Ecological Aspects of Evolution of the Plague Microbe Yersinia pestis ...

Biology Bulletin, Vol. 27, No. 6, 2000, pp. 541–552. Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 6, 2000, pp. 645–657. Original Russian Text Copyright © 2000 by Suntsov, Suntsova.

GENERAL BIOLOGY

Ecological Aspects of Evolution of the Plague Microbe Yersinia pestis and the Genesis of Natural Foci V. V. Suntsov and N. I. Suntsova Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii pr. 33, Moscow, 117071 Russia Received March 20, 2000

Abstract—A new hypothesis of the origin of the plague microbe in the Mongolian bobak (Marmota sibirica Radde, 1862) populations in Central Asia during the Pleistocene is based on the ideas of its relative phylogenetic recency. The Late Pleistocene cooling, which induced a deep freezing of the grounds in southern Siberia, Mongolia, and Manchuria, is considered as an inducer of speciation. The main ecological factors of the plague microbe evolution include the species specific behavior of the Mongolian bobak as it prepared to hibernate related to its occurrence in arid petrophytic landscapes and the larval parasitism of the flea Oropsylla silantiewi Wagn., 1898 in winter. Genesis of the plague foci is divided into two periods: natural-historical and biosocial. During the first period, the primary natural foci in Eurasia were formed and, during the second period, synanthropic (rat) and secondary natural foci appeared with the participation of humans in Africa, The New World, and on some tropical islands.

INTRODUCTION The natural plague foci are now considered ancient landscape-ecological formations that originated as a result of the long-term coupled evolution of members of the epizootic triad “burrow rodent–flea–causative agent.” Adaptive features of the plague microbes provide for the development of a high level of bacteremia in rodents, which is necessary for the invasion of blood sucking vectors, fleas and the transmission of this infection (Ioff, 1941; Klassovskii and Petrov, 1968; Bibikova and Klassovskii, 1974; Vashchenok, 1988). The microbe virulence towards the warm-blooded host is considered an integral expression of the microbe adaptation, and the host response is expressed in a certain level of resistance to the causative agent. Most researchers believe that the continuous circulation of the microbe in the “rodent–flea” system is the only way the microbe can exist in nature. The appearance of the first natural foci is dated within wide limits: from the Mesozoic (Domaradskii, 1998) to Pliocene–lower Pleistocene (Petrov, 1968), i.e., from 100–70 to 1 billion years ago. The preference is often given to the Oligocene–Miocene–Pliocene, the periods of formation of the continental steppe, semidesert and desert landscapes and origin of the faunas of burrow rodents and their fleas (Fedorov, 1960; Kucheruk, 1965; Rall’, 1965). The plague could have originated in Central Asia, The Himalayas, Tibet, Yunnan’, The Caucasus, Central and Eastern Africa, or North America, and marmots, gerbils, pikas, voles, or African grass mice could have been the primary hosts of the microbe (Devignat, 1951; Rall’, 1958; Sultanaev, 1960; Kucheruk, 1965; Achtman et al., 1999).

The wide distribution of the plague foci all over the world is considered indirect evidence of the ancient origin of the plague microbe. However, this does not explain the distribution of the rat subspecies of the microbe alone (Y. pestis ratti) over the vast territory of the New World and the distribution of the rat and marmot (Y. pestis marmota) subspecies in Africa. Rather, the distribution of only two subspecies suggests a recent introduction of the plague to these continents by people. Note that the name of subspecies “rat” is related to the priority of its description: A. Iersin and Sh. Kitazato described the plague causative agent as isolated from humans and synanthropic rats. Later, it was shown that this subspecies circulates in nature in the Indian gerbil (Tatera indica) populations on the Indostan Peninsula. The causative agent can pass from them to the populations of synanthropic black rats (Baltazard and Bachmanyar, 1960). Both marmots, as an object of hunting, and synanthropic rats had and have close contacts with humans and, hence, one cannot deny the role of the anthropogenic factor in the distribution of the plague by these rodents. The specific pathway of transmission of the plague microbe via fleas is considered another essential evidence of the ancient origin of plague. A “block” is formed in the flea proventriculus, when fleas feed on the invaded animals, which is an aggregate of reproduced bacteria mechanically plugging the lumen of the gastrointestinal tract. The “blocked” fleas rapidly die as a result of dehydration and starvation (Vashchenok, 1988). Due to fast elimination of the “blocked” fleas, the chances of transmission of their genetic material to subsequent generations are rather small. Moreover, the infected fleas that were liberated from the block have a markedly shorter life span than the noninfected fleas

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(Vashchenok, 1988). In this case, the bacterial “block” of the proventriculus of an infected flea cannot be considered from the viewpoint of the modern evolutionary theory as a result of long-term coevolution of the microbe and flea, and the transmission of the causative agent via blocked fleas cannot be considered evidence of the ancient origin of the plague. In many foci, mostly on the flatland, long interepizootic periods were found when the microbe was not recorded in the host and vector populations using modern methods. This phenomenon cannot yet be explained from the viewpoint of plague microbe circulation in the system “rodent–flea.” Hypotheses about so-called “telluric” (saprophytic, sapronozic) plague imply the presence of a saprophytic phase in the microbe life cycle, which allows it to survive interepizootic periods outside the warm-blooded animal or flea (Baltazard, 1964; Bukharin and Litvin, 1997; Levi, 1997). In this case, the plague causative agent should be considered a facultative parasite. Extensive studies of the “sapronozic” plague in Russia began after Somov (1988) substantiated the concept of “sapronozes.” The group of sapronozes includes the infections, whose causative agents have a saprophytic phase and can reproduce in both soil organic matter and animals. Pseudotuberculosis of rodents was referred to saprozoonoses, the group of typical zoophilic sapronoses. Pseudotuberculosis is caused by the closest relative of the plague microbe Yersinia pseudotuberculosis widespread in the moderate zone (Litvin et al., 1998). The pseudotuberculosis microbe is psychrophilic and intensely reproduces in both organic residues at 4–10°C and warm-blooded animals at the temperature up to 42°C. Two isozyme systems were found in this microbe which switch on with respect to specific environmental conditions (Somov, 1988). Vashchenok (1988) reported a very intense reproduction of the pseudotuberculosis microbe in the fleas Xenopsylla cheopis kept in a refrigerator at 6–8°C. The amount of microbes rapidly increased from 32 cells per flea to millions of cells within 5–10 days. The fleas kept at 16–24°C did not transmit the infection to the experimental animals. The white mice were experimentally invaded only through the fleas that had been kept in the refrigerator at 6–8°C for several days. The plague and pseudotuberculosis microbes are closely related: the similarity of their genomes is more than 90%, which is unique for bacteria (Bercovier et al., 1980; Ibrahim et al., 1994). The similarity of the genomes of two other intestinal bacteria, Y. pseudotuberculosis and Y. enterolcolitica, amounts to 35% (Moore and Brubaker, 1975) and in different bacterial genera it does not usually exceed 5% (Zavarzin, 1974). On the basis of the high similarity of the genomes and some key morphological and physiological features, Bercovier et al. (1980) united the pseudotuberculosis and plague microbes into one species, while many

researchers referred the plague to the group of sapronoses or saprozoonoses (Levi, 1997; Domaradskii, 1998; Litvin et al., 1998). In this case, possible pathways of its evolution are reconstructed in the open system “rodent–flea–causative agent–environment (soil),” i.e., outside the limits of the closed epizootic parasitic system “rodent–flea–causative agent.” However, these hypotheses lack direct evidence of the capacity of the plague microbe to reproduce outside the host and vector. Most researchers consider it as an obligate parasite and refer to auxotrophs incapable of synthesizing the amino acids that they receive from the host (Aparin and Golubinskii, 1989; Perry and Fetherston, 1997). The hypothesis put forward by Dyatlov (1989) occupies a special place among the hypotheses of the plague origin. On the basis of a comprehensive analysis of interactions between the causative agent and hosts and vectors, Dyatlov came to the conclusion that these interactions are relatively recent. However, he denies the possibility of a slow, through natural selection, evolution of the plague microbe in the parasitic system “rodent–flea–causative agent” in the recent past and believes that it takes its origin from closely related sapronosic microorganisms (of the pseudotuberculosis microbe type) through systemic mutations in rodents in the beginning of every epizootic cycle. Dyatlov understands the imperfection and artificiality of his hypothesis, but he published it in the hope that it might stimulate new ideas. The scientists of western schools recognize the recency of the plague foci in Africa and America and believe that they appeared after the causative agent was introduced by humans from Asia during the 3rd pandemic in the end of the 19th–beginning of the 20th century (Perry and Fetherston, 1997). However, the ecological-geographic aspects of plague origin do not receive sufficient attention against the background of comparative genetic and molecular biology studies. The problems of how and when the marmot subspecies of the plague microbe, which circulates in the marmot populations in the Middle and Central Asian mountains and Tibet, penetrated into the mountain savannas of Eastern and Central Africa need to be studied (Devignat, 1951; Davis et al., 1968; Martinevskii, 1975; Guiyoule et al., 1994). The hypothesis of Pollitzer (1954) about the Central-Asian source of the 2nd pandemic in Europe and Northern Africa seems to be the most reliable. The plague could reach the Nile outflows along its densely populated valley from Egypt through Nubia and Ethiopia, which were also embraced by the 2nd pandemic (Kozlov and Sultanov, 1993). Thus, despite a great bulk of diverse studies, no common concept of the plague origin has so far been developed and the existing hypotheses are rather contradictory. We believe that the idea of phylogenetic recency of the plague causative agent is the most promising. BIOLOGY BULLETIN

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ECOLOGICAL ASPECTS OF EVOLUTION

DEVELOPMENT OF THE IDEA OF PHYLOGENETIC RECENCY OF THE PLAGUE CAUSATIVE AGENT After the phenomenon of the natural focus pattern of the plague had been discovered, the ideas about the ancient origin of this infection became widespread. However, this idea is now questioned in light of the data obtained when studying the biology of the causative agents of plague and other yersinioses. Beklemishev had already put forward the idea of plague in 1956 on the basis of the principle of comparative parasitology he developed, which constituted the basis of classifying the parasitic systems. While characterizing the natural focus diseases according to the mechanism of their transmission, he wrote: “…the bacterioses plague and tularemia, little specialized in the respect of transmission by the arthropods, are phylogenetically recent transmissive diseases, at the boundary between obligate and facultative transmissiveness” (Beklemishev, 1970, p. 346). In the parasitic plague system, the microbe is transmitted from one rodent to another in a specific transmissive way based on the mechanical transmission inherent in recent parasitic systems. The plague microbe occurs in the flea only in a blood clot, a derivative of the warm-blooded host, and does not penetrate in the vector tissues. However, Beklemishev’s conclusions were not noticed at that time. Among specific properties of the plague microbe that indicate to its recency, Dyatlov (1989) stressed the following: the incapacity of hemolysis (the plague microbe is not a true blood parasite); the absence of adaptations for adhesion to the host cell membranes (the system of tissue complementation was not improved); the absence of organotropism; the possibility of transmission only after sepsis development as an unspecialized form of infection; the production of toxins not coupled with a trend to mutualism with the host; the thermal dependence of antigenic properties; the capacity to invade different rodents, double-toothed rodents, and other animals; the unstable capacity for the formation of a proventriculus “block” and the absence of biochemical mechanisms of block prevention or liberation from block in fleas, and the capacity to be transmitted in both transmissive and nontransmissive ways. Dyatlov’s conclusions were not supported, since he explained the microbe recency by its abrupt appearance through mutagenesis. The idea of the plague microbe recency is confirmed by the results of latest molecular biology and molecular genetics studies at the Institut Pasteur in Paris and the Max Planck Institute in Berlin (Achtman et al., 1999; Buchrieser et al., 1999). It was shown by gene sequencing the plague and pseudotuberculosis microbes that the plague microbe had originated from in the recent past (15–20 thousand years ago in the Late Pleistocene–Holocene; the plague is now at the initial stages of evolutionary transformations. In these studies, indicating the onset of a new stage in understanding the BIOLOGY BULLETIN

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phylogeny of yersinioses, structural rearrangements of genes were followed which reflected the sequential transformation of the pseudotuberculosis microbe into the plague microbe. It was also shown that the rat subspecies of the plague microbe is the most recent among the three studied subspecies (marmot, vole, and rat). Such unexpected results made the ecological interpretation of this case of speciation difficult. Achtman et al. (1999) believe that the evolution of the plague, unlike anthroponoses, should not be intimately related to human socialization because the plague is a disease of wild rodents. It was proposed that the microbe could have originated in the populations of African grass mice Arvicanthis niloticus and fleas Xenopsylla cheopis in the regions of ancient land cultivation in the Nile Valley and sources and in eastern Africa. Later, the plague distributed, supposedly, through the Mediterranean in the population of wild rodents of Eurasia (Achtman et al., 1999). The hypothesis of origin of the plague microbe from the pseudotuberculosis microbe in the Late Pleistocene may be supported by two facts: (1) the Late Pleistocene is considered as the time of extreme cooling in the temperate latitudes of the northern hemisphere (Velichko, 1968; Lozhkin et al., 1995) and (2) the successful transmission of the pseudotuberculosis microbe via fleas happens only in the cold (Vashchenok, 1988). Thus, the idea of the plague phylogenetic recency starts to gain more firm positions. Let us stress the most important arguments in its favor: (1) Beklemishev’s concept on the mechanical essence of a specific mechanism underlying the transmissive transfer of the causative agent by the fleas, (2) the absence of pronounced coadaptive features in the microbe and its hosts and vectors, (3) genetic data that confirm the recent origin of the microbe after its late separation from the pseudotuberculosis microbe, and (4) the absence of facts suggesting the evolutionarily ancient plague origin. ECOLOGICAL FACTORS OF THE PLAGUE MICROBE EVOLUTION While accepting the concept of the plague phylogenetic recency, we should explain the causes of its wide distribution on the vast spaces of Eurasia, Africa, and America. In other words, we should analyze the physicogeographical, ecological, and social events on the planet in the relatively recent past which could have led to the appearance of the plague microbe and its foci. The distribution of the pseudotuberculosis microbe around the world was accompanied by the formation of many intraspecific forms (serological types). Among them, only one local population could have become the ancestor of the plague microbe. One rat subspecies of the plague microbe was found in the New World, which is typical for the natural foci on the Indostan Peninsula. Two subspecies were described in Africa, rat and mar-

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mot, which also occur in Eurasia. The greatest number of subspecies were recorded in Eurasia: 3 to 10 (Tumanskii, 1957; Levi et al., 1961; Timofeeva and Logachev, 1978; Aparin and Golubinskii, 1989; Domaradskii, 1998). Hence, it is more logical to search for the sources of plague in Eurasia. There is a hypothesis accepted by a wide circle of researchers concerning the origin of plague in the marmot populations in the arid regions of Central Asia. The first contribution to its formation was made by the Russian physicians and scientists in the second half of the 19th–beginning of the 20th century, who studied the disease bursts and diseases of individual humans in Transbaikalia, Mongolia, and China (Zabolotnyi, 1956; Golubinskii et al., 1987). This idea gained gradually firm positions (Wu Lien Teh et al., 1936; Devignat, 1951; Pollitzer, 1954; Tumanskii, 1957; Rall’, 1965; Petrov, 1968; Burnet and White, 1975; Norris, 1977; Dols, 1978; Gage et al., 1995). While espousing the view of these scientists, Kozlov (1979) wrote: “It is known that the zone of temperate climate is optimal for distribution of pseudotuberculosis. It can be proposed that the conditions of mountain steppes with their fauna were the most suitable for the formation of the plague causative agent from its ancestral pseudotuberculosis form. This could be enhanced by the long-term presence of the plague microbe in hibernating rodents or fleas. The formation of the plague causative agent under these conditions could also be enhanced by abiotic factors, in addition to the biotic ones (marmots and their parasites, fleas). The exceptional aridity of the climate of mountain steppes could not help but create difficulties for the circulation of the ancestral pseudotuberculosis form the alimentary way. Hence, it can be proposed that the first to appear was the variant of the plague causative agent called by Tumanskii (1957) Pasteurella pestis var. marmotae (P. pestis antique after Devignat, 1951)” (Kozlov, 1979, p. 33). Following this hypothesis, let us consider some ecological features of the Mongolian bobak (Marmota sibirica Radde, 1862) and the essence of larval parasitism of the marmot flea Oropsylla silantiewi Wagn., 1898. Behavior of the Mongolian bobak during preparation for hibernation. Aridization of the landscapes in the Northern hemisphere began from the Eocene and continued in the Neogene for tens millions of years. As a result, landscapes with characteristic steppe (hipparionic) fauna formed on vast spaces of North America, Eurasia and northern Africa (Vrba, 1996). The marmots became a component of this fauna. The most ancient fossil findings of the ancestral forms of marmots are known from the Late Miocene (9–11.5 million years ago) in Montana, USA (Black, 1972; Thomas and Martin, 1993). The early rare findings of the Marmota fossil in Eurasia are known from the Late Pliocene (Zimina and Gerasimov, 1973). The most ancient finding of the Mongolian bobak M. sibirica nekipelovi is known only

from the Early Pleistocene in Tologoi (southern Siberia) (Erbaeva, 1970). All other contemporary species of Eurasian marmots are known mostly from the Paleolithic fauna of the Valdai glacier epoch of the Late Pleistocene (Zimina and Gerasimov, 1973). The forest marmot M. monax is considered the most primitive form of contemporary marmots (Hoffmann and Nadler, 1968). Therefore, the origin of the genus Marmota is related to the North America. It is quite likely that the marmots came to Eurasia across Beringia in the Late Pliocene or Early Pleistocene (Black, 1978). The branch of marmots that penetrated in the arid regions of southern Siberia could have formed the species M. sibirica (Mongolian bobak). On the other hand, recent findings of fossil Pliocene and Early Pleistocene marmots in Eurasia confirm the idea of Central Asiatic origin of the genus Marmota (Bibikov, 1989). Bazhanov (1947) believed that Marmota appeared in the arid midmountains of Central Asia that were under the influence of the ancient Gobi Desert. Irrespective of the site of origin of the genus Marmota, the origin of the Mongolian bobak as a species is unambiguously related to the arid regions of Central Asia (Gromov et al., 1965; Zimina and Gerasimov, 1973; Bibikov, 1989). With respect to possible vertical migrations upon climate changes, the boundaries of the ranges of the Mongolian bobak and other mountain marmots suffered no marked changes during the Pleistocene and Holocene (Zimina and Gerasimov, 1980; Bibikov, 1989). The aridity of the landscape where the Mongolian bobak speciation took place determined the species specificity of its behavior. It is known that, before hibernation, the bobaks bury all entrances of the burrows from the outside, except the main entrance which is “plugged” from inside. Among all marmots, only the Mongolian bobak makes “toilet” arms, where feces is accumulated. In the arid petrophytic steppes of Central Asia, typical habitats of the Mongolian bobak, the rubble ground has a low humidity (3–6%) and is hardly suitable for making a strong plug necessary for protection against predators, above all, the numerous Siberian polecats. Therefore, the Mongolian bobak utilizes its metabolic water, i.e., uses the feces as cementing material and makes a plug from their mixture with stones and fine earth. While plugging the main entrance from inside, the bobaks use their teeth to move the “toilet” stones rolled in feces. Some marmots we removed from the dug burrows in winters had their paws, muzzle and teeth covered feces. Thus, the behavior of the Mongolian bobak creates especially favorable conditions for infestation by the pseudotuberculosis causative agent by the fecaloral way and for stable circulation of infection in its populations. Larval parasitism of the marmot flea O. silantiewi. The flea larvae are typical detritophages. Their parasitBIOLOGY BULLETIN

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ism on hosts is a rare phenomenon known in the fleas of nomadic animals such the Arctic hare and Tasmanian marsupial predators, fleas of some Antarctic birds, fleas of sousliks occurring in the severe conditions of Siberia, and marmot fleas O. silantiewi (Freeman and Madsen, 1949; Zhovtyi, 1970; Beaucournu, 1973; Vasil’ev and Zhovtyi, 1974; Rothschild, 1975; Beaucournu and Launay, 1990; Chastel and Beaucournu, 1992; Guiguen et al., 1993). The larval parasitism of O. silantiewi is characterized as “rudimentary” (Vashchenok, 1988) and is so far known only in the Mongolian bobak populations in Transbaikalia and Mongolia (Zhovtyi, 1970). We found this phenomenon in Tuva. O. silantiewi can lay eggs in the fur of hibernating bobaks. The larvae hatched in winter in the burrow do not leave the bobak body and feed on the feces of adult fleas and derivatives of skin cover (Vovchinskaya and Olovina, 1946; Ryabov, 1946; Zhovtyi, 1970). The larvae pupate and complete metamorphosis on the bobaks. The main cause of transition of the O. silantiewi larvae to parasitism on bobaks is the deep freezing of the ground in eastern Siberia, northern Mongolia, and Manchuria related to the sharply continental climate, low winter temperatures (the January isotherm –20°C reaches Ulan-Bator in the south), and snowless winters or winters with little snow. The cold corridor is now extended in the meridional direction from Verkhoyansk and Oimyakon to Manchuria. Most winter burrows of Mongolian bobaks have nest chambers at a depth of 1.6–2.2 m. At this depth, the ground in Transbaikalia and Tuva freezes by the end of December and thaws in June–July. Thus, the bobak burrows are in the frozen grounds for five to six months. In these frozen nests, the bobaks are in the state of interrupted hibernation from January until April. The body temperature does not normally decrease below 5°C and may reach about 1°C in the northern species (Bibikov, 1989; Arnold, 1993; Vasiliev and Solomonov, 1996). The lowest temperatures of the ground around winter chambers of the Mongolian bobak are –6 to –8°C. Following the positive thermotaxis, the flea larvae move from the freezing nest flooring onto the bodies of bobaks hibernating as a familial group. We described a previously unknown phenomenon in Tuva: facultative hemophagy of the O. silantiewi larvae. In winter, after transition of the larvae from the nest flooring onto the hibernating bobaks, some of them come to the oral cavity and begin feeding on the blood and damaging the mucosa. Admittedly, the warm air exhaled by the bobak serves as a peculiar attractant for the larvae and draws them to the nasal and oral orifices. In Tuva, we found eight hibernating and nine active Mongolian bobaks in three burrows dug in February– March and collected more than 230 flea larvae, 60 live cocoons, and 430 cocoon envelopes from the fur of these animals. More than 20 larvae were collected from the oral cavity of the hibernating animals. All larvae BIOLOGY BULLETIN

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removed from the oral cavity had blood inside and their size was increased due to the body fat development. The oral cavity mucosa and tongue were damaged and abundantly bled. Nonscarified wounds in the oral cavity could be found in almost all bobaks caught in April soon after their exit from the winter burrows. We believe that the positive thermotaxis, an individual adaptive behavioral reaction, served a prerequisite for “rudimentary” parasitism of the O. silantiewi larvae. This form of parasitism is expressed locally under the extreme conditions. O. silantiewi is a monotypic species without distinct geographical variability (Ioff, 1941; Ioff and Skalon, 1954; Kapitonov, 1960, 1978; Ioff et al., 1965; Bibikov, 1967; Zhovtyi, 1970; Lewis, 1975; Traub et al., 1983; Goncharov et al., 1989). Monomorphic imago and identity of the characteristics of the life cycles of various populations all over the range, from the Don steppes to Verkhoyan’e and Kamchatka, suggest that behavioral deviations in the larvae of these fleas from southern Siberia and Mongolia fit the reaction norm and are not evolutionary shifts. The parasitism of larvae should only be considered as labile behavior upon the choice of adequate conditions in the accessible thermal continuum “bobak–nest” without a noticeable trend to evolutionary transition of the local population to a new ecological niche with the change of the life cycle. Nevertheless, the larval parasitism of O. silantiewi became a local population event. Hence, one can speak about the appearance of a new parasitic system “bobak–flea larvae,” whose structure is not yet fixed in the population gene pools of the parasite and host. The appearance of the O. silantiewi larval parasitism on the Mongolian bobak should be timed, with a high probability, to the coldest period in southern Siberia and Central Asia, Late Pleistocene. This time agrees quite well with the date of the plague microbe appearance according to the molecular biology data (Achtman et al., 1999). The species specific behavior of the Mongolian bobak related to construction of hibernation plugs and larval parasitism of the marmot flea in winter led to the appearance of conditions for penetration of the pseudotuberculosis microbe from the feces to the blood through the damaged oral cavity of bobaks. On the basis of these facts and well known ecological features of the bobaks and their fleas, a logical model of the pseudotuberculosis microbe transformation into the plague microbe can be easily constructed. This mode is based on the following data: (1) the vast territory densely populated by the Mongolian bobak in Mongolia and adjacent regions of Russia and China characterized by cold winters with little snow; (2) the heterothermic bobaks regularly waking up in the burrows in winter (1–38°C); (3) the familial-colonial mode of life (up to 20 hibernating animals per burrow); (4) the different ages of animals in the family; (5) the large size of bobaks and related features of thermoregulation during hibernation; (6) the long-term use of

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constant and winter burrows by the bobaks (hundreds and thousands years); (7) the year-round parasitism of the marmot fleas O. silantiewi on hosts; and (8) the intense reproduction of the pseudotuberculosis microbe in fleas under the cold conditions. The plague microbe was formed in a discrete thermal field with a gradient from 1 to 38°C. The temperature correlated with the level of blood immunological activity and adaptatiogenesis of newly formed microbe was directed at overcoming of this activity. At temperatures over 26–30°C, the plague microbe starts to produce the antibiotic pesticin (Aparin and Golubinskii, 1989). The pesticin production is usually related to its hostal adaptation–virulence towards the hosts. However, it is known that pesticin suppresses the growth of the initial species, pseudotuberculosis microbe (Martinevskii, 1969). In all likelihood, this product of metabolism regulates interspecific relations between the plague microbe and pseudotuberculosis and other microbes. Pesticin could have ensured the formation of an interspecific chiatus during sympatric speciation of Y. pestis after diverging from Y. pseudotuberculosis in the blood of hibernating bobaks. Thus, according to our concept, the plague microbe formation should be related to: (1) the specific behavior of the Mongolian bobak that occur in dry rubbly steppes, (2) the marked cooling in southern Siberia and Central Asia in the Late Pleistocene, and (3) the larval parasitism of the flea O. silantiewi on bobaks in winter (Suntsov and Suntsova, 2000). We propose that the formation of the Neogene steppe fauna and of many parasitic systems “burrow rodent–flea” in Eurasia was only a landscape-ecological prerequisite for the appearance of prospective natural plague foci. The species specific behavior of the Mongolian bobak in the arid landscapes with a deficit of soil water became its most important component. The plague causative agent appeared much later, only in the Late Pleistocene, when the January isotherms −20 to –30°C moved from eastern Siberia to Central Asia and reached the territories that were densely populated by the Mongolian bobak. An abiotic factor that caused certain ecological shifts in the ecosystem served as an inducer of speciation. The simplest behavioral reaction within the limits of the reaction norm to the temperature oscillations in a component of the ecosystem, the O. silantiewi larvae, was the triggering ecological factor of the plague microbe speciation. Thus, the newly formed epizootic plague system was derived from two ancient stable parasitic systems: “Mongolian bobak–Y. pseudotuberculosis–soil” and “Mongolian bobak–O. silantiewi,” and one recent system: “Mongolian bobak–O. silantiewi larvae,” the relations within which have not yet been fixed genetically. The appearance of a new parasitic system “Mongolian bobak–O. silantiewi larvae” led to the formation of new interspecific relations and, finally, to the appearance of a new species–Y. pestis.

The Mongolian bobak was a coupling element of all parasitic systems involved in coevolution, allowing us to talk about the evolution of the bobak coadaptive complex as a component of biocoenosis. The properties of the microbe as a blood parasite were formed in the bobak coadaptive complex. We consider the marmot subspecies Y. pestis marmotae as an initial form of the polytypic species–plague microbe. MORPHOGENESIS OF THE PLAGUE MICROBE AS A MACROEVOLUTIONARY PROCESS The high genetic similarity of the plague and pseudotuberculosis microbes allowed the taxonomic and ecological convergence of the causative agents of these etiologically different infections. For example, Bercovier et al. (1980) proposed to unite them into a common species as two subspecies: Y. pseudotuberculosis pestis and Y. p. pseudotuberculosis. On the other hand, the hypotheses of a “saprophytic” plague appeared during the recent decades. This led to a search for the microbe in the organic residues in the rodent’s burrows, in soil amoebas, in plants, etc. The mode of existence of the microbe in nature, enzo-epizootic, saprophytic, or as their different combinations, is so far hotly debated. Transformation of the intestinal parasite pseudotuberculosis microbe into the obligate blood parasite plague microbe corresponds to the concepts of adaptatiogenesis and adaptive zones, which many evolutionists consider the main concepts of macroevolution. The mobile prototrophic pseudotuberculosis microbe passes into a new adaptive zone: from circulation in the open parasitic system “rodent–causative agent–soil” to the closed system “rodent–flea–causative agent.” Its life scheme undergoes sharp changes, and the character of metabolism becomes auxotrophic. In this case, the origin of the plague microbe from the pseudotuberculosis microbe may be considered a process of macroevolution according to the ecological criterion of transition to another ecological system, where the microbe enters in radically different relations with a different environment. However, based on molecular biology criteria, Bercovier et al. consider this process microevolutionary, and other researchers (Klassovskii and Petrov, 1968; Aparin and Vershinina, 1984; Aparin and Golubinskii, 1989), consider it speciation. Despite the similarity of the plague and pseudotuberculosis causative agents according to a number of key features, the chromosome of the plague microbe is 20% larger than that of the pseudotuberculosis microbe (Anisimov et al., 1985). This may be considered an essential morphological distinction between these two species. Moreover, the plague microbe has two plasmids that determine its specific properties as an obligate parasite. These organelles are absent in the pseudotuberculosis microbe. Thus, the two species may be considered closely related only in comparison with other species of bacteria. BIOLOGY BULLETIN

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Note that the unique traumatic pathway of the plague microbe origin as a blood parasite puts it in an exceptional position among many intestinal bacteria of the family Enterobacteriaceae, to which it belongs. None other causative agents of the intestinal infections in rodents except pseudotuberculosis has closely related forms that are transmitted by the transmissive way. This exceptional evolutionary fate of the plague microbe allows us to consider its formation as a macroevolutionary process. In the context of the phylogenetic approach in taxonomy, the newly formed species, the plague microbe, can be confidently called a newly formed genus according to the ecological criterion. The progressive features of its evolution were clearly expressed in its wide distribution in Eurasia and adaptive radiation with the formation of specialized subspecies: marmot, gerbil, rat, souslik, pika, and several vole subspecies, in various parasitic systems “rodent–flea.” The disjunction of the subspecies ranges and sympatric existence of some of them, their stable characteristics, and a good microbiological chiatus with possible diverse variability of features in individual strains may satisfy the species rank (Dyatlov, 1989). For example, in Central Africa, there are foci of rat and marmot subspecies in one or another adjacent habitats, and in the south-east of Hagai in Mongolia there are foci of marmot and vole subspecies in the joint colonies of the Mongolian bobak and Brandt’s vole. In the Bayan-Ulegei aimak in Mongolia, two different subspecies, altai and ulegei, occur jointly in the populations of Mongolian pikas. There are zones of intergradation of different microbe subspecies in Caucasus and Transcaucasia (Varshavskii et al., 1972; Martinevskii, 1975; Kozlov, 1978; Logachev et al., 1978; Timofeeva and Logachev, 1978; Sotnikova et al., 1980; Dyatlov, 1989; Kozlov and Sultanov, 1993). Martinevskii (1969) believed that the vole subspecies should at least be isolated as an independent species with several varieties. These facts suggest that there is no theoretical ban for giving the generic ran to the plague causative agent. The plague causative agents may be referred to an independent polytypic genus, which includes three to 10 or more forms with possible geographical sympatric existence. According to the official classification, these forms are now called subspecies. It was experimentally shown that the strains of different subspecies cannot exist in the same animal simultaneously. They are replaced by a dominant subspecies inherent in the given host (Stepanov et al., 1980). This suggests an actual existence of the accepted subspecies. In a specific geographical region (focus), the microbe is adapted to a specific host, and there is no biotypical sympatry of subspecies. Nevertheless, the patterns of population variability of the plague microbe are not yet fully clear, and this complicates elaboration of the principles of its intraspecific classification (Aparin and Vershinina, 1984). BIOLOGY BULLETIN

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Speciation (genus formation) of the plague microbe may be represented as a unique macroevolutionary process, with the present-day coexistence of the ancestral and newly derived forms, which can be studied by the same methods as the microevolutionary process: direct observation, comparative analysis, experimentally, and on a model. The distribution of this microbe may be considered as ubiquitous. However, the evolutionary transformation of the pseudotuberculosis microbe in the plague microbe took place only in the marmot population in a certain geographical region, at a certain time, and at a combination of ecological conditions, i.e., the process of microevolution of a local population of the pseudotuberculosis microbe was terminated by a macroevolutionary effect. In other words, an unlikely combination of conditions proved to be realized in this case, which led to essential ecological transformations. Only the recentness of these evolutionary events and preserved ecological conditions in which these events took place allowed us to reconstitute the true pathway of plague evolution. In the context of the concept of macroevolution and ecological approach to the taxonomy of Yersinioses, the chiatus between the plague and pseudotuberculosis microbes may become more understandable and material. Domaradskii (1998) insists on the ecological principle of classifying the plague microbe and other pathogenic yersinias with a special reference to distinct metabolic differences. Such approach would be useful for the revision of the modern aggregate genus Yersinia, which includes 11 species whose representatives have radically different ecological characteristics: Y. ruckeri is a causative agent of fish disease, Y. pseudotuberculosis and Y. enterocolitica are causative agents of intestinal infections in diverse animals, Y. pestis is a causative agents of plague in rodents and double-toothed rodents; other species may in some cases induce opportunistic infections in humans or are not pathogenic (Howlt et al., 1997). The correctness of their inclusion in the same genus cannot be considered doubtless. According to Achtman et al. (1999), a common ancestral form of the pseudotuberculosis and plague causative agents already existed 0.4–1.9 million years ago, i.e., in the Early–Middle Pleistocene. Despite a relatively long-term relations of this ancestral form with rodents, their interactions did not lead to the formation of the parasitic plague system. Special conditions were required for the pseudotuberculosis microbe to gain access to the system “rodent–flea,” i.e., for its breakthrough in a new adaptive zone. This took place due only to the natural cataclysm: Late Pleistocene cooling. The coevolution of members of the epizootic plague triad began only in the Late Pleistocene. Thus, the evolution of the causative agent proceeded via a relatively fast ecological specialization in the already existing system “rodent–flea” after opening of new “gates” into this system, rather than via long-term phylogenetic specialization in the system “warm-blooded

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host–flea” during the Paleogene–Neogene development of this environment.

(Suntsov et al., 1995; Suntsov and Suntsova, 1999; Suntsova, 1999).

On the example of the plague evolution, we can see with our own eyes that an abstract “inadaptive intermediate zone” is a so called “narrow corridor” to a new adaptive zone. These are populations of Mongolian bobaks hibernating in the winter but regularly waking up with fleas parasitizing them. As the bobaks were waking up and their body temperature increased, the ancestral intestinal form adapted to the life in their blood. The principle of overcoming of the “inadaptive zone” by the evolving form may be represented as the “movement across the bog by hummocks.” The bobak blood has a varying degree of immune “aggressiveness” for the microbe with respect to the hibernation depth and body temperature. In this ecologically unstable intermediate environment, a driving form of selection dominated, which was directed, on one hand, at a selection of the clones capable of maintaining the bacteremia in hosts as their body temperature increases and, on the other, at the reproduction and survival in fleas during the period of activity of the bobaks. The genetic and phenotypic properties of the evolving microbe in this environment were not stabilized, and the “plague of hibernating bobaks” did not appear. Stabilization took place only after the appearance in the newly formed microbe of adaptations to parasitism in the blood of active bobaks with a stable constant body temperature of about 37°C.

Later, the economical activity of humans led to a new biosocial stage of the genesis of foci and their anthropogenic distribution. Synanthropization of rats, predominantly the black rat in Indostan, was the most important ecological factor that started this stage. Vast and numerous colonies of black rats appeared in human settlements and surrounding agricultural landscapes at a certain period of social development. The parasitic exchanges of the black rat and Indian gerbil led to the appearance of synanthropic (rat) foci. At this stage, secondary natural foci were formed through the synanthropic foci in the New World, Africa, on Madagascar, Java, Hawaii and some others. Humans were involved in the distribution of only two subspecies whose hosts are most closely related to humans, marmot and rat subspecies.

NATURAL-HISTORICAL AND BIOSOCIAL STAGES OF THE GENESIS OF THE PLAGUE FOCI IN THE WORLD According to our concept, the plague microbe was formed in the Mongolian bobak populations in Central Asia in the Late Pleistocene. Its further evolution and geographical relay expansion in Eurasia proceeded along the pathway of adaptive radiation and adaptation of the microbe to new ecological niches with the formation of some allopatric and sympatric subspecies, i.e., to the circulation in other parasitic systems “rodent– flea,” but without sharp changes in metabolism. The microbe transition to other parasitic system was realized at the expense of interspecific parasitic contacts, when the fleas were exchanged between different hosts. The natural range of the plague causative agent embraced vast spaces: from the east to the west, from Manchuria to Caucasus and Middle East, and from the north to the south from southern Siberia, northern Kazakhstan, and northern Caspian territory to southern Indostan. The primary range of the microbe did not include Indochina and southern China. There are no ecological conditions for the natural foci formation because specific fleas are scanty, they parasitize only during the dry season, or they are altogether absent on the background animal species, wild rats populating the tropical and subtropical forest and savanna landscapes

The origin of the present-day foci may be classified as primary natural, synanthropic (rat), secondary natural, or a combination. For example, the secondary natural plague foci on the Hawaiian Islands were formed in the beginning of the 20th century in the populations of previously introduced black rats (apparently, from the Zond shelf) and fleas (from Indochina), i.e., on the basis of the human-produced artificial parasitic system “Rattus exulans–Xenopsylla vexabilis.” On the Madagascar Island, the microbe circulates in nature in the populations of black rats introduced from Indostan, on which the local endemic flea Synopsyllus fonquernei parasitizes. In North America, the secondary natural foci are maintained by the populations of endemic wild rodents and their fleas. In Central and Eastern Africa, a complex of synanthropic, anthopurgic, and secondary natural foci formed, where the role of main hosts is played by representatives of the genera Arvicanthis and Praomys (Mastomys) (Muridae), and introduced black rats and the African species of the genus Xenopsylla act as the main vectors (Pollitzer, 1954). Sufficient conditions of the existence of the plague as a recent and little specialized infection include a high and relatively stable abundance of the host and parasitizing fleas and a certain level of the host resistance to the microbe. This is confirmed by the appearance of many secondary natural foci. The synanthropic plague foci may also exist for a long time upon the preservation of constantly high epizootic contact in the host populations. For example, such foci in a network of human settlements still exist more than 100 years after the plague was brought in 1898. CONCLUSION Thus, the natural plague foci are a relatively recent phenomenon. The formation of the infection causative agent and of natural foci took place naturally, without the participation of humans, in the Pleistocene. BIOLOGY BULLETIN

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The human economical activity played an important role in the distribution of the plague over the world from its natural foci. The increased population density, construction of houses and creation of food reserves, tolerance of people to synanthropic rats, assimilation of the territories where the natural foci were located, hunting of marmots, increased trade, wars, and strengthened international and intercontinental relations led finally to the appearance of stable synanthropic and secondary natural foci in the New World, Africa, and in many regions of the tropical zone. The proposed concept of Pleistocene origin of the plague parasitic system, like others, does not explain the mechanisms of “interepizootic” existence of the causative agent of this disease. But it agrees with the postulate of the classical theory of plague natural focus pattern that this period is absent in its functional understanding. According to our hypothesis, the formation of the plague microbe as Y. pestis took place during the development of the closed self-regulating epizootic system “Mongolian bobak–flea O. silantiewi–causative agent.” The initial mode of life of the microbe in nature is its continuous circulation in the system “rodent–flea” as successive enzo-epizootic cycles. The system “rodent–flea” is the microbe environment, and every stable natural system in Eurasia has its own specific subspecies. Therefore, within the context of our position concerning the plague microbe origin and evolution, the dynamics of these cycles should be based on the immune interactions of the heterogenous populations of the causative agent and hosts and, to a lesser extent, vectors or, in other words, in dialectical unity and contradiction of the features “virulence–resistance,” rather than on the existence of an extraorganismic (extrahostal and extravectorial) phase of the microbe. ACKNOWLEDGMENTS The authors express their sincere gratitude to L.P. Korzun, V.S. Lobachev, V.S. Nikol’skii, A.S. Severtsov, B.R. Striganova, and S.A. Shilova for the helpful discussion of the materials on which this paper is based, and to G. Sadler (Wisconsin University, USA) who provided access to Internet and supported the preparation of this manuscript to press. REFERENCES Achtman, M., Zurth, K., Morelli, G., et al., Yersinia pestis, the Cause of Plague, Is a Recently Emerged Clone of Yersinia pseudotuberculosis, Proc. Nat. Acad. Sci. USA, 1999, vol. 96, no. 24, pp. 14043–14048. Anisimov, P.I., Mozharov, O.T., Kondrashin, Yu.I., et al., Estimation of the Degree of Homology of the Y. pestis and Y. pseudotuberculosis Genomes, Immunokhimiya i laboratornaya diagnostika osobo opasnykh infektsii (Immunochemistry and Laboratory Diagnostics of Especially Dangerous Infections), Saratov, 1985, pp. 87–96. BIOLOGY BULLETIN

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