Discovery of the photosynthetic relatives of the "Maltese mushroom" Cynomorium
© Nickrent et al. 2005
Received: 26 April 2005
Accepted: 21 June 2005
Published: 21 June 2005
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© Nickrent et al. 2005
Received: 26 April 2005
Accepted: 21 June 2005
Published: 21 June 2005
Although recent molecular phylogenetic studies have identified the photosynthetic relatives of several enigmatic holoparasitic angiosperms, uncertainty remains for the last parasitic plant order, Balanophorales, often considered to include two families, Balanophoraceae and Cynomoriaceae. The nonphotosynthetic (holoparasitic) flowering plant Cynomorium coccineum has long been known to the Muslim world as "tarthuth" and to Europeans as the "Maltese mushroom"; C. songaricum is known in Chinese medicine as "suo yang." Interest in these plants is increasing and they are being extensively collected from wild populations for use in herbal medicines.
Here we report molecular phylogenetic analyses of nuclear ribosomal DNA and mitochondrial matR sequence data that strongly support the independent origin of Balanophoraceae and Cynomoriaceae. Analyses of single gene and combined gene data sets place Cynomorium in Saxifragales, possibly near Crassulaceae (stonecrop family). Balanophoraceae appear related to Santalales (sandalwood order), a position previously suggested from morphological characters that are often assumed to be convergent.
Our work shows that Cynomorium and Balanophoraceae are not closely related as indicated in all past and present classifications. Thus, morphological features, such as inflorescences bearing numerous highly reduced flowers, are convergent and were attained independently by these two holoparasite lineages. Given the widespread harvest of wild Cynomorium species for herbal medicines, we here raise conservation concerns and suggest that further molecular phylogenetic work is needed to identify its photosynthetic relatives. These relatives, which will be easier to cultivate, should then be examined for phytochemical activity purported to be present in the more sensitive Cynomorium.
Molecular phylogenetics has expanded understanding of relationships among all major angiosperm groups and has thereby strongly impacted their classification . More recently, such advances have also included some nonphotosynthetic holoparasites whose phylogenetic positions had previously been uncertain, such as Hydnoraceae  and Rafflesiales . The latter study documented that Rafflesiales are actually a polyphyletic assemblage of three or four independent evolutionary lineages. The losses and reductions in features that are pervasive in parasitic plants have resulted in remarkable morphological convergences, thereby explaining their erroneous placement by traditional methods. Previous molecular phylogenetic work with such holoparasites highlighted the need to employ gene sequences from different subcellular compartments and analytical methods that accommodate rate heterogeneity, thus avoiding long-branch attraction artifacts . These steps are justified because congruence among different gene trees provides evidence that the organismal tree is being recovered, while incongruence suggests the presence of nonstandard processes such as introgression, lineage sorting, and horizontal gene transfer (HGT) [3–6].
This work documents that Cynomorium and Balanophoraceae are not closely related, a result in conflict with past and present classifications. Assuming our molecular gene trees reflect the organismal tree, these data indicate that morphological features, such as inflorescences bearing numerous highly reduced flowers, are convergent and were thus attained independently by these two holoparasite lineages.
From an historical perspective, Hooker  allied Cynomorium and Balanophoraceae with Haloragaceae (the latter is a family in Saxifragales, see Figure 3) because both groups have epigynous flowers with stamens opposite the valvate perianth lobes. He also noted the striking similarities between the female flowers of Gunnera and Lophophytum (Balanophoraceae). Our analyses that included Gunnera (data not shown) indicate it is not closely related to either Cynomoriaceae or Balanophoraceae, a result in agreement with other work that placed Gunnera as sister to all core eudicots [17, 18]. Other 19th century workers such as Hoffmeister proposed a relationships between Cynomorium and Hippuris (Plantaginaceae, an asterid) because both have a single, epigynous stamen attached to the top of a unicarpellate ovary. This relationship was not confirmed following a detailed floral morphological study  nor was Cynomorium shown to be related to asterids following molecular analyses reported here.
The exact position of Saxifragales within the core eudicot clade remains uncertain because various molecular phylogenetic analyses are in conflict (c. f. [17, 18, 20, 21]). These studies have proposed relationships with rosids, asterids, caryophyllids, and Santalales, i.e. nearly all members of the core tricolpate clade (see review by Judd and Olmstead ). The results reported here also demonstrate uncertainty with regard to the topology of the above clades (Figure 2). Given the variety of ways these clades could resolve, the possibility exists that Balanophoraceae and Cynomorium are more closely related than graphically depicted in Figure 2. Although future molecular phylogenetic studies may support this, it is unlikely that these two holoparasite groups will become sister given that the Balanophoraceae + Santalales clade has BS support of 94% and Cynomorium is sister to Peridiscus with 98% BS support. Moreover, the latter two genera are sister to Hamamelis with a 98% BS value.
Long-branch attraction has long been suggested as a confounding factor when conducting phylogenetic analyses of organisms with marked among-lineage rate heterogeneity . Indeed, our group has shown that Rafflesiales, another holoparasitic angiosperm group, is susceptible to long-branch attraction, particularly when analyzed with MP . The results reported here differ from that study in several respects. First, MP analysis of the global data set does not indicate that Balanophoraceae and Cynomorium exhibit particularly long branches with respect to other angiosperms, nor are these holoparasite clades "attracted" to each other (Additional File 8). This result differs from that obtained when the Rafflesiales molecular data are analyzed with MP where the four distinct lineages (Apodanthaceae, Cytinaceae, Mitrastemonaceae and Rafflesiaceae) appear monophyletic . When the global data set is analyzed with BI, some degree of branch length heterogeneity is seen (Additional File 9), however, Balanophoraceae and Cynomorium still remain on distinct clades. It should be pointed out that for the global data set, branch length comparisons between holoparasites and photosynthetic plants are somewhat biased given that the chloroplast genes were coded as missing for the holoparasites. Taken together, we feel our results are not being influence by long-branch attraction and that they provide strong evidence for the independent evolution of these two holoparasite lineages.
Attempts to find morphological features that link Cynomorium with Saxifragales are complicated, first because the latter order is morphologically heterogeneous and second because of morphological reductions in the holoparasite (see below). The heterogeneity of Saxifragales is evidenced by the fact that pre-molecular classifications placed many of its component families in distinct subclasses . Although a close relationship between Cynomorium and Crassulaceae was not seen on the BI tree, the shortest MP tree showed these genera as sister taxa, thus this possible affinity will here be considered. Tricolporate pollen (Figure 1e), stem succulence, and proanthocyanidins (red pigments, see ) are possible characters that may reflect a relationship between Cynomorium and Crassulaceae, but these are all general features found in many groups. Other than such facies, there are few floral characters that could be considered synapomorphies between the extremely reduced flowers of Cynomorium and those of Crassulaceae. The staminate flower of Cynomorium is composed of four to six distinct perianth parts forming a whorl below the single stamen and a wedge-shaped structure interpreted as a pistillode . The apical portion of the pistillode is concave and accepts the base of an anther theca prior to filament elongation (Figure 1b) and its internal lateral surface has a groove that accepts the filament. Stamen morphology is not unlike many tricolpate angiosperms: the anthers are dorsifixed, tetrasporangiate, dithecal and open by longitudinal slits. The carpellate flower (Figure 1c) is composed of a single carpel with an elongated, grooved style. The perianth (or perigonial scales) are much smaller than in the staminate flower, reduced to small papillae at the summit of the ovary or along its sides. The bisexual flowers are similar to carpellate flowers except for the addition of a stamen at the base of the ovary (Figure 1d). As with many holoparasitic flowering plants, the reduction or loss of morphological features interferes with the comparative morphological approach when searching for relatives among less modified photosynthetic plants.
Although precedents exists for placing Balanophoraceae with Santalales [8, 26–29], recovering this relationship with both nuclear and mitochondrial genes is surprising because any morphological similarities (e.g., reduction in the gynoecium) have usually been assumed to be cases of convergence . Moreover, BI of the Saxifragales data set indicates a derivation of Balanophoraceae from within Santalales, not as its sister (Figure 3 and Additional File 8). Although a multigene analysis of Santalales with robust taxon sampling exists , it was generated with nuclear ribosomal DNA and chloroplast genes. To conduct analyses with Balanophoraceae and Santalales, mitochondrial genes from the latter will be needed to test relationships.
Cronquist  viewed the sandalwoods as the best candidate ancestral group for Balanophoraceae, however, he admitted any similarities might reflect convergent adaptations to parasitism. Of the 17 genera in the family, only Dactylanthus, Hachettea and Mystropetalon were used in this study because they are sister (and basal) to the remaining genera and have lower substitution rates in SSU rDNA, thus avoiding potential long-branch artifacts. The floral morphology of Mystropetalon is less reduced than other genera in the family. Given that its staminate flowers have pistillodes (vestigial carpels) and carpellate flowers have staminodes (vestigial stamens), it is assumed this plesiomorphic condition reflects evolution from an ancestor with bisexual flowers. Floral features in common with Santalales include a valvate perianth and stamens with "typical" morphology (i.e. anther sacs and filaments as opposed to synangia as seen in Balanophora). When examining the haustorial structure of Mystropetalon, Weber  noted the presence of a collapsed zone, graniferous tracheary elements, and "runners," which he associated with Santalales. Similarly, reduction in ovules is seen in several santalalean families, particularly the mistletoes in Viscaceae where no true ovule exists. Either an embryo sac is embedded within central placenta called a "mamelon" or the archesporium develops hypodermally in the ovary, functioning directly as megaspore mother cells . Similarly, ovules have been lost in some Santalaceae (Exocarpos) and Loranthaceae. During the course of floral reduction, the loss of ovules most influenced Fagerlind  who drew a direct ancestor-descendant relationship between Santalaceae and Balanophoraceae.
As mentioned above, such apparent similarities between Balanophoraceae and Santalales have also been interpreted as convergent. Kuijt  suggests that the reduction of santalalean ovules may permit retention of meristematic activity, a requirement for the formation of complex fruits in epiparasites. For root parasites such as Balanophoraceae, he suggests ovular reduction is related to selection for tiny seeds that require host stimulants for germination, as is seen in Orobanchaceae. In addition to ovules, the androecia of both Viscaceae and Balanophoraceae are diverse with both showing trends toward the formation of synandria and porose dehiscence. Whether these trends are convergent or follow from homologous suites of genes may yield to direct testing using floral homeotic mutants.
Although herbaceous root parasites have evolved in Santalaceae (e.g. Thesium, Quinchamalium), most members of Santalales are woody. Completely holoparasitic species do not occur in this order, with the possible exception of Daenikera, an endemic monotypic genus of New Caledonia whose photosynthetic capabilities remain to be determined. As adults, dwarf mistletoes (Arceuthobium, Viscaceae) fix only about 30% of the carbon needed for growth [34, 35] but prior to host attachment seedlings actively photosynthesize. For this reason these mistletoes must be considered advanced obligate hemiparasites, not holoparasites. Thus, to include with Santalales the entirely holoparasitic group Balanophoraceae requires further investigation to identify the exact sister group.
Cynomorium coccineum, known to the Muslim world as "tarthuth," has been harvested from the deserts of north Africa and the Middle East for thousands of years. Arabs and Bedouins eat the interior portions of fresh young stems, prepare infusions of older stems to treat colic or stomach ulcers, or dry and pulverize the plant for use as a spice or condiment with meat dishes . Medicinal uses of tarthuth can be traced to Al-Kindi, Al-Razi (Rhazes), Ibn Masawayh, Ibn Wahshiya, and Maimonides but the plant became known to Europeans only in the 16th century. A group called the Knights Hospitaller of St. John operated a hospital in Jerusalem and learned of the medicinal qualities of tarthuth from local Muslim physicians. When the Crusaders lost Jerusalem to the Muslims, they moved to the island of Malta where Cynomorium was also native. The site where the "Maltese Mushroom" grew (Fungus Rock) was thereafter vigorously guarded and thieves were imprisoned or made galley slaves. The "treasure of drugs," as the Arabs called it, was used for a variety of purposes, including treating apoplexy, venereal disease, high blood pressure, vomiting, irregular menstrual periods and as a contraceptive and toothpaste.
Modern biomedical and phytochemical research on Cynomorium coccineum has demonstrated a variety of activities from plant extracts. Effects of Cynomorium extracts on mammalian reproductive cells modulation of pituitary gonadotrophins  as well as changes in testicular development  and epididymal sperm patterns  in rats. Cynomorium songaricum, known in Chinese medicine as "suo yang," has been shown to contain triterpenes with HIV protease inhibitory activity [40, 41]. Interest in herbal medicines is growing at a rapid pace, and attention has been focused upon Cynomorium as evidenced by its availability via hundreds of distributors advertising (via the internet) herbal remedies for kidney and intestinal ailments as well as for impotence. Although it is not at present clear how many original distributors of the plant exist, and whether plant material being marketed as Cynomorium is actually this plant, what is clear is that it is not being cultivated, thus authentic herbal preparations must be obtained from wild populations. Very little information exists on the cultivation of Cynomorium  and certainly commercial suppliers are not practicing sustainable harvest by cultivating this obligate holoparasite. There is evidence that overexploitation of this plant has resulted in localized extinction . For these reasons, we here raise concerns for the conservation of both species of Cynomorium and strongly voice the need to develop cultivation methodologies. Given the potential and actual biomedical applications of extracts from this plant, and conservation concerns given extensive harvesting from wild populations, information on its phylogenetic position within angiosperms is timely. Further molecular work will likely illuminate its closest relatives within Saxifragales. These taxa should then become the subject of phytochemical analyses to determine whether they also contain compounds of biomedical interest. Cultivation of photosynthetic plants would be more straightforward than the holoparasite, thus possibly relieving some of the pressure to harvest this more sensitive species from the wild.
All previous classifications have allied Cynomorium and Balanophoraceae, likely owing to the holoparasitic habit and the presence of inflorescences with numerous tiny flowers. Molecular phylogenetic analyses using nuclear and mitochondrial gene sequences both indicate that these taxa are not closely related and that perceived similarities are a result of convergent evolution. Cynomorium is strongly supported as being a member of Saxifragales, however, its exact position within this order remains unresolved. Surprisingly, both nuclear and mitochondrial genes place Balanophoraceae with the sandalwood order, a relationship previously proposed but explained by some as a case of convergent evolution. Both species of wild Cynomorium are being harvested from wild populations for use in herbal medicines, and evidence exists for overexploitation. Given that methods to cultivate these holoparasites are not being employed, we here raise conservation concerns. If further molecular phylogenetic work identifies the nearest photosynthetic relatives of Cynomorium, these plants should be examined for phytochemical activity.
Voucher information for all newly sequenced taxa is given in Additional Files 1 and 2. DNA was extracted, PCR amplified, cloned, and sequenced following reported methods [3, 43]. The nuclear and mitochondrial sequences were amplified using primers reported elsewhere [14, 44, 45]. Sequencing was conducted using an ABI Prism® 377 automated DNA sequencer (Applied Biosystems) according to the manufacturer's protocols. Two multiple sequence alignments were constructed. The purpose of the global data set was to test the position of Cynomorium and the three Balanophoraceae genera among angiosperm orders. This matrix included nuclear small-subunit (SSU) rDNA, chloroplast rbcL and atpB, and mitochondrial matR for 67 taxa with Laurus and Cinnamomum as outgroups. Balanophoraceae and Cynomorium lack the chloroplast genes rbcL and atpB, but they were included in the global data set to stabilize the overall tree topology as previously demonstrated [2, 3]. Given the strong support for a relationship between Cynomorium and Saxifragales obtained from this global analysis, a Saxifragales data set was constructed to further resolve the position of the parasites within this clade. Here the genes used were nuclear SSU and LSU rDNA, chloroplast rbcL, atpB, and matK for 62 taxa with Lea and Vitis as outgroups. In both alignments, chloroplast genes were coded as missing for the holoparasites. Accession numbers for newly sequenced genes are: AY957440 – AY957454.
Summary of output from MrModelTest for each data partition in the global and Saxifragales data sets §
Chosen Model (AICc)
Chosen Model (hLRT)
Akaike Weight of Chosen Model
Both global and Saxifragales
GTR+I+Γ (all)/HKY+I+Γ (variable)
Akaike Information criterion
ATP synthase beta subunit
general time reversible model (a model of DNA sequence evolution)
human immunodeficiency virus
invariant sites plus gamma distribution
chloroplast maturase K
mitochondrial maturase R
Markov Chain Monte Carlo (a simulation method used to approximate the posterior probabilities of trees)
ribulose bisphosphate carboxylase/oxygenase, large subunit
nuclear small-subunit ribosomal DNA
nuclear large-subunit ribosomal DNA
tree bisection-reconnection branch swapping
The authors wish to thank all who assisted this project by collecting plant samples or assisting in field work: A. Aparicio and I. Sanchez García (Cynomorium coccineum), C. Ecroyd (Dactylanthus taylori), W. Forstreuter (Benthamina alyxifolia), J.-M. Groult (Hachettea austrocaledonica), R. Narayana (Santalum album), K. Steiner and A. Wolfe (Mystropetalon thomii). Data analysis was greatly facilitated by David Swofford and Peter Foster who generously allowed access to computer clusters at Florida State University and The Natural History Museum (London), respectively. J. Bozzola and S. Schmitt at the SIUC IMAGE facility assisted in SEM photography. Financial support was provided by the National Science Foundation to DLN. The helpful comments of S. Stefanovic improved the final manuscript.
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