On the phylogeny of splenic structure and function

DEVELOPMENTAL AND COMLPARATIVE IM~FUNOLOGY, Vol. 4, pp. 395-416, 1980. 0145-305X/80/030395-22502.00/0 Printed in the USA. Copyright (c) 1980 Pergamon Press Ltd. All rights reserved.

REVIEW ON THE PHYLOGENY OF SPLENIC STRUCTURE AND FUNCTION

RM. Pit chappan Department of Immunology, School of Biological Sciences Madural KamaraJ University, Madurai-625 021, India

Evolution may be defined as 'descent with modification' -- Darwin. When an organ system is studied in diverse representatives of a single phylum, one gets the impression that the system is based upon a prototype which is simply varied from class to class, with finer variations within the class (1,2). Once the variation has set in, selection pressures operate and the fittests survive. A survey of the immune system and their functions in various vertebrate classes may reveal that the immune system is no exception to this. The kigher the status of an animal in the phylogenetic tree, the greater is the organization and efficiency of its immune system. In recent years, a large body of information has accumulated on the immune system of lower vertebrates (3-11). The lower vertebrates are known to elicit a variety of immune responses, similar to those of higher animals. The early lymphoid aggregations appearing in lampreys (ll,12) and developing into definite lymphoid organs in ether lower vertebrates culminate in the development of several lymphoid organs with specific spatial distribution of lymphocytes in higher vertebrates. The architecture of the lymphoid organs also increases in organizational complexity at each evolutlonary level. The main reasons for these may be the principle of division of labour and structural sophistication leading to precision in function. Infact, it is tempting to hypothesize so, on looking at the phylogeny of splenic structure and function in relation to the appearance of ether lymphoid organs.

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The thymus was the first lymphoid organ to appear during ontogeny and phylogeny and remained almost the same through vertebrate evolution. However, the major secondary lymphoid organ, the spleen appeared first in fishes and unlike the thymus the spleen has undergone radical changes through vertebrate evolution both structurally and functionally. This may be correlated with its mesenchymal origin and its resultant role in hemopoiesis, the need to tackle the threat by pathogens prevailing in the environment and the presence of other efficient secondary lymphoid organs. A number of investigators have investigated the splenic structure, function and ontogeny and the role of spleen in the development of other lymphoid organs and in various immune responses (13). However, most of the information is restricted to mammals and birds. Among lower vertebrates sporadic information is available in a few groups and again in many cases, comprehensive and clear picture of different aspects of the spleen is not available. This review aims at consolidating the information available on the spleen of lower vertebrates and thus, describing the phylogeny of splenic structure and function. The appearance of white pulp, thymic dependent periarteriolar region and ~ursal dependent germinal center can be followed in sequence during evolution. Further, the importance of spleen in immune response depends on the presence of other secondary lymphoid organs.

DEVELOPMENT AND ANATOMY OF THE SFLEMN The general pattern of development of the spleen is the same in different animal groups. A splenic rudiment first appears as a mesenchymal condensation in the mesentery close to the pancreatic rudiment. As the rudiment assumes definite shape, capillaries and sinuses appear. This is followed by erythropoiesis, granulopoiesis and lymphopoiesis in subsequent development. The similarity is further extended to the relative time or stage and the origin and histogenesis of the splenic primordium from fishes through mammals (14-19). In all the animals the spleen is situated near the pancreas and towards the duodenal curvature. Nonetheless the shape varies in different animal groups. Thus, the spleen in fishes is a flattened and elongated structure, while globular in amphibians and oval in reptiles and birds (14-18).

SPLENIC ARCHITECTURE A comparative study of the splenic histology of various animals reveals the phylogeny of white pulp follicle, thymus dependent area and germinal center. The development and spatial distribution of these structures determines the extensiveness of white pulp follicles.

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White and red pulps: In all vertebrate groups, the splenic capsule is made up of fibro-muscular tissue. Extending inwards from the capsule, the reticular framework representing the trabeculae traverses the splenic cavity and divides it into compartments. Major arteries entering the spleen through the hilus traverse this framework and ramify. A few arteries enter each compartment. Lymphoid accumulations occur invariably from fishes to mammals surrounding these arteries traversing the compartment: these are called lymphoid or white pulp follicles. The size and shape of these follicles vary depending on the length of the artery traversing the splenic compartment which in turn is determined by the size and shape of the compartment itself. Observation on the histology of various spleens confirms this (Table 1). In all the animals the red pulp lies in the interstices of white pulps.

PhyloKeny of white and red pulps: Fishes As one looks at the spleens of various animals from ~ s h e s through mammals the phylogeny of various structures is quite impressive. The splenic white pulp of the teleost fish, Tilapia mossambica is a rather poorly developed reticular tissue without well defined lymphoid centers (14). Red pulp is very extensive and fully erythroid surrounding the cuff of loose reticular cells enveloping the arteriole (14,22). The lymphoid follicle is not well developed in Tilapia. In the newborn guitar fish Rhinobatue productus, a primitive Elasmobranch, the spleen has only red pulp and the development of lymphoid centers is postponed to the adult stage. In Polyodon spathula, a chondrostean fish, the white pulp follicle is well developed (12). Further detailed analyses of the white pulp, red pulp and other constituents of fish spleen are required. Amphibians Among various amphibians, the white pulp follicle differs in its size and shape (23). In Ascaphus truei the most primitive anuran, lymphocytes are scattered almost evenly throughout the spleen and carbon injection does not circumscribe white pulp. Scaphiophus couchii, Rana pipiens and Bufo powerii have an intermediate arrangement and show definite red and white pulps which are specially distinguishable after carbon injection. In _Scaphiophus lymDhocytes occur as relatively discrete units suggestive of white pulp. The most complex white pulp morphology is found in Xenopus laevis. Lymphocytes are arranged in two compact layers concentric around a central arteriole (23-28). Based on their studies on amphibian spleen, Cooper and Wright proposed that the trend from primitive to more advanced white pulp is from diffuse to a concentrated arrangement of lymphocytee (23). Reptiles In contrast to the extensive red pulp in amphibians, in squamate reptiles it is very much restricted in

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between the globular white pulp follicles. In all the species of lizards studied, Mabuya carinata, Hemidactylus brooki, Calotes versicolor ( 1 7 - ~ d ~ rugosa (32) the splenic pulps are very similar to each other. Our extensive studies on Calotes spleen have revealed that in lizards white pulp exists in the form of globular follicles and the red pulp is represented in the interstices (33). The red pulp overlies the reticular framework of the spleen. One could appreciate the presence of honeycomb like (hexagon and pentagon in allsaggital, transverse and frontal sections of spleens) red pulp, only when the spleen is antigenically stimulated resulting in the prominence of the sinusoidal system. Carbon injection studies also confirms the presence of only a restricted red pulp. However t chelonians, such as the turtles Lessemy s punctatus (17) and Chelydra serpentina (31) have a very definite elongated follicular arrangement of white pulp with extensive red pulp. Reticular cells lie immediately adjacent to the arteriole and small lymphocytes are located more peripherally, surrounding the reticular cells (Ref. Table 1). This type of arrangement may be attributed to their early origin from stem reptiles (Cf. below). In recent years, the presence of very extensive sheathed capillaries and red pulp in the lizards Mabu2a quinquetaeniata and Uromastys ae~yptia has been reported (36). Further, the periarterial lymphatic sheath of a lizard has been depicted, similar to chelonian white pulp (35). Thus, detailed analysis of lizards' spleens is warranted. It is proposed that the presence of a clear reticular area surrounding the central artery as in the case of the teleost fish Tilapia, the amphibians Ascaphus and Sca~hiophus and the turtles Lessemys and Chel2dra and the diffuse arrangement of lymphocytes as in Tilapla and Ascaphus may reflect the primitiveness of their spleens. This correlates well with the early evolutionary origin of these animals. Indeed, bony fish appear earlier in the fossil record than do sharks, during the early Devonian period (Of. 1,2,11). The Chelonians from their earlier origin from stem-reptiles have remained the same with a few changes through all the periods since Permian records (12).

EVOLUTION OF THYMUS DEPENDENT LYMPHOID REGIONS In mammalian and avian spleens, the regions immediately adjacent to the central arteriole have been shown to be thymus dependent, wherein the thymus derived lymphocytes home and carry out their function (34,37). Various investigators working on lower vertebrate systems have looked for such thymic dependent areas in these animals. Although the thymuses

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are well defined in these groups and resemble histologically those of higher vertebrate classes, the antigenic identity and spatial distribution of thymus derived lymphocytes in the secondary lymphoid organs is still uncertain. Nonetheless the importance of the thymus in various immune functions has been established throughout the vertebrate classes (9,14,24,25,

38-41). Among lower vertebrates, very little is known about the selective spatial distribution of T cells. In the fish, Tilapia a uniform periarteriolar accumulation of melanomacrophages is observed following immunization (42). The antigen capturing in this region may thus represent the prospective thymus dependent periarteriolar region. As has been discussed earlier, the question whether there is a definite white pulp follicle in Tilapia itself remains open. The attempts of Ellis and De Sousa (43) to identify the spatial distribution of neural duct lymphocytes also did not contribute to our knowledge of spatial distribution of different lymphocytes. Amphibians, in spite of their very definite white pulp and periarteriolar region, showed no specific periarteriolar aplasia but only generalized aplasia, following thymic ablation (27,44). In Triturus and Xenopus following early thymectomy diminution of" ~ size was observed (27,45). The depletion of lymphocytes was significant in the white pulp and the white to red pulp ratio was reduced. In thymectomized Xenopus toadlets, lymphocyte depletion in the perlfollicular area of red pulp was observed (46). Further, the studies on the localization of antigen using fluorescent antibody have shown thymic dependence of late localization of antigen within the boundary layer of white pulp (47). In the marine toad, Bufo marinus although the antigen is trapped in the splenic red pulp and concentrated around the white pulp, antigen never appears within the white pulp (48). Thus, the thymus dependent area of the spleen in amphibians seems to have more in common with the ma2ginal zone of the mammalian spleen (24,25,47). It was further concluded from these studies that the periarteriolar region is not thymus-dependent in Xenopus (25). While discussing the thymic dependent lymphoid regions of the spleens of frogs, it would be more appropriate to ask another still more fundamental question; is there any T and B cell compartmentalization of lymphoid cells in these animals as in higher animals? In Xenopus both B as well as T cells may be derived from the thymus (4) since thymic lymphocytes of Xenopus larvae are immunoglobulin positive and actively synthesize immunoglobulin (24,39). Furthermore, early thymectomy in larval Xenopus impairs both humoral and cell mediated immunity (25). The available evidence on the seeding of thymic lymphocytes further supports this contention. Studies involving cell marker and thymic anlage transplantation in the leopard frog, Rana pipiens showed that virtually all lymphocytes in the spleen, kidney and bone marrow are ontogenically derived from thymic cells (49). Should these be true, the whole spleen is thymus dependent'

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Among reptiles, studies in the lizard, Calotes involving adult thymectomy and anti-thymocytic serum showed a selective loss of lymphocytes (33). In these animals, the periarteriolar region of the white pulp seems to be thymus dependent resembling that of higher animals. From these observations, it was suggested that during evolution the selective spatial distribution of different populations of lymphocytes and the thymic dependence of the periarteriolar region occurred with the advent of reptiles. Starting from the accumulation of lymphocytes surrounding the arterioles in fishes and the thymic dependence of the red pulp lymphocytes in amphibians (27), the periarteriolar lymphocytes seem to have become thymus dependent in lizards and possibly in other reptiles. However, more direct evidence in several species of modern reptiles is required to convincingly establish this. PHYLOGENY OF GERMINAL CENTERS Analysis of the phylogeny of germinal centers provides some interesting correlations with other aspects of the lower vertebrate immune system, but also poses some questions. In fishes, no germinal centers are found in the spleen of nurse shark Ginglymostoma and snapper LutJanus, a teleost. The antigen induced activation primarily occurs in the head kidney of these animals (50). In spleen and head kidney of another teleost Tilapia, cellular changes occur following antigen injection (51). However, germinal centers were not reported in these studies. Studies on the loci of antibody forming cells in chondrostean fish using immunofluorescence showed the absence of germinal centers (20). In amphibians although immunofluorescent, histochemical and electron microscopic studies have shown the presence of small lymphocytes and pyroninophilic cells, similar to those implicated in the mammalian immune response in the perifollicular region of their spleens, cell clusters resembling the germinal centers of mammalian immune response are absent (29,52). Among reptiles, the tuatara Sphenodon punctatum spleen has neither marginal zone nor germinal center (30). Germinal centers are absent in the spleens of the turtle Chelydra (31), the lizards Tiliqua (32) and Calotes (17,33). The existence of germinal centers has been convincingly proved in birds and mammals. Germinal centers were found in lymphoid follicle especially close to the bifurcating angle of the arteries within the lymphoid tissue, and are characterized by a thin fibrous capsule circumscribing them and complete lack of blood vessels (34). Further these germinal centers are bursa dependent (53). Although the typical germinal centers of homiotherms may not be present in lower vertebrates, the efficiency of lower vertebrates' immune systems might be sufficient for immune surveillance. This can be further appreciated if the correlation between germinal centers and immunoglobulin types is examined.

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GERMINAL CENTERS AND IMMUNOGLOBULIN Pollara et al. (20) proposed that the absence of germinal centers in a chondrostean, the paddle fish Polyodon may be correlated with the absence of 7S antibody synthesis which may require an expansion site in the form of germinal centers. This may hold true for all the lower vertebrate classes, wherein IgG both structurally and functionally analogous to mammalian IgG is absent. Recently, the phylogeny of immunity and humoral antioody production have been extensively discussed by Borysenko (4). Elasmobranch antibodies are represented by both 18S and 7S immunoglobulin and the H and L chains of beth are identical in terms of molecular weight, antigenic properties, amino acid composition and electrophoretic pattern (Cf. 4,20). Based on the distinctive properties of H chains, the lungfish possess two major immunoglobulin classes (20). among amphibians two distinct (18S and 7S) classes of immunoglobulin are present. Based on the larval anuran tadpole immunoglobulin which is exclusively of IgM type and their inability to elicit secondary responses, the association of IgG production with secondary responsiveness was suggested (4,54). Urodeles produced no IgG type of immunoglobulins (55,56). Xenopus is the most primitive tetrapod in which IgM and IgG with antigenically distinct heavy chains have been found and both types of immunoglobulins possessed antibody activity (57). Among reptiles, in the tuatara a 7S immunoglobulin, antigenically related to 18S but with no antibody activity, appears (30). In turtles during the course of antibody production, the 18S antibody was replaced very slowly by a mercaptan sensitive 7S antibody. Mercaptan resistant antibodies appeared following repeated challenges (58-60). Among lizards, both high and low molecular weight antibodies were mercaptoethanol sensitive and prolonged production of 19S in the presence of 7S has been noticed in Tiliqua (32). However, in the lizard Calotes no IgG antibody either 2 ME resistant or susceptible could be identified yet (61). It was proposed by Wetheral __et__al. that having a 7S antibody sensitive to mercaptoethanol and the continuous production of high molecular weight antibody in the presence of low molecular weight ones may be the characteristic feature of the immune responses at this reptilian level (32). It is clear from the literature available that IgG types of immunoglobulin synthesis have occurred during evolution in various animal groups (Cf. 4). Even phylogenetically primitive groups within a class (clawed toad and turtle) possess these antigenically distinct 2ME resistant antibodies whereas the modern groups of the classes lack them and this may be attributed to divergent evolution and genetic drift. It is, in fact, difficult to demonstrate typical immunological memory during a secondary humoral immune response in lower vertebrates (62). However, the typical anamnesis with a copious amount of 2ME resistant, high avidity IgG antibody in quick succession following immunization and the feedback control of

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IgM production by IgG seems to have occurred only in higher vertebrates. The absence of germinal centers in lower vertebrates could be correlated with the aforementioned features of immunoglobulin production in them. This can be supported by the observation made in higher animals that the appearance of 19S antibody producing cells is relatively independent of germinal centers (63) whereas IgG antibody synthesis is dependent on germinal centers (64). Thus, it may be attractive to propose that the germinal center is the innovation of homiothermy and the high avidity IgG and the typical amamneses are the outcome of germinal center function. IS THE EVOLUTION OF THE SPLEEN RELATED TO THAT OF OTHER SECONDARY LYMPHOID ORGANS? A correlation between the presence of various secondary lymphoid organs of animals at various phylogenetic levels, and the evolution of different splenic structures can be made. It seems that adaptation in the form of division of labour and sophistication in the immune system and its resultant functions are two parallels which have contributed to the phylogeny of immunity. From our earlier discussions, it is clear that the homiothermic splenic structure has evolved through different stages in sequence viz, the accumulation of lymphocytes surrounding the arteries, appearance of compact white pulp follicles, the dependence of the periarterial region on the thymus and the appearance of germinal centers. It would be interesting to see whether there is any correlation between the appearance of spleen and other lymphoid organs during evolution and to correlate the splenic structure and function (Ref. Table 2). Fishes In all fishes studied the well organised lymphoid tissues are the head kidney, spleen and thymus. Fishes lack bone marrow (67). Head kidney contained more lymphocytes than spleen. Antigen-induced activation occurs primarily in the head kidney (50). Further, head kidney produced more antibody forming cells than spleen (51,67,68). In a few species of fishes the thymus has been reported to contain antibody producing cells (14,51,67). Further, during phylogeny the spleen attains the ability to perform its immune

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ROLE OF SPLEEN IN THE IMMUNE SYSTEM OF THE LIZARD CALOTES VERS ICOLOR Concerted efforts were made in this laboratory to understand the role of spleen in cell mediated and humoral immune responses of the lizard Calotes versicolor. Splenectomy does not affect cell mediated immunity as adjudged by the skin transplantation studies (38) and migration inhibition assay (96). However, the minimal dose of sensitizing antigen used failed to sensitize splenectomized lizards for migration inhibition (96) and this was suggested to be due to the minimal chances of antigen to interact with immuno competent lymphocytes in the absence of spleen (96). While splenectomy does not affect the cell mediated immune responses markedly, they have a profound effect on humoral immune response. Studies using sheep erythrocyte antigen for eliciting both humoral and cell mediated ~mmune responses showed that splenectomy erased the humoral immune response without affecting the cell mediated migration inhibition response (96). Splenectomy suppressed the humoral antibody production to sheep erythrocytes (78) and bovine serum albumen (77). The humoral immune response was abrogated irrespective of route of immunization (96). Further, splenectomized lizards were not sensitized to anaphylactlc shock to egg albumen (97). The fate of specific antigen recognising (RFC) and plaque forming cells (PFC) to sheep erythrocytes in peripheral blood, peritoneal exudate and bone marrow of splenectomized lizards were also analysed. In splenectomized and immunized lizards~ PFCs were absent in peripheral blood, peritoneal exudate (96) and bone marrow (79). Further splenectomy suppressed the appearance of immune RFC in bone marrow and peripheral blood (79). It was suggested that both RFC and PFC found in these areas originate from spleen, the splenic microenvironment being required for the development and differentiation of these cells. The spleen of the lizard, thus is the major secondary lymphoid organ wherein antigen recognition and cell collaboration takes place and may be the sole saviour of the humoral ~mmune system. This idea of the spleen's central role in the lizard immune system is further supported by the observation on hatchnng spleen. It has a definite follicular arrangement and produced antibody forming cells which outnumbered adult values (98). Further the hatchling resists graft versus host reaction induced by injecting adult cells and rejects allografts (38,98). This precocious development of the immune system of this squamate reptile can be compared with the immune response of the chelonian hatchllng. In turtles, immunological maturity is attained only two months after birth and graft versus host responses can be induced up to this time by adult allogeneic spleen cells (31,92). The inability of turtle hatchlings thus shall be attributed to the primitive phylogenetic position of chelonians, which is

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also reflected in the histology of their splenic white pulps; whereas the precocious development of lizard spleen shall be attributed to its well developed histological architecture and the resultant function (Table 2). H i g h e r vertebrates With the evolution of birds and mammals their spleens have been further adapted to their immunological obligations. Mammals diverging in one line from reptiles specialized with numerous lymph nodes and gut associated lymphoid tissues. Whereas in birds, in the absence of true lymph nodes (mammalian type), spleen is modified further and the highest degree of complexity consisting of Scheweigger-Seidel ellipsoid and perlarterial and perivenous lymphoid tissues in addition to germinal centers is seen. However, in none of these higher vertebrates is the spleen as important as in the lizards. The importance of spleen in the immune system/function of a given animal thus depends on the existence of other well defined immuno-competent lymphoid organs (Table 2) (99). CONCLUSIONS A survey of splenic structure in various lower vertebrates reveals the development of white pulp surrounding the arterioles which traverse the splenic compartments and the elaboration of red pulp depending on the extensiveness of white pulp. The trend from primitive to advanced white pulp morphology is from more diffuse to a concentrated follicular arrangement of lymphocytes surroundin~ the artery (23). Among each class of lower vertebrate (viz. Pisces, Amphibia and Reptilia) one could identify different degrees of development of reticular cells, white pulp follicles and red pulp. Among anuran amphibians, the more primitive members of the family Ascaphidae have primitive spleenss the lymphocytes are scattered evenly and carbon does not circumscribe white pulp (23) and the reticular cells are also extensive. Among reptiles the Chelonians which arose from stem reptiles 230 million years ago (1,2), have a spleen with abundant reticular cells surrounding the central arterioles the white pulp is a thin layer forming the follicle around the reticular area (Figure 1). Further, the degree of splenic development varies among different reptiles, the chelonian having more primitive spleen and the lizard with a well developed one. The divergent evolution of splenic structures has thus taken place in each class of lower vertebrate (Figure 1). A careful study of splenic structure reveals that evolution of spleen has followed different degrees of development. During this evolutionary experimentation leading to progressive evolution, more sophisticated structural organization has occurred more than once. It has occurred within each class and at times in primitive members of the same class (Figure 1). This might have culminated in the well defined white pulp follicles in elasmobranchs and chondrosteans among fishes and in Pipidae (Xenopus) among Amphibia. This kind of advanced structural organization in primitive groups of the same class may be attributed to 'genetic drift'. Nonetheless one can always infer a progressive evolution in splenic s t r u c t u r e as

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evidenced in Figure i. The progressive evolution of spleen in lower vertebrates has thus followed in sequence the appearance of white pulp follicles and thymus dependent areas. The germinal center, Scheweigger and Seidel sheath and peri-ellipsold appear in higher vertebrates. The immunological functions like memory, high avidity antibodies, and the importance of spleen in immune responses depend on the degree of splenic development and the presence of other well organized lymphoid tissues (Table 1,2; Figure 1). In fishes, head kidney being the major antibody producer, spleen has a little role to play in their immune response. Among amphibians 19S antibody synthesis is also not dependent on spleen and this may be attributed to the major role of other lymphoid organs in antibody synthesis. Among reptiles, in lizard Calotes, the role of spleen as a repository of cells responsible for humoral immunity shows its importance in the absence of any other well defined peripheral lymphoid organ. This may be an immunological compensation and shall be correlated with the well developed histology of their spleen. However, in lizard Scincus scincus which has numerous GALT, the spleen is not as much important as in Calotes (99). While summing up this review on the phylogeny of immunity, it is essential that we give a word of caution. In the first place one has to attempt this kind of analysis bearing in mind the paucity of literature in the field. Even among the available literature most of the authors have mentioned the presence of red and white pulps only and no detailed description or photographs were given. In order to draw definite conclusions on the phylogeny of splenic structure and function, experiments on a large number of representatives from various orders of a given class should be attempted under strictly controlled conditions. This is essential in view of the fact that these lower vertebrates being poikilothermic and most of them seasonal breeders, very wide variations in their lymphoid tissue architecture and immune functions may occur in a single animal during various phases of its development and from season to season (36,99).

AC KN OWL EDGEMENT S I thank Prof. E.L.Cooper, Department of Anatomy, University of California, Los Angels, U.S.A. and Prof. VR. Muthukkaruppan, Department of Immunology, School of Biological Sciences, Madurai KamaraJ University, Madurai, India for their encouragement and constructive criticism. I thank Dr. R.T.S.Worley, University of Essex, Colchester, England, for reading the manuscript, and Prof. J.C.B.Abraham, American College, Madurai for his suggestions on the evolutionary aspects. The excellent secretarial assistance by Miss Hema Malini and preparation of figures by Mr. Narayanan and Mr. Seenithasan are acknowledged.

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DODSON, E. O. Evolution, Process and Product. Reinhold Pub. Corp., N . Y ~ Affliated East-West Press Fvt. Ltd., New Delhi, 1968.

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