increasing in Indonesia).

Jaime

https://www.researchgate.net/profile/Jaime_Teixeira_Da_Silva

On Friday, July 3, 2020, 7:53:41 PM GMT+9, Adhityo Wicaksono wrote:

Dear Jaime,

Looking forward for your input!

Best wishes,

https://mail.google.com/mail/u/1?ik=c367ee2064&view=pt&search=all&permthid=thread-f%3A1672034440969950350&simpl=msg-f%3A16720344409… 3/4 7/31/2021 Email Universitas Bengkulu - Rhizanthes paper preFinal Adhit

2 lampiran Rhizanthes, the forgotten relative of Rafflesia preFinal.docx 1519K Supplementary Tables Rhizanthes preFinal.docx 25K

Adhityo Wicaksono 13 Juli 2020 00.56 Kepada: Jaime Silva Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Jaime,

Before I am going to the final edits, I want to inform you that Pak Zahratus Saleh (corresponding author of Saleh et al. 2015) has agreed for us to put his data on our paper. I have our conversation (in Indonesian) saved. Bu Sofi has contacted Bu Rita (other author) and Pak Zuhud (Pak Zahratus’ MSc supervisor). Bu Rita said that we should ask Pak Zuhud. We did, no reply yet. How about the one we should ask to the journal?

Adhit [Kutipan teks disembunyikan]

[Kutipan teks disembunyikan]

Adhityo Wicaksono 13 Juli 2020 17.59 Kepada: Jaime Silva Cc: Ghea Putri Christy , Reza Raihan , Sofi Mursidawati , "Dr. Agus Susatya"

Dear Jaime,

We have added the revision, and some revisions in the figure 1 and 2. Pak Agus and I had a thorough recheck yesterday and based on the tuft hair and ramenta visualization plus ecological distribution, we believe that the photo of the brown Rhizanthes are Rh. infanticida.

We also changed the Rafflesia and Rhizanthes genus abbreviation into Ra and Rh from Ra and R.

Looking forward to your update!

Adhit [Kutipan teks disembunyikan]

2 lampiran Supplementary Tables Rhizanthes preFinal (Rev).docx 22K Rhizanthes, the forgotten relative of Rafflesia preFinal (Rev).docx 1463K

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Agus Susatya

The Botanical Review - Submission Confirmation Rhizanthes, the forgotten relative of Rafflesia in the Rafflesiaceae for co-author 1 pesan

The Botanical Review - Editorial Office 14 Juli 2020 08.10 Balas Ke: The Botanical Review - Editorial Office Kepada: Agus Susatya

Re: "Rhizanthes, the forgotten relative of Rafflesia in the Rafflesiaceae" Full author list: Adhityo Wicaksono, M.Sc; Ghea Putri Cristy, S.Si; Reza Raihandhany, ST; Sofi Mursidawati, M.Sc; Jaime A. Teixeira da Silva, PhD; Agus Susatya, PhD

Dear Dr. Agus Susatya,

We have just received the submission entitled: "Rhizanthes, the forgotten relative of Rafflesia in the Rafflesiaceae" for possible publication in The Botanical Review, and you are listed as one of the co-authors.

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Agus Susatya

Fwd: Your Submission BOTR-D-20-00009R1 - [EMID:4682cf8f429b89ed] 3 pesan

Adhityo Wicaksono 10 April 2021 06.52 Kepada: Reza Raihan , Ghea Putri Christy , Sofi Mursidawati , "Jaime A. Teixeira da Silva" , "Dr. Agus Susatya"

Dear colleagues,

I am happy to share this news with you. Congratulations to us all, and thank you for the great collaboration!

Let us wait for the e-proofing stage.

Adhit

------Forwarded message ------From: The Botanical Review - Editorial Office Date: Sat, Apr 10, 2021 at 3:30 AM Subject: Your Submission BOTR-D-20-00009R1 - [EMID:4682cf8f429b89ed] To: Adhityo Wicaksono

Dear Mr. Wicaksono,

We are pleased to inform you that your manuscript, "Rhizanthes, the forgotten relative of Rafflesia in the Rafflesiaceae", has been accepted for publication in The Botanical Review.

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Jaime Silva 10 April 2021 06.56 Kepada: Reza Raihan , Ghea Putri Christy , Sofi Mursidawati , "Dr. Agus Susatya" , Adhityo Wicaksono

Adhit and colleagues,

Fantastic news!

Well done.

Jaime

[Kutipan teks disembunyikan]

Sofi Mursidawati 13 April 2021 09.37 Kepada: Adhityo Wicaksono Cc: Reza Raihan , Ghea Putri Christy , "Jaime A. Teixeira da Silva" , "Dr. Agus Susatya"

Great news... Thanks Adhit.. [Kutipan teks disembunyikan]

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Agus Susatya

Fwd: Sharing Information for "Rhizanthes, the Forgotten Relative of Rafflesia in the Rafflesiaceae" 1 pesan

Adhityo Wicaksono 19 Mei 2021 11.59 Kepada: Sofi Mursidawati , Ghea Putri Christy , Jaime Silva , Reza Raihan , "Dr. Agus Susatya"

Dear everyone,

Finally, after almost 1 year of submission!

Congratulations to us all. Now, I think, we will wait to get the PDF. Because as usual, I chose “Subscription” mode instead of paying for open access. If we know someone who have access to Springer, I think we can get it sooner.

Best wishes,

Adhit

Begin forwarded message:

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REVIEW

Rhizanthes, the Forgotten Relative of Rafflesia in the Rafflesiaceae

Adhityo Wicaksono1,6 & Ghea Putri Cristy1 & Reza Raihandhany1,2 & Sofi Mursidawati3,6 & Jaime A. Teixeira da Silva4 & Agus Susatya5,6

1 Generasi Biologi Indonesia Foundation, Jl. Swadaya Barat no. 4, Gresik 61171, Indonesia 2 School of Life Sciences and Technology (SITH), Institut Teknologi Bandung, Jl. Ganeca no. 10-12, Bandung 40132, Indonesia 3 Research Center for Plant Conservation and Botanic Gardens – Indonesian Institute of Science (LIPI), Jl. Ir. H. Juanda no.13, Bogor 16003, Indonesia 4 Independent researcher, P.O. Box 7, Ikenobe 3011-2, Kagawa-ken 761-0799, Japan 5 Department of Forestry, Faculty of Agriculture, Universitas Bengkulu, Jl. Raya Kandang Limun, Bengkulu 38371, Indonesia 6 Author for Correspondence; e-mail: [email protected]; [email protected]; [email protected]

# The New York Botanical Garden 2021

Abstract Rhizanthes is a holoparasitic plant of the Rafflesiaceae, and, just like Rafflesia, its host is Tetrastigma (Vitaceae). Unlike Rafflesia, very little research has been conducted on Rhizanthes other than a few studies focusing on its taxonomy and anatomy, and limited studies on its propagation, despite some ethnomedicinal uses in several regions of Indonesia. Wild populations of Rhizanthes are declining due to deforestation and overharvesting by locals. Artificial pollination and possible seed spread, which are similar to Rafflesia, may be useful for future propagation-based studies, which are generally very difficult and challenging for members of the Rafflesiaceae. This paper emphasizes the cultural and ethnomedicinal importance of Rhizanthes and seeks to define a conservation road-map that incorporates a scientifically-based approach to research while also seeking a four-pronged approach to the conservation of Rhizanthes: 1) conventional and biotechnology-based conservation; 2) germplasm multiplication and preservation; 3) reintroduction into the wild and conservation of wild populations; 4) policy-based protective measures.

Keywords Biotechnology . Conservation . Ethnobotany . Holoparasite . In vitro propagation . Parasitic plant

Introduction

Rhizanthes belongs to the Rafflesiaceae, alongside Rafflesia and Sapria (Meijer, 1997). The plant also holoparasitizes some species of Tetrastigma (Bänziger & Hansen, 2000). Unlike Rafflesia, Rhizanthes has not been studied much. A Google Scholar search on A. Wicaksono et al.

June 11, 2020 revealed 4920 results for “Rafflesia”, 259 for “Rhizanthes”,and804for “Sapria”. Not much information exists on its propagation, unlike in Rafflesia and Sapria where artificial pollination resulted in fruit production (Bänziger, 2004). Initial- ly, Rh. lowii and Rh. zippelii were respectively called Brugmansia lowi (sic) Beccari in 1868 and Brugmansia zippelii Blume in 1841 (Bänziger & Hansen, 2000)aslater Brugmansia is a homonym for a species name in the Solanaceae. The four species of Rhizanthes in the world are spread across the Malesian region, from Peninsular Malaysia, to Sumatra, Borneo (Kalimantan), and Java (Meijer, 1997). The species are Rhizanthes deceptor Bänziger & B. Hansen, Rh. infanticida Bänziger & B. Hansen, Rh. lowii (Beccari) Harms, and Rh. zippelii Blume (Spach) (Bänziger & Hansen, 2000). Some species can be observed in Fig. 1. Rh. zippelii is distributed in Sumatra, Peninsular Malaysia, Java (specifically on West Java; Bänziger & Hansen, 2000), and Borneo (Meijer & Veldkamp, 1988), Rh. lowii in Sumatra, Peninsular Malaysia, and Borneo (Meijer & Veldkamp, 1988; Bänziger & Hansen, 2000), Rh. infanticida in Thailand, Peninsular Malaysia, and Sumatra (Bänziger & Hansen, 2000), and Rh. deceptor in the west of Sumatra (Bänziger & Hansen, 2000)(Fig.2). Research on Rhizanthes can be traced back to the early nineteenth century, when the German botanist, Alexander Zippelius, found a specimen at Gunung Salak, Bogor, Indonesia which was later described by Karl Ludwig von Blume as a new genus within the Rafflesiaceae (Meijer, 1997). Meijer and Veldkamp (1988) revised the genus Rhizanthes and Meijer (1997) then established two species Rhizanthes zippelii (Blume) Spach and Rhizanthes lowii (Beccari) Harms in a published revision of the entire Rafflesiaceae family. Bänziger and Hansen (2000), after reviewing the Meijer and Veldkamp article, focused on variation of ramenta structures, but also considered color, size, and sexuality of the flower, and observed more available herbarium specimens. They established two new additional species, Rh. infanticida and Rh.

Fig. 1 Rhizanthes upon anthesis. Two Rhizanthes infanticida flower buds emerged and grew in the same spot a; enlarged view of Rh. infanticida flower bud b; fully bloomed Rh. infanticida (sex unidentified) c;theclose- up view of the distal region of the perigone with tuft hairs region (TH), ramenta region (Ra), and proximal part of caudal appendage (Ca) where nectar pad can be found as seen with the ant harvesting nectar as in Bänziger (1996) d; and the post-anthetic flower of Rh. infanticida with flower bud underneath (yellow arrow) e.Fully bloomed Rh. deceptor (sex unidentified) f and a zoomed view of a male flower pseudo-brown colored (color is actually from the hairs) central area with anther region exposed (blue arrow) g; Fully bloomed Rh. lowii (sex unidentified) h. Scale bars = 5 cm (a-c, e-h), 1 cm (d). Photos taken by Arbi Wiguna (a-e), Sofian Rafflesia (f), Neka Afnidarti (g), and Inama Ahmad (h) (used with kind permission via personal communication) Rhizanthes, the Forgotten Relative of Rafflesia in the...

Fig. 2 Map of Rhizanthes distribution: red – Rh. zippelii, green – Rh. infanticida, Rh. lowii,andRh. zippelii, purple – Rh. infanticida, blue – Rh. lowii and Rh. zippelii, magenta – all four species. This map was modified from Plants of The World Online website (http://www.plantsoftheworldonline.org/). Data information is licensed under Creative Commons Attribution CC BY v4.0 and © copyright The Trustees of the Royal Botanic Gardens, Kew (retrieved March 31, 2021) deceptor. Existing studies on Rhizanthes have mainly focused on morphology and taxonomy, and rarely on ecological aspects, even though ecological information is important for future conservation of the genus. Ecological studies of Rhizanthes are limited to Rh. zippelii (Bänziger, 1995)andRh. lowii (Susatya, 2003). Similar to Rafflesia species, Rhizanthes species are holoparasitic and do not have any leaves, or trunk. The only visible structures are either flower buds or opening flowers which are diagnostic features to each species. Studies on population ecology generally observe the mortality, new recruitment, and growth of flower buds. Bänziger (1995) observed the population and life cycle of Rh. zippelii, estimating that the plant could take as long as 200–255 days to emerge from its host plant, shorter than the life cycle of Rafflesia patma Blume (Hidayati et al., 2000)andRa. arnoldii R. Br. (Susatya, 2020). It was estimated that Ra. patma and Ra. arnoldii required 256–512 days (Hidayati et al., 2000) and 339–497 days (Susatya, 2020), respectively to complete their life cycles. However, Bänziger (1995) did not provide information of the mortality of individual flower buds of Rh. zippelii. Susatya (2003), who observed the mortality and life history of Rh. lowii, found that the population size of this species consisted of 32 individual flower buds, higher than that of Ra. arnoldii, which only forms 12.5 flower buds (Susatya, 2020). Within 4 months, Rh. lowii lost 25% of its flower buds A. Wicaksono et al. with bud mortality occurring within a very small diameter (0.50–0.94 cm), and the local Rh. lowii population would likely go extinct within 30 months (Susatya, 2003). In some regions of Indonesia, Rhizanthes is used for its ethnomedicinal properties, especially by locals in Borneo island (Uluk et al., 2001; Syaifuddin et al., 2018), which will likely result in uncontrolled harvesting of flowers in the field, rapidly declining wild populations. Unfortunately, none of the four Rhizanthes species (Rh. deceptor, Rh. infanticida, Rh. lowii,andRh. zippelii;Bänziger&Hansen,2000) are listed in the IUCN Red List or in the CITES Appendix. Moreover, there are no techniques available for the propagation of Rhizanthes, although some propagation techniques involving biotechnology and metabolite testing had been conducted on Rafflesia since 2001 (Wicaksono et al., 2016). The objective of this paper is to develop greater awareness of Rhizanthes biology to establish conservation measures.

Biology

Rhizanthes is derived from the Greek words rhiza (root) and –anthes (flowering) because it was believed to bloom from roots (Bänziger & Hansen, 2000). According to Bänziger and Hansen (2000), Rhizanthes is also known as the “deceptive flower” because of its tendency to deceive visiting organisms. For example, by emulating the scent of a rotting corpse, it leads pollinating insects to lay many eggs with doom-fated hatchlings. In addition, the flower center is a suspected location where bees, wasps, ants, and butterflies can steal scarce to non-existing nectar. The flower is also a vantage ground for male flies to find a female. The morphological terms of Rhizanthes (Fig. 3) are similar to those of Rafflesia and Sapria since the plant belongs to the Rafflesiaceae, but some scientists disagree about the assigned terms. The petal-like organ of Rhizanthes is referred to as the tepal (Bänziger & Hansen, 2000), but is sometimes also called the perianth (Nikolov et al. 2013), which is synonymous to the perigone (Meijer, 1997). Comparatively, in Raffle- sia, the petal-like organ is referred to, and is mostly known as, the perigone (Wicaksono et al., 2016). The unique part of Rhizanthes is its caudal appendage in the distal region of the perigone, which folds together in the hole of the central column prior to anthesis and extends upon anthesis (Bänziger et al., 2007; Saleh, 2015). According to Bänziger (1996), some insects (i.e., bees and ants) often visit the distal region of the perigone where the ramenta exists and located near the caudal appendage, as it possesses a region with nectar pads as observed in Rh. zippelii (and from observations in Rh. infanticida; Fig. 1d). The outer organ that protects the young flower bud is the bract (Meijer, 1997; Bänziger & Hansen, 2000). The flower has a column in the center, a gonadal region in the lateral part surrounding the column, and a hole or ‘crater’ in the distal region (Bänziger & Hansen, 2000). Rhizanthes possesses three types of “hairs” or modified epidermal features (Fig. 4), which serve as important identification features between the four species (Bänziger & Hansen, 2000): bristles, brush-like tuft hairs, smaller and densely packed furry hairs, and tiny ramentae. Rh. deceptor and Rh. infanticida are dioecious plants with separate male and female flowers while Rh. zippelii (Bänziger and Hansen 2000)andRh. lowii (Bänziger et al., 2007) have separate male and female flowers, as well as bisexual flowers. Rh. lowii flowers have another unique feature, endothermy and the ability to self- regulate flower temperature (Patiño et al., 2000). Unlike the other Rhizanthes species Rhizanthes, the Forgotten Relative of Rafflesia in the...

Halved late bud

Tepal/Perianth/Perigone Late bud Caudal (abaxial surface) appendage Early bud Young bract Bract Matured bract

Tetrastigma (host) root Scale bars = 5 cm

R. infanticida (full anthesis) - Scale bar = 5 cm Crater

Proto bud Early bud Late bud Tepal/Perianth/Perigone Scale bars = 5 cm Anther (without epidermal features) Crater Crater

Ampulla Ampulla Column Caudal Column Globular head Globular head appendage Stigmatic fascia Stigmatic fascia

Reduced Reduced anther Anther ovary Style Style Bract Female flower Bisexual flower Male flower column column (In R. zippelii and in R. lowii) Fig. 3 The anatomy of Rhizanthes (modelled using Rh. lowii and Rh. infanticida buds), showing the proto bud (prior to emergence from the host periderm), early (using Rh. lowii sample), and late bud (with halved bud in the inset, showing the inward-folded caudal appendage; using Rh. lowii bud sample), full organ male flower (perigone lobe surface without epidermal features) and inset of female flower column. Additionally, inset of a bisexual flower column is also shown (but only available in Rh. zippelii and Rh. lowii). Anatomic figures of the flower were inspired from Bänziger and Hansen (2000), and Bänziger et al. (2007). Note: The Rh. infanticida flower in fully anthesis and the Rh. infanticida late bud are the same flowers from Fig. 1c and b, respectively. Photos were taken by Arbi Wiguna (used with kind permission via personal communication). Photos of proto bud, early bud, and halved late bud were taken by Sofi Mursidawati whose perigones are brown to yellowish, the Rh. deceptor perigone is whitish (Bänziger & Hansen, 2000). The deceptive nature of Rh. deceptor is due to its apparent brown coloration of the proximal area of the perigone which lies opposite to the central column and is actually caused by the overshadowing effect of the increasingly dense bristles and the convergence of the radial lines, while the perigone wall is in fact white (see Fig. 1f, g; Bänziger & Hansen, 2000). According to Cammerloher (1920), Rh. zippelii (at that time, still known as Brugmansia zippelii) possesses stomata with two guard cells on its epidermis but this feature requires further investigation. This because Cammerloher also referred to the existence of stomata with 3–5 guard cells in Ra. rochussenii Teijsm. & Binnend. Until recently, this epidermal feature was thought to be universal, but a study by Mursidawati et al. (2020) in Ra. patma revealed the absence of stomata. Cytologically, as recorded by Meijer and Veldkamp (1988), who cite older references (van der Pijl 1933, cit. Harms 1935), the pollen of Rh. zippelii has 11 haploid chromosomes (i.e., 2n = 22). Prior to anthesis, Rhizanthes grows as unicellular strands inside the host (Nikolov et al., 2014a). It grows near the phloem and xylem vessels of its host plant as can be seen in transversal sections. However, longitudinal sections are required for future studies to obtain a clearer picture of the endophyte and its possible behavior at the vegetative or A. Wicaksono et al.

0.5 mm 20 mm

Bristles

M F B Rh. lowii Rh. deceptor Rh. zippelii 0.5 mm 20 mm

Furry hairs

Rh. zippelii Rh. infanticida M F 0.5 mm Rh. lowii 20 mm Ramenta

Rh. lowii Rh. infanticidaRh. deceptor M F 0.5 mm Rh. infanticida 20 mm Tuft hairs (no details on male ﬂower from Banziger and Hansen, 2000) Rh. zippelii Rh. lowii F Rh. infanticidaRh. deceptor Rh. deceptor

Perigonal epidermal hair tips Columnal epidermal hairs Furry hairs Tuft hairs Bristles

Ramenta Tuft hairs 20 mm 20 mm Rh. zippelii Rh. lowii Furry hairs Bristles

Tuft hairs 20 mm Ramenta Tuft hairs 20 mm Ramenta

Rh. infanticida Rh. deceptor Perigonal epidermal hair conﬁgurations Fig. 4 A comparison of epidermal hair features in four species of Rhizanthes. Picture was redrawn and inspired from Bänziger and Hansen (2000) Rhizanthes, the Forgotten Relative of Rafflesia in the... generative stages of development, as were performed in Rafflesia (Mursidawati et al. 2020). As the Rhizanthes flower bud develops, the perigone lobes show specific gene expression in the sepal, petal, and specific ring derivatives unlike only the sepal in Rafflesia and both the sepal and petal in Sapria (Nikolov et al., 2013). The Rhizanthes perigone cup or ring (proximal area of the perigone) is derived from a specific ring derivative, Rafflesia has no perigone ring but forms a perigone tube or chamber derived from petal whorls and sepal while the ring derivative forms the inner annuli (as described in Nais, 2001), and Sapria also has a perigone ring derived from a specific ring derivative (possibly the inner ring (annulus) found in the base (proximal) of the central chamber/ perigone tube), similar to Rhizanthes (Nikolov et al., 2013; Nikolov et al., 2014b). After anthesis and pollination, Rhizanthes produces a fruit with tiny microseeds. Rh. zippelii seeds are circa. 0.75 × 0.3 mm (Meijer 1997)whileRh. lowii seeds are 0.70– 0.78 mm long and 0.33–0.40 mm wide (Bänziger et al., 2007). As observed in Rh. lowii, the fruit has labyrinth-like lines with thousands of brownish seeds inside (Bänziger et al., 2007). Rhizanthes seeds have similar morphology to Rafflesia seeds (Bouman & Meijer 1994;Nais,2001; Mursidawati et al., 2014), namely bilobed with irregular sized bee-hive-like holes or pores over the tegmic surface and covered with a testa, as observed in Rh. lowii (Bänziger et al., 2007;Fig.5)andRh. zippelii (Bouman &Meijer1994).

Key Species for Rhizanthes Biology: Hosts, Pollinators, and Seed Dispersal Agents

Rhizanthes grows in several Tetrastigma species that can serve as a host. Rh. zippelii grows in T. papillosum Planch. (Meijer, 1997), T. pedunculare (Wallich ex Lawson) Planch. (Bänziger & Hansen, 2000), and T. tuberculatum Latiff (syn. T. coriaceum (DC.) Gagnep.) (Meijer 1997). Rh. lowii grows in T. papillosum (Meijer, 1997)andT. pedunculare (Patiño et al. 2000). Rh. infanticida grows exclusively in T. papillosum (Bänziger, 1995; Bänziger &Hansen,2000). Rh. deceptor grows in T. pedunculare (Bänziger and Hansen 2000)and T. papillosum (Bänziger & Hansen, 2000; Saleh et al., 2015). Rhizanthes emits a rotting flesh-like odor, like Rafflesia, so pollination relies on carrion insects (flies), making it sapromyophilous. Rh. lowii is pollinated by carrion flies (Calliphoridae), specifically Hemipyrellia ligurriens (Wiedemann) (Bänziger et al., 2007). Rh. infanticida acquired its name “infanticide” due to its extremely effective deceptive nature, appearing as a mammalian carcass to its pollinators (carrion flies, 10 species of Calliphoridae) which lay hundreds of eggs but then die by starvation

Fig. 5 A SEM image of the seed of Rhizanthes lowii, revealing the bilobed structure a, and the enlarged surface with the pores tegment still covered with testa layer. Scale bars = 200 μmina and 100 μminb.Taken by Sofi Mursidawati, unpublished data Table 1 Metabolites produced by two of the isolates found in Rafflesia cantleyi in the study by Refaei et al. (2011) and the organisms inhibited by the metabolites

Fungal genus Metabolites produced Inhibited microorganisms References

Colletotrichum 6-isoprenylindole-3-carboxylic acid Bacillus substillis, Staphylococcus aureus, Sarcina lutea, Pseudomonas sp. Lu et al. (2000) 3β,5α-dihydroxy-6β-acetoxy-ergosta-7, 22-diene Bacillus substillis, Staphylococcus aureus, Sarcina lutea, Pseudomonas sp. 3β,5α-dihydroxy-6β-phenylacetoxy-ergosta-7, 22-diene Bacillus substillis, Staphylococcus aureus, Sarcina lutea, Pseudomonas sp. 3β,5α-dihydroxy-6β-acetoxy-ergosta-7, 22-diene +3β, Candida albicans, Aspergillus niger 5α-dihydroxy-6β-phenylacetoxy-ergosta-7, 22-diene +3β-hydroxy-ergosta-5-ene+3β-hydroxy-5α, 8α-epidioxy-ergosta-6, 22-diene Colletotric acid Bacillus substillis, Staphylococcus aureus, Sarcina lutea Zou et al. (2000) Colutellin A Helminthosporium sativum, Botyris cinerea, Sclerotina sclerotium Ren et al. (2008) Cytospora Cytoskyrin A Gram-positive bacteria, Escherichia coli Singh et al. (2007) Cytosporon D Gram-positive bacteria, yeast Cytosporon E Gram-positive bacteria, yeast 3,5-dimethyl-8-hydroxy-7-methoxy-3,4-dihydroisocoumarin Gram-positive bacteria, fungi Kokubun et al. (2003) 3,5-dimethyl-8-hydroxy-3,4-dihydroisocoumarin Gram-positive bacteria, fungi .Wckooe al. et Wicaksono A. Rhizanthes, the Forgotten Relative of Rafflesia in the...

Fig. 6 Commercially sold dried bud of Rhizanthes lowii (also known locally as “ulur-ulur”) a, and the clones of Tetrastigma leucostaphyllum (Dennst.) Alston ex Mabb (the host of some species of Rafflesia) propagated via stem cuttings (specimens are currently found in Bogor Botanical Garden, Indonesia) b. Photos taken by Adhityo Wicaksono. Scale bar in a = 5 cm after hatching (Bänziger & Hansen, 2000). The insects that pollinate Rh. deceptor include the carrion flies Chrysomya defixa (Walker), Lucilia porphyrina (Walker), and Hypopygiopsis fumipennis (Walker) (Bänziger & Hansen, 2000). Rh. zippelii pollination, as observed by Bänziger (1996), often involves carrion flies, mostly Lucilia porphyrina (Walker), Chrysomya pinguis (Walker), Ch. chani (Kurahashi), Ch. villeneuvei (Patton), Ch. megacephala (Fabricius), and Hemipyrellia ligurriens (Weidemann), while others (Hypopygiopsis infumata (Bigot), Hy. fumipennis (Walker), Hy. tumrasvini (Kurahashi), and Lu. papuensis (Macquart) are considered exceptional and potential pollinators. After fruit has formed, seed are assumed to be dispersed by small mammals like rats, similar to Rafflesia and Sapria (Bänziger, 2004). Rh. lowii fruit was bitten by a giant rat (Leopoldamys or Sundamys) (Bänziger et al., 2007). It is unclear if Rhizanthes seed dispersal is exozoochorous (i.e. if it sticks on fur, animal snouts, etc.) or endozoochorous (via the digestive tract) (Bouman & Meijer, 1994). The discovery of squirrel (Callosciurus notatus (Bodaert)) and tree shrew (Tupaia tana (Raffles)) eating Ra. keithii fruit in Borneo may indicate endozoochory as a possibility. It is unknown if any Rhizanthes species harbor endophytic fungal species. Colletotrichum, Cytospora,andGliocladiopsis were isolated from bud parts of Ra. cantleyi Solms-Laubach then cultured in potato dextrose agar medium (Refaei et al., 2011). These fungi produced bioactive metabolites (Table 1) which might contribute to plant defense and ethnomedicinal properties in addition to natural secondary metabolite biosynthesis.

Ethnobotany, Ethnomedicinal Properties, and Secondary Metabolites

Rh. lowii is used as a folk medicine in Banjarmasin, a South Borneo (South Kaliman- tan) province of Indonesia, where the fruit is known as “ulur-ulur”,althoughitis actually the flower bud prior to anthesis that is removed from the host stem, left to dry, then boiled (Fig. 6a). The boiled water of flower buds is traditionally used to cure Table 2 Rhizanthes deceptor and Tetrastigma papillosum extract test

Ethanolic extract samples Physical description Presence (+) or absence (−)TPCvaluesBSLT LC50 Antioxidant of extract (mg GAE/g) (μg/mL) IC50 (μg/mL) Alkaloids Flavonoids Phenolics Terpenoids Saponins Steroids

Rh. deceptor flower buds Blackish brown, ++ ++ ––433 283 32 viscous solids, with flavor T. papillosum roots Dark red-black, solid + + + + + – 324 720 22 T. papillosum stems Blackish brown, solid + + + – + + 271 278 35

Data was based on, and compiled and modified from, Saleh et al. (2015). BSLT = brine shrimp lethality test; GAE = gallic acid equivalent; IC50 = concentration that inhibits to 50% of biological processes; LC50 = lethal concentration causing 50% mortality; TPC = total phenolic content .Wckooe al. et Wicaksono A. Rhizanthes, the Forgotten Relative of Rafflesia in the...

Table 3 Plant secondary metabolites and their medical properties

Metabolite Property: prsence (+) or absence (−) References groups Anti- microbial Antiviral Anti - Anti-inflammatory Antioxidant cancer

Alkaloids + + + + – Adamski et al. (2020) Phenolics + – + + + Kumar and Pandey (2013) Triterpenoids ––+ ––Du et al. (2014)

Note: The information for this table was taken from the journal articles and represents some identified and tested metabolites hemorrhages, coronary disease, cholesterol, and used as a female reproductive organ supplement. Rh. lowii “ulur-ulur” is used to cure hemorrhages, back pain, stomachache (acid reflux), and hematuria by people from Murung B Village, South Kalimantan, Indonesia by boiling dried flower buds in about 500 mL of water, reducing it by boiling the water to about 250 mL, and consuming periodically (Syaifuddin et al., 2018). On Sumatra Island, Rh. deceptor flower buds are used to treat diarrhea and stomachache by charring them on an open flame, grinding them to a powder, mixed into water, then consumed (Quattrocchi, 2012). An ethnobotanical study by Uluk et al. (2001)noted that Rhizanthes sp., known locally by people of the Dayak tribe near Kayan Mentarang National Park as “aka kepun”, is used locally as rope, but could be mistaken for its liana host, Tetrastigma sp. Rh. deceptor flower buds and host T. papillosum roots and stems taken from a site near Universitas Andalas, West Sumatra, Indonesia were phytochemically analyzed (Saleh et al., 2015;Table2). Table 2 shows the existence of alkaloids, phenolics (including flavonoids), and terpenoids from Rh. deceptor flower buds (but no triterpenoid glycosides, saponin, or steroids) and T. papillosum roots (has saponin but no steroids), while T. papillosum stems have alkaloids, phenolics (including flavonoids), saponin (although the terpenoid test was negative), and steroids. Antiox- idant activities in R. deceptor bud extracts rank second after T. papillosum root extracts, and along with T. papillosum stem extract (ranked third) all are categorized as having very high activities (IC50 < 50). The prospective use of ethanolic extracts as a biopesticide, shown by the brine shrimp lethality test, was an LC50 value of 283 μg/mL for Rh. deceptor flower buds, 720 μg/mL for T. papillosum roots, and 278 μg/mL for T. papillosum stems. Despite these findings, the exact properties of alkaloids, phenolics, flavonoids, and triterpenoids in the Rh. deceptor extract have not yet been described. Some plant metabolite groups have benefits (Table 3). Comparatively, in Rafflesia, Sofiyanti et al. (2008) detected two alkaloids (nicotine and caffeine) and three phenolic compounds (catechin, proanthocyanidin/ leucoanthocyanidin, and phenolic acid) in Ra. hasseltii and T. leucostaphyllum.Apart from Saleh et al. (2015), no studies on secondary metabolites exist for Rhizanthes,but they are needed to better appreciate the ethnomedicinal properties. Propagation of the host (Tetrastigma) and of the parasite (Rhizanthes) are needed for effective parasite growth, and detailed phytochemical analyses of both host and parasite are needed to A. Wicaksono et al. reduce exploitation. This is necessary because, if the secondary metabolites in Rhizanthes and T. papillosum are similar, as in Ra. hasseltii and T. leucostaphyllum (Sofiyanti et al., 2008), then focus can be placed on the conservation of Rhizanthes, since Tetrastigma canbeeasilypropagated(Fig.6b).

Propagation Technology

There is very little research on the propagation (in vivo and in vitro seed germination, and tissue culture) of Rhizanthes, so cues could be taken from more extensive studies on Rafflesia, including host grafting (Mursidawati et al., 2015), seed germination (in vivo – both in situ and ex situ – and in vitro; Mursidawati et al., 2015;Molina et al., 2017; Wicaksono et al., 2016), and tissue culture (Sukamto, 2001; Mursidawati &Handini,2009; Sukamto & Mujiono, 2010; Wicaksono & Teixeira da Silva, 2015). Rafflesia is agamospermous, i.e., produces fruit without pollination, as seen in Ra. tengku-adlinii and Ra. cantleyii (Nais, 2001), but it is unclear if this is the case also for Rhizanthes. Artificial pollination in Rafflesia would allow fertile seeds to be produced (Bänziger, 2004). Bänziger (2004) conducted artificial pollination on S. himalayana by collecting pollen from the anthers, transferring it to the female flowers using aluminum strips. As the result, the fruiting of S. himalayana increased from 8 to 12% in natural pollination to 78%.

Conclusion and Future Prospects

Some Rafflesia species are in the IUCN Red List and CITES Appendix as rare or endangered species. Unlike Rafflesia, Rhizanthes is still understudied, and its life cycle is as enigmatic as other Rafflesiaceae. Detailed phytochemical studies are needed to validate the current ethnomedicinal uses. Procedures for propagation, conventional or tissue culture, either to sustain important germplasm, or for release into the wild, are needed. Reintroduction requires integrated conservation policies and education of local tribes and conservation groups, researchers, and the government to prevent the overexploitation of Rhizanthes.

Acknowledgements The authors thank Arbi Wiguna (Universitas Negeri Padang, West Sumatra, Indonesia; and Division of Zoology, Genbinesia Foundation, Indonesia), Inama Ahmad (SMK Kehutanan Negeri Kadipaten, West Java, Indonesia), Sofian Rafflesia and Neka Afnidarti (Komunitas Peduli Puspa Langka, Bengkulu, Indonesia) for providing the figures used in this paper. The authors also thank Dra. Inggit Puji Astuti, M.Si and Ngatari from Research Center for Conservation and Botanic Gardens, LIPI, Bogor, Indonesia for helping one of the authors (Sofi Mursidawati) to gain access to the Rh. lowii samples in West Borneo, Indonesia in 2009, allowing the author to obtain some photographs.

Author Contributions All authors contributed equally to all parts of the paper development.

Declarations

Conflict of Interest All authors declare no conflicts of interest. Rhizanthes, the Forgotten Relative of Rafflesia in the...

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