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Despite being the world's largest single-flower, Rafflesia's biology and life history are still poorly understood due to its cryptic growth strategy on Tetrastigma vines. Previous studies have been mostly short-term, contrary to Rafflesia's long development period before blooming. Bud development and flower phenology of R. cantleyi was studied in a dipterocarp forest in Lata Jarum, Peninsular Malaysia. Seven populations, consisting of 247 buds, were monitored fortnightly for 65 months in two discrete studies between 2009 and 2018. The bud size distribution of R. cantleyi is dynamic, progressively changing from small flower buds to larger buds before flowering. Buds < 5.0 cm across had the slowest growth and highest mortality rates, while those > 15.0 cm across demonstrated accelerated growth. The bud growth profiles of the same site clustered distinctively regardless of sex with successful blooming rate that varied greatly between sites, prompting speculation about their relatedness to the sites' physical attributes. We reported the first female-dominated population in Rafflesia's life history. Rafflesia cantleyi's blooming rate at Lata Jarum is moderate to high, with non-seasonal flowering phenology as evident by the lack of synchronisation and consistency between flowering and local rainfall patterns. Based on the field data of the present study and the published information of other Rafflesia species, R. cantleyi's life cycle was estimated between 4.0 and 5.3 years. Our findings further explain Rafflesia's biology and life history and highlight the gap in knowledge of the natural habitats on the endoparasite's growth and fate potentially for future conservation and study. [ABSTRACT FROM AUTHOR]

Premise: Rafflesia are rare holoparasitic plants. In the Philippines, all but one species are found only on single islands. This study aimed to better understand the factors contributing to this distributional pattern. Specifically, we sought to determine whether narrow environmental tolerances of host and/or parasite species might explain their island endemicity. Methods: We used Maxent species distribution modeling to identify areas with suitable habitat for R. lagascae, R. lobata, and R. speciosa and their Tetrastigma host species. These analyses were carried out for current climate conditions and two future climate change scenarios. Results: Although species distribution models indicated suitable environmental conditions for the Tetrastigma host species in many parts of the Philippines, considerably fewer areas were inferred to have suitable conditions for the three Rafflesia species. Some of these areas are on islands from which they have not been reported. All three species will face significant threats as a result of climate change. Conclusions: Our results suggest that limited inter‐island dispersibility and/or specific environmental requirements are likely responsible for the current pattern of island endemicity of the three Rafflesia species, rather than environmental requirements of their Tetrastigma host species. [ABSTRACT FROM AUTHOR]

Background The molecular evolution of organellar genomes in angiosperms has been studied extensively, with some lineages, such as parasitic ones, displaying unique characteristics. Parasitism has emerged 12 times independently in angiosperm evolution. Holoparasitism is the most severe form of parasitism, and is found in ~10 % of parasitic angiosperms. Although a few holoparasitic species have been examined at the molecular level, most reports involve plastomes instead of mitogenomes. Parasitic plants establish vascular connections with their hosts through haustoria to obtain water and nutrients, which facilitates the exchange of genetic information, making them more susceptible to horizontal gene transfer (HGT). HGT is more prevalent in the mitochondria than in the chloroplast or nuclear compartments. Scope This review summarizes current knowledge on the plastid and mitochondrial genomes of holoparasitic angiosperms, compares the genomic features across the different lineages, and discusses their convergent evolutionary trajectories and distinctive features. We focused on Balanophoraceae (Santalales), which exhibits extraordinary traits in both their organelles. Conclusions Apart from morphological similarities, plastid genomes of holoparasitic plants also display other convergent features, such as rampant gene loss, biased nucleotide composition and accelerated evolutionary rates. In addition, the plastomes of Balanophoraceae have extremely low GC and gene content, and two unexpected changes in the genetic code. Limited data on the mitochondrial genomes of holoparasitic plants preclude thorough comparisons. Nonetheless, no obvious genomic features distinguish them from the mitochondria of free-living angiosperms, except for a higher incidence of HGT. HGT appears to be predominant in holoparasitic angiosperms with a long-lasting endophytic stage. Among the Balanophoraceae, mitochondrial genomes exhibit disparate evolutionary paths with notable levels of heteroplasmy in Rhopalocnemis and unprecedented levels of HGT in Lophophytum. Despite their differences, these Balanophoraceae share a multichromosomal mitogenome, a feature also found in a few free-living angiosperms. [ABSTRACT FROM AUTHOR]

Societal Impact Statement Summary Rafflesia is a genus of parasitic plants with the largest flowers in the world, unique to the threatened forest habitats of tropical Asia. Here, we report on genes that are active (the transcriptome) in Rafflesia seeds as part of a larger effort to understand Rafflesia. Rafflesia has never been grown successfully outside of its native range. Consequently, seed banking is not yet possible, precluding a critical management strategy for conservation. The study of Rafflesia seed biology is a critical step to improve its cultivation, which will educate the public about unique species and the importance of conserving their habitats. Rafflesia is of great interest as one of the only two plants known to have completely lost its chloroplast genome. Rafflesia is a holoparasite and an endophyte that lives inside the tissues of its host, a tropical grape vine (Tetrastigma), emerging only to bloom—with the largest flower of any plant. Here, we report the first Rafflesia seed transcriptome and compare it with those of other plants to deepen our understanding of its extraordinary life history. We assembled a transcriptome from RNA extracted from seeds of the Philippine endemic Rafflesia speciosa and compared this with those of other plants, including Arabidopsis, parasitic plants Striga and Cuscuta, and the mycoheterotrophic orchid Anoectochilus. Genetic and metabolic seed pathways in Rafflesia were generally similar to the other plant species. However, there were some notable exceptions. We found evidence of horizontal transfer of a gene potentially involved in circumventing host defenses. Moreover, we identified a possible convergence among parasitic plants because Rafflesia, Striga, and Cuscuta shared important similarities. We were unable to find evidence of genes involved in mycorrhizal symbiosis, suggesting that mycoheterotrophy is unlikely to play a role in Rafflesia parasitism. To date, ex situ propagation of Rafflesia by seed has been mostly unsuccessful. Our research is a bold step forward in understanding the fundamentals of Rafflesia seed biology that will inform the continued propagation and seed‐banking efforts concerning this recalcitrant plant. We discuss our findings in the broader context of the conservation of a genus in peril. [ABSTRACT FROM AUTHOR]

The Rafflesiaceae family consists of three genera of parasitic plants -- Rafflesia, Rhizanthes, and Sapria -- with purported ethnobotanical and ethnomedicinal properties. In this study, the inhibitory properties of 21 characterized metabolites associated with Rafflesia and Sapria were tested against eight proteins linked to human diseases -- including seven pathogenic-associated HMGCR, VEGFR2, acetylcholinesterase, NMT, H1N1 neuraminidase, GSK3-β, and estrogen receptor α, and one plant-pathogenic associated Colletotrichum chitin deacetylase. Each metabolite was tested using drug-likeness screening, screening metabolite activity, and molecular docking to eight diseases and microbial physiological processes. Hydrogen bonds and hydrophobic interactions between metabolite ligands and protein residues were characterized. Molecular dynamics were also assessed to analyze the stability of the protein-ligand interaction. Our results indicate that the gallotannins and flavonol phenolics from Rafflesia and Sapria display high inhibitory potential against disease proteins. All metabolite-protein pairs displayed stable fluctuations. However, some compounds disobeyed LRO5 drug-likeness and displayed moderate bioavailability and synthetic accessibility, so an improved drug delivery method is required. All 21 metabolites are available in other popular edible plants (mainly tea and certain berries) and could be used to create artificially mixed metabolite-based medicine to prevent the exploitation and endangerment of wild Rafflesia and Sapria populations. Our activity likelihood screening and molecular docking data indicate that Rafflesia and Sapria metabolites possess considerable potential as anti-cholesterol, respiratory antiviral, wound-healing, and antifungal properties. To protect Rafflesiaceae plants in the wild, metabolites can be assessed from other plant sources and combined as an artificial herbal mix. [ABSTRACT FROM AUTHOR]

Sapria, a holoparasitic plant of Rafflesiaceae is still understudied in term of anatomical, even more in histoanatomical study. This study aimed to perform preliminary observation on Sapria himalayana Griff. f. albovinosa morphology on its early and late stage of flower bud development inside its host, Tetrastigma laoticum Gagnep. (Vitaceae). The results show the progression from the early flower bud as it starts to grow in the host vascular cambium area towards proximal direction to reach the host xylem and distal direction to reach the host phloem and to reach the next stage of the life cycle, into the late flower bud where differentiation occurs. In late flower bud, some primordial of the organs are visible as the flower bud grows larger distally towards the periderm. This development pattern is similar to the progression of development in the previous studies in Rafflesia and Rhizanthes, where endophytic growth was observable in both proximal (xylem area) and distal (peridermal). Future comparative study is encouraged, especially to compare between Sapria species and between different stages of growth. Although, an efficient method and less invasive way of sampling is encouraged to prevent decline of Sapria species in the wild.

The flower bud of Rafflesia patma Blume following the protocorm stage of the flower will undergo rapid cell growth followed by the differentiation stage which lead to the later stage of the flower bud morphogenesis into the floral organs. During this transitional period of the flower bud, we revisited our R. patma flower bud microscope slide samples and some images from the previous study in [MURSIDAWATI & SUNARYO, 2012] to examine how the R. patma tissue behave prior to the late differentiation stage. We discovered that there are several types of meristematic cells in the R. patma early flower bud tissue: The elongated cells in the basal/proximal region of the bud where it in proximity with host xylem, then a column of non-elongated cells where ovary will develop in the later stage (in female flower), and in the apical/distal region of the flower bud, we found a densely packed meristematic cells where in the later flower bud this area will be developed into the protective bracts, perigone lobes, and central disc as later seen in the late flower bud tissue. During the late stage of the flower bud growth, the flower bud also inhibits growth of 1-2 vascular bundles, altering few others host vascular bundles surrounding the flower bud, while on the other side the root vascular bundle growth is just normal. This growth mechanism can minimize the host vasculature damage if multiple buds are growing the same growth direction.

Summary: Endoparasitic plants are the most reduced flowering plants, spending most of their lives as a network of filaments within the tissues of their hosts. Despite their extraordinary life form, we know little about their biology. Research into a few species has revealed unexpected insights, such as the total loss of plastome, the reduction of the vegetative phase to a proembryonic stage, and elevated information exchange between host and parasite. To consolidate our understanding, we review life history, anatomy, and molecular genetics across the four independent lineages of endoparasitic plants. We highlight convergence across these clades and a striking trans‐kingdom convergence in life history among endoparasitic plants and disparate lineages of fungi at the molecular and physiological levels. We hypothesize that parasitism of woody plants preselected for the endoparasitic life history, providing parasites a stable host environment and the necessary hydraulics to enable floral gigantism and/or high reproductive output. Finally, we propose a broader view of endoparasitic plants that connects research across disciplines, for example, pollen–pistil and graft incompatibility interactions and plant associations with various fungi. We shine a light on endoparasitic plants and their hosts as under‐explored ecological microcosms ripe for identifying unexpected biological processes, interactions and evolutionary convergence. [ABSTRACT FROM AUTHOR]

Tetrastigma (Vitaceae) are a genus of tropical lianas that are hosts for parasitic plants in the family Rafflesiaceae. Since the stem of Tetrastigma is creeping and often devoid of leaves, it is often mistakenly visually identified as a root. Rafflesia flowers at the ground level (i.e. terrestrial flowers) have been documented to grow on Tetrastigma roots, but it is unclear whether those that arise aerially emerge from host root or stem organs. In this study, we present a new, less-destructive means of sampling Tetrastigma leucostaphylum, which is a host of many Rafflesia species. Histological observation of two 250 cm above-ground aerial samples confirmed that both were stems, as shown by distinct anatomical features (compactly arranged vascular bundles with simple phloem bundles) in contrast to roots, which had loosely arranged vascular bundles with ramified phloem bundles. Clearer identification of Tetrastigma vine infected with aerial Rafflesia is needed to confirm whether the parasitic flower grows on a stem or on an erect aerial root to better understand the biology of this enigmatic Rafflesia species that have evaded cultivation and ex situ conservation efforts. This new sampling method may be a useful way to identify the Tetrastigma vine in the future due to its less invasive nature compared to severing the whole vine. [ABSTRACT FROM AUTHOR]

Rafflesia produces the largest single flowers in the world, at the expense of its host vine, Tetrastigma, yet it begins as an inconspicuous endophyte. It is unknown how the Rafflesia seed gets into the host and germinates. Multiple locals claim to have successfully grown the holoparasitic flower from seeds resulting in blooms. Using available morpho-histological studies, combined with descriptions from local Rafflesia seed growers, as well as unpublished details of our own work, we filled in the missing details of Rafflesia's life cycle from seed germination to endophyte inside the host before it transitions to its flowering stage. Post-germination, the Rafflesia endophyte forms a clonal network of vegetative meristematic cells, separated by the dividing host tissue, each meristematic cell cluster eventually developing into the primordial floral bud or protocorm. We propose future work involving mass spectrometry imaging to characterize the metabolites that allow communication between distant endophytic clusters and floral bud induction without destroying the histology of the sample. [ABSTRACT FROM AUTHOR]

Premise: A subset of parasitic plants bear extremely reduced features and grow nearly entirely within their hosts. Until recently, most of these endoparasites were thought to represent a single clade united by their reduced morphology. Current phylogenetic understanding contradicts this assumption and indicates these plants represent distantly related clades, thus offering an opportunity to examine convergence among plants with this life history. Methods: We sampled species from Apodanthaceae, Cytinaceae, Mitrastemonaceae, and Rafflesiaceae spanning a range of developmental stages. To provide a broader comparative framework, Santalaceae mistletoes with a similar lifestyle were also analyzed. Microtomography and microscopy were used to analyze growth patterns and the ontogeny of host–parasite vascular connections. Results: Apodanthaceae, Cytinaceae, Mitrastemonaceae, and Rafflesiaceae species demonstrated a common development characterized by late cell differentiation. These species were also observed to form direct connections to host vessels and to cause severe alterations of host xylem development. Apodanthaceae and Rafflesiaceae species were additionally observed to form sieve elements, which connect with the host phloem. Endophytic Santalaceae species demonstrated a dramatically different developmental pattern, featuring early cell differentiation and tissue organization, and little effect on host anatomy and cambial activity. Conclusions: Our results illuminate two distinct developmental trajectories in endoparasites. One involves the retention of embryonic characteristics and late connection with host vessels, as demonstrated in species of Apodanthaceae, Cytinaceae, Mitrastemonaceae, and Rafflesiaceae. The second involves tissue specialization and early connection with host xylem, as exemplified by Santalaceae species. These differences are hypothesized to be related to the absence/presence of photosynthesis in these plants. [ABSTRACT FROM AUTHOR]

Flower and fruit development of Rafflesia consueloae were studied between February 2014 and April 2016 in Pantabangan, Nueva Ecija, Philippines. Flower development was divided into five distinct phases: (1) emergence, (2) post-emergence, (3) bract, (4) perigone, and (5) anthesis. Fruit development was monitored from flower senescence until fruiting and maturation. A total of 512 individual buds were monitored - discovered at different stages of bud development. Only nine buds were monitored from post-emergence until the perigone phase. A bloom rate of 19.73% and an overall mortality rate of 77.34% were recorded. Mortality was highest during the early phases (post-emergence and bract) and lowest at the perigone phase. R. consueloae exhibited nocturnal flowering; wherein anthesis usually begins at dusk, signaled by the detachment of the first lobe, and from there on, full bloom took 15 ± 5.85 h to complete. Flowering was at its highest during the coldest and driest months of the year - between December and April. The growth rate is exponential, with an overall diameter growth of 1.80% every 4 d. The highest growth rate was recorded during the perigone phase. Six fruits, considered as dehiscent berries, ripened mostly in July - within 145 ± 11.03 d from anthesis. [ABSTRACT FROM AUTHOR]

Sapria, a holoparasitic plant of Rafflesiaceae is still understudied in term of anatomical, even more in histoanatomical study. This study aimed to perform preliminary observation on Sapria himalayana Griff. f. albovinosa morphology on its early and late stage of flower bud development inside its host, Tetrastigma laoticum Gagnep. (Vitaceae). The results show the progression from the early flower bud as it starts to grow in the host vascular cambium area towards proximal direction to reach the host xylem and distal direction to reach the host phloem and to reach the next stage of the life cycle, into the late flower bud where differentiation occurs. In late flower bud, some primordial of the organs are visible as the flower bud grows larger distally towards the periderm. This development pattern is similar to the progression of development in the previous studies in Rafflesia and Rhizanthes, where endophytic growth was observable in both proximal (xylem area) and distal (peridermal). Future comparative study is encouraged, especially to compare between Sapria species and between different stages of growth. Although, an efficient method and less invasive way of sampling is encouraged to prevent decline of Sapria species in the wild. [ABSTRACT FROM AUTHOR]

The flower bud of Rafflesia patma Blume following the protocorm stage of the flower will undergo rapid cell growth followed by the differentiation stage which lead to the later stage of the flower bud morphogenesis into the floral organs. During this transitional period of the flower bud, we revisited our R. patma flower bud microscope slide samples and some images from the previous study in [MURSIDAWATI & SUNARYO, 2012] to examine how the R. patma tissue behave prior to the late differentiation stage. We discovered that there are several types of meristematic cells in the R. patma early flower bud tissue: The elongated cells in the basal/proximal region of the bud where it in proximity with host xylem, then a column of non-elongated cells where ovary will develop in the later stage (in female flower), and in the apical/distal region of the flower bud, we found a densely packed meristematic cells where in the later flower bud this area will be developed into the protective bracts, perigone lobes, and central disc as later seen in the late flower bud tissue. During the late stage of the flower bud growth, the flower bud also inhibits growth of 1-2 vascular bundles, altering few others host vascular bundles surrounding the flower bud, while on the other side the root vascular bundle growth is just normal. This growth mechanism can minimize the host vasculature damage if multiple buds are growing the same growth direction. [ABSTRACT FROM AUTHOR]

Rhizanthes lowii (Becc.) Harms., a new species record from the Lanjak Entimau Wildlife Sanctuary, Sarawak is re-described and illustrated. The blooms show outstanding features that include its orange-white batik pattern half-way through every tepal with very dense hairs covering the entire inner side of the tepals. Its host is Tetrastigma diepenhorstii (Vitaceae). [ABSTRACT FROM AUTHOR]

Main conclusion: A histological study of Rafflesia patma revealed the simplicity of a flower’s vascular tissue and epidermal features of flower organs, including their structures and pigmentation. Rafflesia is an endophytic holoparasitic plant that infects Tetrastigma. In a previous study, we characterized the shape of the strands of an endophyte (Rafflesia patma Blume) and hypothesized their distribution. In this study, we deepened our analysis by assessing parts of flower tissue sampled during anthesis, performed surface casting of the abaxial and adaxial sides of the perigone lobe to profile their surface features, and histologically characterized the perigone lobe, perigone tube, and central column base, including the anther and cupula region. The objective of these observations was to compare tissues from different organs and the distribution of cells staining positive for tannin, suberin, and lignin. Observable features in this study were vascular and epidermal tissue. We also observed reduced vascular tissue with xylem and vascular parenchyma in multiple organs. The adaxial epidermis found in the perigone lobes and tube had papillate cells, and their function might be to assist with the emission of odor through chemical evaporation. The abaxial epidermis, also found in perigone lobes and tube, had flattened cells. These, combined with the nearby flattened parenchyma cells, especially in the outermost, early perigone lobe, might provide a tougher (stiffer) outer protective barrier for the flower. The accumulation of tannin in perigone lobes might offer protection to the flower from herbivores prior to anthesis. Although a previous observation indicated the possibility of stomata on the surface of Rafflesia flowers, no stomata were found in this study. [ABSTRACT FROM AUTHOR]

This study provides a hypothetical discussion about the growth of Rafflesia patma Blume (syn. R. horsfieldii R. Br. (1821); Rafflesiaceae), an endophytic parasite, within a grafted host, a woody vine (Tetrastigma leucostaphylum (Dennst.) Alston). Based on the observation of a 10-year old (2007 to 2017) R. patma – T. leucostaphylum graft, we hypothesize that R. patma moves away from its host rather than remaining in an anchored position from which it flowers, although flower knobs that emerge later may vary in range from the grafting point, i.e., flower knobs may develop close to or far away from the grafting point. Our provisional macroscopic observations point towards a gradual creeping motion of the endophyte towards new host tissues, flowering opportunistically when sufficient nutrients have been found. Much has yet to be discovered about the growth and flowering behavior of R. patma and about the dynamics of the R. patma – T. leucostaphylum interaction.

In the course of our intensive floristic inventories for the flora of Myanmar, a new species of the genus Sapria (Rafflesiaceae), S. myanmarensis Nob. Tanaka, Nagam., Tagane & M.M. Aung is described and photographed. In addition, S. himalayana Griff. f. albovinosa Banziger & B. Hansen is newly recorded in the country. A key to the species of Sapria presently occurring in Myanmar is provided. [ABSTRACT FROM AUTHOR]

The early life history of Rafflesia R.Br. is hidden by its cryptic nature. Being a holoparasitic plant, early growth and development of species in this genus have been suggested to occur inside the host plant. In this study, using microscopic analyses, we examined how Rafflesia patma Blume grows from a simple clump of cells or protocorm inside the cambium tissue of Tetrastigma leucostaphylum (Dennst.) Alston, its host plant. We further show how the protocorm expands in about 2 months into an early form of a flower knob or bud, just before the flower begins to emerge from the host plant vine. Our study suggests that the endophyte, R. patma , spreads within T. leucostaphylum vascular cambium tissue in a linear manner, but not as a continuous strand. We suspect that the parasitic endophyte spreads inside the host vascular cambium and is pushed farther away from its point of origin to another part of the host as the host vine cambium fusiform initial cells divide and enlarge over months. Moreover, the protocorm matures in the cambium region of the T. leucostaphylum vine which lies close to the xylem, and grow outwards, bursting through the host phloem, then the host cortex, and finally towards the host periderm as it begins to emerge. Although we believe this might be the closest prediction on how Rafflesia develops at an early stage and is distributed within its host body, a future analysis of endophytic tissue growth in a controlled environment, such as the use of tissue culture, is required to strengthen our findings. • Rafflesia patma Blume is a holoparasitic plant. • Rafflesia patma parasitizes the cambium tissue of Tetrastigma leucostaphylum. • We use microscopic analyses to assess protocorm growth and flower knob formation. • Rafflesia patma spreads within T. leucostaphylum vascular cambium linearly. • The protocorm matures in the cambium region of the T. leucostaphylum vine. [ABSTRACT FROM AUTHOR]

The plants of the enigmatic genus Rafflesia are well known for their gigantic flowers and their floral features such as pungent floral scent and vivid dark color, which mimics the food/brood sites of carrion. However, information on the pollination biology of this plant group remains limited and mostly anecdotal. In the present paper, we studied the floral volatiles of R. cantleyi Solms-Laubach and their role in pollinator attraction. To achieve these aims, the floral scent was collected in situ in the field using a dynamic headspace method followed by chemical analysis via GC-MS. The olfactory preferences of pollinators to the identified chemical compounds, were tested singly and in blends, in flight tunnel bioassays and compared with responses to headspace floral extracts. In addition, flower-visiting calliphorid flies and the local carrion fly community were sampled and identified. Five species of calliphorid flies (subfamilies of Chrysomyinae and Calliphorinae), all females, were found on the flowers, whereas nine species were found in the traps that were baited with tainted meat in the surrounding habitat. However, only flower visitors of one blow fly species, Chrysomya chani Kurahashi, were observed to carry R. cantleyi pollen after visiting male flowers. The floral volatiles emitted by male flowers in full bloom were dominated by two sulphur-containing compounds, dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS). These were accompanied by other minor compounds such as benzenoids (4), monoterpenoids (4), trace amounts of aliphatic compounds (1), and sesquiterpenes (1). In flight-tunnel bioassays, a female-specific positive response of C. chani flies to individual DMDS, DMTS, and a blend of DMDS and DMTS was evident. Our findings suggest that R. cantleyi biochemically mimics carrion and that relative ratio of oligosulfides in the floral scent play a key role in sex-biased pollinator specialization, attracting only female C. chani carrion flies to the flowers. [ABSTRACT FROM AUTHOR]

Rafflesia is a holoparasite in the tropical rainforest of Southeast Asia. In Sarawak, Rafflesia tuan-mudae Becc. grows on Tetrastigma sp.. For conservation purposes, we studied microbes from the soil and parts of R. tuan-mudae located in designated block A, B and C areas of Gunung Gading National Park. A total of 35 Tetrastigma stems were reported. However, a high percentage of death at 70% was observed in R. tuan-mudae between the time when the bud swells up to bloom. Biological samples were sampled from active and inactive buds to identify the microbes and their relationships. Isolates from the soil and the debris produce many bacterial colonies and fungi. Thus, the microbial activities associated with Rafflesia tuan-mudae and Tetrastigma sp. need further attention. [ABSTRACT FROM AUTHOR]

The large-flowered parasitic genus Rafflesia R.Br. (Rafflesiaceae) has long fascinated naturalists and scientists and is an iconic symbol for plant conservation. Techniques to effectively propagate members of the genus outside of their natural habitat are sparse, and grafting infected Tetrastigma K.Schum.(Vitaceae) host plants has previously been reported as a successful strategy for ex situ conservation of Rafflesia. Here we report our attempts in the United States to propagate host cuttings infected with Rafflesia speciosa Barcelona & Fernando and R. lagascae Blanco collected from the Philippines, as well as uninfected host material. We also describe efforts to germinate R. speciosa seeds in vitro using various plant growth regulators (PGRs). After rooting, infected host cuttings survived for a maximum of 11 months, but did not produce shoots. However, an uninfected cutting of T. cf. magnum grafted onto an established Malaysian species of Tetrastigma in June 2017 has succeeded in the commencement of new growth. Three propagules of a second potential host, T. harmandii Planch., have also been vigorously growing at the United States Botanic Garden since June 2017. However, Rafflesia seeds did not germinate with the application of PGRs, even though the seeds were viable according to tetrazolium (TZ) testing. These ex situ propagation attempts have revealed challenges in propagating these species outside of their native ranges, but our incremental success in rooting infected Tetrastigma, as well as grafting interspecific Tetrastigma species, bodes well for further advances. With Philippine host species, T. harmandii and T. cf. magnum in cultivation, we can begin using these specimens for future experimentation involving grafting of infected material and Rafflesia seed inoculation trials. Furthermore, we describe new avenues of propagation techniques for Rafflesia as practised by Marius Gabin, one of the owners of the Vivian Rafflesia garden, which contains a natural Rafflesia forest habitat at Poring Springs, Sabah, Malaysia. Gabin openly shared his successes in artificially inoculating Rafflesia seeds into a mature Tetrastigma vine. Gabin’s willingness to share his experience highlights the importance of collaborating with practitioners who have developed local knowledge of Rafflesia horticulture and conservation.

Rafflesiaceae, crowned 'the greatest prodigy of the vegetable world', produce the largest flowers in angiosperms. They are also holoparasites residing inside their vine hosts, and emerge only during flowering. The floral gigantism and obligate parasitism of Rafflesiaceae have rendered their structure unrecognizable to most plant biologists. The vegetative body is composed of highly reduced strands of cells embedded in the host tissue, and does not differentiate into leaves, stems, or roots. The flowers look and smell like decaying animal flesh and exhibit numerous features unknown in the vast majority of flowering plants. This unusual combination of characters, alongside their generally elevated rates of molecular evolution among commonly used phylogenetic markers and propensity for host-to-parasite horizontal gene transfer, has obscured the phylogenetic affinities of Rafflesiaceae since their discovery two centuries ago. Here, we review the phylogenetic placement of Rafflesiaceae and how it has informed a deeper understanding of the pattern and magnitude of horizontal gene transfer in parasitic plants. We also examine the vegetative and reproductive morphology of Rafflesiaceae to provide insight into how these unusual plants are constructed, and offer clues on their evolution from tiny flowered ancestors to floral giants. [ABSTRACT FROM AUTHOR]

Parasitic plants demonstrate a diversity of growth strategies, life histories, and developmental and physiological characteristics. Most research to date has focused on a narrow range of parasitic taxa, particularly in the Orobanchaceae, while the other independent origins of parasitism have largely gone unstudied. One type of parasite that has received relatively little attention are the endophytic parasites, which have a fascinating growth strategy where the parasite is embedded within the host tissue, with the flower the only externally visibly plant part. Endophytic growth makes it challenging to understand basic aspects of species biology, such as the size of a given parasite, the number of parasites per host, and the genetic diversity of populations. Recent studies by Barkman et al. (2017) and Pelser et al. (2017) have used microsatellite genotyping to investigate the population biology of endoparasitic Rafflesiaceae species in Asia. They show the potential for extensive parasite spread within a host vine and the strong partitioning of genetic diversity by host. These species are also shown to have an outcrossing mating system. However, these studies suggest different reproductive strategies, one supporting monoecy and one suggesting dioecy. Overall, these studies partly "lift the lid" on the cryptic biology of Rafflesia and the Rafflesiaceae and open the door for future comparative studies between endophytic and free-living parasitic plants. [ABSTRACT FROM AUTHOR]

PREMISE OF THE STUDY: Partitioning of population genetic variation in plants may be affected by numerous factors including life history and dispersal characteristics. In parasitic plants, interactions with host populations may be an additional factor infl uencing partitioning. To test for hierarchical population genetic patterns related to obligate endoparasitism, we studied three species of Rafflesiaceae, which grow as extremely reduced endophytes infecting Tetrastigma vines in Southeast Asia. METHODS: Microsatellite markers were developed and multilocus genotypes were determined for Rafflesia cantleyi, Rafflesia tuan-mudae , and Sapria himalayana and each of their Tetrastigma hosts. Relatedness among parasite individuals was estimated, and AMOVAs were used to determine levels of population genetic subdivision. KEY RESULTS: Microsatellite genotypes for 340 paired parasite and host samples revealed that host vines were infected by numerous Rafflesiaceae individuals that may spread for up to 14 m within stem tissues. Surprisingly, Rafflesiaceae parasites within a given host are significantly more closely related to each other than individuals of the same species in other host individuals. The pattern of hierarchical population genetic subdivision we detected across species is likely due to limited seed dispersal with reinfection of natal host vines. CONCLUSIONS: These findings demonstrate common population genetic patterns between animal and plant parasites, potentially indicating advantages of close relatives infecting hosts. This study also has important conservation implications for Rafflesiaceae since our data suggest that destruction of a single infected host vine could result in large genetic losses. [ABSTRACT FROM AUTHOR]

This study provides a hypothetical discussion about the growth of Rafflesia patma Blume (syn. R. horsfieldii R. Br. (1821); Rafflesiaceae), an endophytic parasite, within a grafted host, a woody vine (Tetrastigma leucostaphylum (Dennst.) Alston). Based on the observation of a 10-year old (2007 to 2017) R. patma -- T. leucostaphylum graft, we hypothesize that R. patma moves away from its host rather than remaining in an anchored position from which it flowers, although flower knobs that emerge later may vary in range from the grafting point, i.e., flower knobs may develop close to or far away from the grafting point. Our provisional macroscopic observations point towards a gradual creeping motion of the endophyte towards new host tissues, flowering opportunistically when sufficient nutrients have been found. Much has yet to be discovered about the growth and flowering behavior of R. patma and about the dynamics of the R. patma -- T. leucostaphylum interaction. [ABSTRACT FROM AUTHOR]

Rafflesia tuan-mudae Becc. (Rafflesiaceae) is endemic to Borneo and was recorded from Sarawak and probably Kalimantan. Previous records showed that Tetrastigma rafflesiae (Miq.) Planch. (Vitaceae) is the only host plant for R. tuan-mudae. In this study the host plants were collected each time R. tuan-mudae was observed or collected. Out of 20 Tetrastigma specimens collected infected by R. tuan-mudae, 14 were identified as T. diepenhorstii (Miq.) Latiff while 6 belonged to T. rafflesiae. Therefore, a new host for R. tuan-mudae is recorded and descriptions for each host are presented. [ABSTRACT FROM AUTHOR]

Background and Aims Species in the holoparasitic plant family Rafflesiaceae exhibit one of the most highly modified vegetative bodies in flowering plants. Apart from the flower shoot and associated bracts, the parasite is a mycelium-like endophyte living inside their grapevine hosts. This study provides a comprehensive treatment of the endophytic vegetative body for all three genera of Rafflesiaceae (Rafflesia, Rhizanthes and Sapria), and reports on the cytology and development of the endophyte, including its structural connection to the host, shedding light on the poorly understood nature of this symbiosis. Methods Serial sectioning and staining with non-specific dyes, periodic–Schiff's reagent and aniline blue were employed in order to characterize the structure of the endophyte across a phylogenetically diverse sampling. Key Results A previously identified difference in the nuclear size between Rafflesiaceae endophytes and their hosts was used to investigate the morphology and development of the endophytic body. The endophytes generally comprise uniseriate filaments oriented radially within the host root. The emergence of the parasite from the host during floral development is arrested in some cases by an apparent host response, but otherwise vegetative growth does not appear to elicit suppression by the host. Conclusions Rafflesiaceae produce greatly reduced and modified vegetative bodies even when compared with the other holoparasitic angiosperms once grouped with Rafflesiaceae, which possess some vegetative differentiation. Based on previous studies of seeds together with these findings, it is concluded that the endophyte probably develops directly from a proembryo, and not from an embryo proper. Similarly, the flowering shoot arises directly from the undifferentiated endophyte. These filaments produce a protocorm in which a shoot apex originates endogenously by formation of a secondary morphological surface. This degree of modification to the vegetative body is exceptional within angiosperms and warrants additional investigation. Furthermore, the study highlights a mechanical isolation mechanism by which the host may defend itself from the parasite. [ABSTRACT FROM AUTHOR]

Rafflesia kerrii has been used in Thai traditional remedies for treatment of several diseases. However, scientific data particularly on biological activities of this plant is very rare. The present study explores an antioxidant activity of R. kerrii flower (RKF). Extracting solvent and extraction procedure were found to play an important role on the activity of RKF extract. The extract obtained from water-ethanol system showed higher antioxidant activity than that from water-propylene glycol system. Fractionated extraction using different solvents revealed that methanol fractionated extract (RM) possessed the highest antioxidant activity with Trolox equivalent antioxidant capacity (TEAC) and inhibitory concentration of 50% inhibition (IC50) values of approximately 39 mM/mg and 3 μg/mL, respectively. Phytochemical assays demonstrated that RM contained extremely high quantity of phenolic content with gallic antioxidant equivalent (GAE) and quercetin equivalent (QE) values of approximately 312 mg/g and 16 mg/g, respectively. Ultraviolet-visible spectroscopy (UVVIS) and high-pressure liquid chromatography (HPLC) indicated that gallic acid was a major component. RM which was stored at 40°C, 75% RH for 4 months showed slightly significant change (p < 0.05) in phytochemical content and antioxidant activity with zero order degradation. The results of this study could be concluded that R. kerrii flower was a promising natural source of strong antioxidant compounds. [ABSTRACT FROM AUTHOR]

Premise of the study: The holoparasitic plant family Rafflesiaceae include the world's largest flowers. Despite their iconic status, relatively little is known about the morphology and development of their flowers. A recent study clarified the organization of the outer (sterile) floral organs, surprisingly revealing that their distinctive floral chambers arose via different developmental pathways in the two major genera of the family. Here, we expand that research to investigate the structure and development of the reproductive organs of Rafflesiaceae. Methods: Serial sectioning, scanning electron microscopy, and x-ray tomography of floral buds were employed to reconstruct the structure and development of all three Rafflesiaceae genera. Key results: Unlike most angiosperms, which form their shoot apex from the primary morphological surface, the shoot apex of Rafflesiaceae instead forms secondarily via internal cell separation (schizogeny) along the distal boundary of the host-parasite interface. Similarly, the radially directed ovarial clefts of the gynoecium forms via schizogeny within solid tissue, and no carpels are initiated from the floral apex. Conclusions: The development of the shoot apex and gynoecium of Rafflesiaceae are highly unusual. Although secondary formation of the morphological surface from the shoot apex has been documented in other plant groups, secondary derivation of the inner gynoecium surface is otherwise unknown. Both features are likely synapomorphies of Rafflesiaceae. The secondary derivation of the shoot apex may protect the developing floral shoot as it emerges from within dense host tissue. The secondary formation of the ovarial clefts may generate the extensive placental area necessary to produce hundreds of thousands of ovules. [ABSTRACT FROM AUTHOR]

Rafflesiaceae, which produce the world's largest flowers, have captivated the attention of biologists for nearly two centuries. Despite their fame, however, the developmental nature of the floral organs in these giants has remained a mystery. Most members of the family have a large floral chamber defined by a diaphragm. The diaphragm encloses the reproductive organs where pollination by carrion flies occurs. In lieu of a functional genetic system to investigate floral development in these highly specialized holoparasites, we used comparative studies of structure, development, and gene-expression patterns to investigate the homology of their floral organs. Our results surprisingly demonstrate that the otherwise similar floral chambers in two Rafflesiaceae subclades, Rafflesia and Sapria, are constructed very differently. In Rafflesia, the diaphragm is derived from the petal whorl. In contrast, in Sapria it is derived from elaboration of a unique ring structure located between the perianth and the stamen whorl, which, although developed to varying degrees among the genera, appears to be a synapomorphy of the Rafflesiaceae. Thus, the characteristic features that define the floral chamber in these closely related genera are not homologous. These differences refute the prevailing hypothesis that similarities between Sapria and Rafflesia are ancestral in the family. Instead, our data indicate that Rafflesia-like and Sapria-like floral chambers represent two distinct derivations of this morphology. The developmental repatterning we identified in Rafflesia, in particular, may have provided architectural reinforcement, which permitted the explosive growth in floral diameter that has arisen secondarily within this subclade. [ABSTRACT FROM AUTHOR]

A new species of the genus Rafflesia, Rafflesia sharifah-hapsahiae, is described, with illustrations. A key to 2 related species of Rafflesia of Peninsular Malaysia is also provided. A discussion on its differences and similarities with Rafflesia azlanii and Rafflesia hasseltii is presented. [ABSTRACT FROM AUTHOR]

Rafflesia, a holoparasitic genus that produces the largest flower in the world is characterized by the absence of leaves, stem and other macroscopic organs. To better understand the molecular regulation of flower development in this genus we isolated and characterized a floral MADS-box gene, namely, RcMADS1 from Rafflesia cantleyi. Heterologous expression analysis in Arabidopsis was chosen because Rafflesia is not amenable to genetic manipulations. RcMADS1 shares sequence similarity with AGAMOUS-LIKE 24 (AGL24) and SHORT VEGETATIVE PHASE (SVP) of Arabidopsis. Ectopic expression of RcMADS1 in Arabidopsis caused early flowering and conversion of sepals and petals into leaf-like structures, and carpels into inflorescences. In 35S::RcMADS1 plants SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), a downstream target gene of AGL24, was upregulated. 35S::RcMADS1 plants exhibit early flowering and conversion of the floral meristem into inflorescence meristem, as in 35S::AGL24 plants. Similar to AGL24, RcMADS1 could rescue the late flowering phenotypes of agl24-1 and FRIGIDA, but not the early flowering of svp-41. Based on these results, we propose that RcMADS1 is a functional ortholog of Arabidopsis AGL24. [ABSTRACT FROM AUTHOR]