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Cleavage patterns and mesentoblast formation in the Gastropoda: an evolutionary perspective.

Key words.--Cleavage pattern, gastropods, mesoderm, mesodermal stem cell, Mollusca, phylogeny.

Received July 3, 1995. Accepted September 29, 1995.

Variations in ontogeny have enabled the evolution of the various metazoan body plans (Garstang 1928; de Beer 1940). In the extant phyla, the development from the zygote to the fully formed new individual is realized by a limited number of cleavage styles. Holoblastic cleavage is practiced in each of the approximately 31 phyla. The three main types of holoblastic cleavage are spiral, radial, and bilateral. The greatest number of phyla, collectively indicated as the Spiralia, has adopted spiral cleavage. It is very unlikely that the spiralian phyla have convergently developed the spiral style of cleavage. In closely related spiralians, the zygote is not only partitioned according to geometrically similar patterns of blastomeres, but these blastomeres have equivalent developmental fates. In all spiralian embryos studied so far, the mesodermal bands originate from a corresponding stem cell (Stewing 1969). In many spiralians, the embryo passes through a trochophore or a trochophore-like larval stage, characterized by a transverse belt of ciliated, trochal cells. In all spiralians these trochal cells are derived from the comparable trochoblast cell lines. The conservation of the early cleavage patterns and the specification of the developmental fate of blastomeres with identical geometrical positions strongly argue for the assumption that spiral cleavage has been adopted only once, and is very refractory to evolutionary modifications (Buss 1987). As the cleavage patterns are so conservative, minor differences may be characteristic for the different spiralian phyla.

Whether the cleavages are spiral or not, a major aspect of the evolution of diploblastic into triploblastic Metazoa is the formation of a third germ layer, the mesoderm. In protostomes, as well as in deuterostomes, the first cleavages initially result in the separation of the cells of the presumptive ectodermal and endodermal germ layers. The formation of the mesodermal germ layer requires an inductive interaction between presumptive ecto- and endodermal cells. In mollusks (van den Biggelaar and Guerrier 1979; Martindale et al.1985; Boring 1989), ectodermal cells induce one of four equivalent presumptive endodermal cells to deviate from the original endodermal fate and to produce the mesodermal stem cell from which the two mesodermal bands arise. In deuterostomes, e.g., in vertebrates, however, the opposite occurs. Endodermal cells induce a number of ectodermal cells to deviate from their original ectodermal fate and to produce mesoderm (Nieuwkoop 1973). Irrespective of the origin of the mesoderm, once it is formed it functions as an organizer, and specifies the developmental fate of the endo- and ectodermal cells. For instance, in vertebrates the formation of epidermal or neural ectoderm depends upon the presence of the prechordal and chordal mesoderm (Nieuwkoop 1952). Similarly in mollusks the stem cell of the mesentoblast (macromere 3D) and its daughter cell (mesentoblast 4d) act as organizers and specify the developmental fate of a large number of blastomeres (Damen 1994, Damen and Dictus 1994a,b).

Because of the crucial significance of macromere 3D and mesentoblast 4d for the specification of the developmental fate of the embryonic cells, it may be assumed that heterochronic changes in 3D induction may have far reaching morphogenetic consequences. The later this induction occurs, the later the developmental fate of the other blastomeres is adapted to their final destination, and the longer the period of autonomous development will be. The earlier the mesentoblast is formed, the earlier the blastomeres will receive their final developmental instructions, and the shorter the period of autonomously specified development will be. As a rule, specification of 3D and formation of the mesentoblast occur as soon as the presumptive ectodermal micromeres and endodermal macromeres have been separated. At the third cleavage the four blastomeres divide into the first quartet of ectodermal micromeres (1m) and the four macromeres of the first generation (1M). At the fourth cleavage the 1M macromeres form a second quartet of ectodermal micromeres (2m) and a second generation of macromeres (2M). At the fifth division the 2M macromeres produce the third and last quartet of ectodermal micromeres (3m) and the third macromere generation (3M). Thus, by the fifth cleavage the separation of the ecto- and endodermal lineages is completed. During the interval preceding the sixth cleavage, one of the 3M macromeres attains a central position in the embryo, and is induced to form mesentoblast 4d during the sixth cleavage cycle.

If the proliferation rates of micro- and macromeres are equal, then all blastomeres divide synchronously, mesoderm induction occurs during the interval between the 32- and the 64-cell stage, and the mesentoblast is formed at the transition to the 64-cell stage. However, in far the greatest number of species, division synchrony is an exception. Heterochronic differences in mesentoblast formation are the rule, and according to the species, the mesentoblast is formed at different cell stages. When the ectodermal micromeres divide faster than the macromeres, then 3D induction and mesentoblast formation occur at a more advanced cell stage. When the micromeres divide slower than the macromeres, 3D induction and mesentoblast formation are relatively accelerated, and occur at a younger cell stage. Heath (1899) and Pelseneer (1911) already observed that in primitive mollusks, like the polyplacophorans and the archaeogastropods, mesentoblast 4d is formed at a relatively late cell stage, 63- and 72-cell stage, respectively, whereas in the most derived euthyneuran gastropods this is accelerated up to the 24-cell stage. As the separation of the ectodermal micromeres (1m, 2m, and 3m) and the endodermal macromeres 3M requires five cleavage cycles, 3D induction and subsequent specification of the developmental fate of all other cells, similarly requires five division cycles. The formation of the third generation of macromeres (3M) is a developmental constraint for a further acceleration of the specification of the mesodermal stem cell and, henceforth, for the specification of the dorsal D quadrant out of four initially equal quadrants.

In caenogastropods the specification of the dorsal quadrant is no longer associated with the induction of 3D out of four equal 3M macromeres, but with the segregation of a polar lobe. Synchronously with the first cleavage, a protrusion, the so-called polar lobe, is formed at the vegetal pole of the egg. After cleavage the lobe fuses with only one of the sister blastomeres. The smaller cell represents the presumptive AB cell and the lobe containing cell the presumptive CD cell. At the second cleavage CD again forms a polar lobe, which again fuses with only one of the two sister cells, the presumptive D cell. Finally a 4-cell stage is reached in which the polar lobe substances are segregated into one of the four quadrants, the presumptive dorsal or D quadrant. Thus, in gastropods the D quadrant may be specified by two different mechanisms. The first is a conditional specification resulting from of an inductive interaction between the animal micromeres and a centralized 3M macromere. The second is an autonomous specification by the segregation of polar lobe substances (van den Biggelaar and Guerrier 1983; Freeman and Lundelius 1992; van den Biggelaar 1996a). The developmental significance of a precocious D quadrant specification, however, appears to be limited. Although in polar lobe forming species the D quadrant is apparent as early as the 4-cell stage, the D quadrant does not start its organizer function prior to the separation of the ectoblast and entoblast cells (Clement 1952).

The above described differences in time and mode of D quadrant specification are the main variations in the early development of gastropods, and Conklin (1907, p. 325) was right when he wrote "the early cleavage pattern may be reckoned as one of the most conservative features in the development of any gastropod." One may even extend Conklin's conclusion from the level of the species to the level of the taxon, and hypothesize that "the early cleavage pattern may be reckoned as one of the most conservative features in the development of any gastropod taxon." Indeed, we assume that the cleavage pattern of a species has a high predictive value for its taxonomic and evolutionary ranking. As there is almost no doubt about the monophyletic origin of the gastropods, these mollusks offer an ideal system to test the hypothesis that the evolution of the main gastropod taxa from an ancestral form has been associated with progressive deviations of the early cleavage patterns from the architypical pattern.

In the present paper, we will test the above hypothesis, and have attempted to arrange the early gastropod cleavage patterns in a series of progressive deviations from a presumed ancestral pattern as observed in the lower archaeogastropods to the patterns found in more derived gastropods. Therefore, data obtained from the literature have been supplemented with new data on the cleavage patterns in embryos of Theodoxus fluviatilis, Ampullaria cf. gigas, Viviparus viviparus, and Valvata piscinalis. Finally, the obtained series has been compared with the phyletic position of the main taxa in phylograms proposed by Haszprunar (1988), Ponder (1991), Ponder and Lindberg (1995), and Tillier et al. (1992, 1994). Our conclusion is that the spatiotemporal variations in the early cleavage patterns are a powerful guide for the reconstruction of the phylogenetic history of the gastropods (van den Biggelaar 1993, 1996a).

MATERIALS AND METHODS

Valvata piscinalis was collected from clear and slowly streaming freshwater canals in the neighborhood of Utrecht, The Netherlands. The animals were kept at 20 [degrees] C. Development has been followed at 25 [degrees] C. The egg masses were deposited on the glass wall. Each egg mass contained about 15-25 elliptic egg capsules. At the two ends of the longer axis, the capsules had a threadlike extension, the chalaza. The capsules closely surrounded the large, yolk rich eggs. There was no capsule fluid. Staining with gallocyanin demanded that the embryos be decapsulated soon after fixation.

Viviparus viviparus was collected from the same regions as Valvata. About 20-50 egg capsules can be collected from the uterus of one animal. The most advanced embryonic stages are found in the distal part, and the youngest are found in the proximal part of the uterus. No more than one or two early cleavage stages can be found in one and the same animal. The capsules are very large and contain only one egg. The eggs are oligolecithal and very small. The diameter is about 60 micron. As in Valvata, the capsules have chalazae. In vivo observations were not possible, because soon after decapsulation, the eggs cytolyze.

A hybrid resembling Ampullaria gigas was obtained from a local pet shop and was kept in fresh water at approximately 20 [degrees] C. Observations are as follows: Slightly above the surface of the water, the capsules are deposited one after the other. The capsules firmly adhere to each other and together they form a large, pink colored egg mass. The size of the individual capsules is about 2 mm. The eggs of Ampullaria are small. The diameter is about 90 micron. Each capsule contains one egg. The outer layer of the capsule is thin and calcified. The inner capsule is thick, transparent and gelatinous. The capsule "fluid" of freshly laid eggs is granular and opaque. It looks as if it has been laid down in several layers around the egg. In the center of these layers, the egg is found in a small amount of hyalin fluid. The whole content is extremely tough, and by decapsulation the eggs are destroyed easily. After decapsulation the eggs soon cytolyze. In vitro observations are excluded, and no precise data have been obtained about the duration of the successive cleavages. At 20 [degrees], the duration of a cell cycle is about 5 h, at 25 [degrees], approximately 3 h. Within one and the same egg mass the development of the embryos is relatively synchronous.

Theodoxus fluviatilis were collected from the southern border of the "5e Plas" parallel to the "Tienhovens Kanaal," about 15 km north of Utrecht. Together with the animals a number of stones covered with algae were transported to the laboratory. The algae on the stones appeared to be sufficient to feed the animals for a few weeks. The animals were kept in aquaria with copper free tapwater at 20 [degrees] C. Fresh capsules were opened with forceps. Each capsule contains several tens of eggs of which only one will develop normally and hatch, whereas the others function as food eggs. As there is only one normally cleaving egg per capsule, and the eggs cytolyze after decapsulation, it was not possible to follow the successive cleavages in the living embryo. Nothing definite can be said about the duration of the cleavages. A rough indication can be derived from the observation that if a freshly laid capsule is opened about 24 h later, often no more than five or six cleavage cycles have taken place. This indicates that each cycle takes about 5 h.

The embryos of all three species were fixed in Zenker's fixative and stained with gallocyanin. Whole mount preparations were made as described earlier for Patella (van den Biggelaar 1977). Of each species the cleavage pattern of about 40 embryos was reconstructed. For a reconstruction, first a projection of all the nuclei of an embryo was made. Then, in the same position and seen from above, the cell pattern was drawn. In the same position, a second drawing of the cell pattern of the opposite hemisphere was made as seen from the inside. In the absence of polar bodies, and in the absence of obvious differences between the blastomeres, the cross-furrows offered a reliable landmark for the reconstruction of the embryo. Mistakes in the definition of the cross-furrows could be excluded, as then the final cell patterns did not fit with the rule of alternating right and left handed cleavages

RESULTS AND DISCUSSION

The data obtained on the cleavage patterns of Theodoxus, Viviparus, Ampullaria, and Valvata will be discussed together with data about the cleavage patterns of other gastropod species derived from the literature. First, we will attempt to construct a seriatim of cleavage patterns based upon the cleavage stage at which stage between the 64- and the 24-cell stage the mesentoblast is formed. Finally, we will discuss the evolutionary direction and phyletic significance of that series.

A. Docoglossa (Patellogastropoda)

The cleavage pattern of Patella vulgata is very regular (Fig.1). Up to the fifth cleavage the divisions are synchronous, and the embryo successively passes from the 1-into the 2-, 4-, 8-, 16-, and 32-cell stage. Up to then the embryo is radially symmetrical, and the four quadrants have equal developmental properties. During the interval between the fifth and the sixth cleavage, one of the 3M macromeres is centralized and then induced to produce the mesentoblast 4d, (van den Biggelaar and Guerrier 1977; Arnolds et al. 1983). After the 32-cell stage, the sixth cleavage cycle is initiated by the two tiers of primary trochoblasts, and completed by the division of the macromeres 3A-3D at the transition of the 60- to the 64-cell stage. This division is not synchronous; 3A, 3B, and 3C divide first. With a delay of about 18 min 3D divides last at the transition of the 63- to the 64-cell stage (Fig. 1) (van den Biggelaar 1977).

[Figure 1 ILLUSTRATION OMITTED]

B. Vetigastropoda

Detailed cleavage patterns are available of Gibbula magus (Robert 1902) and Haliotis tuberculata (Crofts 1937; van den Biggelaar 1993). The cleavage patterns of both species differ only slightly. The cleavage pattern of Haliotis is given in Fig. 2. The 16-cell stage is reached by a synchronous division of all blastomeres. In Gibbula, Robert (1902) occasionally observed a negligible intermediate 12-cell stage, as the first quartet cells divide with a minor delay. In both species the fifth cleavage is definitely asynchronous. The derivatives of the 1m quartet divide slightly later than the derivatives of 1M. As a result, a 24-cell stage of only a few minutes precedes the transition to the 32-cell stage. Division asynchrony is much more pronounced during the sixth cleavage cycle. Whereas in Patella the primary trochoblasts (1[m.sup.21] and 1[m.sup.22]) derived from the first quartet of micromeres start the sixth cycle, in Gibbula and Haliotis the trochoblasts divide significantly later, just prior to the division of macromeres 3A-3D (Fig. 2). Again, this division is not synchronous; with a minor delay of only a few minutes, 3D divides last at the transition of the 63- to the 64-cell stage.

[Figure 2 ILLUSTRATION OMITTED]

C. Architaenioglossa

First, the cleavage pattern of Viviparus viviparus was reinvestigated, as according to Tonniges (1896) and Dautert (1929) mesentoblast formation in Viviparus deviates from the normal gastropod pattern. During the interval between the fifth and sixth cleavage, no asymmetries occur, and up to gastrulation the embryo remains radially symmetric. Second, because of the deviating cleavage pattern in Viviparus, we wanted to analyze whether the cleavage pattern in a related representative of the Architaenioglossa-Ampullarioidea, Ampullaria cf. gigas, would also deviate. Third, both species are representatives of a taxon with an uncertain phyletic position, somewhere between the lower Gastropoda and the bifurcation of the gastropod tree into the caenogastropod and heterobranch branches (Haszprunar 1988; Ponder and Lindberg 1995).

The Cleavage Pattern of Viviparus viviparus.--The cleavage pattern is shown in Figure 3. The first two cleavages are equal. As none of the embryos appeared to be fixed exactly at the first or second cleavage, it remains uncertain whether polar lobe formation takes place. If a lobe is formed, it cannot be large, as no size differences between the blastomeres A--D have been observed. From the third cleavage the divisions are no longer synchronous. The cleavage rhythm of the cells derived from 1m is slower than of the derivatives of 1M. This leads to the formation of an intermediate 12-cell stage prior to the transition to the 16-cell stage. Whereas the cells 2m and 2M divide, the 1[m.sup.1] and 1[m.sup.2] cells remain undivided, and the embryo passes to a 24-cell stage (Fig. 3). During this stage, a bilateral symmetric pattern is attained as one of the 3M macromeres is centralized, and therefore, can be denominated as 3D. After the 24-cell stage, the 1[m.sup.2] cells, the primary trochoblasts, resume mitosis and the embryo attains a 28-cell stage. Then the 1[m.sup.1] cells divide almost simultaneously with the two tiers of second quartet cells, followed slightly later by the micromeres of the third quartet and the

[Figure 3-4 ILLUSTRATION OMITTED] macromeres 3A-3C (Fig. 4). The embryo has then reached the 47-cell stage. Macromere 3D divides significantly later. Both, the central position of 3D and its cleavage delay strengthen the bilateral symmetry of the embryo (Fig. 5). 3D divides and forms the mesentoblast at the transition to the 48-cell stage (Fig. 3).

[Figure 3-6 ILLUSTRATION OMITTED]

The Cleavage Pattern of Ampullaria cf. gigas.--The cell lineage is given in Figure 6. The first two cleavages are equal. Although several embryos have been fixed during cytokinesis, polar lobe formation has not been observed. In a few embryos a protrusion has been observed at the vegetal pole of one or both blastomeres. As these protrusions were not associated with cytokinesis, they cannot be considered as true polar lobes. Presumably they were the first indications of cytolysis.

From the 8-cell stage the cleavages are no longer synchronous. Because of a lower mitotic rhythm of the Im cells than of the derivatives of the 1M macromeres, from the 8-to the 16-cell stage the embryo passes an intermediate 12-cell stage. After the 16-cell stage the 2m and 2M cells divide first, whereas the I[m.sup.1] and I[m.sup.2] cells follow much later. As a consequence, there is a relatively long intermediate 24-cell stage. Again during that 24-cell stage, one of the four 3M macromeres attains a central position just underneath the animal micromeres, and hence can be denominated as 3D. After the 24-cell stage mitotic activity is resumed by the second and third quartet cells, and the embryo reaches a 36-cell stage. Only then the two tiers of first quartet cells follow, increasing the number of blastomeres to 44. Meanwhile 3D remains in contact with the majority of the animal micromeres (Fig. 7). ] The 48-cell stage is reached by a synchronous division of the macromeres 3A-3D (Fig. 8).

[Figure 7-8 ILLUSTRATION OMITTED]

Tanaka et al. (1987) observed that in the ampullarioid Sinotaia quadratus historica (Viviparidae), the majority of the embryos form a protrusion during the first cleavage. There are three reasons to doubt whether this protrusion really corresponds with a characteristic polar lobe. (1) In Sinotaia lobe formation was aberrant as not all embryos did form a polar lobe. (2) In contrast to all other lobe-forming species, in Sinotaia no polar lobes were detected at the second cleavage. (3) The region where the lobes were formed did not exactly correspond with the region of the cleavage furrow.

D. Neritimorpha

We reinvestigated the cleavage pattern of Theodoxus flu-viatilis primarily because of Blochmann's (1882) observations that in Theodoxus (formerly Neritina) the general rule, that in equal cleavers the embryo attains bilateral symmetry in connection with mesentoblast formation, was violated. The first two cleavages in the embryo of Theodoxus are equal. Nevertheless, radial symmetry appears to be lost as early as the 4-cell stage, whereas in species with an equal 4-cell stage this does not occur prior to the fifth cleavage in association with mesentoblast formation. During the first two cleavages of the embryo of Theodoxus, a typical kind of granules, so-called "lichtbrechende Kornchen," are segregated exclusively to the two lateral quadrants. Both, the animal and vegetal cross-furrow are formed by the presumptive median quadrants.

We have confirmed that the first two cleavages are equal and not associated with polar lobe formation. Therefore, it may be assumed that in Theodoxus the D quadrant is specified by induction. The succession of the cleavages is shown in Fig. 9. It appears that the period of synchronous divisions is short. From the 8-cell stage the cleavage rhythm of the Im micromeres is slower than of the IM macromeres. As a consequence, an intermediate 12-cell stage precedes the 16-cell stage. The fourth division is not synchronous either, and the embryo passes to a 24-cell stage composed of eight derivatives of Im (four l[m.sup.1] and four l[m.sup.2] cells), and 16 cells derived from the four IM macromeres: four 2[m.sup.1], four 2[m.sup.2], four 3m and four 3M cells. During this 24-cell stage the embryo loses its radial symmetry, as one of the 3M macromeres attains a central position, and touches the overlying animal micromeres. The animal daughter cells of lm (l[a.sup.1]-[d.sup.1]) then resume mitotic activity whereas the vegetal daughter cells, the primary trochoblasts l[a.sup.1]-l[d.sup.2], remain undivided. The embryo thus attains the 28-cell stage. Then, the micromeres of the second and third generation divide more or less synchronously, simultaneously with the central 3D macromere. Thus, 3D divides and produces the mesentoblast at the transition of the 28- to the 37-cell stage, long before the division of macromeres 3A, 3B, and 3C.

[Figure 9 ILLUSTRATION OMITTED]

Special attention has been paid to the segregation of the so-called lichtbrechende Kornchen. After staining with gallocyanin these particles are barely visible. However, in methylene-blue stained sections they can easily be recognized as concentrations of blue granules in a field of clear cytoplasm. Prior to the first division all granules are located around the animal pole. At the first cleavage they are segregated into both blastomeres. In the 4-cell embryo the majority is found in the two lateral quadrants, although a few may be present in the median quadrants. During further division the granules are segregated into the second quartet cells, mainly into 2a and 2c. These findings roughly confirm Blochmann's (1882) observations. It must be emphasized, however, that these lichtbrechende Kornchen are not exclusively, but predominantly segregated to the two lateral quadrants.

E. Caenogastropoda

The cleavage pattern of the Caenogastropoda is unique among gastropod taxa. Only in the caenogastropods are the first cleavages associated with polar lobe formation. Lobe formation always occurs in relation with cytokinesis. During mitosis the lobe-forming region protrudes from the vegetal pole, and is constricted off maximally at the end of cytokinesis, when the blastomeres are in late telophase or early interphase. After maximal constriction of the two blastomeres, the lobe fuses with the blastomere with which it remained connected, with CD after the first and with D after the second division (Fig. 10). Development of bilateral symmetry and D quadrant specification are no longer the result of the induction of 3D and mesentoblast formation, but with the segregation the polar lobe into one of the four quadrants.

[Figure 10 ILLUSTRATION OMITTED]

From a literature survey on lobe formation in gastropods provided by Freeman and Lundelius (1992), it appears that in 15 out of 25 caenogastropod species lobe formation has not been mentioned. However, if papers on the early cleavage pattern do not report lobe formation, it should not be concluded that the species in question does not form a polar lobe. Often no special attention has been paid to lobe formation. In addition, in the majority of species the lobes are small and the formation of a lobe can easily be missed if it is not explicitly studied. For instance, Delsman (1914) did not report lobe formation in Littorina obtusata, whereas Moor (1973) observed the formation of a small lobe during the first five cleavages in Littorina littorea. Neither von Erlanger (1891) nor Sachwatkin (1926) described lobe formation in Bithynia, whereas in a careful analysis of the first two cleavages, Hess (1956) and Verdonk (1973) observed the formation of a very small polar lobe. Ostergaard (1950) describes the first cleavage stages of Conus tahitiensis and C. omaria without mentioning lobe formation, whereas for C. sumatrensis Ostergaard has mistaken a small polar lobe for a polar body. One of the most complete studies on gastropod development is Conklin's (1897) famous paper on the embryology of Crepidula fornicata. Conklin described that he frequently found a rounded mass of hyaline substance which persists until after the first two cleavages. He was not satisfied as to the significance of that body, and thought it was a remnant of the stalk with which the ovum was attached to the basal membrane of the ovarian follicle. These examples clearly demonstrate that lobe formation can easily be missed if it is not explicitly investigated. We think that lobe formation is characteristic for the all caenogastropods and that in so-called lobeless caenogastropods lobe formation has not been noticed.

The assumption that lobe formation within the gastropods is distinctive for the Caenogastropoda is strengthened by recent observations on the early development of the archetypical caenogastropods Bittium latreillei and Cerithium vulgatum (van den Biggelaar, unpubl. obs.). In both species a polar lobe has been observed during the first three cleavage cycles.

Great spatio-temporal and structural differences appear to exist in polar lobe formation. The relative size of the lobe may vary considerably (Verdonk and van den Biggelaar 1983; Freeman and Lundelius 1992). The formation of small polar lobes is characteristic for primitive caenogastropods such as Littorina (Moor 1973) and Hydrobia (van den Biggelaar, pers. obs.). The formation of large polar lobes is limited to the more derived stenoglossan caenogastropods, e.g., Ocenebra erinacea (Fioroni 1979), Purpura lapillus (Pelseneer 1911), and Ilyanasa obsoleta (Clement 1952). Also the frequency of polar lobe formation varies. In some primitive caenogastropod species, such as Littorina littorea (Moor 1973) and Hydrobia ulvae (van den Biggelaar, pers. obs.), lobe formation is repeated during the fourth and fifth cleavage. In other caenogastropods lobe formation during the first and second cleavage is the rule, whereas in some species it occurs once more during the third cleavage cycle.

Although the size and the architecture of the lobe forming region may differ (Dohmen 1983), great similarities appear to exist in the developmental stage at which macromere 3D starts its organizing role (van Dongen 1976a,b). Clement (1952) has shown that in the caenogastropod Ilyanassa up to a short period after the formation of 3D, the development of embryos from which 3D has been deleted strongly resemble lobeless embryos. Thus, irrespective of the specification of the D-quadrant as early as the 4-cell stage, its organizing role is not accelerated, but postponed until the normal number of cell cycles required for the separation of the ecto- and endodermal lineages has been completed.

The cleavage pattern and cell lineage of a number of caenogastropods has been investigated, e.g., in Littorina littorea (Moor 1973), Crepidula fornicata (Conklin 1897), Ilyanasa obsoleta (Clement 1952; Craig and Morrill 1986). The cleavage rhythm of the D macromeres only slightly differs from the other macromeres. As a representative of the caenogastropod cleavage pattern the cell lineage of Crepidulafornicata is shown in Figure 11. After the 8-cell stage, the animal micromeres divide later than the macromeres IA-ID, and a short 12-cell stage precedes the following 16-cell stage. The fifth cleavage cycle is also very asynchronous, the micromeres 2a-2d and the macromeres 2A-2D divide long before the first quartet cells, and thus the embryo passes a 24-cell stage. Before the first quartet cells complete the fifth cleavage cycle, macromere 3D divides and forms the mesentoblast 4d at the transition to the 25-cell stage. The other macromeres 3A-C divide much later at the transition to a stage of about 53 cells.

[Figure 11 ILLUSTRATION OMITTED]

From the 24-cell stage the cleavage pattern in the different caenogastropod species varies enormously, and mesentoblast formation takes place at different cell stages. In Rissoa parva, this occurs at about the 40-cell stage (Pelseneer 1911), in Pterotrachaea coronata at the 32-cell stage (Fol 1876); in Hydrobia ulvae (van den Biggelaar, pers. obs.), Bithynia tentaculata (van Dam 1986), and Ilyanasa obsoleta (Clement 1952) from the 28- to the 37-cell stage; in Littorina obtusata (Delsman 1914), and Crepidula fornicata (Conklin 1897) at the 24-cell stage.

It is a general rule that macromere 3D divides significantly earlier than its counterparts 3A, 3B and 3C. Clement (1952) has shown that in Ilyanasa the precocious division of 3D depends upon the presence of the polar lobe substances. After deletion of the polar lobe, all four macromeres divide synchronously at the transition from the 44- to the 48-cell stage. Similarly, in non-gastropod mollusks, like the scaphopod Dentalium, deletion of the polar lobe results in the formation of four equal quadrants (van Dongen and Geilenkirchen 1976a,b). Therefore, it may be assumed that segregation of the lobe substances into the D quadrant is one of the factors which causes the heterochronic shifts in the formation of the mesentoblast.

F. Valvatoidea

The Valvatoidea represent the earliest clade of the Heterobranchia (Allogastropoda, Opisthobranchia and Pulmonata). In the literature, data about the cleavage pattern of allogastropods are limited to Pelseneer's observations on the first cleavages of two representatives of the Pyramidelloidea: Odostomia pallida and O. rissoides (Pelseneer 1914). Also in Odostomia the first two cleavages are equal, and polar formation has not been reported. As no data were available about mesentoblast formation in allogastropods, it was decided to investigate the early cleavage pattern of the valvatoid Valvata piscinalis.

The Cleavage Pattern of Valvata piscinalis.--The cleavage pattern of Valvata is shown in Figure 12. The first two cleavages are more or less equal, and at the 4-cell stage no obvious size differences between the quadrants have been observed. There is no polar lobe formation. Because of a lower mitotic rate of the lm cells, cleavage synchrony is lost after the 8-cell stage, and a short intermediate 12-cell stage precedes the 16-cell stage. Then, again the first quartet cells lag behind and remain undivided, whereas the 2m and 2M cells cleave, and a 24-cell stage is formed during which one of the 3M macromeres is centralized. From that moment this macromere can be denominated as 3D. Then the two tiers of second quartet cells divide. Thus, the reached 32-cell stage does not last long; the animal tier of first quartet cells divide almost synchronously with the third quartet of micromeres. At the attained 40-cell stage, macromere 3D divides and forms the mesentoblast (Figs. 13, 14).

[Figure 12-14 ILLUSTRATION OMITTED]

G. Opisthobranchia

In opisthobranchs the formation of four equal quadrants is the rule, with the exception of the Thecosomata, Aplysiomorpha and Umbraculomorpha, where the quadrants are unequal. In the Thecosomata Cavolina tridentata and Cymbulia peronii, the D quadrant is smaller than A, B, and C (Fol 1875). In the aplysiomorph Aplysia punctata (Carazzi 1905) and the umbraculomorph Umbraculum mediterraneaum (Heymons 1893), C and D are both smaller than A and B. From all the available data on the cleavage patterns in opisthobranchs, it appears that the embryo passes a 24-cell stage because of a cleavage delay of the first quartet cells. The mesentoblast seems to be formed as a result of an inductive interaction between the animal micromeres and the centralized macromere (Fig. 15). This pattern appears to be characteristic for the Opisthobranchia and Pulmonata, and it strengthens the assumption that the Euthyneura (Opistho-branchia plus Pulmonata) form a single holophyletic group, in which the mesentoblast is formed at the 24-cell stage, distinctively earlier than in an allogastropod outgroup such as the Valvatoidea. It will be interesting to perform a comparative analysis of the cleavage patterns of other allogastropods with uncertain phyletic relations with the Euthyneura, e.g., the Pyramidelloidea or Architectonicoidea.

[Figure 15 ILLUSTRATION OMITTED]

The phenomenon of different size relations between the quadrants within distinct groups of Opisthobranchia needs further comment. Although the systematics of the Opisthobranchia is still a matter of debate (see Salvini-Plawen 1991a,b; Salvini-Plawen and Steiner 1995 for recent reviews), it is unquestionable that, together with the Pulmonata, they all belong to the Euthyneura. The stem group of Opisthobranchia from which probably all other taxa are derived are the "cephalaspids" (sensu stricto, Architectibranchia and Bullomorpha; Schmekel 1985; Salvini-Plawen 1991a,b). Haszprunar (1985) considered the archtitectibranch groups (Acteonoidea, Ringiculoidea, Diaphanoidea) as the earliest opisthobranch offshoots. In all investigated members of Architectibranchia and Bullomorpha, the cleavage pattern is plesiomorphic in that the four quadrants are equal. Boring (1989) has shown that in the bullomorph Haminea mesentoblast formation is induced at the 24-cell stage. If Haminea is representative for the cephalaspids, then also mesentoblast formation by induction is a plesiomorphic character. In the opisthobranchs Cavolinia tridentata (Fol 1875) and Cymbulia peronii (Pelseneer 1911), representatives of the Thecosomata, it has been observed that the A, B, and C quadrants are larger than D. This is unique among the gastropods and probably an autapomorphy of the clade. In Aplysiomorpha (Carazzi 1905) and Umbraculomorpha (Heymons 1893), the quadrants A and B are larger than C and D. The unique similarity in the cleavage pattern of these two groups supports the ideas of Schmekel (1985), who proposed a close relationship between the Umbraculomorpha and the Aplysiomorpha, and the diphyletic origin of the Notaspidea. Indeed, in the notaspid pleurobranchomorphs Berthellina citrina (Usuki 1969) and Pleurobranchus sp. (Rao and Alagarswami 1961), no size differences between the macromeres have been reported.

The formation of quadrants with remarkable size differences is neither unique for opisthobranch gastropods, nor for mollusks in general. However, the formation of a smaller D, or a smaller C and D quadrant has only been observed in opisthobranchs. In non-opisthobranch mollusks with unequal cleavage, the D quadrant is always larger. Also the way in which the size differences arise, are different. In caenogastropods the larger size of D is due to the segregation of the polar lobe substance. In lamellibrach species the formation of unequal quadrants is the result of direct unequal division or lobe formation, in both cases D is larger.

H. Pulmonata

In all pulmonate species the first two cleavages are more or less equal and polar lobe formation has never been observed. The cleavage pattern of Lymnaea stagnalis has been chosen as a representative for the pulmonates (Fig. 16). As far as the cleavages have been followed precisely, it appears that the third cleavage is the last synchronous division. At the fourth cleavage the macromeres lA-lD divide first, slightly later followed by the micromeres la-ld. Thus, the embryo passes an intermediate 12-cell stage before reaching the 16-cell stage. At the fourth division the second quartet cells 2a-2d and the macromeres 2A-2D divide, and the 24-cell stage is reached, during which macromere 3D is centralized. Just as in the opisthobranchs the interval prior to the following cleavages is a relatively long resting stage, after which cleavage is resumed by the formation of the mesentoblast by macromere 3D.

[Figure 16 ILLUSTRATION OMITTED]

In Lymnaea it has been demonstrated that during this interval one of the two cross-furrow macromeres is induced to develop the mesentoblast (van den Biggelaar 1976a,b; Arnolds et al. 1983; Martindale et al. 1985). The observations of Kofoid (1894) and Meisenheimer (1896) that in Limax the mesentoblast is formed at the 32- or at the 36-cell stage, have been corrected by later observations of Guerrier (1970). The cleavage pattern of Limax does not deviate from the rule that in the euthyneurans the mesentoblast is formed at the 24-cell stage.

Summarizing, it may be concluded that a pulmonate is a gastropod in which four equal quadrants are formed, in which the embryo becomes definitely bilaterally symmetric by centralization and subsequent induction of macromere 3D, followed by the formation of the mesentoblast at the 24-cell stage. This pattern is not unique for the Pulmonata, but is shared by a great number of Opisthobranchia, especially the cephalaspid stem group. This is not surprising, as it is assumed that Opisthobranchia and Pulmonata belong to a monophyletic taxon, the Euthyneura or Pentaganglionata (Haszprunar 1985, 1988).

Evolutionary Considerations

The main purpose of this paper was to investigate the validity of the assumption that the cleavage patterns of gastropod species have a high predictive value for their taxonomic and evolutionary ranking. The pivotal aspect of the early development in gastropods, and presumably in all spiralian embryos, is the determination of the D quadrant and the associated specification of 3D, the stem cell of the mesentoblast (4d). As the specification of the developmental fate of all blastomeres depends upon the specification of the dorsal quadrant (D), it is not astonishing that 3D specification and mesentoblast formation are the most conservative features in the development of any gastropod clade. As 3D acts as an organizer, minor changes in the spatio-temporal aspects of 3D induction must have far reaching consequences on the early development. The later the organizer 3D is induced, the later the developmental fate of all other blastomeres can be determined, and the longer the phase of autonomous development will last. The earlier 3D is specified, the earlier the developmental fate of each blastomere can be specified, and the shorter the phase of autonomous development will be. Therefore, our attention has been focused on the spatio-temporal aspects of 3D specification and mesentoblast formation.

In Figure 17 the various gastropod taxa have been arranged according to the cell stage at which 3D forms the mesentoblast. For outgroup comparison, the Polyplacophora have been included in this series. In none of the gastropod species studied so far is the mesentoblast formed later than the 63-cell stage, and in none of the species is it formed earlier than the 24-cell stage. In the ancestral-like forms, such as the Docoglossa and the Vetigastropoda, the mesentoblast is formed last, i.e., at the 63-cell stage. In the Docoglossa, the 63-cell stage lasts relatively long, whereas in the Vetigastropoda it is short. In some Caenogastropoda, and in all Euthyneura (Opisthobranchia and Pulmonata) mesentoblast formation is accelerated up to the 24-cell stage. This demonstrates that the evolutionary radiation from an archaeogastropod-like ancestor to each of the more derived clades has been associated with an accelerated specification of 3D, and a precocious formation of the mesentoblast.

[Figure 17 ILLUSTRATION OMITTED]

As the mesentoblast is formed by one of the four initially equal, presumptive endodermal macromeres 3M, an accelerated formation of the mesentoblast can either be achieved by an accelerated formation of the 3M macromeres, or by a retardation of the cleavage rhythm in the ectodermal lineages. If the cells in the endodermal lineage (IM, 2M, and 3M) divide faster than the ectodermal micromeres Im and 2m, then a heterochronic shift causes a precocious formation of the competent 3M macromeres. In archaeogastropods the macromeres IM and 2M, and the micromeres Im and 2m divide almost synchronously or with a minimal time difference, and the macromeres 3M are formed at the 32-cell stage. In euthyneuran gastropods, the IM macromeres divide faster than the lm cells, and the formation of the competent 3M macromeres is accelerated to the 24-cell stage. Whereas in none of the Gastropoda the mesentoblast is formed later than the 63-cell stage, in the more primitive Polyplacophora, such as Ischnochiton (Heath 1899) and Acanthochiton (van den

Biggelaar 1996b), this occurs at the 72-cell stage. Below it will be discussed whether the heterochronic shift of mesentoblast formation shown in Figure 17 has been realized along a single orthogenetic line, or independently along several evolutionary lines.

In addition to the introduction of division asynchrony as a mechanism for an advanced formation of the competent 3M macromeres, a second mechanism influences the developmental stage at which the mesentoblast is formed. The induction out of one of the 3M macromeres appears to influence the proliferation rate of the induced macromere (3D). The induction may either delay or fasten its division in comparison with the other macromeres. In archaeogastropods, the induction delays the division of 3D. The non-induced macromeres 3A, 3B, and 3C divide at the 60-cell stage, whereas after being induced 3D divides later at the 63-cell stage. In euthyneurans the induction significantly accelerate the division of the induced macromere. 3D divides already at the 24-cell stage, whereas 3A, 3B, and 3C only divide at the 41-cell stage. If 3D induction is suppressed, all 3M macromeres divide simultaneously, in archaeogastropods at the 60- and in euthyneurans at the 40-cell stage. It is not evident why induction causes a cleavage delay in the lower gastropods and an acceleration in the more advanced forms.

A second remarkable variation in the gastropod cleavage pattern in distinct taxa, is the size relation between the quadrants. During evolution from the ancestral forms (Docoglossa and Vetigastropoda) with late mesentoblast formation to the advanced Euthyneura, the first two cleavages not only divide the embryo into four equal quadrants, but also into quadrants with equal developmental capacities. Only after the fifth cleavage, the dorsal quadrant is determined by centralization of one of the 3M macromeres. In the Caenogastropoda, however, from the early onset the quadrants are not equal. The first two cleavages are accompanied by the formation of a polar lobe and its segregation into one of the quadrants. As early as the 4-cell stage, the quadrant enriched with the polar lobe is specified as the dorsal quadrant. As there are no caenogastropods in which the mesentoblast is formed later than the 40-cell stage, it may be assumed that mesentoblast formation had been accelerated up to the 40-cell stage when polar lobe formation was introduced in the caenogastropod lineage. Therefore, from the 40-cell stage the series of cleavage patterns has to be split into two series: a caenogastropod line with polar lobe formation, and a heterobranchian line including the Allogastropoda (Valvatoidea) and Euthyneura (Opisthobranchia and Pulmonata) in which the system of four equal quadrants is initially maintained (Fig. 18). In both lines 3D specification and mesentoblast formation has been independently accelerated to the 24-cell stage.

[Figure 18 ILLUSTRATION OMITTED]

As in Valvatoidea the first two cleavages are equal, and the first overt indication of the dorsal quadrant is centralization of 3D (Figs. 13, 14), they have to be incorporated in the heterobranch line. In Valvata, the retardation of the proliferation rate in the first quartet cells is less pronounced than in euthyneuran species. In valvatoids as well as in euthyneurans the cells the la2-ld2 (the primary trochoblasts) have only passed the fourth cleavage cycle when 3D passes the sixth (Figs. 12, 13). In Valvata, however, the cells la'-ld' are less retarded than in euthyneurans and pass the fifth cleavage round, when macromere 3D passes the sixth and forms the mesentoblast (Fig. 12). In euthyneurans la'-ld' divide significantly later than 3D. As the retardation of the cleavage rhythm in the first quartet cells and the concomitant relatively accelerated formation of the generation of 3M macromeres and of the mesentoblast 4d is less advanced in the Valvatoidea than in the Euthyneura (compare Figs. 12, 16), the Valvatoidea have to be included in the heterobranchian line below the Euthyneura (Fig. 18).

It remains to be discussed to which cell stage the formation of the third generation of macromeres (3M) and the formation of the 4m micromeres was accelerated when polar lobe formation was introduced, and the caenogastropod and the heterobranchian lines were separated. From a comparison of the early cleavage patterns in Caenogastropoda and Architaenioglossa, it appears that, although the division of the 3M macromeres in Caenogastropoda may vary, no caenogastropods are known in which this division has been accelerated less far than to the transition to the 44-48 cell stage (e.g., Ilyanassa, Clement 1952). This exactly corresponds with the cell stage at which the division of the 3M macromeres occurs in the two architacnioglossan species Viviparus (Fig. 3) and Ampullaria (Fig. 6). Apparently, in Architaenioglossa as well as in Caenogastropoda, the formation of the 3M generation has been accelerated to the same cell stage. Thus, from an ontogenetic point of view, the two groups are closely related. As we did observe lobe formation neither in Viviparus nor in Ampullaria, and lobe formation in Sinotaia (Tanaka et al. 1987) is doubtful, it must be concluded that the series of cleavage patterns has to be divided into a caenogastropod and an euthyneuran branch just above the level of the Architaenioglossa as shown in Figure 18. If after a repeated analysis of the cleavage pattern in Sinotaia lobe formation will be affirmed, then the caenogastropod and the heterobranchian lines must be separated above the level of the Vetigastropoda (Fig. 19).

[Figure 19 ILLUSTRATION OMITTED]

Because of the absence of any overt indication of D quadrant specification in the embryo of Theodoxus prior to centralization of one of the 3M macromeres, and because of mesentoblast formation at the transition of the 28- to the 37-cell stage, there is developmental reason to incorporate the neritimorphs in the heterobranchian line between the Valvatoidea and the Euthyneura (Fig. 19).

Once we have arranged the main gastropod taxa in a sequence of patterns according to equal versus unequal cleavage, heterochronic differences in their early cleavage patterns and in mesentoblast formation as shown in Figures 18, 19, these arrangements have to be compared with phyletic patterns of the main gastropod taxa derived from other criteria. With the exception of the Neritimorpha, the two ontogenetic series represented in Figures 18, 19 remarkably resemble phyletic trees based upon adult morphologies proposed by Ponder (1991) and Ponder and Lindberg (1995) and represented in our Figure 22. Curiously enough, the uncertainties in the morphogenetic as well as in the morphologic trees, pivots around the differences in the position of the Architaenioglossa. With the exception of the Neritimorpha, the morphogenetic trees are also compatible with molecular phylogenetic trees based on comparisons of ribosomal RNA (Tillier et al. 1992, 1994; Tillier and Tillier 1995). These correspondences demonstrate that comparative studies of the early cleavage patterns are powerful tools for the construction of the evolutionary relation between the main gastropod taxa. It may be expected that additional data on the early cleavage patterns in Architaenioglossa and architypical caenogastropods may elucidate at which level the caenogastropod and heterobranch lines diverged.

No agreement exists about the phyletic position of the Neritimorpha. Based on morphological characters, Haszprunar (1988) and Ponder and Lindberg (1995) conclude that the Neritimorpha emerged between the Docoglossa and the Vetigastropoda. However, according to the analysis of 28S rRNA, Tillier and Tillier (1995) assume that the neritimorphs emerge between the Vetigastropoda and the Caenogastropoda, in correspondence with our ontogenetic trees shown in Figures 20, 21, and 22. If polar lobe formation in Sinotaia and other Architaenioglossa will be affirmed, then the Neritimorphs may represent an independent and early offshoot in the cacnogastropod lineage, as shown in Figure 20. If none of the Architaenioglossa, Sinotaia included, forms a polar lobe, then the Neritimorpha might also be incorporated in the caenogastropod line before the development of a polar lobe, as shown in Figure 21. Additional developmental criteria, such as presence or absence of food eggs, will be needed before definite conclusions can be drawn. For instance, in Theodoxus the eggs of a single spawn are deposited in a single capsule. Only one of these eggs develops into a complete embryo, whereas the others only pass a limited number of divisions, and function as food eggs. Apart from the Neritimorpha, this phenomenon is exclusively found to the Caenogastropoda.

[Figure 20-22 ILLUSTRATION OMITTED]

One may wonder why "the early cleavage pattern may be reckoned as one of the most conservative features in the development of any gastropod" (Conklin 1907, p. 325). One of the constraints that may have limited the heterochronic shifts in the cleavage patterns is that a minimal and fixed number of five cleavage cycles is required for the separation of the ecto- and endodermal cells, independent of the amount of yolk or environmental adaptations. Only then the animal micromeres may become potential inducers, and the endodermal macromeres 3M may become responsive. This corresponds with the observation that the organizing role of the D quadrant in lobe forming species is not accelerated but is postponed to the same developmental stage as in species with four equal quadrants.

It is a matter of debate which kind of selective pressure may have led to the heterochronic shifts in the succession of cleavages related with the accelerated mesentoblast formation. How might a precocious formation of the mesentoblast have been more useful for the embryo than a more delayed formation? In gastropods with equal division the induction of 3D and mesentoblast formation definitely determine the dorsal quadrant, and therefore, one may ask what might have been the benefit of an accelerated specification of the dorsal quadrant? This question is the more crucial in polar lobe forming gastropods, and in taxa with an unequal division, where D quadrant specification has been further advanced up to the 4-cell stage. In a paper on the evolutionary implications of the mode of D quadrant specification, Freeman and Lundelius (1992) discussed the evolutionary implications of an accelerated specification of the D quadrant. According to these authors the precocious specification of the D quadrant by segregation might have been the result of a selective pressure to shorten the transition of the larval into the adult phase. One way to shorten that transitional period would have been to start the development of adult organs prior to metamorphosis. That would have led to adultation, the formation of veliger larvae with adult features like shell and foot. In species in which 3D is specified by induction of one of the 3M cells, initiation of the Anlagen for adult organs cannot start before the fifth cleavage, not before the 24-cell stage. In species with polar lobes, the D quadrant is specified as early as the 4-cell stage. That might have accelerated the formation of teloblasts from which adult structures are formed. However, as discussed above, accelerated D quadrant specification appears not to be accompanied by an acceleration of the organizer function of the dorsal quadrant.

A quite different and independent selective pressure may have led to a similar heterochronic shift in the succession of cleavages. During gastropod evolution the mode of reproduction has changed. Ancient gastropods like the Docoglossa and Vetigastropoda are free spawners; all other gastropods produce egg masses. The eggs are encapsulated, and the capsules are surrounded by several kinds of gelatinous protective surroundings. We assume that the production of protective coverings has changed the kind of selective pressure on the formation of the different cell lines, which in turn may have led to heterochronic shifts in the succession of cleavages. As the ancient archaeogastropods are free spawners, the embryos are deprived of any protection and a short free-swimming phase must have been required. The early cleavages are fast, with intervals of about 30 min. Within one day a free-swimming trochophore-like larva is formed. In all other gastropods the eggs are protected by capsules, eventually provided with additional food reserves, and the capsules are often surrounded by gelatinous coverings. The onset of development is intracapsular, a rapid formation of the locomotory prototroch is no longer necessary, and the free-swimming phase is postponed to later developmental stages. When the larva hatches, it is already provided with adult organs like a shell, foot, and eyes. The original prototroch then no longer suffices and is modified by the development of two lateral velar lobes into a much more powerful locomotory organ, the velum (Garstang 1928). The lower cleavage rhythm in the trochal cell lines may be the result of a decreased pressure on the fast formation of the prototroch. The majority of the trochal cells is derived from the first generation of micromeres formed at the 8-cell stage, and it is not surprising that the cleavage rhythm of these 1m cells is decelerated in comparison with the cleavage rhythm of the IM cells. As a consequence the formation of the 3M macromeres, 3D specification and mesentoblast 4d formation is relatively accelerated. An additional indication for the reduced significance of the trochoblasts is that in a great number of species their size is also strongly reduced.

Finally, if the different taxa of gastropods may be characterized by a specific cleavage pattern, the question arises if there is a specific gastropod cleavage pattern, different from the patterns found in other mollusks, and different from the patterns found in other spiralian embryos. If so, then the significance of the early cleavage patterns strongly increases and might allow the reconstruction of the phyletic relations between the main molluscan taxa as well as between the main spiralian phyla. To determine the phyletic relations between higher animal taxa, one has to compare ancestral representatives of these taxa. It then appears that in primitive mollusks such as the chitons, in ancestral representatives of the bivalves such as the protobranchs, and in archaeogastropods the first two cleavages are equal. In more derived forms, for instance within the class Bivalvia, the cleavages are unequal, or associated with polar lobe formation. As Scaphopoda form polar lobes, and it is generally assumed that the Scaphopoda are the sister group of the Bivalvia, this may imply that polar lobe formation is a matter of convergence. This is the more likely, as lobe formation has been developed independently in gastropods and annelids. Just as equal cleavage is characteristic for architypical mollusks, it appears to be so in Annelida and Platyhelminthes.

Although a complete discussion of the cleavage patterns in non-gastropods and non-mollusks is out of the scope of this paper, here it may suffice to mention that remarkable differences exists between the cleavage patterns of the various molluscan classes. For instance, in bivalves and scaphopods the cleavage pattern of the 2d cell, the first somatoblast, completely differs from the cleavage pattern of 2d in gastropods and polyplacophores. In the former two groups, 2d produces two ectodermal teloblasts, a right and left one, preceded by a typical series of very unequal divisions (Lillie 1895; Herbers 1914; van Dongen and Geilenkirchen 1974). Only in polyplacophores the first two divisions are equal, and in contrast to any other mollusk, the mesentoblast is formed at the 72-cell stage (Heath 1899; van den Biggelaar 1996b).

It is has been reported that just as in mollusks, also in annelids the formation of four equal quadrants is a primitive developmental feature (Freeman and Lundelius 1992). In primitive annelids like Podarke (Treadwell 1901), and Polygordius (Woltereck 1904), the first two cleavages are equal, but D quadrant specification presumably does not occur by specifying 3D, but does occur one cleavage cycle later by specifying one of the fourth quartet cells.

In polyclad flatworms again mesentoblast formation is unique. The mesentoblast is not a daughter cell of macromere 3D as it is in mollusks and annelids, but its formation is postponed one cleavage cycle. In Polyclada the mesentoblast is the animal daughter cell of 4d (Surface 1907; van den Biggelaar, pers. obs.). These characteristic differences are encouraging enough to explore the significance of the early cleavage patterns for the reconstruction of the phyletic relations between the major spiralian taxa.

In a paper on gastropod phylogeny and systematics, Bieler (1992, p. 330) concluded that "gastropods have remained surprisingly underutilized as models for and objects of evolutionary studies. No other animal group offers an equal opportunity to combine the findings of comparative morphological and molecular studies on the diverse extant fauna with data derived from the extensive fossil record." In addition to this remark, we conclude that the early ontogeny of gastropods has remained surprisingly underestimated as a guide in the reconstruction of their evolutionary history. It may be assumed that an extensive comparative embryological study, supported by research on pattern controlling genes, will strongly contribute to our understanding of gastropod, molluscan and spiralian phylogeny. This assumption corresponds with the observations that in the annelid Platynereis, the protein encoded by the homeobox containing gene engrailed is limited to the trochoblasts (Dorresteijn et al., 1993), whereas in Patella engrailed mRNA is limited to the trochoblasts (Damen et al., unpubl.). It is our conviction that comparative and experimental studies on spiralian developmental patterns, supplemented with studies on the expression of pattern controlling genes, especially genes involved in mesoderm formation, will elucidate the evolutionary history of spiralian animals.

ACKNOWLEDGMENTS

The first author wishes to thank P. Lasserre, the former director of the Station Biologique in Roscoff for his hospitality during part of this work and the "marine de la Station" for providing part of the material; G. de Jong for valuable discussions; B. Wagemaker for his enthusiasm in making the series of whole mount preparations; D. Smit, L. W. Landman, and especially F. Gerards-Kuijer for the translation of rough drafts of cleavage patterns and lineages into definite figures. Part of this work has been enabled by the financial support of the Commissie voor buitenlandse marien-biologische Instituten van de Koninklijke Nederlandse Akademie van Wetenschappen, Amsterdam. I am also grateful to Miep for her contribution.

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JO A. M. VAN DEN BIGGELAAR, Department of Experimental Zoology, University of Utrecht, Padualaan 8, 3584 CH Utrecht, The Netherlands. E-mail: J.A.M.van den Biggelaar@biol.ruu.nl

G. HASZPRUNAR, Zoologische Staatssammlung Munchen, Munchhausenstrasse 21, D-81247 Munchen, Germany
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Author:Biggelaar, Jo A.M. van den; Haszprunar, G.
Publication:Evolution
Date:Aug 1, 1996
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