by Natalia KUTLUNINA, Cand. Sc. (Biol.), Assistant Professor, Botany Department, Ural Federal University; Mikhail KNYAZEV, Cand. Sc. (Biol.), Botanical Gardens laboratory head, Ural Branch, Russian Academy of Sciences, Yekaterinburg, Russia

Clonal organisms are not only a product of science laboratories, they are common to living nature, too. Natural clones, however, harbor quite a few riddles. Say, how a plant not capable of seminal reproduction-and this happened long ago-could have propagated over a territory hundreds of square kilometers large, getting ahead of fully sound rivals. Why do some clones find themselves outside the ravages of time and ambient environment? Clonal organisms are not destroyed by age, they are not devoured by all kinds of protean enemies. Sluggish and immobile, they survive in this ever changeful world. How come? No cogent explanations so far. Even though our hypotheses are not self-evident, they may prod one toward further research.

Parthenogenesis (virginal reproduction) occurs both in invertebrates and in large vertebrates. Photo: VARANUS KOMODOENSIS hatching from a parthenogenetic egg.

Polyploid parthenogenetic races of the weevil (OTIORHYNCHUS NODOSUS) are of much wider occurrence in Europe (hatched) than bisexual diploid races (black). (C. Grebelny, 2009.)

Cloning means getting several genetically identical organisms by asexual reproduction, either under natural or laboratory conditions. Such organisms are called "clones". The word-"clone", "cloning"-came into Russian from English, though originally it springs from Greek (кλωυ) standing for a "twig", "shoot", "sprout". At first it denoted a group of plants evolved from one "parent" vegetatively. Subsequently this term came to be applied to bacteria and animals, and, hypothetically, to man and identical lines of his common ancestor. Most often it denotes the technology of obtaining genetically identical organisms, a procedure known as the cell nucleus substitution. The topic of cloning has now become very popular among the science community as well as in the mass media, fiction and motion pictures. The most thrilling subject is what concerns the cloning, or "copying", of human individuals. Although this matter has great scientific and moral implications, it will not be considered in the present article.

While the technologies of artificial cloning-animals in particular-is just coming to be and has to confront too many difficulties, Mother Nature has mastered this process over millions of years and is going on with her experiments. In the animal kingdom cloning is most typical among most primitive taxa (corals, starfishes), though it occurs in rather "advanced" species as well. Some invertebrates and vertebrates multiply through parthenogenesis, or virginal reproduction, when the egg cell is not fertilized.

Such organisms beget a genetically identical progeny. In their fantastic novel The Snail on the Slope (written long before present sensational cloning experiments), the brothers Arkady and Boris Strugatsky depicted a fancy world populated by witches giving birth to daughters conceived without males. These sorceresses ruled the world, they subjugated wild nature and made history. Such kind of phantasms are not so much far-fetched, however. Parthenogenesis occurs among rotifers, lower crustaceous, insects, reptiles, sharks, certain domesticated birds like guinea fowl. In most animals virginal reproduction is confined to a definite life cycle period, making them adapt to ambient environment conditions (cyclic parthenogenesis of Daphnia, or water fleas). Occasionally some taxa do without fertilization at all, like a class of Bdelloidea rotifers that, for about 80 mln years, have been reproducing asexually.

As found in wood lice and weevils, both bisexual and parthenogenetic races of one species may be far apart geographically, with forms multiplying sexually retaining ancient (relict) land tracts, with parthenogenetic forms colonizing new, and often rather large territories like those that got free from the ice shield ten thousand years ago. So, parthenogenesis plays an important adaptive role in animals as a stage in their life cycle or as they colonize free territories.

In many perennial plants natural cloning is connected with vegetative reproduction. Those having short rhizomes (tap roots) form a tight clone, the

courtine, or phalanx. And those with long tap roots, stolons* or other propagules**, propagating over long distances, form extensive loose clones; this kind of growth is designated "guerilla".

Man has learned to make good use of cloning playing a big part in farming with its cloned potatoes, gladioli, begonia, grape, vine and the like.

The Martians described by Herbert G. Wells in the novel The War of the Worlds had a heel of Achilles-they were more predisposed to diseases than populations reproducing sexually. These are more diverse and in case of epidemic diseases (epizooties, epiphioties) only a few misfits perish. However numerous, populations born through vegetative of virginal reproduction are foredoomed to total extinction. In Britain, for instance, the elm Ulmus procera was killed en masse in the 1920s by the parasitic fungus transmitting the "Dutch disease" (Ceratocysis ulmi) destroying millions of trees. Only a few, genetically identical species, survived. Overseas, on the continent, this elm species are more resistant to the "Dutch disease" since it propagates by seed largely.

Plants capable of vegetative reproduction only get wonderful odds in life's "game of chess" compared with their rivals burdened with "family chores". They sacrifice just a little thing-diversity. In this connection some questions come up. How often do clonal species acquire full sterility? What are the causes and consequences of this phenomenon? How far can clones spread? And how long can they live? In an attempt to answer these queries, we have made a study of the genotypic diversity and reproductive system of some clonal species belonging to different families and having dissimilar types of vegetative reproduction and living spaces. These include the Lemna trisulca L., Gladiolus tenuis Bieb., Phlox sibirica L., Glycyrrhiza glabra L., Glycyrrhiza korshinskyi Grig., Glycyrrhiza uralensia Fisch, Trifolium repens L., Poa bulbosa s.1., Cardamine trifida (Lam. et Poiret) B.M. Jones, Tulipa biebersteiniana Schult et Schult and Tulipa riparia Knjas. Kulikov et Philippov. To evaluate genotypic diversity and identify clones we used isoenzymic and DNA analysis, in particular, ISSR (Inter Simple Sequence Repeats) marking, i.e. for detecting the polymorphism of intermicrosatellite regions of the genome.

In most species we found a combination vegetative and seed reproduction, with polyclonal populations formed as a result. Given certain conditions, clones spread over large territories ranging from tens to hundreds of sq. meters. Thus, the Biberstein tulip (Tulipa bibersteiniana) was found under the forest canopy. This plant makes use of the advantages of vegetative reproduction under shadowing conditions inhibiting insemination. On open dry stretches seminal propagation (by seeds) recovers to the detriment of vegeta-

* An offshoot with elongated internodules and underdeveloped leaves giving rise to tubules, bulbs and rosette sprouts.-Auth. ** Vegetative reproduction structure in higher plants (buds, bulbs, offsets).-- Auth.

ISSR analysis involves a comparison of bands on gel. Each band stands for a DNA region between two microsatellites (short repeats). The first position on gel is a mol.wt. marker, and the other positions take in samples of three populations of G. TENUIS. Full identity of samples according to all 10 primers (fragments) was evaluated as the genetic identity and, consequently, as belonging to one clone.

tive reproduction, and highly polymorphous clones are found on small land patches. In this particular case the "unity of opposites", i.e. the sexual and clonal reproduction, ensures a survival success of the species. The Biberstein tulip and other species studied by us confirm the now classical conceptions about the sufficiently high level of genetic variability in vegetatively mobile plant species.

One kindred, closely related tulip species, Tulipa riparia, is a triploid*. It occurs in the flood-lands of the river Belaya and its tributaries flowing down the western slope of the Ural range. Triploid species, which are genetic "uglies", carry an extra third set of chromosomes that cannot be divided harmoniously among sex cells. This leads to complete or partial sterility. Such species (races) can live on only under effective vegetative reproduction. The reproductive system of T. riparia shows a lower level of fertility in the male and female domains. Yet around 50 percent of viable pollen and seed buds are formed nonetheless. Occasionally T. riparia bears fruit but the seeds contained in the boll do not germinate. Apparently T. riparia clones are self-sterile, though seeds are formed now and then through the cross-pollination of different clones. Although we have not observed seminal propagation of these plants under natural conditions, it does take place every now and then, sub rosa, so to say, as seen in the variegated coloring of the flowers and in the rather high genotypic diversity (no less than 6 clones in each of the populations studied by us). Strangely enough, the tulip's triploid cytotype has retained seminal reproduction.

The Gladiolus tenuis Bieb was another object of our research. This perennial bulbotuberiferous plant multiplies both by seed and vegetatively, by daughter bulbs. As we found, east of the Volga G. tenuis is sterile, it does not fructify. Selective studies in six eastern populations in a transect strip about 1,000 km long (stretching from Lake Kandry-kul in western Bashkiria to the southern termination of the Mugodzhar Mountains in Kazakhstan) have shown them to belong to one giant sterile clone.

In the major part of its propagation area G. tenuis is tetraploid*, while sterile clone plants exhibit stable triploidy. Tetraploid populations in the Chernysh-kovsky district of the Volgograd oblast (region) show a high fertility of pollen and seed buds; pollen sprouts profusely in the stigma accounting for seminal reproduction and high genetic polymorphism. Odd polyploidy** of Ural plants results in partial sterilization of pollen and total sterilization of seed buds.

To explain the emergence of sterile clones we have proposed two hypotheses. The first one: the sterile

* An organism with three single, unpaired (haploid) sets of chromosomes in somatic cell nuclei; this phenomenon is known as triploidy.--Ed.

* An organism with four sets of chromosomes in the nuclei of somatic cells.--Ed.

** Cell state in which some multiple of the usual number (diploid) of chromosomes is present in the nucleus. Triploids are also among odd polyploids.--Ed.

Science in Russia, No.4, 2012

GLADIOLUS TENUIS occurs in the Crimea, in Caucasia, and in European Russia's east; its colonies are also found in the South Urals and in Kazakhstan.

clone is of hybrid origin and appeared through a crossing of the tetraploid gladiolus G. tenuis and hypothetical diploid (with two sets of chromosomes) species now extinct. Ancient diploid gladioli do not occur in Europe and are found in Southern Africa only. Thus, the triploid clone could be very, very old. According to the other hypothesis, it could have mutated, lost one of the four genomes and changed its status. The triploid gladiolus proved to be more viable than those capable of seminal propagation, and it propagated widely east of the Volga. There might have been two mutually complementary mechanisms of its propagation (a phenomenon true of many other plant species for that matter).

One is hydrochorous, that is realized through transportation of seeds and other rudiments by water currents in heavy rainfall or high water. Yet another scenario is possible when soupy soil forms a viscous "mud dough" carried away in the fur of animals along with seeds, tap root fragments, spores and sprouts. Animal wallowing in mud could be very good carriers. Today these are wild boars and in the Early Pleistocene, i.e. 1.8 mln-11,000 years ago, those were wooly rhinoceroses and mammoths. The arcwise area of sterile gladiolus propagation could have been confined to the principal pathways of migrations of ancient animals.

Cardamine trifida is yet another instance of wide propagation of a sterile clone. The main population area of C. trifida is in Siberia, though some of its spots are also found in the Urals and in the Mid-Russian Highlands. In the Urals we detected a broad occurrence of plants with total male sterility. Remarkably, such plants are genetically original in different parts of the population area, i.e. these mutants emerged independently. Most likely, natural selection promotes these forms. The Ural C. trifida plans are partially polyclonal (8 to 12 genotypes), and in part they are represented by just one clone ("Berda" and "Aghidel" populations). The population of the "Aghidel" clone in the Belaya River valley is of the greatest interest. It is distinguished by full male sterility and is capable of forming germinal tubucles in cauline leaf axils. This is a highly remarkable mor-

C. TRIFIDA in the Urals, and populations studies (white spots). Boxed, propagation of the "Aghidel" sterile clone.

phological characteristic. No doubt, this anomaly largely facilitates its propagation and gives substantial competitive advantages. Apparently in the climatically unfavorable epoch of the Late Pleistocene the original population in this part of the propagation area contracted down to one single clone but, as tolerable conditions were back in the Holocene (during the last 11,000 years) it has disseminated throughout the Belaya River valley.

Sterile populations are a predominantly local phenomenon confined to the marginal part of a population area; it indicates the isolatedness of propagation of this or that species with the exception of water plants like the Posidonia oceanica and the aquatic hyacinth Eichornia crassipes. We have demonstrated the existence of sterile clones of land herbaceous plants (devoid of seminal propagation and genetic recombination) in large areas. It is rather difficult to tell the age of clones since, for example, different scientists put the age of the American poplar (Populus tremuloides) at 8,000, 20,000 and even 1 mln years. In herbaceous plants the life span of an individual plan is much shorter than that of woody plans; be that as it may, the age of clones we have studied is at least 10,000 years. Only separate clones best adapted and having a high level of heterzygosis* survive in the acid test of natural selection.

Sexual reproduction may kill happy combinations-nature "takes a rain check" on prodigies, and this is one of the causes accounting for tactical advantages of clones. The tight bounds of their absolute standardization may be overcome through somatic mutations. Yet such mutations affect just one gene, they can hardly beget a race superior to the mother model. These mutations are of interest to research scientists since they make it possible to tell the approximate age of a clone and trace stages of its population. In our works on two individual plants of the giant clone of the Gladiolus tenuis we were able to identify the occurrence of these very mutations. Further research will allow to read the chronicle of the giant clone of Gladiolus tenuis.

To conclude, there are opposite viewpoints on prospects of asexual organisms: some researchers will exaggerate the advantages or just negate the significance of sexual reproduction (Maynard Smith, a British evolutionary biologist and geneticist, 1978), while others will contend sterile clones have no evolutionary prospects at all (Verne Grant, a US botanist and evolutionist, 1974). Now the existence of clones like that for thousands of years and large areas of their propagation invite the following suggestion: tactical advantages suddenly become significant and long-term even under the conditions of ecological variability and instability. It may be that the decisive advantages of sexually multiplying species over clones become manifest only in case of viral and fungal infections to which clones show but poor resistance.

The present work is supported by grants of the Russian Fund of Basic Research 10-04-00989-a and 11-04-10091-k.

* With reference to a heterzygote--a cell or organism in which homologous pairs of chromosomes carry changed forms of genes (alleles).-Ed.