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With this study, we explored the identity and chemistry of fungal endophytes from the roots of yerba mansa [Anemopsis californica (Nutt.) Hook. & Arn. (Saururaceae)], a botanical traditionally used to treat infection. We compared the diversity of fungal endophytes isolated from a wild-harvested A. californica population, and those from plants cultivated for one year in a greenhouse environment. The wild-harvested population yielded thirteen fungal strains (eleven unique genotypes). Of the extracts prepared from these fungi, four inhibited growth of Staphylococcus aureus by >25% at 20 µg/mL, and three inhibited growth of Pseudomonas aeruginosa by =20% at 200 µg/mL. By comparison, A. californica roots after one year of cultivation in the greenhouse produced only two unique genotypes, neither of which displayed significant antimicrobial activity. The fungus Chaetomium cupreum isolated from wild-harvested A. californica yielded a new antimicrobial spirolactone, chaetocuprum (1). An additional 14 known compounds were identified using LC–MS dereplication of the various fungal endophytes. This study provides new insights into the identity and chemistry of A. californica fungal endophytes, and demonstrates the importance of considering growing conditions when pursuing natural product drug discovery from endophytic fungi.

The climate conditions and chemical composition of root essential oils for 17 populations of Anemopsis californica in New Mexico were examined. The objective of this study was to observe the effect of environmental conditions and management conditions on essential oil composition in different populations of A. californica. Chemical concentrations of three abundant compounds—methyleugenol, thymol, and piperitone—were determined. Maximum accumulations of each compound were associated with different mean annual temperatures, precipitation, and elevation. Similar chemical profiles were detected in root samples recollected for four populations, suggesting retention of unique chemical profiles in different populations. Vegetative propagation of wild plants under cultivated conditions did not significantly alter the chemical profile of the root essential oil. The chemical concentrations for six essential oil components of A. californica roots were determined under field conditions with varying irrigation and nitrogen (N) fertilizer regimens. The concentration of only two compounds, thymol and piperitone, was increased by increasing irrigation. The concentration of all other compounds, methyeugenol, elemicin, 1,8-cineole, and myrtenol, were independent of the irrigation rates and N fertilizer rates used in the study. These results suggest that the chemical variability observed among different populations of A. californica is primarily genetically controlled and the environmental conditions in New Mexico are conducive to the production of this medicinal plant as a high-value crop.

Isolation and characterization of leaf volatiles in Anemopsis californica (Nutt.) Hook. and Arn. (A. californica) was performed using steam distillation, solid-phase microextraction, and supercritical fluid extraction. Thirty-eight compounds were detected and identified by gas chromatography; elemicin was the major component of the leaf volatiles. While the composition of the leaf volatiles varied with method of extraction, alpha-pinene, sabinene, beta-phellandrene, 1,8-cineole, piperitone, methyl eugenol, (E)-caryophyllene, and elemicin were usually present in readily detectable amounts. Greenhouse-reared clones of a wild population of A. californica had an identical leaf volatile composition with the parent plants. Steam-distilled oil had antimicrobial properties against 3 (Staphylococcus aureus, Streptococcus pneumoniae, and Geotrichim candidum) of 11 microbial species tested. Some of this bioactivity could be accounted for by the alpha-pinene in the oil.

The perianthless members of the Piperales are unique among the basal lineages of angiosperms because they are mainly herbaceous plants with over 2000 species possessing highly reduced flowers. There have been several attemptstoaddresstheevolutionoftheflowermorphologyinthegroup,butnopreviousstudyhasincludedaDNAbased estimate of phylogeny. Here we present a robust reconstruction of the phylogenetic relationships of the genera in the perianthless Piperales using DNA sequence data from three genes (rbcL, atpB, and 18S) and two genomes(nuclearandchloroplast).Weestimatedthelikelihoodvaluesofancestralcharacterstatesofmaturefloral structures.Developmentalsequencesalsowereanalyzedusingstepmatricesunderspecificmodelsofcharacterstate change to examine the origin of meristic differences of the androecium and gynoecium. Developmental lability is the general theme in the evolution of floral merosity in the perianthless Piperales, with different developmental processes giving rise to morphologically identical mature stages. Our findings confirm this notion based on several distinctpatterns:(1)nonidenticalontogeniesgiverisetohomoplasious,six-staminateandroeciaofSaururaceaeand some Piperaceae most likely through terminal addition; (2) identical ontogenies produce the homoplasious twostaminate flowers in Peperomia and Piper umbellatum through deletion; and (3) nearly identical ontogenies produce homplasious tricarpellate gynoecia of Piper and Anemopsis þ Houttuynia clade, and the apocarpous gynoecium of Saururus may be secondarily derived, both end products occurring through deletion. We note that changesinorgannumberandthedegreeofcarpelfusionhavebeenimportantduringtheevolutionaryhistoryofthe perianthless Piperales as well as the basal angiosperms in general and should be studied more extensively. Overall we emphasize the lability of developmental pathways, especially in the flower, and endorse the methodological utility of developmental sequences for directing future investigations of floral evolution.

The amount of total phenols and flavonoids and the antioxidant activity of leaf, stem, and rhizome methanolic extracts from a commonly consumedAnemopsis californicaunder different storage conditions were investigated. Storage conditions were at 50, 25, 4, and −20°C, protected or not from light, during 180 days. The inhibition of the elastase enzyme was also evaluated. The results demonstrated that leaf, stem, and rhizome methanolic extracts ofAnemopsis californicamaintain approximately up to 97 and 95% stability in phenolic content and antioxidant activity, respectively, when stored during 60 days at −20°C in the dark. Additionally, these extracts, principally from leaf and rhizome, showed an elastase inhibitory effect by 75 and 71.8%, respectively. Therefore, this study provides the basis for further research on the anti-inflammatory activity. On the other hand,Anemopsis californicacould comprise a good alternative of use as antioxidant in foods.

Vessel elements in Anemopsis have simple perforation plates and alternate (sometimes scalariform) lateral wall pitting; Anemopsis has tracheids with large, densely placed pits. These conditions are in contrast with tracheary element features of Gymnotheca, Houttuynia, and Saururus, in which perforation plates are scalariform (many with notably slender bars) and with scalariform lateral wall pitting. Porose pit membrane remnants, which cannot be seen with light microscopy, are newly reported in Houttuynia and Saururus. These porose pit membranes underline the primitive nature of vessels in Gymnotheca, Houttuynia, and Saururus. The highly specialized vessels of Anemopsis may relate to entry into seasonally dry habitats, whereas Gymnotheca, Houttuynia, and Saururus may have experienced unbroken occupancy of mesic habitats.

ADDITIONAL INDEX WORDS. cost and return estimates, Echinacea purpurea, Valeriana officinalis, Anemopsis californica, yerba mansa SUMMARY. Cost and return estimates are presented for selected medicinal herbs grown in a plant-spacing study at two sites in New Mexico. The selected herbs were echinacea [Echinacea purpurea (L.) Moench], valerian (Valeriana officinalis L.), and yerba mansa (Anemopsis californica Nutt.). Significant returns to land and risk were observed in the crops grown at the closest plant spacing, 12 inches (30 cm). Return to land and risk after two growing seasons from echinacea was estimated for a 10-acre (4-ha) farm to be $16,093/acre ($39,750/ha) in Las Cruces and $14,612/acre ($36,092/ ha) in Alcalde.

Anemopsis californicahas been used empirically to treat infectious diseases. However, there are no antimutagenic evaluation reports on this plant. The present study evaluated the antioxidant activity in relation to the mutagenic and antimutagenic activity properties of leaf (LME) and stem (SME) methanolic extracts ofA. californicacollected in the central Mexican state of Querétaro. Antioxidant properties and total phenols of extracts were evaluated using DPPH (1,1-diphenyl-2-picrylhydrazyl) and Folin-Ciocalteu methods, respectively. Mutagenicity was evaluated using the Ames test employingSalmonella entericaserovar Typhimurium strains (TA98, TA100, and TA102), with and without an aroclor 1254 (S9 mixture). Antimutagenesis was performed against mutations induced on the Ames test with MNNG, 2AA, or 4NQO. SME presented the highest antioxidant capacity and total phenolic content. None of the extracts exhibited mutagenicity in the Ames test. The extracts produced a significant reduction in 2AA-induced mutations inS. typhimuriumTA98. In both extracts, mutagenesis induced by 4NQO or methyl-N′-nitro-N-nitrosoguanidine (MNNG) was reduced only if the exposure of strains was μg/Petri dish.A. californcaantioxidant properties and its capacity to reduce point mutations render it suitable to enhance medical cancer treatments. The significant effect against antimutagenic 2AA suggests that their consumption would provide protection against carcinogenic polycyclic aromatic compounds.

Stern and rhizome anatomy is reported for Anemopsis californica Hook., Houttuynia cordata Thunb., and Saururus cernuus L. Secondary growth is reported for the first time in Saururaceae. Cambia function indefinitely in Anemopsis in both fascicular and interfascicular areas. Interfascicular cambium is minimal in Saururus and absent in Houttuynia; fascicular cambium is present in both genera and produces a finite quantity of vessels, fiber-tracheids, and axial parenchyma but no rays. Anemopsis has vessels with simple perforation plates plus tracheids in wood, suggestive of adaptation to fluctuating water availability. The scalariform perforation plates of Houttuynia and Saururus suggest an unbroken history of occupancy of mesic habitats. Ethereal oil cells are reported for rays of Anemopsis, and for pith and cortex of the three genera studied. Stern idioblasts and other histologieal details are ineluded along with wood data in anatomical deseriptions of sterns. DNA data as weIl as maeromorphologie al data implicate Saururaeeae, along with Aristolochiaceae, Lactoridaceae, and Piperaceae as paleoherbs elose to the origin of monoeotyledons. Key wood features that unite these families are cited. Chloranthaeeae, whieh share stern endodermis and sealariform perforation plates with Saururaeeae, are also considered elose. Features of Saururaceae that are analyzed with respeet to monoeotyledon origin inelude loss of interfaseieular cambium, minimization of faseieular cambium, lack of imperforate traeheary elements in monocotyledon bundles, trimery, the sympodial habit, and production of adventitious roots.

Zantedeschia aethiopica (calla lily) and Anemopsis californica (yerba mansa) are plant species capable of accumulating arsenic (As) and therefore proposed as phytoremediation for removal of As from drinking water. The effects of a continuous 6 month As exposure (34±11 μg/L) from local contaminated groundwater on the antioxidant response of Z. aethiopica and A. californica were evaluated in leaves and stems of the plants bimonthly in a subsurface flow constructed wetland. As increased the activities of the antioxidant enzymes ascorbate peroxidase, glutathione reductase and catalase where higher levels were observed in Z. aethiopica than A. californica. No significant differences were detected on lipid peroxidation levels or antioxidant capacity evaluated by ORAC and DPPH assays or total phenol contents in any part of the plant, although in general the leaves of both plants showed the best antioxidant defense against the metal. In conclusion, Z. aethiopica and A. californica were able to cope to As through induction of a more sensitive enzymatic antioxidant response mechanism.

Anemopsis californica, commonly known as yerba mansa, is an important medicinal plant that has traditionally been used to treat a variety of digestive, respiratory and skin ailments, and has been reported to possess anticancer properties. The increase in demand for this medicinal plant puts wild populations at risk due to over-harvesting; therefore, the development of alternatives such as in vitro propagation may help reduce this pressure on natural plant resources. The aim of this work was to develop protocols for the in vitropropagation of A. californica through axillary bud proliferation and somatic embryogenesis. Shoot bud multiplication was achieved using nodal explants from axenic seedlings, reaching the highest multiplication rate (on average 15 shoots per explant) when the culture medium was supplemented with 4 mg/L kinetin. Propagated shoots produced roots in medium containing 1 mg/L indole-3-butyric acid. Best induction of somatic embryogenesis (on average 19.75 embryos per explant) was obtained from leaf explants cultured on medium supplemented with 4 mg/L naphthalene acetic acid. These protocols may be used either for the commercial mass propagation of the species, studies on the production of secondary metabolites or as a basis for the development of genetic improvement strategies. Key words: Multiple shoot proliferation, regeneration of embryos, plant growth regulators,yerba mansa.

Three different extract conditions (aqueous, EtOH and EtOAc) of four different parts (bracts, leaves, roots and stems) of the plant Anemopsis californica (A. californica) were evaluated for their effect on the growth and migration of human colon cancer cells, HCT-8, and the breast cancer cell lines Hs 578T and MCF-7/AZ. Our aim was to identify potential anticancer activity in crude A. californica extracts, given that this plant is used by Native Americans to treat a variety of diseases, including cancer. Our results demonstrated that for each of the cell lines tested, the majority of ethyl acetate extracts of all the plant parts are more toxic than the aqueous and ethanol extracts. Furthermore, significant growth inhibitory activity against the three cell lines was found for the ethyl acetate extract of the roots, while the aqueous extract of the roots influenced the migratory capacity of the three cell lines. This study provides evidence for the anticancer properties of A. californica when extracted in water and ethyl acetate, and supports the importance for further purification of the crude extracts and isolation of potential new anticancer compounds through bio-guided fractionation.

Anemopsis californica (Saururaceae) commonly called yerba mansa, is an important medicinal plant in many deserts in the southwestern region of North America. Populations of A. californica, collected throughout New Mexico, were examined for chemical variability in roots and rhizomes for select monocyclic (cymene, limonene, piperitone and thymol) and bicyclic (alpha-pinene, 1,8-cineole and myrtenol) monoterpenoid and phenylpropanoid (methyleugenol, isoeugenol and elemicin) derived essential oil components. Three distinct chemotypes were detected using a hierarchical clustering analysis on the concentration of 10 different analytes in three individuals from each of 17 populations. One chemotype was characterized by high elemicin concentrations, a second chemotype by high methyleugenol concentrations and the third by high piperitone and thymol concentrations. Steam distilled oil was used to screen for anticancer bioactivity. A. californica root oils demonstrated anti-proliferative activity against AN3CA and HeLa cells in vitro but no activity against lung, breast, prostate or colon cancer cells. The IC(50) values for the root oil were 0.056% and 0.052% (v/v) for the AN3CA and HeLa cells, respectively.

Phylogenetic relationships of the six extant species of four genera of the Saururaceae are resolved based on sequence data: 18S and ITS from the nuclear genome; rbcL and trnL-F from the chloroplast genome; and matR from the mitochondrial genome. Zippelia begoniaefolia, from a genus of Piperaceae, is used as an outgroup. Results are presented in separate and combined analyses of sequence data. Forty-nine morphological characters reconstruct the phylogeny in this family, again using Zippelia as outgroup. Whether the topologies of Saururaceae are based on individual genomic data sets, the combined DNA sequence data sets, morphological data sets, or the combined DNA sequence and morphological data sets, all are closely congruent. In all analyses, the monophyly of Saururus and Gymnotheca, respectively, is strongly supported, and the sister relationship between Gymnotheca and Saururus is well supported. In the analysis of nuclear DNA data sets, Anemopsis is the sister group to all other Saururaceae, with Houttuynia then sister to Saururus and Gymnotheca, and with Saururas sister to Gymnotheca; however, in the analyses of the other data sets, Anemopsis is the sister group of Houttuynia, and the Anemopsis-Houttuynia clade lies sister to the Saururus-Gymnotheca clade. The result that the Anemopsis-Houttuynia clade comprises the sister group of Saururus-Gymnotheca clade is novel and differs from previous phylogenetic opinion.

All flowers of Anemopsis californica, the most specialized taxon of the family Saururaceae, are initiated as individual primordia subtended by previously initiated bracts, in contrast to the common-primordium initiation of all flowers of Saururus cernuus and of most flowers of Houttuynia cordata. Floral symmetry is bilateral and zygomorphic, and the sequence of initiation among floral parts is paired or whorled. In A. californica, the six stamens arise as three common primordia, each of which later bifurcates to form a pair. The three common primordia occupy sites corresponding to the positions of the three stamens in H. cordata flowers. In Anemopsis, the filaments of each pair are connate. Each stamen pair is vascularized by a single bifurcating vascular bundle. The three carpels per flower are usually initiated simultaneously although there may be some variation. Adnation between stamens and carpels results from zonal growth. Downward extension of the locule, and proliferation and expansion of receptacular tissue and inflorescence cortical tissue around the locule below the bases of the carpels produce the inferior ovary. The inflorescence terminates its activity as a flattened apical residuum, surrounded by

Karyomorphological observations were carried out on three genera belonging to the Saururaceae and four genera of the Piperacea. All of the genera of Saururaceae show the same karyomorphological characteristics from interphase to metaphase in the somatic cell divisions. However there are two types of the karyomorphology in Piperaceae, i) the first type observed inPiper, Pothomorphe andZippelia, and ii) the second type inPeperomia. Each group corresponds to Thorne's two subfamilies (1974, 1976), Piperoideae and Peperomioideae. The basic chromosome numbers of the genera are confirmed or newly proposed as follows:Saururus x=11,Houttuynia x=12,Anemopsis x=22 (Saururaceae),Peperomia x=11,Piper andPothomorphe (=Heckeria) x=13,Zippelia x=19 (Piperaceae). The relationships of these basic chromosome numbers are presumed to be as shown schematically in Fig. 4. The original basic chromosome number of the common ancestral stock of Saururaceae and Piperaceae is presumed to be x=11.

1. The flower of Anemopsis californica, subtended by a small floral bract, consists of six stamens, three carpels, and an ovary completely buried in the inflorescence axis. 2. The vascular supply of the individual flower and its bract originates in a single bundle of the ring of bundles in the inflorescence axis. This bundle splits up into seven: the bract bundle and three stamen bundles alternating with three placental ones. 3. Above the ovary the stamen and placental bundles branch and anastomose in such a way as to provide one bundle for each stamen and three for each style. 4. The ovule is orthotropous, with two integuments and an eventually massive nucellus. The outer integument is initiated first. Both integuments exhibit certain differentiations in their cell layers. 5. The megaspore mother cell, which with two parietals arises from a hypodermal cell, divides twice, but a wall is formed only after the first division. 6. The pair of megaspores in the upper cell degenerate and the megametophyte is fo...

The constituents of the hydrodistilled essential oil from the roots and rhizomes of Anemopsis californica have been examined. Thymol was identified from the phenolic fraction of the oil. Gas chromatography on Hyprose SP-80 followed by collection of effluent fractions and comparison of their IR spectra with those of pure samples confirmed the presence of methyleugenol as the major constituent. A third constituent of the oil was found to be piperitone. Methyleugenol (approximately 55 percent v/v of the oil), thymol (13 percent), and piperitone (5 percent) together make up approximately 74 percent of the oil. A fourth constituent, tentatively identified as an aromatic unsaturated ether, and other constituents are being investigated.

Anemopsis californica was the subject of a phytochemical and pharmacological investigation to determine the plant principle responsible for its reported antispasmodic activity. The extraction procedure yielded among various fractions, leucoanthocyanidins, an unidentified crystalline fraction, and 4-allylveratrole, which was the most active antispasmodic agent. The possibility of antitumor activity attributed to this plant could not be verified by the screens employed.

The minor constituents of the essential oil hydrodistilled from the roots and rhizomes of Anemopsis californica (Nutt.) Hook and Arn. (Fam. Saururaceae) have been isolated by gas chromatography on two stationary phases: polypropylene glycol adipate and polyethylene glycol. The isolated compounds were identified by the comparison of their LR spectra with those of pure authentic compounds. Esdragol, thymolmethylether, linalool, p-cymene, 1,8-cineol, d-limonene, camphene and α- and β-pinene have been shown to be present in the oil. In addition, the presence of two ketones, a sec alcohol, tentatively identified as 1-methyl-4-isopropyl-6-hydroxy-bicyclo (3:1:0) hexane, an ester, and six hydrocarbons is also indicated on the basis of the IR spectra of the corresponding fractions.

Light petroleum extract of the roots and rhizomes of Anemopsis californica (Nutt.) Hook and Arn. yielded a compound identified as (+)-asarinin from its spectral and other analytical data.

Writers, in their descriptions of Anemopsis californica, have differed as to the pubescence of the plant, when they mentioned it at all. Hooker and Arnott (Bot. Beechey Voyage, 390) do not describe the pubescence but the figure (Tab. xcii) shows a moderately dense, long-villous pubescence on scape and petioles and extending onto the basal margins of the leaves. Nuttall (Ann. Nat. Hist. 1: 136. 1833) describes the scapes and petioles as being pubescent. Casimir De Candolle (Linnaea 37: 333. 1871) described A. Bolanderi as glabrous, petioles 8 in. long and sheathing for one-third their length. The same writer (Prodromus xvil, 237.) described the leaves of the typical form as ciliolate on the bases and the petioles as pilose. Watson (Geol. Surv. Cal. 2: 78. 1880) says the plant is "more or less floccose".

The present paper deals with certain aspects of morphology and anatomy of the Saururaceae. Floral anatomy and embryology suggest a close relationship among the genera. In floral anatomy the genus Saururus is the least specialized and Anemopsis the most highly specialized. Structure and morphology of inflorescence and also flower suggest the manifestation of certain evolutionary trends, probably guided by reduction, cohesion and adnation. Origin of inferior ovary in the Saururaceae is discussed. Carpels and nature of placentation show certain primitive features. Origin and evolution from apocarpous condition to syncarpous condition of carpels are briefly discussed. Further details will be presented in a separate paper. M. V. S. RAJU, Department of Biology, University of Saskatchewan, Saskatoon, Saskatchewan, Canada.