Senior Honors Thesis Research Proposal

Albert B. Ulrich III
Thesis Advisor: Dr. Wayne Leibel
11 September 1998


Neotropical fish of the family Cichlidae are a widespread and diverse group of freshwater fish which, through adaptive radiation, have exploited various niches in freshwater ecosystems. One such evolutionary adaptation employed by numerous taxa is miniaturization, an evolutionary process in which a large ancestral form becomes reduced in size to exploit alternative niches. A considerable amount of research has been conducted on the effects of miniaturization on amphibians (Hanken 1983), but although miniaturization has been found to occur in 85 species of freshwater South American fish, little has been done to investigate the effects which miniaturization imposes on the anatomy of the fish (Hanken and Wake 1993).


Evolution is the process by which species adapt to environmental stresses over time. Nature imposes various selective pressures on ecosystems causing adaptive radiation, where species expand and fill new niches. One such adaptation for a new niche is miniaturization. Miniaturization can be defined as “the evolution of extremely small adult body size within a lineage” (Hanken and Wake 1993). Miniaturization is observed in a variety of taxa, and evolutionary size decreases are observed in mammals and higher vertebrates, but it is more common and more pronounced in reptiles, amphibians and fish (Hanken and Wake 1993). Miniaturization evolved as a specialization which allowed the organisms to avoid selective pressures and occupy a new niche. Miniaturization as a concept is dependent on the phylogenetic assumption that the organism evolved from a larger predecessor. Over time, the miniature organism had to adapt to the new conditions as a tiny species. All of the same basic needs had to be met, but with a smaller body.

In miniature species there is a critical relationship between structure of the body and body size, and frequently this downsizing results in structural and functional changes within the animal (Harrison 1996). Within the concept of miniaturization is the assumption that the species evolved from a larger progenitor. It is necessary then to explore the effects of the miniaturization process. “Miniaturization involves not only small body size per se, but also the consequent and often dramatic effects of extreme size reduction on anatomy, physiology, ecology, life history, and behavior” (Hanken and Wake 1993).

Hanken and Wake 1993 found that the adult skulls of the salamander Thorius were lacking several bones, others were highly underdeveloped, and many species within the genus were toothless. Several invertebrate species display the wholesale loss of major organs systems as a result of the drastic reduction in body size (Hanken and Wake 1993). Hanken and Wake also have shown that morphological novelty is a common result of miniaturization. Morphological novelty, in essence, is the development of new structures in the miniature organism. For example, as body size decreases, certain vital organs will only be able to be reduced by a certain amount and still function. As a result organs such as the inner ear remain large relative to the size of the miniature skull, and structural innovations have to occur in order to support the proportionately large inner ear.

In 1983, James Hanken, at the University of Colorado determined that the adult skull of the Plethodontid salamanders could be characterized by three observations: 1) there was a limited development or even an absence of several ossified elements such as dentition and other bones; 2) there was interspecific and intraspecific variability; 3) there were novel mophological configurations of the braincase and jaw (Hanken 1983).

In his experiments, Hanken found that cranial miniaturization of the Thorius skull was achieved at the expense of ossification. Much of the ossified skeleton was lost or reduced, especially in the anterior elements, which are seen typically in larger adult salamanders (Hanken 1983). In contrast to this ossified downsizing, many of the sensory organs were not diminished in size — therefore present in greater proportion to the rest of the reduced head. He also reported that due to the geometrical space availability, there is a competition for space in reduced sized skulls, and the “predominant brain, otic capsules, and eyes have imposed structural rearrangements on much of the skull that remains” (Hanken 1983).

Hanken proposed that paedomorphosis was the mode of evolution of the plethodontid salamanders (Hanken 1983). Paedomorphosis is the state where the miniaturized structures of the adult salamanders can be described as arrested juvenile states. To support this theory, Hanken showed data where cranial skeletal reduction was less extreme in the posterior regions of the skull. One of the hallmarks of paedamorphosis is the lack of conservation in structures derived late in development. Early developed structures are highly conserved, and the latter derivations become either lost, or greatly reduced. Again, Hanken has shown that elements appearing late in development exhibit greater variation among species than do elements appearing earlier in ontogeny (Hanken 1983). But the presence of novel morphological features cannot be accounted for merely by truncated development and the retention of juvenile traits. Miniature Plethodontid salamanders display features that are not present in other species, juvenile or adult. These novel morphological features are associated with the evolution of decreased size and are postulated to compensate for the reductions occurring in other areas (Hanken 1983).

In 1985, Trueb and Alberch published a paper presenting similar results in their experiments with frogs. They explored the “relationships between body sizes of anurans and their cranial configurations with respect to the degree of ossification of the skull and two ontogenetic variables‹shape and number of differentiation events” (Trueb and Alberch 1985). Trueb and Alberch examined three morphological variables: size, sequence of differentiation events, and shape changes in individual structures. Size and snout length were measured, and the data showed that the more heavily ossified frogs tended to be smaller, whereas the less-ossified species were of average size, contrary to what was hypothesized. But Trueb and Alberch also attributed the diminution in size to paedomorphosis, citing that the smaller frogs lacked one or more of the elements typically associated with anuran skulls‹these missing elements were typically late in the developmental sequence. It is significant to note, however, that although there was an apparent paedomorphic trend, it could not be “applied unequivocally to all anuans” (Trueb and Alberch 1985). Very little research has been done on the effects of miniaturization on fish. In 1993, Buckup published a paper discussing the phylogeny of newly found minature species of Characidiin fish, but the extent of the examination was merely an acknowledgment that the species were indeed miniatures so that they could be taxonomically reclassified ( Buckup 1993). It is this deficit of knowledge with regard to miniaturization in fish that prompts this research.

Statement of the Problem:

How does miniaturization affect other vertebrates, such as fish? There are over 85 species of freshwater South American fish which are regarded as miniature, spanning 5 orders, 11 families and 40 genera (Hanken and Wake 1993). One such species, Apistogramma cacatuoides, is a South American Cichlid native to Peru. It lives in shallow water bodies in the rainforests, where miniature size is necessary. Males in this species reach approximately 8cm, and females only 5cm. This makes A. cacatuoides an ideal specimen for examination. In this senior honors thesis, I intend to examine the effects of miniaturization on cranial morphology of A. cacatuoides.

Plan of Research:

In this thesis, I will compare the cranial anatomy of A. cacatuoides to that of “Cichlasoma” (Archocentrus) nigrofasciatum, a commonly bred fish reared by aquarists known as the Convict Cichlid, a “typical” medium-sized cichlid also of South American origin. The Convicts will be examined at various stages in development, from juvenile to adult, and will be compared to A.cacatuoides.

The first part of this project will involve whole mount preparation of A. cacatuoides, utilizing the staining and clearing procedures described by Taylor and Van Dyke, 1985. This procedure involves the use of Alizarin Red and Alcian Blue to stain bone and cartilage, and takes into account the adaptations and recommendations Proposed in an earlier paper (Hanken and Wassersug 1981). The Taylor and Van Dyke procedure is specifically for the staining and clearing of small fish and other vertebrates. I tested the procedure during last semester¹s Independent Study and made a few minor adjustments to the protocol.

First, the specimens will be placed serially into an absolute ethyl alcohol solution and stained with Alcian Blue. The fish will then be neutralized in a saturated borax solution, transferred to a 20% hydrogen peroxide solution in potassium hydroxide, and then bleached under a fluorescent light. The unwanted soft tissues will then be cleared using trypsin powder, and then stained in KOH again with alizarin red. The final preparation of the fish involves rinsing the fish, and placing them serially into 40%, 70%, and finally 100% glycerin.

Following the above preparation of the specimens, the crania of the A. cacatuoides specimens will be examined for morphological variation and compared to the cranial anatomy of the Convict cichlid as a progenitor reference point examined at various developmental stages to see if paedomorphosis in indeed the mechanism of miniaturization in A. cacatuoides.

Expected Costs:

The project is estimated to cost no more that five hundred dollars for chemicals and supplies for the entire year.

Literature Cited:

Hanken, J., 1983. Miniaturization and its Effects on Cranial Morphology in Plethodontid Salamanders, Genus Thorius (Amphibia: Plethodontidae). I. Osteological Variation”. Biological Journal of the Linnean Society (London) 23: 55-75.

Hanken, James, 1983. Miniaturization and its Effects on Cranial Morhology in Plethodontid Salamanders, Genus Thorius (Amphibia, Plethodintidae): II.The Fate of the Brain and Sense Organs and Their Role in Skull Morphogenesis and Evolution . Journal of Morphology 177: 255-268.

Hanken, James and David Wake, 1993. Miniaturization of Body Size: Origanismal Consequences and Evolutionary Significance. Annual Review of Ecological Systems 24: 501-19.

Harrison, I. J., 1996. Interface Areas in Small Fish. Zoological Symposium No. 69. The Zoological Society of London: London.

Miller, P. J., 1996. Miniature Vertebrates: The Implications of Small Body Size. Symposium of the Zoological Society of London. No. 69: 15-45.

Taylor, William R. and George Van Dyke, 1985. Revised Procedures for Staining and Clearing Small Fishes and Other Vertebrates for Small Bone and Cartilage Study. Cybium. 9(2): 107-119.

Trueb, L. and P. Alberch, 1985. Miniaturization and the Anuran Skull: a Case Study of Heterochrony. Fortschritte der Zoologie. Bund 30.

Williams, T. Walley, 1941 Bone and Cartilage. Stain. Tech. 16:23-25.