renids, 111 pg in Lepidosiren and 142 pg in Protopterus; ceratodontids, 80 pg in Neoceratodus; ref. 15), but sala- manders have the second largest genomes, ...
Proc. Nati. Acad. Sci. USA Vol. 91, pp. 4796-4800, May 1994 Neurobiology
Cell size predicts morphological complexity in the brains of frogs and salamanders GERHARD ROTH*t, JENS BLANKE*,
AND
DAVID B.
WAKEO
*Brain Research Institute, University of Bremen, D-28334 Bremen, Federal Republic of Germany; and tMuseum of Vertebrate Zoology and Department of Integrative Biology, University of California, Berkeley, CA 94720
Communicated by Stephen Jay Gould, February 14, 1994
the tectum opticum (3, 4), the torus semicircularis (5), and a number of diencephalic nuclei (2). The brain of salamanders long has been known to be morphologically much simpler than that of frogs and other vertebrates (6, 7). It has a compact periventricular cellular layer (gray matter) and a superficial fiber layer (white matter). Very few migrated nuclei can be recognized on morphological grounds. Few to very few migrated cells are found in the superficial fiber layers of the mesencephalic tectum (8). The tegmentum mesencephali, including the torus semicircularis, resembles the tectum in that it has a relatively compact periventricular layer (9). Here, we report the results of a comparative study of brain complexity in salamanders and frogs. We concentrate on the tectum mesencephali (optic tectum). Both frogs and salamanders are predators that depend on vision, and the tectum is the most important visual center for localization and identification of prey objects. In addition, the tectum exhibits the most distinctive morphology and cytoarchitecture of any part of the amphibian brain. A priori, one expects the tectum to present the most clear-cut influence of function on form, if such an influence exists. We demonstrate that cell size is the most likely determinant of tectal morphology in frogs. In salamanders, brain size is an additional important factor. These findings show that alternatives to strict functionalism must be considered in explaining differences in brain morphology among taxa.
ABSTRACT The morphological organization of the brain of frogs and salamanders varies greatly in the degree to which it is subdivided and differentiated. Members of these taxa are visually oriented predators, but the morphological complexity of the visual centers in the brain varies interspecifically. We give evidence that the morphological complexity of the amphibian tectum mesencephali, the main visual center, can be predicted from knowledge of cell size, which varies greatly among these taxa. Further, cell size is highiy correlated with genome size. Frogs with small cells have more complex morphologies of the tectum than do those with large cells independent of body and brain size. In contrast, in salamanders brain-body size relationships also are correlated with morphological complexity of the brain. Small salamanders with large cells have the simplest tecta, whereas large salamanders with small cells exhibit the most complex tectal morphologies. Increases in genome, and consequently cell size, are associated with a decrease in the differentiation rate of nervous tissue, which leads to the observed differences in brain morphology. On the basis of these findings we hypothesize that important features of the structure of the brain can arise independently of functional demands, from changes at a lower level of organismal organization this case increase in genome size, which induces simpllifcation of brain morphology. The morphological organization of the brain varies among vertebrates in the degree to which it is subdivided and differentiated. Parts of brains exhibit, among other features, differences in lamination, presence of distinct nuclei, numbers of different cell types, and degree of complexity of neuronal connectivity. There is little understanding of the processes that lead to the observed differences, although the most prevalent explanations are forms of functionalism (i.e., the observed differences are the result of environmental selection regarding the specific function of brain parts) and phylogenetic history (older lineages generally have less complex brains). We have examined an alternative view: that the simple brain morphology of salamanders is secondary, derived in large part by pedomorphic evolution associated with increases in genome and cell size (1). We here argue that variation in morphological complexity in the brains of frogs and salamanders is based predominantly on such intrinsic factors and is likely to be independent of direct selection. The brains of frogs (Order Anura) and salamanders (Order Caudata) differ considerably within and among these orders in the degree of morphological complexity. In general, frogs have more complex brain morphology than do salamanders, having morphologically distinct nuclei that often lie in migrated positions in the diencephalon, the pretectum, and the mesencephalic tegmentum (2). In addition, multiple lamination (an alternation of cellular and fibrous layers) is found in
MATERIALS AND METHODS Brains of 22 species of salamanders (3 families) and 17 species of frogs (11 families) were used in the present study (Tables 1 and 2). Heads fixed in formalin were cut in 10-,um serial sections and silver-impregnated by using the KlueverBarrera method (10). In addition, two specimens of the salamander genus Parvimolge stained with the Giemsa method (11) were used. To determine brain volume, we drew equidistant cross sections of the brain (30-50 sections) with a Zeiss camera lucida. Cell-size measurements were taken for 50 neurons from three different tectal areas and from as many specimens per species as were available. Information on genome size was obtained from the literature, and all values were converted to pg of DNA per haploid genome. For correlational analysis regarding cell size, brain size, and morphological complexity, we established five classes in frogs and six classes of morphological complexity of the tectum in salamanders (including an undifferentiated state). The classes of morphological complexity for the frog tectum are as follows (descriptions of layers are from refs. 3 and 5). Class 1. Separation of layers was indistinct and not continuous in mediolateral extent. Layers 7-9 were diffusely arranged; there was no distinct formation of a layer 8.
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tTo whom reprint requests should be addressed.
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Neurobiology: Roth et aL Table 1. Salamanders n GS* CSt BS* MC Classification Family Plethodontidae Subfamily Desmognathinae Desmognathus wrighti 4 14.1 7.7 1.2 1 Desmognathus aeneus 2 7.5 1.8 1.5 Desmognathus ochrophaeus 2 14.0 7.2 2.2 2.3 1 15.0 9.2 10.1 4 Desmognathus monticola Desmognathus quadramaculatus 1 15.0 9.5 12.9 5 Subfamily Plethodontinae Tribe Hemidactyliini 1 20.8 8.8 3.0 1.5 Eurycea bislineata Tribe Plethodontini Plethodon cinereus 3 22.3 8.9 3.2 3 1 29.0 11.8 6.5 4 Plethodon jordani Tribe Bolitoglossini 2 76.2 15.1 12.5 0 Hydromantes italicus 2 15.1 16.3 0.5 Hydromantes genei 2 37.0 11.3 2.3 0 Batrachoseps attenuatus 3 20.3 8.1 1.4 1 Parvimolge townsendi 2 25.2 10.5 1.3 0.5 Thorius narisovalis Thorius pennatulus 2 9.0 0.5 0.5 2 68.9 13.4 7.8 0 Bolitoglossa subpalmata 1 59.4 13.2 17.2 1 Bolitoglossa dofleini Family Ambystomatidae 2 20.5 12.2 8.1 3 Ambystoma opacum 2 21.9 11.0 28.1 4.3 Ambystoma mexicanum Family Salamandridae 1 20.5 11.0 1.8 3 Salamandrina terdigitata 2 33.0 10.1 19.3 3 Salamandra salamandra Pleurodeles waltl 1 19.7 9.7 10.9 3 Triturus alpestris 1 23.7 11.0 GS, genome size; CS, cell size; BS, brain size; MC, morphological complexity. *Data are reported as pg of DNA haploid genome. tData are reported as ,m3. tData are reported as mm3.
Class 2. Layer 6 was well separated, but layers 1-5 were diffusely arranged, particularly in the lateral portion; layers 7-9 were arranged as in class 1. Class 3. Layers 1-6 were well separated from each other; layer 8 was separated from layers 7 and 9 and contained 60-80% of the migrated neurons with the rest dispersed throughout layers 7 and 9. Class 4. Layers were separated rostrocaudally, as well as mediolaterally but were without sharp borders, except for layer 8, which contained 80-95% of the migrated neurons. Class 5. There was a clear and sharp separation of all layers in all parts of the tectum; layer 8 contained >95% of the migrated neurons. In salamanders, the tectum is never as differentiated as in frogs, so a different classification of morphological differentiation is used (description of layers is from ref. 12). Class 0. There was a periventricular layer of densely packed cells with no further subdivisions; no migrated cells appeared in the superficial white matter. Class 1. Gray matter was subdivided by short fiber bands extending mediolaterally up to one-fourth of the width of the tectum; no migrated cells were present. Class 2. Gray matter was subdivided by short fiber bands extending mediolaterally up to one-half of the width of the tectum; very few migrated cells were present. Class 3. Gray matter was more loosely arranged; cellular islands had formed, in addition to short bands of fibers; up to 5% of the neurons had migrated. Class 4. Fiber bands extended mediolaterally over the entire width of the tectum; gray matter was loosely arranged; up to 5% of neurons had migrated into superficial layers 1-4.
Proc. Natl. Acad. Sci. USA 91 (1994) Table 2. Anurans Family and species Bombinatoridae Bombina orientalis Discoglossidae Discoglossus pictus Pipidae Xenopus laevis Ranidae Rana temporaria Mantella aurantiaca Mantella cowani Hyperoliidae Hyperolius quinquevittatus
Afrixalus fornasinii
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n
GS*
CSt
BSt
MC
3
10.3
9.9
10.9
2
3
5.3
8.0
15.6
3.7
1
3.0
6.2
30.9
4
3 2 2
4.2 4.8
7.9 6.2 6.8
18.5 5.3 6.5
3 4.5 4.2
6.4 6.9
8.8
5 4.5
2 2
Hylidae 1 Hyla septentrionalis 6.5 20.9 4 Gastrotheca riobambae 2 3.7 7.3 19.2 3.5 Bufonidae Bufo bufo 2 5.8 4 6.4 Myobatrachidae Limnodynastes tasmaniensis 2 2.3 6.6 9.8 3.5 Arenophryne rotunda 1 19.0 10.0 5.5 1.5 Dendrobatidae Dendrobates pumilio 2 7.0 5.0 3 Leptodactylidae Eleutherodactylus coqui 2 5.8 14.0 4 Sminthillus limbatus 2 5.1 1.4 3.5 Rhacophoridae Rhacophorus leucomystax 1 7.2 18.6 3.8 GS, genome size; CS, cell size; BS, brain size; MC, morphological complexity. *Data are reported as pg of DNA haploid genome.
tData are reported as ,um3.
tData are reported as mm3.
Class 5. Fiber bands extended over the entire rostrocaudal and mediolateral width of the tectum, separating deep cellular layers 6 and 8 and continuing into the tegmentum; 3-10%1 of the neurons had migrated. Specimens were ranked by complexity class. In cases where specimens of the same species ranked differently, means were calculated. Statistical analysis (13) included Pearson's product moment correlation, used for analysis of the continuous variables of brain size, cell size, and genome size, and Kendall's rank correlation, used for comparisons of degree of morphological differentiation. Felsenstein's contrast method was used for phylogenetic comparisons (not presented in detail here) (14).
RESULTS We present results for frogs and for salamanders separately
(Fig. 1).
Frogs. We recognize four groups based on our criteria for morphological complexity of the tectum. The first group contains frogs with small to very small brains and large to very large cell sizes-i.e., Arenophryne rotunda and Bombina orientalis (9.8 ,um average cell diameter). Their tecta, with scores of 1.5-2, rank as the least differentiated among anurans. In addition, the mesencephalic tegmentum and torus semicircularis in these species show little, if any, lamination. The next group contains species that have intermediate cell sizes-i.e., Gastrotheca riobambae, Dendrobates pumilio, and Discoglossus pictus (average cell diameter, 7.4 ,um). Tectal morphology ranks 3-3.7 in complexity. The next group consists of frogs with medium to small cells-i.e., Mantella cowani, Rhacophorus leucomystax,
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FIG. 1. Cross sections through brains of frogs and salamanders at the level of the tectum and the dorsal tegmentum/torus semicircularis showing differences in cell size and morphological complexity. (A) Hydromantes italicus. (B) Arenophryne rotunda. (C) Desmognathus quadramaculatus. (D) Limnodynastes tasmaniensis. A and C represent the simplest and most complex morphologies among salamanders of the family Plethodontidae; B and D represent the simplest and most complex morphologies among frogs of the family Myobatrachidae. These cross sections also represent the extremes of morphological complexity encountered within the orders Caudata and Anura. Within each group, morphological complexity of the tectum, as well as the tegmentum and torus semicircularis, correlates negatively with cell size, which is positively correlated with genome size. TE, tectum; TG, tegmentum/torus semicircularis. (Bar = 100 ,um.)
Rana temporaria, Limnodynastes tasmaniensis, and Hyla septentrionalis (average cell diameter, 7 am). Tectal morphology ranks 3-4.2 in complexity. We include Sminthillus limbatus in this group based on its complexity score, but note that it has the smallest cells found among frogs (cell diameter, 5.1 pum). Sminthillus is a miniaturized species that has by far the smallest brain among the anurans sampled. The last group consists of frogs that have small to very small cells: Hyperolius quinquevittatus, Xenopus laevis, Eleutherodactylus coqui, and Mantella aurantiaca (average cell diameter, 6.2 ,um). Their tectal morphology ranks 4-5. In frogs, brain size is neither correlated with cell size nor with morphological complexity of the tectum (0.1, P < 0.6; n = 15). Thus, larger brains do not necessarily have more complex morphologies. However, cell size is significantly negatively correlated with morphological complexity of the tectum (-0.5, P < 0.01; n = 16). Thus, frogs with smaller cells have more complex tecta (as well as other brain centers), independent of brain size. Salamanders. We recognize four groups based on the combined criteria of rank in morphological complexity of the tectum and body size. The least differentiated tectum (average rank per species, 0-0.5) is associated with cells of medium to large size (diameters, 9-11.3 pm) and found in small species (13- to 42-mm snout-to-vent length): Thorius narisovalis, Thorius pennatulus, and Batrachoseps attenua-
tus. The second group also has a relatively undifferentiated tectum (0-1) and even larger cells (diameters, 13.2-15.1 jam); species in this group are medium-sized to large (>45-mm snout-to-vent length): Bolitoglossa subpalmata, Bolitoglossa dofleini, Hydromantes italicus, and Hydromantes genei. A third group has a more differentiated tectum (rank, 1-3), but cells that are small to medium in size (diameters, 7.2-11.0 pum); members ofthis group are small (64-mm snout-to-vent length): Ambystoma opacum, Ambystoma mexicanum, Desmognathus monticola, Desmognathus quadramaculatus, Plethodon jordani, Salamandra salamandra, and Pleurodeles walti. The neotenic Ambystoma mexicanum has a well differentiated tectum that is more complex than that of its metamorphosed congener, Ambystoma opacum, which has slightly larger cells and a much smaller brain than Ambystoma mexicanum. In salamanders, in contrast to frogs, brain size (which is positively correlated with body size; 0.90, P < 0.01; n = 20) and cell size correlate significantly (0.49, P < 0.02, n =
Neurobiology: Roth et A 21)-i.e., salamanders with larger brains tend to have larger cells, and those with smaller brains tend to have smaller cells. Furthermore, brain size and body size are significantly positively correlated with the degree of morphological complexity of the tectum (0.49, P < 0.01, n = 20). Holding cell size constant, salamanders with larger brains have more complex tecta. In contrast, cell size is significantly negatively correlated with the degree of morphological complexity of the tectum. Holding brain size constant (i.e., by dividing our sample into small, 0.5-3 mm3, and large, 3.2-28.1 mm3, brain-size categories), salamanders with smaller cells have more complex tecta (small brains: -0.8, P < 0.01, n = 8; large brains: -0.53, P < 0.01, n = 13).
DISCUSSION Our analysis demonstrates that, for frogs, cell size is significantly correlated with morphological complexity of the tectum (as well as other brain parts). Thus, frogs with the largest cells have the simplest brains, regardless of body and brain size. Bombina orientalis has a much larger brain than do Hyperolius, Mantella, Sminthillus, or Dendrobates, yet it has a much simpler brain. We attribute simplification (as opposed to retention of an ancestral simplicity as determined by our phylogenetic analysis, data not shown) of the brain in this species to its large cells. The most complex brain morphologies are found in frogs that have intermediate to small brains. An important exception is the case of Xenopus laevis, which has by far the largest brain among the taxa studied but has a relatively differentiated tectum (rank, 4). In contrast, Sminthillus also has a relatively complex brain morphology (rank, 3.5), although its brain is the smallest among the taxa studied. Both species have small cells. Although the degree of morphological differentiation of the salamander tectum is significantly negatively correlated with cell size, as in frogs, there is also a significant positive correlation between brain size and tectal complexity. Small cells and large brains are both important factors contributing
to increased differentiation of the brain in salamanders and appear to exert a counteracting influence on brain morphol-
ogy. Thus, in salamanders, the following generalities appear (i) If two species have equal brain sizes, the species with smaller cells has the more complex brain morphology. (ii) If two species have cells of equal sizes, the species with the larger brain has the more complex brain morphology. Accordingly, the least differentiated brains are found in small species (with small brains) having large cells and in mediumsized to large species (with large brains) having large cells. Brains become increasingly complex in degree of morphological differentiation in the following order: small species with small cells, medium-sized to large species with medium to small cells, and large species with small cells. An extraordinarily broad range of cell sizes is found in amphibians; within our samples both of frogs and salamanders, cell diameters double. The simplest explanation for variation in cell size is variation in genome size, which is known to correlate positively with cell size and which shows nearly an order of magnitude variation within each group (15). The smallest genome among vertebrates is found in teleost fishes, with
