Inventory The Microcosmos!

Bringing love and order to the taxonomy of lower organisms

Although no one knows, nor ever has known, the extent of biodiversity at any one given time, formal scientific attempts to catalog the known living world have been made since Carolus Linnaeus (1707-1778). The vast majority of scientists have described mammals, birds, reptiles, fish, and flowering plants. In general, people love these creatures (especially when they resemble children). We are repulsed by spiders, mildew, and bacterial slime. We may even have an innate tendency to respond positively to those organisms (the "biophilia hypothesis") with which we share a strong and positive past history pandas, Coho salmon, grapevines, and woodland shrubs. But to complete the Linnean task, or even to approach its completion, a detailed systematics of the less charismatic species, especially the microbial world, needs to be undertaken, especially before habitat destruction and extinction render this impossible.

AN EMBARRASSMENT OF MICROBIAL RICHES

The real news of twentieth-century science is that the greatest diversity of life on Earth is not in any plant or animal group. Rather it lies squarely in the province of those organisms that are always wrongly forced into the plant or animal kingdoms, or whose very existence is simply ignored. These are the inhabitants of the planet's microcosm, organisms that require a microscope for their proper visualization. Bacteria (unless they swim) and fungi, for instance, traditionally have been squeezed into the inferior portions of the Kingdom Plantae, whereas protoctists (algae, slime molds, foraminifera, ciliates, water molds: some thirty major groups) have been split in many irrational and pragmatic fashions into either Animalia and Plantae.

The first Linnean rule of taxonomy--that all organisms of the world are either plants or animals and should be cataloged into one of these two groups--ironically became a major impediment to the fulfillment of his dreams. This pigeonholing has greatly thwarted attempts at completing an inventory of all the planet's species.

Many brilliant biologists tried to overcome this taxonomic impediment, but were ignored, dismissed, or worse, ridiculed. The German Ernst Haeckel (1834-1919) tried to establish a third kingdom for many microorganims, but was ignored. John Hogg (1860) tried to separate out all creatures with a nucleus that were not obviously plants or animals. He was ignored until 1956 when Herbert Copeland (an American at Sacramento City College) resurrected Hogg's work as the Kingdom Protoctista. But it wasn't until Robert Whittaker (1924-1980) persuasively argued for five kingdoms (separating out fungi, protoctists, and non-nucleated bacteria) that the Linnean mind-set began to crumble. Today, some believe that as many as twenty kingdom will be declared once the protoctists are figured out!

In the biological community, a great tolerance for contradiction and inconsistency within microcosmic taxonomy persists, because of the separation of practices by at least three classes of practitioners and the microcosmic challenge to the species concept. Zoologists use the International Code of Zoological Nomenclature; botanists follow the distinct rules of the International Code of Botanical Nomenclature; and bacteriologists, having seceded from the botanists, follow the rules of the International Code of Nomenclature of Bacteria. Meanwhile, the mycologists, who study the fungi kingdom, have begun their own international mycological congresses; they tend to use rules of botanical nomenclature, but confine discussion of the taxonomy of the organisms to their own journals and societies. Virtually no taxonomic communication exists between members of these communities.

A second Linnean rule was that each distinct kind of life form have a unique name: a genus name and a species name. The same genus name is allowed for both an animal and a plant (e.g., Proteus the fish and Proteus the South African flower), because of the separation of the kingdoms. Having the same name presents no difficulties whatsoever to the zoologist nor to the botanist. But names become a serious source of confusion for the student and investigator of "lower organisms," when an organism is forced into the animal or plant kingdom, or tossed between the eubacteria, fungi, and protoctist kingdoms. (There is even a bacterium, Proteus vulgaris!)

Without the commitment, scientific dialogue, and funding for study of the microorganisms that existed for animal and plant study, the taxonomic study of microbes slowed almost to a halt. Algologists, protozoologists, and protistologists (whose titles still reflect the old taxonomy, but who actually study protoctists), have become disenfranchised by the continued dominance of the plant/animal mind-set. Students did not see mysteries of microcosmic taxonomies as opportunities and did not become lower-organism taxonomists. The Cambridge Culture Collection for Algae and Protozoa was closed in the 1980s; the American Type Culture Collection (ATCC) protistology division has no academic program for training taxonomists. Indeed, since the "lower organisms" have been mostly orphaned from their parent zoological and botanical social institutions, almost no opportunity for advanced taxonomic training covering any major group of protoctists exists today. The only professional graduate studies that depend on expert taxonomic information and the ability to identify live organisms to the species level are organized around pure practicality--such as study of the plant pathogens (especially of the turf grass used in golf courses); the fossil foraminifera used--indeed, decreasingly used--in stratigraphic analysis of oil-bearing sediments; and "pathogenic protozoa" and pollution-indicating algae and ciliates likely to be found on beaches or in public water supplies.

REVITALIZING MICROBIOLOGY

To inspire microbial enthusiasm, we encourage graduate students to investigate the excitement and the complexities of microbial discovery. Students in Lynn Margulis's Symbiosis seminar wrote about one of the great unknowns in the species inventory: How many, and what, are the microbes in the digestive tracts and other body parts of animals and plants?

Joseph Leidy of Philadelphia (the "last man who knew everything," according to a recent biography) was the first person in the United States to ask what a termite encased in wood actually eats. From vertebrate paleontology to freshwater amoeba, Leidy, who is also credited as being the founder of parasitology in the United States, found all life interesting.

In 1881 he examined Reticulitermes flavipes, the eastern subterranean termite, and reported his great astonishment that the gut was "swarming with myriads of parasites," and that, "some animals are so habitually and constantly infested with multitudes of various parasites that it would appear to be their normal condition."

When the intestine of the termite is withdrawn from the abdomen and submitted to gentle pressure, under the microscope, the brownish matter in the interior is observed as a swarming mass, in which the largest parasites are distinguished pushing their way through the crowd. If the intestine is ruptured, myriads of the living occupants escape, reminding one of the turning out of a multitude of persons from the door of a crowded meeting-house.

While he went on to begin the process of naming and describing the microbes of Reticulitermes (Trichonpmpha agilis, Pyrsonympha vertens, Dinenympha gracilis), Leidy would have needed to do some experiments to realize that these parasitic protozoa are actually obligate wood-digesting symbionts of the termite. If deprived of them, the insects die, their hindguts filling up with undigested wood.

Over the years, hundreds of species of these protists have been described from wood-eating termites around the world. (They're not found in the fungal-gardener soil-eating termites, mainly of the tropics). Much of this descriptive work was done by Professor Harold Kirby of the University of California, Berkeley, who described and wrote monumental taxonomic accounts of more than 100 species of them.

His work, by today's standards, was fairly straightforward. A slide-and-stain taxonomist removes the termite gut by pulling off the end of its abdomen and then gently smearing the gut contents onto a glass microscope coverslip. This specimen is then "fixed" by immersion in an appropriately noxious chemical compound, aldehyde, acid, or alcohol, or some combination of them. This preserves the specimen, preventing the cellular structure from disintegrating or rotting.

The specimen must then be stained so the little fibers and filaments and other structures characteristic of each species can be seen. The coverslips are then dehydrated in alcohol so that they can remain inert, and mounted on a microscope slide. Then the taxonomist must spend many hours obsessively studying the slides under the microscope, gently focusing up and down to see the little details, some of which can be resolved to the level of a single micrometer or two.

Finally, the taxonomist makes some lovely drawings or photographs of the new organisms, writes a paper describing them, and deposits the slides in a museum as the type specimens, against which all others will compare their own specimens.

But because of the confusion surrounding microbial taxonomy and how to classify specimens in collections, the Linnean quest can easily suffer setbacks. When Kirby died suddenly, his 20,000 slides traveled around the country to various people before ending up in Amherst, Massachusetts in the hands of his former student Bronislaw Honigberg, who had the foresight to deposit them in the American Museum of Natural History in New York. But when Michael Yamin, then a graduate student at Rockefeller University (he subsequently published the most comprehensive list of termite symbionts throughout the world), examined the collection, he couldn't find 1,000 type specimen slides.

After many years, Yamin finally found them. After Kirby died, he was replaced by one of his students, Bill Balamuth. When Balamuth died suddenly in 1981, his students came back to clean out his lab. One of them, Tom Gong, realized the value of Kirby's slides, saved them from the trash, and had stored them in his house near Berkeley ever since.

PLANETARY MICROBIOLOGY AND DIGITAL TECHNOLOGY

The inventory of the Earth's living creatures has been slowed by a bias toward big, charismatic, and more loveable animals and plants; and by trying to jam all organisms into two kingdoms. It has been slowed by a prejudiced filter that searches for and describes only those microbes involved in disease and medicines, or horticulture and agriculture, or eating wooden homes. Add to this still another barrier. Living beings taken out of their native places of residence do not survive long, and so methods need to be developed to preserve the salient features of their structures after death.

Back to Linneaus, who insisted that drawings and publications about life do not properly suffice to document living forms, and that therefore "type specimens" needed to be preserved in places of higher learning. But how to best preserve microbial specimens? The old and elegant technique of the fixed and stained typeslide preparations, e.g.L.R. Cleveland (1892-1969) and Harold Kirby (1900-1952), missed too much. With the light microscope, only features bigger than about [10.sup.-6] meters could be seen. With scanning electron microscopes, new features as small as [10.sup.-8] meters, and more three-dimensional features, became visible. Membrane structures and organelles became clearer. With the transmission electron microscope, features even smaller ([10.sup.-9] meters) became apparent, and the larger features more vivid. With protein and DNA/RNA analysis, the creature's morphology was lost but its genome(s) could be tallied to help taxonomists clarify relationships.

But all this never equalled the live images of protoctists. It is life that is far more communicative and informative. Microvideo recording of all stages of the life history can reveal such taxonomic features as developmental timing, motility, sexual and asexual acts, propagule formation, and kinetid structure, which help enormously in classifying the microbial world. The films by Cleveland, taken with the phase contrast microscope in 1956-1957, document the organisms in question in a far superior and more useful manner than slides of single life stages. If transferred to digital imagery, for instance, Cleveland's films can serve as the type material for about thirty species of unique cellulolytic microorganisms of wood-eating cockroach and termite hindgut like those first described by Leidy.

High-quality footage of the type specimens of live organisms would help investigators more easily resolve the rampant taxonomic disputes and confusion. Does Takakia belong in the phylum bryophyta or does it deserve higher taxonomic status on its own? Should the fungi Candida and Taphrina be placed into the phylum Asco- or Basidiomycota? Or, should the Irish potato blight, Phytophtora infestens (an oomyceti) be properly classified as a protoctist, not a fungus? While perhaps sounding obscure to those only familiar with charismatic species, these questions are at the heart of Linneaus's passion to bring order and harmony into our knowledge of all life.

Wissenschaftlichen fur Film (IWF, Gottingen, Germany), has made an unprecedented beginning in this endeavor. They should be financially and intellectually supported by the international community of scholars. Their pristine insistence on live material only, with an absolute minimum of interpretation, should be emulated worldwide. Their work deserves coordination with all organizations dedicated to the completion of the Linnean task.

In the present world, replete with computers and CD-ROMs, primary science could wonderfully avail itself of the use of the film-to-video-to-digital transfer and video technology itself. The deposit of original footage (or digital copies) as biological "type material," subject to international standards and review by appropriate taxonomic experts, would accelerate the spread of biodiversity knowledge and the accomplishment of an inventory of all species. At thirty frames per second, five to ten seconds per scene, and approximately ten minutes per species, the amount of high quality taxonomic information that can easily be stored on disk would be immediately increased by a factor of about 100,000 (the estimated increase over the usual ten photographs per monograph).

Our knowledge is very poor. The odd collection of microorganisms in the Kingdom Protoctista may include anywhere from 210,000 species to 1.2 million. Only 80,000 have been described. The difficulty of handling this diversity of nucleated organisms that are "too large" and "too irrelevant" to be of interest to bacteriologists and "too small" and "too confusing" to concern zoologists and botanists is stunning. The serious accomplishment of the dream of Linnaeus will require a political will, ingenious organizational skills, compromise, commitment, and international collaboration that is unprecedented among biologists. Reminiscent of the kinds of coordination required to return a spacecraft from Mars, this inspiration, the Linnean mega-effort, would do well to imitate the techniques of the metanational "Big Science" organizations that successfully rendezvoused the Comet Halley, installed the Deep Space Net monitoring system, and mapped the surface of Venus in the radar and visible regions of the electromagnetic spectrum.

BY RICARDO GUERRERO, LYNN MARGULIS, AND MICHAEL DOLAN, and from notes from Lynn Margulis's Symbiosis class in the Organismic and Evolutionary Biology graduate program at the University of Massachusetts, Amherst

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