Rhizanthes, the Forgotten Relative of Rafflesia in the Rafflesiaceae

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.


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 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; the closeup 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) 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) and Rh. 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) and Ra. 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 Fig. 2 Map of Rhizanthes distribution: red -Rh. zippelii, green -Rh. infanticida, Rh. lowii, and Rh. zippelii, purple -Rh. infanticida, blue -Rh. lowii and Rh. zippelii, magentaall 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) 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, and Rh. 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 Rafflesia, 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) and Rh. 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 selfregulate flower temperature (Patiño et al., 2000). Unlike the other Rhizanthes species 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  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 Fig. 4 A comparison of epidermal hair features in four species of Rhizanthes. Picture was redrawn and inspired from Bänziger and Hansen (2000) 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).
It is unknown if any Rhizanthes species harbor endophytic fungal species. Colletotrichum, Cytospora, and Gliocladiopsis 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 Kalimantan) province of Indonesia, where the fruit is known as "ulur-ulur", although it is 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 Data was based on, and compiled and modified from, Saleh et al. (2015). BSLT = brine shrimp lethality test; GAE = gallic acid equivalent; IC 50 = concentration that inhibits to 50% of biological processes; LC 50 = lethal concentration causing 50% mortality; TPC = total phenolic content 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; Table 2). 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. Antioxidant 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 (IC 50 < 50). The prospective use of ethanolic extracts as a biopesticide, shown by the brine shrimp lethality test, was an LC 50 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  (2014) Note: The information for this table was taken from the journal articles and represents some identified and tested metabolites 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 can be easily propagated (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 vivoboth in situ and ex situand 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.