The isolation and characterization of the active compounds in bryophytes has largely been conducted by Dr. David G. I. Kingston, a Distinguished Professor at the Virginia Polytechnic Institute and State University (VPISU) in Blacksburg, Virginia, and to a limited extent by Dr. John Cassady, Chairman at Purdue University and later Dean at Ohio State University (now at Oregon State University), and to a much lesser extent by chemists at the University of Illinois at Chicago.  Additionally Dr. Kingston screened several hundred species during 1989–1991, employing engineered yeast bioassays.  These later investigations were summarized by R. W. Spjut, D. G. I. Kingston and J. M. Cassady (1992), Systematic screening of bryophytes for antitumor agents, Tropical Bryology 6: 193–202.

     Further details on the collection of bryophytes for antitumor screening are discussed below, chronologically and by Institution.

Bryophytes collected for the National Cancer Institute (NCI) prior to 1982.

    As reported by Spjut et al. (1986), the first discovery of significant biological activity in bryophytes was KB (an in vitro assay) activity in a sample of ~500 g of the moss Polytrichum ohioense, one of nine mosses collected for antitumor screening during April 1976 (but only 7 were accounted for in screening) from deciduous forests on the Beltsville Agricultural Research Center (BARC) and adjacent forested lands extending to Laurel, Maryland.  It was not until about a year later when a quarterly Cumulative Plant and Animal Materials (CPAM) reported the KB activity—in the average time span for extraction and preliminary screening after samples were shipped to the extraction lab in Madison, Wisconsin (RALTECH).  This report did not show the actual screening data; it only listed plants and animal products with confirmed activity; however, screening data for bryophytes were later reviewed by Spjut et al. (1986).  The inactive bryophyte samples collected by Spjut in 1976 were Callicladium haldanianum, Ceratodon purpureus, Dicranum scoparium, Leucobryum albidum, L. glaucum, Polytrichum commune, and Thuidium erectum(synonym T. delicatulum).  Samples from three other species, Bryum caespiticum, Leucobryum glaucum and Plagiomnium cuspidatum, two of which were also collected on BARC in 1976, were not mentioned in Spjut et al. (1986); they may have not been screened (SPJ-4300 numbers).  These collections were experimental to determine whether bryophytes were a promising source for new antitumor compounds, and if so to what extent large quantities were required for isolation of active agents.

      This experiment came to a quick end, but only temporarily.  Spjut, after collecting samples from ten species of moss, was asked to not to collect any more bryophyte samples because the Chief of the USDA Medicinal Plant Resources Laboratory, Robert E. Perdue, Jr., feared that if a novel compound discovered in a bryophyte species reached clinical studies, it would be impractical to recollect 20,000 kg; thus, from this point of view a lot of money could be spent on screening without being able to advance the (bryophyte) compound to clinical trials.  The USDA Medicinal Plant Resources Laboratory was in the process of procuring ~18,000 kg (40,000 pounds) of Maytenus buchananii (Celastraceae) stems from the Shimba Hills in Kenya for isolation of 8 g of maytansine.  Ironically, active maytansinoids were later discovered in mosses— Isothecium, Thamnobryum, Claopodium and Anomodon (Sakai et al. 1988; Suwanborirux et al. 1990).

     The KB activity in Polytrichum ohioense was not considered a priority for recollection.  Preliminary screening criteria at that time stressed in-vivo activity—in the P-388 Leukemia assay. 

     In 1979, Spjut, while collecting in southern Baja California Sur (Mexico) for the National Cancer Institute's anticancer screening program, felt he had to immediately go to McKinleyville, California when he learned his father, who had terminal cancer, was not expected to live more than another week.  Since he did not want to risk leaving a rented vehicle unattended in Mexico to fly to McKinelyville, as well as 150 samples that he had collected thus far, he arranged to ship the samples to Maryland and obtained permission to drive to northern California, a distance of nearly 2,000 miles, collecting along the way (Spjut et al. 1988).  The leadership of the Medicinal Plant Resources Laboratory, which had a changed in May 1978 (from Robert Perdue to James Duke, Chief and Arthur Barclay, Leader of the Cancer project, in concurrence with Matthew Suffness, Chief of the NCI Natural Products Branch; Spjut et al. 1988), approved Spjut conducting field work in northern California, provided that samples he collected were according to current guidelines, which were not to collect SLOP (Species Low On Priority—see Spjut 1985).  Spjut further suggested he could increase the novelty in his samples by collecting bryophytes. The program leaders also approved the collection of bryophyte samples, but they also expressed concern for recollections if activity was discovered. 

     A USDA accession record and shipping list detailed 17 bryophytes collected in northern California during March 1979, along with samples of vascular plants from California and Nevada, and one moss sample from Baja California Norte, Homalothcium arenarium.  The 18 bryophyte samples were shipped to RALTECH (from Beltsville, MD) on May 18, 1979.  It might be noted that a new extraction procedure adopted in 1976 required more sample weight, an increase from 1.00 lb. to 2.75 lbs (dried).  This new procedure—that added a "simple solvent fractionation" step—appeared to detect more active species based on study of 500 samples from many areas in the world (Statz & Coon 1976) and a retesting of previous samples, which included many collected by Robert Perdue, Jr. from the Southern Highlands of Tanzania.  Especially active were stem-bark samples (Spjut Memorandum 1979). Estimating dry weight of 2.75 lbs from fresh material is not easy, while finding bryophytes available in that quantity was even more difficult.  There were two lighter weight samples among the 18 moss samples, Orthotrichum lyellii (2.25 lbs.) and Hypnum subimponens (2.00 lbs.).

     Among the 17 moss samples collected in northern California were many samples of vascular plants that were collected for various reasons.  One example was stem-bark (sb) of tan oak, Lithocarpus densiflorus.  Spjut in reviewing previous plant collections for the NCI from the western United States found that bark and root had rarely been collected, while native Indians often employed these plant parts in medicinal remedies.  Also, he did not collect a twig-leaf sample of tan oak because undoubtedly these parts had been previously collected by other botanists before him.  There were also samples of Petasites palmata and Buddleja utahensis collected for special screening based on chemotaxonomic and folklore data; such samples were assigned to the PR-80,000 series.  The bulk of the samples were from Baja California—shipped to RALTECH after the bryophyte samples because the shipper in Mexico had not shipped them to Maryland—until assistance from the American Embassy in Mexico City led them to personally contact the Baja shipper who was "persuaded" to ship the samples. Thus, the 17 bryophytes samples from northern California represented about 10% of several hundred samples that Spjut had collected from states of Baja California Sur, Baja California Norte, California and finally Nevada during February and March 1979.

     Only a relatively short time later, January 1980 (9 months), when Matthew Suffness. upon reviewing the monthly reports of screening results noted significant P-388 activity results (TLC > 150) from an extract of Claopodium crispifolium (Thuidiaceae).  Dr. Suffness felt such activity was likely due to a novel compound—as he expressed in a memo he sent to the Economic Botany Laboratory—requesting that a recollection be given high priority.  The policy at that time for recollections was 100 pounds for low priority, 300 pounds for medium priority, and 500+ pounds for high priority. Details on the procurement of Claopodium crispifolium are provided under a separate web page, claopodium.htm.  Within the following month, another moss species, Plagiomnium venustum, was also found to have P-388 activity with a TLC between 130 and 150%; it was assigned medium priority for recollection.

    The discovery of P-388 antileukemic activity in Claopodium crispifolium led to more collecting and screening of bryophytes during 1980 and 1981 (Spjut et al. 1986).  In retrospect, the collection of bryophytes in quantities of 1-2 kg from several hundred species in the United States was a remarkable achievement in itself (e.g., USDA accession record and shipping list for bryophyte samples).  Today, it does not appear feasible to repeat such a collection of bryophytes because, as discussed below, the abundance of bryophyte species appears to have diminished dramatically since the 1980's.  Additionally, there was an extraordinary amount of work involved in removing soil from samples.  Bryophyte samples often had to be laid out on plastic to facilitate the separation of soil, dead leaves from vascular plants, root and rhizomes, and gravel, a very dirty and tedious job (see Polytrichum pallidisetum).  Metal screens were also employed to further remove soil and rock. While some species took as little as 5 minutes to gather in sufficient bulk needed for 1-2 kg (dried), four or more hours were often required to remove the debris from the sample. In cleaning a recollection of Polytrichum ohioense, for example, it was estimated that ~500 pounds of dirt were removed to yield just 6.75 lbs of dried clean moss (Spjut ltr to Cassady, 22 Oct 1986).  Even after extensive cleaning, bryophytes were still found to have microscopic organisms; for example, P. ohioense after cleaning was reported to have colonies of green algae among the leaves (Spjut ltr to Cassady, 29 Oct 1984).  Nevertheless, if activity was due to contaminants, one might expect a more random distribution of antitumor activity among bryophyte species, but as shown by Spjut et al. (1986), the occurrence of activity in species such as Polytrichum ohioense and P. pallidisetum in the Polytrichaceae, and Anomodon rostratus, A. attenuatus, and Claopodium  crispifolium in the Thuidiaceae suggested that activity was due more to the moss than to some contaminant.  Another example is the near absence of activity in the moss family Grimmiaceae except Racomitrium sudeticum that showed only marginal activity in two separate samples from widely separated geographical areas, California and New Hampshire.  Other examples are described in Spjut et al. (1986).

     In October 1981, while Spjut was in Western Australia, the NCI terminated its agreement with the USDA Economic Botany Laboratory, an agreement that had begun in 1960 under the New Crops Research Branch (Spjut 1985).  In winding down the program, only recollections of active plants were wanted.  Spjut, after returning from Australia, arranged to undertake an exploration trip to Mexico for recollections of active plants, especially those from Baja California that he had collected in 1979 and 1980.  None of five moss species collected from Jalisco and one from Baja California Norte were active, only vascular plants were targeted for recollection. The American Embassy assisted in obtaining the permit.  However, during the spring of 1982, it became apparent from an ARS reorganization, that a former lab chief who had objected to Spjut's collecting bryophytes had evidently convinced others that travel money should not be spent on plant collections for cancer research (even though there was money for the recollections, there was the "travel ceiling" factor).  Spjut's travel plans to Mexico were terminated.  Consequently, the collecting permits were returned; see memoranda and reports referenced under Baja California plants screened.  Spjut was also asked to supply another recollection of Plagiomnium venustum, but plans for this recollection were also terminated by the ARS management.

     Spjut then decided to collect Poytrichum ohioense.  This was available on the Beltsville Agricultural Research Center.  Obtaining  this recollection obviously would not impact any "travel ceiling." During the spring of 1982, Spjut collected ~18 kg of this moss over a period of several months.  It was one of the last recollections Spjut made for the NCI under the 22-yr cooperative agreement ARS had with the Agricultural Research Service.

Bryophytes collected for the University of Illinois at Chicago, 1983—Plagiomnium venustum

     Although the NCI had temporarily suspended screening of crude extracts from natural products, Spjut had obtained a memorandum of understanding from the Chairman of the Plant Genetics and Germplasm Institute that he would be allowed to make recollections if he could get his travel paid for.  Also, the ARS had an agreement from the Chief of the Plant Taxonomy Laboratory, Robert E. Perdue, Jr., that he would not get involved in the cancer program; instead, he would devote his full time effort to the acquisition of germplasm for crop improvement.  But that was not case.  During the early spring of 1983, Monroe Wall at the Research Triangle Institute in North Caroline submitted a request for Spjut to obtain recollections of KB active plants from Western Australia, in which the screening results for some of Spjut's 1981 collections were published in the Journal of Natural Products (1983).  Perdue, instead of forwarding Wall's request to Spjut, asked Spjut to prepare a memorandum addressed to him on the subject of where in Western Australia each species occurred and where it could be recollected in quantity.  Spjut also had submitted a request to publish a paper in Economic Botany on the limitations to collecting plants for cancer research; this request was denied.  As a result, Spjut felt he had no choice but to go to the outside.  World Botanical Associates was thus created.  It was a legal partnership with partners Richard Spjut and Charles Edson.  Spjut subsequently obtained permission from the USDA to conduct plant explorations and publish papers outside of the USDA, the first of which was on the limitations to collecting plants at random that appeared in Economic Botany (1985).

     The first samples obtained by the WBA (World Botanical Associates) were recollections of a moss, Plagiomnium venustum, for Charles Phoebe and Doel Soejarto at the University of Illinois at Chicago.  In July 1983, Spjut and Edson returned to the original collection site near Willow Creek in Humboldt Co., CA and obtained a small recollection (WBA-1).  A second recollection was obtained from a new site along Maple Creek in Humboldt Co. (WBA-2).

     As Spjut later learned, these recollections were not just for isolation of the active agents.  Drs. Soejarto and Phoebe were looking for associated organisms in the bryophyte samples that may be responsible for the antitumor activity. In August 1984, they shared copies of their correspondence with Timo Koponen, a specialist in the taxonomy of the Mniaceae.  Their correspondence revealed that 16 samples they screened of Mniaceae—collected by Timo Koponen in Norway since the summer of 1982—gave negative results, in contrast to a sample of Plagiomnium venustum collected by Spjut that was active. However, the Timo Koponen samples weighed only 15 to 40 grams compared to the ~1.5 kg sample and the ~80 kg recollection (180 lbs, PR-53630) of Plagiomnium venustum collected by Spjut and Yakko Koponen—from near the town of Willow Creek, California.

     The correspondence shared by the University of Illinois at Chicago also revealed that they found other bryophytes in the WBA samples of Plagiomnium venustum (WBA-1 & 2), referred to as contaminants.  This included the mosses Metaneckera menziesii and Claopodium crispifolium and liverwort, Porella navicularis at the Willow Creek site (WBA-1), and mosses, Homalothecium pinnatifidum and Antitrichia californica at the Maple Creek site (WBA-2). A sample of Metaneckera menziesii that Spjut had collected at the Willow Creek site in 1979 showed only marginal KB activity (Spjut et al. 1986), no P-388 activity, while a sample of the liverwort Porella navicularis gave negative results (but other samples of this species later screened by Kingston at VPISU were active in genetically engineered yeast strains).  Similarly, samples of Homalothecium pinnatifidum and Antitrichia californica collected earlier by Spjut were negative (Spjut et al. 1986).  Thus, these contaminants were not likely responsible for the anticancer activity in the original Plagiomnium sample.

    It might be further noted that the recollection of Plagiomnim venustum was laid out to dry at Dan Norris' residence. He and his wife, Carol Norris, packed and shipped the sample to Beltsville Maryland from where it was later sent to Norman Farnsworth at the University of Illinois at Chicago.  Spjut has always been impressed with Norris' abilities in the field to discern species of not only bryophytes but also vascular plants, particularly being able to identify species of Carex in their vegetative condition; moreover, with his excellent visual acuity he had an uncanny ability to identify bryophyte species at considerable distance.  He had also traveled and published extensively with Timo Koponen on bryophytes of New Guinea, and is the foremost authority on California mosses (Norris & Shevock 2004).  If there was any unusual bryophyte contamination in the sample of Plagiomnium venustum, Norris would have certainly seen it and removed it.  Dan Norris' assistance and interest in bryophyte collections for the NCI antitumor screening program is further evident in Carol Norris' romantic novel, Lost Letters, where she writes about a botanist named Brandy who recounts the dialogue of questions commonly encountered from airport clerks when shipping large samples of mosses air freight to the National Institutes of Health, and who had to go out early in the morning hours looking for Plagiomnium to avoid conflicts with teaching schedules (C. Norris 1985).

     The University of Illinois chemists may have learned of frequent antitumor activity in Mniaceae from the NCI screening monthly reports, which were summarized by Spjut et al. (1986) for all bryophytes; the Mniaceae were represented by eight species screened, five of which showed activity (Spjut et al. 1986).   The University of Illinois correspondence file also indicated that ~50 hours were spent cleaning 16 tiny samples supplied by Koponen from Norway.  The university chemists also felt that the Plagiomium samples Spjut had supplied were heavily contaminated with rhizomes of the fern Polypodium glycyrrhiza.   What these chemists had not realized, however, was that Spjut had earlier accounted for this in his recollection of Claopodium crispifolium, and that he had continued to pursue other possible contaminants in his bryophyte collections.  As he reported by USDA Memorandum, and elsewhere (Spjut et al. 1986, 1988, 1992), the rhizome of this fern, which had been removed entirely from a 50 kg (110 lb) sample of Claopodium crispifolium, constituted ~1.5% of the sample by weight.  This was reiterated in his letter to Doel Soejarto of September 4, 1984, indicating that samples of the fern rhizomes were screened with negative results. In regard to the occurrence of other bryophytes in the sample of Plagiomnium venustum, this was estimated at < .001%.  Thus, Spjut felt that it was not really necessary to spend a fortune on obtaining pure bryophyte samples.  Instead, Spjut's focus was on reproducing activity that would lead to discovery of new antitumor agents, whereas the association of other organisms in bryophytes was more of academic interest. As Timo Koponen explained to Dr. Soejarto in one of his letters, "Mniaceae commonly grow mixed with other mosses."  Indeed, Spjut et al. (1992) stated that "a bryophyte sample might be viewed as a whole community of organisms dominated by a particular species of moss, liverwort or hornwort."

     Bryophytes collected for John Cassady at Purdue University, later at the Ohio State University, 1984–1992.

    Polytrichum ohioense and P. pallidisetum. In addition to requests received from Charles Phoebe for Plagiomnium venustum, we (WBA) also received requests from John Cassady for more Polytrichum ohioense.  This species generally occurred in rounded tight clumps that were scattered in mixed pine and hardwood forests, often just inside the forest edge.  The best material on the Beltsville Agricultural Research Center had already been collected in the spring of 1982, primarily on soil over broken bottles along an old agricultural road near Glendale MD; it appeared that littered bottles that had accumulated at one time in one small area had become naturally or intentionally covered by soil, and perhaps over a period of 30 years, a dense growth of Polytrichum ohioense had developed.  Gloves had to be worn in gathering the moss at this site because of the sharp glass, the moss often growing inside the lower half of broken wine bottles and other glass jars filled with soil.  The loose soil in this particular environment seemed to make it easier to obtain relatively clean moss; a total of ~18 kg were collected.

     In 1984, Cassady requested more P. ohioense.  An additional 6.75 ("7.5") pounds (WBA-6) of Polytrichum ohioense could only be obtained near the first recollection site; thus, it was necessary to find a new site.  Another 19. 5 lbs (WBA-5) were collected along the eastern shore of Maryland near the state line of Delaware.  Because of the concern raised by Charles Phoebe and his associates on contaminants in Plagiomnium venustum, Cassady was also sent samples designated as not cleaned and partially cleaned just in case they wanted to experiment further on whether activity was mostly in the dirtier samples (WBA-15, 16).  Another small sample was supplied from Pennsylvania.

     In 1989, Cassady requested another recollection of Polytrichum ohioense—100 pounds. The WBA supplied 45 kg from the eastern shore of Maryland.

     A number of papers were eventually published on the active agents of Polytrichum ohioense, the first of which appeared seven years after the initial recollection (Zheng et al. 1989).  The novel benzonaphthoxanthenones were named "ohioensins," the chemical name adopted from the epithet name of the moss, which was for the state of Ohio.  Ironically, Cassady had accepted a position as Dean of the College of Pharmacy at the Ohio State University.  Spjut recalls being asked about what he thought about the new name for P. ohioense active compounds while attending the 30th Annual Meeting of the American Society of Pharmacognosy in San Juan, PR; he suggested that compounds should reflect the genus name rather than the epithet. But obviously Cassady preferred the epithet, a methodology many other chemists follow in naming new compounds isolated from natural products.

     In contrast to the KB activity originally reported for P. ohioense in Maryland, it was also indicated to be active in P-388 from a sample collected in New Hampshire (Spjut et al. 1986); however, Spjut subsequently regarded the New Hampshire sample as P. pallidisetum.  After collecting extensive quantities of P. ohioense in Maryland, and conducting floristic forays in the White Mountains of New Hampshire and Maine for bryophytes during 1982–1985, P. pallidisetum was usually found to be slightly larger and as a result easier to collect clean.  Its mats are similar to those of P. ohioense, also occurring in patches but in coniferous forests instead of hardwood forests, and this was in contrast to less sharply defined mats of P. commune with more loosely connected individual plants.  Samples of P. pallidisetum were best collected in habitats where other Polytrichaceae were less common (WBA 1990 Memorandum).

     The first recollection of Polytrichum pallidisetum was sent to Cassady in October 1984 (WBA-16, 5.5 lbs.).  Later recollections included 8.5 lb from Vermont (WBA-455) and 4.5 lbs from the state of New York (WBA-456).  In 1990 Cassady requested 100 pounds.  The WBA employed Patricia Gilliland for this large recollection in which Spjut and Gilliland managed to obtained 74 lbs from the White Mountains National Forest in Aug 1990 (WBA-1215, S & G 11779).  A small supplementary sample, 805 g, was collected from the Smoky Mountains in Tennessee in Oct 1990 (WBA-1287, SPJ-11840-41).  Although P. pallidisetum was easier to obtain clean, a great deal of time was still spent laying out the moss on plastic and screening out the dirt; see Polytrichum page. 

     All recollections of P. ohioense and P. pallidisetum were active (Spjut et al. 1992). Despite the close taxonomic relationships between P. ohioense and P. pallidisetum, the active agents in the latter species included novel ohioensins, and also novel cinnamoyl bibenzyls referred to as pallidisetins (Cassdy et al. 1990, Zheng 1990; Spjut et al. 1992).  The pallidisetins, which may be derived from a different biosynthetic pathway, showed activity in RPMI-7951 and U251MG cell lines (Spjut et al. 1992). 

Anomodon attenuatus, A. rostratus and A. viticulosus.  Three samples of A. attenuatus were screened during the NCI-ARS program.  Two were active in P-388, one moderately from Tennessee and another marginally from West Virginia, both collected by Spjut.  A third inactive sample collected by C. B. Jones from Missouri was extracted under a procedure used earlier in the NCI program.

     Anomodon attenutatus was the most available species in the genus for large recollection.  The best sites were boulders on steep rocky slopes of hardwood forests in West Virginia, although collectable quantities were sometimes found near base of hardwoods.  Anomodon rostratus was primarily collectable from base of old hardwoods at widely scattered places, but occasionally collectable on rock.  It was less predictable in its occurrence than A. attenuatus.

     Because the best activity in Anomodon was from a sample in Tennessee, Spjut returned to the original site and nearby areas in the Smoky Mountains in Tennessee and North Carolina in August 1984 to collect small quantities for comparative purposes.  Samples were cleaned and then examined under a microscope for the presence of algae and sorted according to frequency of algae in the samples (WBA-47-57).  There were 6 samples of A. attenuatus, ranging in weight from ~300 g to 1.3 kg, and 4 samples of A. rostratus, ranging in sample size from ~100 g to 1 kg.  Additionally, one large sample of ~9 kg of A. attenuatus was collected in West Virginia.  In 1985, Cassady requested more material of A. attenuatus and A. rostratus.  Another ~8 kg (WBA-224, SPJ-9306) of A. attenuatus was obtained from West Virginia in September and October 1985 along with 1 kg of A. viticulosus (WBA-225, SPJ-9307), and in 1986 additional recollections were obtained—1 kg of A. rostratus (WBA-457, SPJ-10151 and ~50 kg of A. attenuatus from which ~25% of dead moss and soil were separated as another sample (WBA-459).  Despite the experimental effort to screen Anomodon samples according to algal content and other materials (dead moss and soil), the screening results reported primarily on the isolation of ansamitocin P-3 from A. attenuatus (Suwanborirux et al. 1990; Cassady et al. 1990; Spjut et al. 1992).

Bryophytes collected for David G. I. Kingston at Virginia Polytechnic Institute and State University.

     Perhaps the most extensive screening on bryophytes for antitumor activity has been done by Dr. Kingston at Virginia Polytechnic Institute and State University (VPISU) in Blacksburg, surpassing that done earlier under the NCI program.  Not all recollections will be discussed as it still appears, even after 22 years, some of the bryophytes remain of interest for further study, particularly those screened during 1989–1992.  Most species mentioned below are in regard to screening in older bioassays—KB, ASK and P-388—that now appear outdated and of academic interest.

     Bryophyte samples collected 1984–1988.  Initially, Kingston was sent recollections of nearly all bryophytes that were active (Spjut et al. (1986), which had not already been assigned to Cassady or Farnsworth during the NCI-ARS procurement era (1960–1982).  The first shipment, July 1984, contained seven recollections representing six species; included were 1–5 kg samples of Pleurozium shreberi, Diphysium foliosum, Dicranum fuscescens, Bazzania trilobata, Hylocomium splendens and Dicranum fulvum (WBA 7–13). 

     A second shipment in October 1984 (WBA 17–46) included 11 bryophyte recollections in 1–4 kg amounts, and also 18 general samples of lichens in 100–300 g quantities with some exceptions, and one marine alga sample collected by Spjut and Edson in northwestern Australia along Shark Bay in August 1981.  Four of the moss samples were collected by Chuck Edson in Oregon.  He was given voucher material to work with and obtained other information at the University of Oregon herbarium where he consulted with Professor David Wagner who was very helpful in providing access to herbarium material and suggesting good locations on where to find active bryophyte species of interest in Oregon.  Edson then conducted exploratory field work, collecting some material for later identification by Dave Wagner, or by Spjut, and then obtained a recollection.  The identification of Edson's recollections were rechecked by Wagner before shipped to Kingston.  The lichens collected by Spjut were identified by Mason Hale at the Smithsonian Institution, and the one marine algae was identified by another specialist at the Smithsonian, James Norris.  The lichen samples were considered exploratory as was the case with bryophytes collected by Spjut in 1976.  One of the Lecanora samples did show activity.  The small quantity in the original sample justified further screening of lichens by the NCI during 1985 and 1986, initially against HIV and later in antitumor cell lines.

     In January 1985, Kingston reported KB activity in 9 bryophyte species that were previously reported active in Spjut et al. (1986) and two new species were found active, Peltigera, a lichen, and the moss Hylocomium brevirostre.  It was encouraging to learn that Hylocomium splendens showed KB activity at three different sites, which were in three different states, Oregon, Maine and West Virginia; the West Virginia location was also the site for P-388 activity reported in Spjut et al. (1986). Another 4 kg of H. splendens was supplied from the West Virginia site in Oct 1986.  Recollections of this species were also supplied to Alice Clark at the University of Mississippi for isolation of active antibiotic agents.

     Another species of moss that showed KB activity was Diphysium foliosum.  It was collected at the same site in Maine where it was first reported to have PS activity (Spjut et al. 1986).  Later recollections were obtained from New York.  This species was of special interest because of soil that could not be easily separated from the moss.  It was also the first strong evidence of any activity that might be associated with soil microbes as suggested by Spjut in a letter to Kingston, 17 October 1986.  In that letter, Spjut further mentioned that possible contamination might be considered in the lichen recollections that were found to be associated with soil and mosses of the Mniaceae and Pottiaceae.

     Another species, Dicranum fulvum, that showed marginal activity in P388 from one of two samples screened by the NCI from two different locations, was reported by Kingston to show KB activity in the 1984 recollection.  A second recollection in 1986 from New Hampshire was also active with activity concentrated in one fraction.  A third recollection was obtained in 1988.

     Other KB actives reported by Kingston included Porotrichum bigelovii, Ptilium crista-castrensis, and Thamnobryum alleghaniense.  Porotrichum bigelovii also had showed moderate activity in P388 (Spjut et al. 1986).  Of four species of Neckeraceae previously screened, three were active (Spjut et al. (1986), and recollections for two were also active.

     A liverwort, Bazzania trilobata, which showed KB activity from New Hampshire was previously active in only one of three samples; the original active sample and active recollection came from the same site in New Hampshire.

     Recollections that failed to show activity included Pleurozium shreberi, one from Maine, and another from New Hampshire.  However, this species was only marginally active in one of three samples collected by Spjut for the NCI, while also negative for one earlier sample collected by Lloyd Spetzman from Alaska in 1969, extracted under an older procedure.

     The overall screening results by Kingston appeared consistent and encouraging, although active compounds had yet to be isolated.

Bryophytes collected 1989–1992.

     In 1988–1989, Kingston applied for funds to screen up to 500 species of bryophytes.  In contrast to collections obtained for the NCI in 1-2 kg quantities, it was becoming difficult to supply bryophytes in 0.5 kg quantities.  We had already found that we could get by with as little as 25 g for lichen samples, so it was suggested we work with smaller size samples for preliminary screening of bryophytes.  The new sample size was 100 g as proposed in a WBA letter of late November 1988.  Dan Norris who helped Spjut tremendously during 1980-81 in obtaining general samples and recollections from California was anxious to help again.  This time we collected in Oregon as well as California.  And as before, Spjut collected alone in the eastern United States.

     Bryophytes vanishing from our forests.  The difficultly in collecting bryophytes in quantities seemed to be due to less luxuriant growth for many species. For instance, a 5.75 lb. sample of Scleropodium obtusifolium ( (PR-50957)) that was collected for the NCI in 1979 (Spjut et al. 1986) from just west of the town of Willow Creek was recalled to have been obtained within less than 5 minutes and within a few meters along a stream.  Upon returning to that site 8 years later, Scleropodium could not be found.  One explanation might be related to a nearby US Forest Service campground where campers, over time, may have impacted the survival of Scleropodium by bathing, washing clothes, and cookware in the stream, or perhaps logging activity on the hill above the road may changed the chemistry of the stream that later affected the bryophyte growth.  As another example, in the White Mountains National Forest of New Hampshire, boulders that were once recalled as being covered by bryophytes, particularly Paraleucobryum and Dicranum fulvum, seemed rather stark.  During the summer months of  1980's traffic had noticeably increased in which there was often long traffic jams due to more people driving to factory outlet stores.  Also, when staying in motels, it seemed that people no longer went to bed at night as traffic could be heard all night long.  In the Smoky Mountains National Park a similar trend was observed.  These and other observations led to the following abstract submitted as part of annual report for a collecting permit (Spjut 1991).

Vanishing Bryophytes
What is happening to the mosses on the rock in left photo?  Definitely dying by the brownish color.  Grayish-blue rock surface appears be a recently exposed surface.  Paler color moss on left part of rock is Paraleucobryum, some of the dark green is undoubtedly Dicranum fulvum. Compare with rock on right that has a healthier growth of mostly Paraleucobryum. Photos taken in northern Vermont, 1991.	Comparison of two rocks with Thuidium erectum.  The rock on the left has dark green growth of Thuidium that appears to be algae. It could be just an association, or it could be a dying moss, or it could be affected by the successional growth of vascular plants.  The Thuidium on rock in the right photo appears healthier. Photos taken in northern New York and Vermont in 1991	Taxus canadensis on bare rock.  This yew spreads vegetatively by layering, but it cannot be expected to establish its roots in solid rock.  So why is it growing so close to the rock?  Could it be that a mixed bryophyte and fern community with soil once covered the rock and the acid rain has since washed it away?  The yew itself appear to be dying.  Photo taken the White Mountains National Forest, Wild Creek, state line of Main and New Hampshire, Sep. 1991.

     An estimated 200 samples of bryophytes were nevertheless collected for screening by David Kingston during 1989–1992 from which ~11% were active in genetically engineered yeast strains (Spjut et al. 1992).  As also reported in Spjut et al. (1992), "regrettably many of the active species failed to reconfirm."  Active sesquiterpenes were nevertheless isolated from two species of liverworts, Porella cordeana (Harrigan et al. 1993; Spjut et al. 1992) and also from Chiloscyphus rivularis (Chongming et al. 1997).   Here it might be noted that Chiloscyphus rivularis is an aquatic that grows under water attached to rocks, and upon collecting the liverwort a lot soil had to be removed by rinsing the plant in the stream from which it was collected.  While one may suspect contamination due to muddy soil that was closely associated with the plant, the active agents isolated, 5 novel sequiterpenes along with other known sesquiterpenes, were clearly a product of the liverwort—based on chemotaxonomic studies in liverworts and hornworts (Asakawa et al. 1979).  Also, “bioassay-directed fractionation of the MeCOEt extract of Porella cordeana yielded drimenin [1] and aristolone [4], which were moderately toxic towards DNA-repair-deficient mutants of Saccharomyces cerevisiae. Three inactive sesquiterpenes, 7-ketoisodrimenin [2], 7-ketoisodrimenin-5-ene [3], and norpinguisanolide, were also obtained. Compounds 2 and 3 are new” (Harrigan et al. 1993).

     Overall, the number of novel antitumor active compounds that have been isolated from bryophtyes appears relatively few, somewhere between 0.5 and 1% of the species screened.  This is perhaps not all that unusual when compared to other natural product groups that have been screened by the NCI, but the quantities of bryophyte samples that had to be recollected to obtain these results is discouraging.  When this study first began in 1976, it was hoped that active agents could be isolated from recollections of not more than 1 kg.  Ironically, it seems that the larger size samples may explain reconfirmed activity and the chemotaxonomic relationships that have been evident in bryophytes (Spjut et al. 1986), whereas the smaller sample sizes may lead to more erratic results if activity is due to highly cytotoxic compounds such as maytansinods that have been found in some of our active samples and are believed to the result of an association with actinomycetes such as Nocardia  (Suwanborirux et al. 1990), or other cytotoxic compounds that have yet to be isolated, which may be associated with blue green algae (Spjut et al. 1988).  Keep in mind that at one time the NCI needed ~18,000 kg of Maytenus buchananii (= Gymnosporia) to obtain just 8 g of maytansine.  One might then ask the question whether the tiny samples (15–40 g) obtained by Timo Koponen of Mnaceae in Norway were simply insufficient for assays to detect the active agents that may have been present.

     To what extent the apparent decline in bryophyte growth has contributed to a loss of antitumor activity in our samples cannot be easily evaluated.  A decline in bryophyte species has been noted recently by Morgan and Sperling (2006) for several parks on Long Island using studies conducted between 1975 and 1986 as a base.  They indicated more than 40% of the bryoflora could not be found.  Especially impacted were liverworts, 13 of 21 species apparently extirpated.  Their data supports Spjut's observations for the moss families Dicranaceae, Thuidaceae, Brachythecicae, and Pottiaceae.  A notable difference was the Hypnaceae, which was indicated to have only one loss in 6 species.  It was interesting to find among the antitumor active mosses that Polytrichum ohioense was a new addition, while Dicranum fulvum was extirpated.  In a moss inventory for the Beltsville Agricultural Research Center (BARC) in Maryland, Terrell et al. (2000) reported Pogonatum penilvanicm was formerly frequent along a road cut in a hardwood forest but it had not been seen there since 1985.  This species was also reported as having been extirpated from Long Island (Moss and Sperling 2006).  Another species still surviving on Long Island and on BARC, Thuidium erectum, has notably diminished in abundance on BARC, especially with Plagiomnium cuspidatum; in the 1970's both species were observed as forming extensive carpets along stream banks (Terrell et al. 2000).

     Another factor that is undoubtedly impacting the luxuriant growth of bryophytes is their commercial harvest in the United States for decorative use in the florist trade (Muir et al. 2006).  The quantities that have been collected historically are not known, but the recent study by Muir et al. (2006) reported ~55,000 kg of dried bryophytes were removed from various places across the U.S. for the year 2001.  Among the species they indicated in the harvest are antitumor active bryophytes such as Eurhynchium oreganum, Fullania tamarisci ssp. nisquallensis, Porella navicularis, and Hypnum curvifolium.

     As a follow-up study to the bryophytes collected for antitumor screening, it would be useful as well as interesting to return to the locations where each species was collected and determine to what extent the species and other bryophytes still survive.  The Claopodium crispifolium site near Willow Creek CA—where ~50 kg was collected in 1980—was revisted by Spjut in 1984 who found the vacated surfaces still bare, but Norris also revisted the site sometime later, before 1991, and found that the surfaces had been filled in with Encalypta (Spjut et al. 1992). The large boulders along Willow Creek upon which Claopodium crispifolium and other bryophytes have flourished are suspected to have been the result of a road construction project in the mid 1950's.  Another collection site in Oregon where an usual abundance of Claopodium crispifolium also appears to have been the product of a highway construction at about the same time.  If this is true, then the age of the successional bryoflora that was on these boulders in 1980 was ~25 years.  Today (Nov. 2006), it has been ~26.5 years since recollections of Claopodium crispifolium were obtained from the California and Oregon sites (May 1980).  Have these rocks been recolonized by Claopodium crispifolium or other bryophytes?  To what extent bryophytes still survive along the streams where Spjut collected during 1980 and 1981?  We do have comparative base—weights that were recorded on the shipping list, and the voucher specimens and their labels to indicate more or less precisely where each species was collected.  We just need the funding to do a follow-up survey.

References

Asakawa, Y., N. Tokunaga, M. Toyota, T. Takemota, S. Hattori, M. Mizutani and C. Suire.  1979.  Chemosystematics of bryophytes 11.  The distribution of terpenoids in Hepaticae and Anthocerotae.  J. Hattori Bot. Lab. 46: 67–76.

Cassady, J. M., W. M. Baird and C-j. Chang.  1990. Natural products as a source of potential cancer chemotherapeutic and chemopreventive agents.  J. Nat. Prod. 53: 23–41.

Chongming, W., A. A. Leslie Gunatilaka, F. L. McCabe, R. K. Johnson, R. W. Spjut, and D. G. I. Kingston. 1997. Bioactive and other sesquiterpenes from Chiloscyphus rivularis. J. Nat. Prod. 60 (12): 1281-1286. http://pubs.acs.org/cgi-bin/abstract.cgi/jnprdf/1997/60/i12/abs/np970251u.html.

Harrigan G. G., A. Ahmad, N. Baj, T. E. Glass, A. A. Gunatilaka and D. G. Kingston.  1993. Bioactive and other sesquiterpenoids from Porella cordeana. J Nat Prod. 56: 921-925.

Morgan, E. and J. A. Sperling. 2006.  Changes in the bryophyte flora of Cunningham Park and Alley Pond Park, Queens County, Long Island, New York City. Evansia 23: 56–60.

Muir, P. S., K. N. Norman and K. G. Sikes.  2006. Quantity and value of commercial moss harvest from forests of the Pacific Northwest and Appalachian regions of the U.S.  The Bryologist 109: 197–214.

National Institutes of Health, National Cancer Institute: Correspondence, Letter from Acting Chief, Matthew Suffness to John Cassady, 19 May 1982, regarding assignment of Polytrichum ohioense for fractionation, with cc to R. Spjut.

Norris, D. H. and J. R. Shevock.  2004. I.  A specimen-based catalogue of mosses. II. Contributions towards a bryoflora of California.  A key to the mosses. Madroño 51: 1–269.

Norris, C.  1985. Lost Letters. A Candlelight Ecstasy Romance, Dell Publishing Co., New York. 191 pp.

Sakai, K., T. Hikawa, U. Amada, M. Yamashita, M. Animoto, A. Hikitay, A. Uin, and K. Oksoin Do.  1988. Antitumor principles in mosses: The first isolation and identification of maytansinoids, including a novel 15-methoxyansamitocin-P-3.  J. Nat. Prod. 51: 845–850.

Spjut, R. W.  1985.  Limitation of a Random Screen: Search for New Anticancer Drugs in Higher Plants.  Econ. Bot. 39(3): 266–288.

Spjut, R. W., M. Suffness, G. M. Cragg, and D. H. Norris 1986. Mosses, hornworts and liverworts screened for antitumor activity. Econ. Bot. 40: 310-338.

Spjut, R.W., J. M. Cassady, T. McCloud, D. H. Norris, M. Suffness, G. M. Cragg, and C. F. Edson. 1988. Variation in cytotoxicity and antitumor activity among samples of a moss, Claopodium crispifolium (Hook.) Ren. & Card. (Thuidiaceae). Econ. Bot. 42(1): 62-72.

Spjut, R. W. 1991. Bryophytes Vanishing from our National Forests and National Parks. Abstract, Seventeenth Annual Meeting on Scientific Research in the National Parks of the Upland Section for the Southeastern Region, May 1991.

Spjut, R. W., D. G. I. Kingston, and J. M. Cassady.  1992. Systematic screening of bryophytes for antitumor agents.  Trop. Bryology 6: 193–202.

Statz, D. and F. B. Coon.  Preparation of plant extracts for antitumor screening.  Cancer Treatment Rep. 60(8): 999–1005.

Suwanborirux, K., C.-J. Chang, R. W. Spjut, and J. M. Cassady.  1990.  Ansamitocin P-3, a maytansinoid, from Claopodium crispifolium and Anomodon attenuatus or associated actinomycetes.  Experientia 46: 117–120.

Terrell, E. E., J. L. Reveal, R. W. Spjut, R. F. Whitcomb, J. H. Kirkbride, Jr., M. T. Cimino, and M. T. Strong. 2000. Annotated list of the flora of the Beltsville Agricultural Research Center, Beltsville, Maryland. USDA ARS-155, Natl. Tech. Info. Serv., Springfield, VA.

University of Illinois Letter, Charles Phoebe to Timo Koponen, University of Helsinki, August 30, 1983, with attachments: (1) summary of screening results for 16 species of Mniaceae and (2) copy of voucher labels for Plagiomnium venustum (WBA-1, WBA-2), cc to D. D. Soejarto, J. M. Pezzuto.

USDA Agricultural Research Service, Accession Record and Shipping List:
PR-50951-997, pro parte.
PR-54382-446
PR-54745-55039

USDA ARS Medicinal Plant Resources Lab/Economic Botany Laboratory active sheet record for Plagiomnium venustum.  Shows collection data and dates, collection numbers.

USDA ARS Memorandum (reproduced from carbon copy), Adjustment to Cost Estimate for Procuring 40,000 Pounds of Stems of Maytenus buchananii.  R. W. Spjut to R. E. Perdue, Jr., Mar 24, 1976.

USDA ARS Memorandum, Activity and Plant Parts as Related to Vegetation and Climate. R. Spjut to A. S. Barclay, Nov. 6, 1979. 2 pp.

USDA Memorandum, Spjut to POSI (Plants of Special Interest) File, Claopodium crispifolium (Hook.) R. & C. (Thuidiaceae), 16 July 1980.

WBA (World Botanical Associates) Memorandum: Original and Re-collections of Polytrichum ohioense and Polytrichum pallidisetum for Antitumor Screening, 16 July 1990.

WBA Correspondence:

          1984. Sep. 4, 1984, Richard Spjut to D. Soejarto, University of Illinois at Chicago, with cc to C. Edson, A. D. Kiinghorn, D. Norris, M. Suffness,
1984 29 Oct. 29, Richard Spjut to John Cassady, Purdue University, with cc to D. Norris, M. Suffness and C. Edson.
1986 Jan. 22,  Richard Spjut to John Cassady at Purdue University, with copy of WBA shipping list for Polytrichum ohioense (WBA-320).
1986 Oct 22, Richard Spjut to John Cassady at Purdue University
1986 Oct 17, Richard Spjut to David Kingston at Virginia Polytechnic Institute & State Univ.

WBA Shipping List Records:

          WBA 7–13, 26 July 1984. Bryophyte recollections to David Kingston
WBA 14-16, 29 Oct 1984. Polytrichum pallidisetum and P. ohioense to John Cassady.
WBA 17–46, 18 Oct 1984. Bryophyte recollections and lichen general samples to Kingston
WBA 47-57, 22 Oct 1984. Anomodon attenuatus and A. rostratus to John Cassady.

Zheng, G-q., C-j. Chang, T. J. Stout, J. Clardy and J. M. Cassady.  1989.  Ohioensin-A: A novel benzonaphthoxanthenone from Polytrichum ohioense.  J. Amer. Chem. Soc. 111: 5500.

Zheng, G-q.  1990. Part 1. Cytotoxic agents from Polytrichum ohioense and Polytrichum pallidisetum; Part 2, Design and synthesis of inhibitors of myristol CoA: Protein n-myristoyltransferase as potential anticancer drugs.  Ph.D. Thesis. Purdue Univ. West Lafayette, IN.