NSF FINAL REPORT

NSF AWARD NUMBER 9510228

PERIOD: 9/15/95 - 8/31/99

TITLE:
TIMING AND 3-D ANALYSIS OF MUSCULOSKELETAL DEVELOPMENT

PI: DR. RAFAEL O. DE SA
DEPT. OF BIOLOGY
UNIVERSITY OF RICHMOND

CO-PI: DR. GARY RADICE
DEPT. OF BIOLOGY
UNIVERSITY OF RICHMOND

CO-PI: DR. MICHAEL KERCKHOVE
DEPT. OF MATHEMATICS AND
COMPUTER SCIENCE
UNIVERSITY OF RICHMOND.

NOVEMBER, 1999.
TABLE OF CONTENTS.

1. Participants 4
A. Who has been involved? 4
B. What people have worked on the project? 4
C. Where are those students now? 5
D. What other organizations have been involved
as partners? 6
E. Have you had other collaborators or contacts? 7

2. Activities and Findings: 8
What have you done? What have you learned? 8
A. Describe the major research and education
activities of the project. 8
Mathematical analyses of shape 8
Heterochronies and life histories in amphibian
skeletal muscle development 8
Chondrocranial Anatomy 9
B. Describe the major findings resulting from
these activities. 10
Mathematical work 10
Myogenesis 12
Patterns of somite cell rotation in pipids 13
Chondrocranial Anatomy 14
3 dimensional (3D) reconstruction of
chondrocrania from serial sections 16
Literature Cited 19
C. Relevance of the Research Accomplishments 21
D. Describe the opportunities for training
and development provided by your project 23
E. What other educational and outreach activities
have you undertaken? 24

3. Publications, Presentations, and Products 25
Summary of output 25
A. What have you published as a result of this work? 25
B. What have you presentations as a result of this work? 26
Presentations at UR. 26
Presentations at Professional Meetings. 28
C. What Web site or other Internet site have you created? 31
D. Other anticipated contributions. 31

4. Project Contributions? 32
A. To the development of your own discipline(s)? 32
B. To other disciplines of science or engineering? 32
C. To education and development of human resources? 32
D. To physical, institutional, and information resources for 33
science and technology

5. Appendix I. Progress Report submitted
A. Progress Report 1 (1995-1996)
B. Progress Report 2 (1996-1997)
C. Progress Report 3 (1997-1998)

6. Appendix II.
A. Publications
B. Manuscript in review.

1.Participants.
A. Who has been involved?
Rafael O. de Sá, PI
Gary Radice, CO-PI
Michael Kerckhove, CO-PI

B. What people have worked on the project?

Students whose work was directly supported by NSF Funds: 11 undergraduate women, 11 undergraduate men, 2 graduate men
Undergraduates:
Ms. Carmen Beitzer Mr. Joseph Oppong
Ms. Mary Kate Boggiano Ms. Erin Pingelski
Ms. Elaine Bucheimer Mr. Tim Riley
Mr. Jon-Eric Burgess Mr. Matthew Smetanick
Mr. Craig Cameron Ms. Lee-Ann Smith
Mr. Mark DeSantis Mr. Stephen Spear
Ms. Shou-Yuan Fan Mr. Todd Stevens
Mr. Michael Franchella Mr. James Tripp
Ms. Shannon Hill Ms. Rebecca Weber
Ms. Erin Hines Ms. Rebecca Weingard
Mr. Peter Larson Ms. Robin Wilburn

Graduate Students:
Mr. Charles Swart
Mr. Will Turner

Students that work on related areas of the project and whose work was indirectly supported b NSF Funds: (3 undergraduate women, 4 undergraduate men, 1 graduate woman, 1 graduate man)

Mr. Diego Arrieta Mr. Peter Matthews
Ms. Anne T. d'Heursel Ms. Kandace Peterson
Mr. Jeffrey Eastman Mr. Geoffrey Schwartz
Mr. Joe LaCroix Ms. Andrea Stigall
Ms. Barbara Summers

C. Where are those students now?

Ms. Carmen Beitzer unknown
Ms. Mary Kate Boggiano undergraduate senior year at U. of Richmond, majoring in Mathematics and Chemistry, plans to attend graduate school in industrial chemistry.
Mr. Elaine Bucheimer undergraduate junior year at U. of Richmond, majoring in Biology and Mathematics with plans to pursue graduate study in Biology.
Mr. Jon-Eric Burgess unknown
Mr. Craig Cameron undergraduate sophomore year at U. of Richmond, majoring in Biology
Mr. Mark DeSantis working for the consulting firm of Deloitte and Touche in Pittsburgh, PA.
Ms. Shou-Yuan Fan undergraduate senior year at U. of Richmond, majoring in Biology with plans to attend graduate school in Biology
Mr. Michael Franchella unknown
Ms. Shannon Hill graduate student in Computer Science at Virginia Commonwealth University
Ms. Erin Hines graduate Student (Masters Program) in Wildlife and Forestry at Ohio State University
Mr. Peter Larson graduate Student (Ph.D. Program) in Biology at Ohio State University
Mr. Joseph Oppong undergraduate senior U. of Richmond, majoring in Chemistry with plans to pursue Medical School.
Ms. Erin Pingelski undergraduate senior U. of Richmond, majoring in Biology with plans to pursue Veterinary School.
Mr. Tim Riley Medical School, Syracuse University
Mr. Matthew Smetanick working in Biotechnology in Maryland, pending admission to medical school
Ms. Lee-Ann Smith undergraduate junior year at U. of Richmond, majoring in Chemistry with plans to pursue Medical School.
Mr. Stephen Spear undergraduate junior year at U. of Richmond, majoring in Biology with plans to attend graduate school in Biology
Mr. Todd Stevens Medical School, Georgetown University

Mr. James Tripp undergraduate senior at U. of Richmond, majoring
in Mathematics with plans to attend graduate school in Mathematics
Ms. Rebecca Weber graduate student (Ph.D. Program) in Mathematics at Notre Dame University
Ms. Rebecca Weingard working for Bell Atlantic in Arlington, VA.
Ms. Robin Wilburn graduate student (Ph.D. program) in Biology at Purdue University
Mr. Will Turner completed MS in Biology at U. of Richmond, teaching high school in Atlanta.
Mr. Chris Swart completed MS in Biology at U. of Richmond,
graduate student (Ph.D. Program) at Louisiana State University.

Students indirectly supported by NSF-CRUI:

Mr. Diego Arrieta undergraduate junior, College of Science, University of Uruguay
Ms. Anne T. d'Heursel graduate student in evolutionary biology (Ph.D. Program), University of Sao Paulo, Brazil.
Mr. Jeffrey Eastman Unknown
Mr. Joe LaCroix completed MS in Biology at U. of Richmond, working in finance/banking in Richmond, Virginia
Mr. Peter Matthews Medical School, Georgetown University.
Ms. Kandace Peterson Medical School, Eastern Virginia Medical School.
Mr. Geoffrey Schwartz working in a pharmaceutical company in New Jersey.
Ms. Andrea Stigall Medical School
Ms. Barbara Summers graduate student in Biochemistry (Ph.D. Program) at Emory University, Atlanta.

D. What other organizations have been involved as partners?

None

E. Have you had other collaborators or contacts?

During the duration of the grant, the three PI's and our students benefited from contacts and collaborations with several colleagues from other institution. We interacted with Dr. Ray Keller, University of Virginia, and one of his Post-Doctoral Associates, Dr. David Shook. Dr. Keller's laboratory focuses on vertebrate development and they are working on different aspects of mesoderm formation in amphibians. As a result of our interactions, Dr. Rafael de Sá gave a seminar at the Biology Department, University of Virginia, and Dr. David Shook gave a seminar at the Biology Department, University of Richmond. In addition, Dr. Shook generously provided specimens of Xenopus tropicalis that were used in our research.
Dr. de Sá collaborated with Dr. Esteban Lavilla from the Instituto Miguel Lillo, Tucuman, Argentina, who works on development of anuran larvae. Dr. Lavilla visited Dr. de Sá laboratory for a 2 months period (1998). In addition, Dr. de Sá interacted via e-mail with Dr. Alexander Haas from the Institut für Spezielle Zoologie, Jena, Germany. Dr. Haas has an extensive research program on evolutionary morphology of anurans. As a result of this interactions, Dr. Haas has recently visited (September 99) Dr. de Sá's laboratory for a period of one month to initiate collaborative research. Dr. de Sá plans to spend one month in the summer of 2001 in Dr. Haas's laboratory.
Dr. Kerckhove and his students have extensive interactions with the University of North Carolina Medical Image Display and Analysis Group led by Dr. Stephen Pizer. Dr. Kerckhove visited Chapel Hill several times in order to exchange ideas concerning the extraction of medial loci from images. Dr. Pizer visited the University of Richmond as one of the speakers in our CRUI funded "3D quantitation workshop" (see below). Additionally, Dr. Kerckhove has maintained contact with two UNC graduates, Dr. Jacob Furst and Dr. Jason Miller, concerning the general structure of critical sets in scale space, of which medial loci are an important example. Furthermore, Dr. Kerckhove established important contacts with: Dr. Bart ter Haar Romeny, Utrecht University; Dr. John Russ, Dr. Chris Russ.
Dr. ter Haar Romeny is a leader in the scale space community who ran a tutorial on this subject at the Computer Vision and Pattern Recognition Conference (June 1999). Dr. Kerckhove attended this tutorial with support from the University of Richmond. Dr. ter Haar Romeny is enthusiastic about the use of differential geometric ideas in image analysis, particularly. Dr. Kerckhove met up with Dr. ter Haar Romeny again at the Scale Space Conference (September 1999). Dr. John Russ is a Professor of Materials Science and Engineering at North Carolina State University and author of the Image Processing Handbook. Dr. Chris Russ, Reindeer Games Software, is the author of IPToolKit, a collection of plug-ins for image processing that run under Adobe PhotoShop. Dr. John Russ and Dr. Chris Russ ran a basic image processing tutorial in which Dr. Kerckhove participated during May of 1997. Dr. C. Russ has been a valuable contact in conjunction with the work that Ms. Lee-Ann Smith has been doing on skeletonization algorithms.
Dr. Kerckhove plans to attend a NATO-sponsored Summer School on Advanced Topics in Scale-Space Theory in July of 2000.

2. Activities and Findings:
What have you done? What have you learned?

A. Describe the major research and education activities of the project.

Mathematical analyses of shape
Dr. Kerckhove has learned a tremendous amount concerning both theory and practice of geometric methods of shape description and image analysis. Originally, working in this area was somewhat of a stretch for Dr. Kerckhove given that his previous work had focused on the mathematics of relativity and geometric optimization problems. Consequently, it took Dr. Kerckhove some time to develop his ideas into publication-quality papers, however Dr. Kerckhove believes that he will continue to contribute to this field during the coming years.
The two publications that Dr. Kerckhove recently produced represent contributions to the problem of shape description from the perspective of differential geometry. In the first paper, "A Shape Metric for Blum Ribbons", Dr. Kerckhove was able to transfer a bi-invariant metric on the group of circle-preserving transformations to a metric on shapes with boundaries presented as envelopes of one-parameter families of circles. This gives a way to quantify shape difference, at least for the component of shape that depends on the radius of the inscribed circles (it is harder to account for the component of shape that depends on the curvature of the locus of centers of these circles).
In the paper entitled "Computation of Ridges via Pullback Metrics from Scale Space," Dr. Kerckhove was able to make an improvement to existing algorithms for ridge extraction by positing a hyperbolic metric on scale space, then using the induced metric on the surface consisting of points where the measurement of medialness has a local maximum in scale to extract the ridge. Again, familiarity with the computational techniques of differential geometry was key.

Heterochronies and life histories in amphibian skeletal muscle development
Heterochronies may provide phylogenetic information or they may reflect historical constraints. Previous reports had shown differences in myogenesis regarding the relative stage at which muscle first expresses muscle-specific proteins, first twitches, and first becomes multinucleated. However, these differences have not been studied systematically nor in a phylogenetic context. We have compared the sequence and timing of these indicators of skeletal muscle development with life history mode of several anurans.
We grouped species in three categories based on their reproductive modes. The non-direct developers-, i.e., Hymenochirus boettgeri, Rana sylvatica, R. utricularia, Xenopus laevis, and X. tropicalis; lay large clutches of eggs in lentic water. Embryos hatch into free-swimming tadpoles before developing into juvenile frogs. Direct developers, i.e., Eleutherodactylus coqui, lay arboreal eggs that lack the intermediate free-swimming tadpole stage. Instead, embryos are positioned on top of a yolk sac from which they obtain nourishment throughout development and hatch directly into froglets. "Intermediate developers", i.e., Agalychnis callidryas, have a reproductive mode intermediate from those previously described. Eggs are arboreal and intracapsular development is extended beyond the embryo stage into the early tadpole stages. Upon hatching, the tadpoles drop into the water below and become free-swimming larvae.
Historically, different tables of normal development have been used with different anurans making cross-taxa comparisons of development a challenge. For our study we initially used Nieuwkoop and Faber (1975) for staging the pipids X. laevis, X. tropicalis and H. boettgeri. We used Gosner (1960) for the ranids R. utricularis and R. sylvatica, and for A. callidryas. For the direct developer E. coqui, we used Townsend and Stewart (1985). We discuss the correlation between stages as it applies to muscle development in a paper in review in J. Herpetology (Smetanick et al., in review).

Chondrocranial Anatomy
Most studies of cranial morphology in anurans have focused on variation and characteristics of the ossified adult skull. Whereas characters derived from the study of adult osteology have played an important role in anuran systematics, few authors have studied characters of the larval chondrocranium in an evolutionary context. In part, this can be attributed to a lack of available comparative baseline data needed to understand character diversity and chondrocranial evolution (de Sá and Trueb, '91). For example, chondrocranial data for entire anurans families is missing, e.g., Pseudidae, and available data for other families is limited to single or few species. During the NSF-CRUI grant we wanted to contribute chondrocranial descriptions for previously unreported genera and families in order to increase the base line comparative data that can be use in higher order comparisons. Furthermore, we intended to address chondrocranial variation among closely related taxa. The neotropical frog genus Leptodactylus currently consists of approximately 61 recognized species, which are clustered into four species group--the fuscus Group, the ocellatus Group, the melanonotus Group, and the pentadactylus Group (Heyer, 1969). The current clustering of species of Leptodactylus into these four "species groups" points to the presence of morphological variation in the genus; at least enough variation to recognize "species groups" within the genus. Consequently, Leptodactylus is an ideal candidate to examine phylogenetic variation of chondrocranial morphology among closely related species and its possible correlation to currently recognized species groups. However, there was no complete description of the chondrocrania of any species of Leptodactylus. Brief references to individual chondrocranial structures are available for Leptodactylus chaquensis (Sokol, 1981), L. pentadactylus, and L. ocellatus (Haas, 1995). Analysis of chondrocranial variation within Leptodactylus may provide additional characters to support or refute the monophyly of the species groups; furthermore, it may help to resolve relationships among the species groups. Additionally, such analysis may help to determine whether chondrocranial anatomy provides useful diagnostic characters to differentiate closely related species.
In addition, characteristics of the tadpole chondrocranium have been used to argue a diphyletic origin of Anura. The rostral area of most living tadpoles (e.g., non-pipoid tadpoles) has the cornua trabeculae and suprarostral cartilages; whereas in pipoid tadpoles this area is occupied by a single, continuous, and cartilaginous-the suprarostral plate (Sokol, 1975; Trueb and Hanken, 1992). Previous developmental work showed that the rostral elements found in pipoid and non-pipoid larvae have a common origin in the cranial neural crest (Sadaghiani and Thiébaud, 1987; Olsson and Hanken, 1996; Reiss, 1997). However, Rocek and Vesely (1989) suggested that these "are not fully corresponding structures" (=not homologous). Trueb and Hanken (1992) argued that the suprarostral plate of pipoid larvae is formed by either the fusion and simplification of the cornua trabeculae of other Anura, or that in pipoids, the cornua trabeculae are missing and a continuous cartilaginous plate develops anteriorly from the planum internasale. However, no additional evidence was provided to support the homology of rostral structures in pipoid and non-pipoid larvae. In order to address this controversy we wanted to analyze the previously unreported chondrocranium of Rhinophrynus dorsalis and that of Hymenochirus boettgeri. These whole-mounted descriptions, together with cross serial sections of early developmental stages of the three genera (Rhinophrynus, Hymenochirus, and Xenopus) could provide new insights on the formation of the suprarostral plate in pipoid tadpoles and the homology of the rostral structures of pipoid and non-pipoid larvae.

B. Describe the major findings resulting from these activities.

Mathematical work
The principal task that occupied the mathematics group during the course of this project was the quantification of shape difference. There were two components to our work, one theoretical and one applied.
On the theoretical side, Dr. Kerckhove contributed two papers to the literature of shape representation. In the paper "A Shape Metric for Blum Ribbons", he developed a novel approach to the problem of quantifying shape difference that focuses on the group of circle-preserving transformations of the plane. Since Blum's description of shape (Blum, 1973) is based entirely on inscribed circles, the metric for shape that Dr. Kerckhove proposed brings the problem back to its foundations. Whereas, the paper entitled "Computation of Ridges via Pullback Metrics from Scale Space" addresses the problem of how to extract medial loci from general images in the context of Scale Space Theory. Building on work of Pizer, et. al. (1994, 1998), Eberly (1996), and Furst et al. (1997), Dr. Kerckhove work presumes that an object has been identified within an image and that its medial locus is the desired feature to be extracted. In the context of scale space theory, a property called medialness is defined and quantified by means of convolution integrals involving certain geometric kernels. A point lies on the medial locus of the object if the value of medialness at the point is high in relation to medialness values at nearby points. The innovation that Dr. Kerckhove proposed was to induce a metric on the graph of the medialness function and to compare local values of medialness by means of this metric. The paper was presented at the 2nd International Scale Space Conference in September of 1999.
On the applied side, we had proposed to make a 3-dimensional description of the shapes of various chondrocranial elements of the tadpole skulls and then to compare these 3-D shapes across species and across time as the tadpoles developed. The main problem we encountered involved registration of serial sections after tadpole slices had been obtained. Despite our best efforts, we could not produce sufficiently smooth and precise boundaries for the reconstructed cartilages. The methodological problems are described in a paper that is currently in press (Radice et al, 1999). Consequently, we re-directed our attention to the largest of the cartilages to be considered, the muscular process. This cartilage is most prominent when viewed laterally, so we constructed 2-D lateral views from the serial sections, using NIH-Image to perform rotations on stacked 2-D tadpole slices. Boundary jaggedness remained very high, and subjecting the 2-D image to morphological smoothing operations (combinations of erosions and dilations) produced images that could not be identified as muscular processes. This opened our investigation into medialness measures based on convolution integrals (Morse et al, 1998); that is integrals whose effect is to smooth the boundary for the purposes of computing a medial locus, without distorting the raw boundary data. These investigations led to the paper described above (Kerckhove, 1999). Results obtained by applying these processing techniques to our images (as opposed to the attempts to modify tadpole processing techniques described above) did not significantly improve our impression of the reliability of the subsequent calculations we performed in order to measure shape differences. As a result, we abandoned the idea of working with serial sections and developed a protocol for capturing lateral views of the muscular process from stained, whole-mounted, specimens. This cartilage was chosen because of its relatively large size and because it is not obscured by other parts of the tadpole's skull. A catalog of roughly 30 such lateral views was produced. Students outlined the muscular process within each of these lateral views and used skeletonization and distance map utilities within Adobe PhotoShop in conjunction with Mathematica to compute values of several shape indices for each muscular process. These shape indices included the isoperimetric ratio, the elongation index, and curl (Russ, 1995). Additionally, pairwise comparison of shapes using the metric described in Kerckhove (1999) was performed. Results were disappointing --- we could not identify any combination of shape measures that would distinguish cartilages belonging to one family from those of another family of frogs. We conclude that the shape differences traditional describe in the literature in the examinations of actual 3-D muscular processes are more subtle than what can be captured on and quantified from a 2-D projection. Further progress in quantifying those perceived differences via the procedures we developed will be possible only when the problem of registration for serial sections has a more satisfactory solution.

Myogenesis
Overall, with the exception of Hymenochirus, the non-direct developing taxa have an earlier expression of muscle protein and muscle function than species representing other reproductive modes. The myogenic pattern of Agalychnis is delayed in its entirety relative to Rana, but it exhibits muscle protein expression, muscle function, and multinucleation earlier than in Eleutherodactylus. Initially, we ranked Agalychnis as an intermediate developer, based on its extended intracapsular development. Our analysis showed that the myogenic pattern of Agalychnis is intermediate between that of non-direct and direct developing anurans. Myogenesis in Agalychnis occurs faster than in Eleutherodactylus, but it is generally slower relative to Rana.
Our work suggests that myogenic events seem to vary with reproductive modes. Furthermore, it suggests a progressive delay of myogenesis associated with a correlated delay in hatching, i.e., extended intracapsular development. If this is correct, we would predict that myogenic events in other anuran taxa such as Dendrobates and Centrolenids would resemble the pattern described for Agalychnis whereas myogenic events in Cophixalus, Ceratobratrachus, and some Gastrotheca, (all direct developers) would resemble that of Eleutherodactylus.
Although life history may correlate with patterns of myogenesis, some myogenic events may be better understood in the light of evolutionary relationships. For example, among the anurans with free swimming larvae, the pipid taxa studied showed delayed multinucleation of axial muscle, further delayed than multinucleation in Eleutherodactylus. However, within pipids Hymenochirus differ from X. laevis and X. tropicalis in the delay detection of muscle protein expression and first twitch. The departure of Hymenochirus from the pattern of myogenesis found in other pipids is not surprising since this taxa has been shown to differ in the characteristics and development of other musculoskeletal structures (de Sá and Swart, 1999).

Patterns of somite cell rotation in pipids
During amphibian somitogenesis presumptive myotomal cells change shape from rounded and randomly oriented in the unsegmented dorsal mesoderm, then become elongated and aligned parallel to the notochord. The final orientation of myotomal cells is always axial but the movements that achieve this final arrangement can differ greatly between species. There is as yet no explanation for the existence of so many diverse mechanisms to achieve apparently identical results. It is possible that each pattern provides different functional advantages. Alternatively, the different patterns could represent historical constraints within specific lineages, but to date only single species within a family have been studied-comparative studies within lineages had not been done. In work performed under this grant we compared the pattern previously seen in Xenopus laevis with two additional members of the Pipidae, Xenopus tropicalis and Hymenochirus boettgeri (Fan et al., in review).
Observation of somitic cellular rearrangement patterns were made on sections of fixed embryos embedded in wax or glycolmethacrylate plastic. Observation of X. tropicalis and H. boettgeri specimens revealed the same cellular movement as those seen in X. laevis. Posterior unsegmented mesoderm cells lie perpendicular to the notochord, begin rotation at segmentation, and complete rotation by the time that two more somites have segmented. In all three species the entire rotation occurs in about an hour at room temperature (Nieuwkoop and Faber, 1975, and our observations).
This unique process of myotome rearrangement may represent a synapomorphy for Pipidae. It has not been found in other species of anurans or urodeles studied thus far, including Pelobates fuscus (Kielbowna, 1981), a member of the Pelobatidae, a taxon that is relatively closely related to pipids (Ford and Cannatella, 1993).
It is possible that different patterns of somite rearrangement are required for mechanical reasons. Perhaps, for example, different patterns are more efficient for cells of different sizes. We observed that X. laevis myotome cells are nearly twice as long as those of both X. tropicalis and H. boettgeri, yet all three species showed the same orientation pattern of somite formation. Therefore, at least within the size range present in these species, cell size does not seem to affect the mechanics of rotation.
Furthermore, X. laevis and H. boettgeri have similar patterns of somitogenesis despite a distinct earlier difference in the mechanics of mesoderm formation (Minsuk and Keller, 1996). Once the dorsal mesoderm has formed, however, the pattern of somitogenesis (the present study) is nearly identical, and the sequence of later myogenic patterns is also similar though they vary somewhat in timing (Smetanick et al., 1999).
The common pattern of rotation rather than direct elongation may be related to different rates of development. Previous studies have shown that X. laevis exhibits early myogenesis. Xenopus. tropicalis and H. boettgeri also develop relatively rapidly compared with other anurans (our unpublished observations) and as well have comparatively rapid myogenesis (Smetanick et al., in review). As yet, however, the relationship between developmental rate, timing of myogenesis, and myotomal cell rotation is simply a correlation, not an explanation.
If this pattern of rotation is indeed synapomorphic for the pipidae then it should also occur in the genus Pipa, a prediction that can be confirmed when embryos of the appropriate stages are obtained. The genus Pipa will be particularly interesting to examine because there are both free swimming larvae (P. carvalhoi) and direct developing (P. pipa) members of the genus. This could allow one to determine whether somitogenesis patterns are related to life history rather than historical events.

Chondrocranial anatomy
Chondrocranial morphology (i.e., the tadpole's skull) is known only for a small percentage of living frog species. During the NSF-CRUI, we contributed several papers on chondrocranial anatomy. This work provided the opportunity to train undergraduate students in evolutionary morphology, developmental biology, and phylogenetics. At the same time, undergraduates were trained on diafanization methodologies as well as scientific illustration.
The manuscript by de Sá and Hill (1998) contributes to our knowledge of chondrocranial morphology among poison-dart frogs (family Dendrobatidae). This paper identified three chondrocranial characters as unique synapomorphies for the genus Dendrobates.
While working on the analysis of the chondrocranial anatomy of the South American frog Pseudis minuta (Lavilla and de Sá, 1999), we made interested observations that lead us to a second manuscript on this taxa. This second manuscript (de Sá and Lavilla, 1997) focused on the characteristics of the larval biology of Pseudis minuta and argued that the coloration pattern of P. minuta tadpoles and early P. paradoxa tadpoles have an ecological/defensive role that evolved under natural selection. Furthermore, we suggested that a heterochronic mechanism, either acceleration or hypermorphosis, played a role in the speciation resulting in two living species, P. minuta and P. paradoxa. Dr. de Sá is continuing this work at the endocrinological level (see below, 3.C. Other anticipated contributions).
A similar situation developed in our work on the chondrocranial anatomy of pipoid frogs. We initially were interested in understanding the previously unreported chondrocranial morphology of Rhinophyrnus dorsalis. We considered this species critical since it represent a basal species and the sister taxa to all other living Pipidae (e.g., Xenopus, Hymenochirus, Pipa). Our analyses of whole-mounted as well as cross sections provided interesting observations about the suprarostral plate of R. dorsalis (Swart and de Sá, 1999). The presence of the suprarostral plate in pipoids has been the source of a long-standing controversy about the monophyly of the Anura. A suprarostral plate is characteristic of the chondrocranium of pipoids. Rocek and Vesely (1989) argued that the suprarostral plate of pipoids and the cornua trabeculae of non-pipoid larvae are not homologous, i. e., they cannot be derived from one another. Subsequently, Trueb and Hanken (1992), while describing the suprarostral plate of Xenopus laevis, suggested that the suprarostral plate of pipoids and the cornua trabeculae of non-pipoid frogs are homologous, but provided no evidence to support their claim. During the CRUI we first asked: Do the cornua trabeculae of non-pipoid anurans appear in pipoids? Our study showed that in early stages of Xenopus, Hymenochirus, and Rhinophrynus, rod-like anterior projections of the ethmoid plate are present (de Sá and Swart, 1999). Although they are small and transitory, we consider these structures to be homologous (i.e., in position and origin) with the cornua trabeculae of non-pipoid larvae. Then we asked: Can the suprarostral plate of pipoid larvae be derived from the cartilaginous structures found in the rostral area of non-pipoid larvae? The data we showed that this is actually the case (de Sá and Swart, 1999). Our serial cross-sections showed that during development, the intertrabecular space is occluded with cartilage forming a continuous plate between the cornua trabeculae, the suprarostrals, and the anterior process of the ethmoid plate. Along the way we also reported the unique characteristics of the chondrocranium of Hymenochirus (de Sá and Swart, 1999).
A few studies have used chondrocranial characters in a phylogenetic context (Sokol '77, '81; de Sá and Trueb, '91; Haas, '95, '96, '97); however, those papers compared chondrocrania among distantly related taxa. No study had attempted a comparative morphological analysis of a large number of congeneric species. Understanding the variation of chondrocranial characters among closely related species represents the other end of the phylogenetic spectrum. Furthermore, it is important if we are going to use chondrocranial characters in phylogenetic studies. The CRUI provided the opportunity to do exactly that resulting in two different papers. The first one compared chondrocranial anatomy in two sister species of the frog genus Hyla (d'Heursel and de Sá, 1999), while the second paper focused on the genus Leptodactylus and it represents the first to examine phylogenetic variation in the chondrocrania of 22 closely related species (Larson and de Sá, 1998).
The work accomplished on chondrocranial anatomy under the NSF-CRUI has implications at different levels. It showed the utility of chondrocranial characters in phylogenetic analyses; however, it also pointed to two aspects that need to be considered carefully while using chondrocranial characters for phylogenetic reconstructions. First, it demonstrated that a large amount of variation is present in the same chondrocranial characters when several congeneric species are considered; that is, it is not appropriate to use a single species as representative of a genus, or worse of a whole family, as has been traditionally done. Second, the NSF-CRUI work also suggested that some elements of the chondrocranial anatomy are highly homoplasious, probably as a result of larval adaptations (caenogenesis). Consequently, chondrocranial characters should be used as part of "total evidence" analyses instead of an independent data set.
The analysis of chondrocranial variation in closely related taxa was also applied to other groups such as Xenopus and Rana, preliminary results were presented at national meetings (Swart et al., 1999; Larson and Swart, 1999) and these students are continuing with this work.

3 dimensional (3D) reconstruction of chondrocrania from serial sections
Tadpole chondrocrania may be useful characters for phylogenetic studies. Comparisons of such complex 3D structures between species would be aided by reliable methods for constructing high-resolution 3D images made from histological sections. We sought to develop a system for generating 3D reconstructions (3DR) of histological serial sections using relatively simple, low cost, and widely available hardware and software (Radice, et al., 1999).
Fixed tadpoles from various species were embedded in wax. Specimen blocks were sectioned at 10 µm transverse to the body axis, beginning at the anterior tip of the head. Sections were stained with Milligan's trichrome stain. To measure distortion introduced by histological technique, chondrocranial dimensions were measured both before embedding and after sectioning and mounting. In some cases the fidelity of our reconstructions were assessed by comparing with whole chondrocrania in cleared and double-stained specimens (Dingerkus and Uhler, 1977).
Images were captured using a stereomicroscope with phototube and 1x TV relay eyepiece connected to a RGB digital cameras and Scion framegrabber cards in Apple Macintosh PowerPC computers. We used public domain image capture and analysis software NIH Image, v1.59 or 1.62b (available on the Internet at http://rsb.info.nih.gov/nih-image) and a modified version of NIH Image called Object Image v.1.62b (http://simon.bio.uva.nl/object-image.html) in combination with Rotater 3.5 (available by anonymous FTP from ftp://raru.adelaide.edu.au/rotater/rotater-3.5.ctp.hqx). We also used SURFdriver" to create create reconstructions with more life-like surface textures (a demonstration version is available from www.surfdriver.com) Most reconstructions were done using Object Image or SURFdriver".
The simplest reconstruction was that performed by NIH Image, which simply combines the individual image files into a stack and renders the completed stack of outlines as seen in a series of views from different angles (a projection). We obtained the best projections by starting with outlines (wireframes) of chondrocranial elements. Outlines were made using either a mouse to trace the screen image or a digitizing tablet and pen to trace a camera lucida projection of the image. Object Image allows one to separately color code various features in our outlined stacks, and then export those data to the program Rotater 3.5. Rotater permits mouse-controlled rotation in any axis, constructs stereo pairs, adjusts depth cues and perspectives, and several other features that aid visualization of wireframe objects.
SURFdriver" creates surfaces by interpolating a set of points along each wireframe contour. It does this by generating a mesh of triangles among the coordinates of points on contours of adjacent sections and then covering the triangular mesh with a texture map, colored and shaded to enhance the illusion of depth. The most recent version of SURFdriver has a variety of texture maps that simulate muscle, bone, nerve and other tissues as they might look in gross anatomical specimens, adding to the realistic impression.
There were 5 problems that we addressed in our work: (1) section distortion. (2) section loss (3) image registration (4) feature segmentation (5) rendering the 3DR.
(1) We found that paraffin sectioning produced extensive distortion, mostly from section compression during sectioning. This compression was as much as 40%, even after floating the sections on water at 46 oC. A better method is the erosion procedure used in mineralogy, whereby a stereomicroscope and camera are mounted at the microtome to capture an image of the specimen within the block face prior to each section and therefore undistorted and in register (Lozanoff, 1992). This method can be applied to structures that can be visualized unstained or stained en bloc and viewed with epi-illumination, such as cartilage elements. It is not as good for softer tissues or those that cannot be easily stained en bloc. For this reason we used conventional serial sectioning for most of our work.
(2) Section loss was minimal. Missing sections were accommodated by incorporating a blank placeholder image in the series, by duplicating the image of the section prior to the missing one, or by adding an image that was an average of the sections before and after the missing slice.
(3) Image registration was the most difficult problem to overcome. We chose to use the dorsal and ventral midpoints of the CNS as internal registration markers and software to assist rotating and aligning successive images with respect to these two points. This method required careful attention by the user and was not completely reliable since aligned reconstructions still showed some misplaced sections, as judged by the smoothness of the reconstructed surface. For tissues without internal symmetry, it is necessary to co-embed an external fiducial marker. The ideal marker has a uniform cross-sectional dimension much smaller than the object of interest, is straight and does not change position from one section to the next, is oriented parallel to the direction of sectioning, and is pigmented or stainable. Having two or more such markers in each section increases their reliability. Given a stable set of fiducial marks, section rotation, lateral translocation, and even compression distortion can be compensated. We tried a number of recommended fiducial markers including strips of liver, silk thread, boar hair from a paint brush, broom straw, and needles from the white pine, Pinus alba. In practice, none of these markers was completely satisfactory.
(4) Image segmentation, e.g., recognizing cartilage tissue in each section, proved relatively easy. Cartilage tissue structure is distinctive and among the easiest to recognize in histological section, even by novices and even in black and white.
(5) Image rendering was handled differently by the two software programs. Both programs provide useful 3D information but render it in different ways. NIH Image creates 3DR using a projection routine that renders the object by drawing the view that would be seen if parallel rays of light passed through the object and draws a an image on a screen of the pixel values intercepted along the way. Then the software rotates the stack incrementally around an axis, and draws another view. Such reconstructions are simple to prepare and look best when shown in animation rather than in static images in which it can be difficult to differentiate those lines that are in the foreground from those in the back. The additional visualization abilities of Object Image and Rotater allow one to create stereo images that can be viewed with 3D glasses, or false coloring of selected outlines to help distinguish connected regions.
Because SURFdriver" can add a textured surface that covers the wireframe view, its reconstructions can appear more life-like than those of NIH Image. The increased realism comes at the cost of much more tedious preliminary data entry, since the user must not only outline each contour but also identify and keep track of which contours belong to distinct objects and which are connected by branch points. This may require users to have a more sophisticated knowledge of the tissue.
Large reconstructions require large amounts of memory and rapid processors. Although we were able to create 3DR with low-end PowerPC CPUs with processors running at 100-120 MHz and 64 MB RAM, such a configuration is barely adequate to create a reconstruction of 200 slices, which can occupy many tens of megabytes. For example, a 100 MHz processor rendered a 17 megabytes NIH Image projection in about 20 minutes, but this same operation took less than three minutes on a computer with 333 MHz G3 processor and graphics accelerator. Fortunately as processor speeds continue to rise and memory prices to decline, it will become easier and cheaper to create larger 3DR faster on standard desktop machines.

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de Sá, R.O., and C.C. Swart. 1999. Development of the suprarostral plate of pipoid frogs. J. Morphol. 240:143-153.
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C. Relevance of the Research Accomplishments

1. Scale Space Theory holds great promise for the modeling of low-level tasks in human vision; tasks such as the segmenting of images into objects and the detection of shape features such as corners within images. The paper "Computation of Ridges via Pullback Metrics from Scale Space" addresses the problem of how to extract medial loci from general images in the context of Scale Space Theory
2. Comparison of myogenesis interpreted from phyologenetic/life history perspective. Previous work had not considered enough different species to detect consistent patterns. We now have a clearer context in which to interpret the evolution of myogenic programs, at least in anurans.
3. Similarly, demonstration of a common pattern of myotomogenesis in pipids shows that X.. laevis is not unique among anurans. This is the best indication (to our knowledge) that a feature of myogenesis might be useful as a phylogenetic character. Also, the observation that earlier steps of muscle specification can vary while the end product is virtually identical between species further demonstrates that early stages may have their own distinct selection pressures, and that earlier stages of development are necessarily more similar than later stages of development.
4. The main problems in obtaining useful 3DR are not related to computing hardware or software, which are quite good and can produce useful images, but in basic histology. Section compression, warping, loss, and registration are all significant problems that are the main obstacles to creating useful 3DR. Using serial erosion rather than serial section is most useful for cartilage elements. More effort should be expended on designing staining methods for intact tissues to make the erosion method more generally useful.
5. The study of heterochrony in Pseudis has the potential to become a classic study on heterochrony. A genus consisting of only two species represents the ideal model to study developmental shifts during development and its role in speciation.
6. Our data on the development of the suprarostral plate showed that: first, the rostral region of pipoids and non-pipoids are homologous, refuting Rocek and Vesely's ('89) argument for a diphyletic origin of Anura. Secondly, the cornua trabeculae do form in pipoid larvae. Furthermore, the suprarostral plate develops not from a single fusion and simplification of the cornua trabeculae, as suggested by Trueb and Hanken (1992), but from a complex fusion of the cornua trabeculae, anterior process of the ethmoid plate, and suprarostrals, complemented by the appearance of new cartilage between these elements.
7. The analysis of interspecific variation of chondrocranial characteristics is at the leading edge of evolutionary morphological research. It has been recently quoted as: ".important interspecies comparisons are being analyzed phylogenetically both among closely related species (e.g. Haas, 1993, Larson and de Sá 1998) and at higher levels" (Chapter 4, Tadpoles: The biology of Anuran Larvae, Ed. R. McDiarmid and R. Altig, Chicago Press, 1999).

D. Describe the opportunities for training and development provided by your project

A major component of this grant was the training of undergraduates, particularly extending their experience with mathematics and biology beyond their discipline and beyond the classroom setting.
The faculty involved worked hard, especially with the summer students, to have them figure out the problems related to the situation they were trying to explore as opposed to just applying mathematics or biological techniques.
Some students made good progress in their ability to use their knowledge in a new setting, whereas other students were less inquisitive and got less out of their summer experiences.
In terms of faculty development, we feel that we have become better at recognizing the differences among student's abilities quickly and we are better at assigning appropriate short-term projects to students based on their interests and abilities.
The project provided opportunities for students to learn basic vertebrate histology and also modern microscopy using digital imaging. Digital imaging includes image capture from digital cameras, scanners, and drawing boards, image processing and enhancement, 3-dimensional reconstruction, and digital image archiving.
Students were trained in the use of microscopes and image capture equipment; in the use of NIH-Image, Adobe PhotoShop, and IPToolKit software, and in the use of Mathematica.
Of particular importance from the mathematics end of the project was the conceptual and empirical progress made by students in writing Mathematica programs. For example, by the summer of 1999, a student was able to incorporate most of the code that had been produced into the format of a Mathematica Package, which made running the code more transparent to the average user. From the point of view of training, it is comparatively rare for an undergraduate math major to be writing Mathematica Packages.
The two graduate students that were supported with the CRUI completed their MS degrees. One of them, Will Turner, is currently teaching biology at the secondary education level. The other Master student, Chris Swart, has initiated his Ph.D. work in biology. Chris intends to apply the evolutionary morphology training of his MS to ecological questions. Most of the students that worked on the CRUI are now pursuing professional or graduate school. One particular student, Peter Larson, has completed his first year of a Ph.D. program. Peter has chosen to expand what he learned during the CRUI and for his doctoral dissertation Peter is working on the application of geometric morphometrics in the analysis of sequences of cranial development in frogs.
Related to the development of student communication skills, most students benefited tremendously from the exercise of presenting their work at meetings. Whether they presented posters or gave talks, the students made progress in figuring out how to convey the major results of their work without either assuming too much knowledge on the part of their audience or becoming bogged down in too much detail. Furthermore, students gained confidence and became more self-assured about their capabilities to do research, their work, and their organization and presentations skills. These skills are very important in scientific communication.

E. What other educational and outreach activities have you undertaken?

Based on our experience with the NSF-CRUI, Dr. Kerckhove will be presenting a seminar on "Undergraduate Research in Mathematics and Computer Science at the University of Richmond" in January, 2000. The main purpose of this contributed paper session is to foster discussion about engagement of undergraduates in scientific research. This presentation will be given as part of the AMS/MAA joint meeting.
Dr. Radice presented some of the techniques and methodologies used in the CRUI project as well as part of our results at a University of Richmond campus wide workshop on "Using new Technologies in the Classroom" in May, 1999.
Dr. Radice now includes 3D reconstruction projects in his upper division course on microanatomy, using the techniques that were developed during this project. This benefits an additional 16 students per year.
In 1997, Dr. Kerckhove began to develop a new general education course under the symbolic reasoning field of study. The course is Math 104: Symmetry in Tilings and Patterns, and it begins with the study of bilateral symmetry. While the equations governing the medial axis transform would be beyond the mathematical capabilities of most students in this course, the idea of local symmetry and the Blum description of shape should be accessible to them as should the content of Leyton's symmetry-curvature duality theorem. Both the medial axis transform and the symmetry-curvature duality theorem were basic to the shape description paradigm that was used throughout the NSF-CRUI grant. Through this theorem, Math 104 students will see some principles of the gestalt school of the psychology of perception expressed in terms of a mathematical theorem. The result of this exercise will be in complete agreement with the description of the symbolic reasoning requirement in that students will have first-hand experience with the formal, symbolic nature of mathematics and with its wide applicability to other fields of inquiry. This course is scheduled to be offered in Spring 2002.
At the mid-point in the duration of the grant, we hosted a workshop at the University of Richmond. This workshop was title "3D reconstruction and quantitation", the workshop was held at the Science Building, University of Richmond, on September 13, 1997, and it was open to the University community. The goal of the workshop was to provide a forum to discuss our work with selected guests from off campus.

Workshop speakers and schedule was:
8:30 am Continental Breakfast in the Science Center E-107.
9:00-10:00 An Overview of the CRUI Project Rafael de Sá (UR, Biol)
Chondrocranial Anatomy. Rafael de Sá (UR)
10:00-11:00 The chondrocranial anatomy of a primitive frog: Rhinophrynus dorsalis (Anura: Rhinophrynidae). Charles Swart (UR, Biol)
11:00-11:30 Some Technical Issues in Preparing Serial Sections Gary Radice (UR, Biol)
11:30-12:00 Heterochronies in Muscle Development. Matt Smetanick (UR, Biol)
12:00-1:00 Lunch Break
1:00-2:00 pm Describing the Muscular Process of the Palatoquadrate. Mike Kerckhove (UR, Math)
2:00-3:00 Figural Representation of Shape in Image Analysis & Graphics Stephen Pizer (Keenan Professor of Computer Science, University of North Carolina, Chapel Hill)
3:00-4:00 Imaging Structures in the Brain Princy Susan Quadros (UR, Psych)
4:00-5:00 Seven Years of Teaching with Digital Microscopy
Robert Blystone (Professor of Biology, Trinity University, San
Antonio TX)
5:00-8:00 Reception at the Deanery.

3. Publications, Presentations, and Products.

Summary of output: 16 publications, 24 intramural presentations, 30 presentations at National and International meetings.

A. What have you published as a result of this work?
* indicates undergraduate student
_ indicates graduate student

1. de Sá, R. O. and E. O. Lavilla. 1996. Caracteristicas de la osificación craneal en Phyllomedusa boliviana. (Anura: Hylidae). Cuadernos de Herpetologia 9(2):69-73
2. de Sá, R. O. and E. O. Lavilla. 1997. An apparent case of heterochrony, the tadpole of Pseudis minuta (Anura: Pseudidae). Amphibia-Reptilia 18:229-240
3. de Sá, R. O. and S. Hill*. 1998. Chondrocranial anatomy and skeletogenesis in Dendrobates auratus (Anura: Dendrobatidae). Journal of Herpetology 32(2):205-210
4. Larson, P*. and R. O. de Sá. 1998. Chondrocranial morphology of Leptodactylus larvae (Leptodactylidae: Leptodactylinae): its utility in phylogenetic reconstruction. Journal of Morphology 238:287-305
5. Swart, C. C_. and R. O. de Sá. 1999. The chondrocranium of the Mexican Burrowing Toad Rhinophrynus dorsalis (Anura: Rhinophrynidae). Journal of Herpetology 33(1):23-28
6. Lavilla, E. O. and R. O. de Sá. 1999. Estructura del condrocraneo y esqueleto visceral de larvas de Pseudis minuta (Anura: Pseudidae). Alytes,16 (3-4): 139-147.
7. Smetanick*, M. T. R. O. de Sá, and G. P. Radice. 1999. The timing and pattern of myogenesis in Hymenochirus boettgeri. Journal of Herpetology 33(2):33-334.
8. de Sá, R. O. and C. Swart_. 1999. Development of the suprarostral plate of pipoid frogs. Journal of Morphology 240:143-153.
9. d'Heursel_, A. and R. O. de Sá. 1999. Comparing the tadpoles of Hyla geographica and Hyla semilineata . Journal of Herpetology 33(3):353-361
10. Kerckhove, M. 1999a. Computation of Ridges via Pullback Metrics from Scale Space, Scale-Space Theories in Computer Vision, Lecture Notes in Computer Science Volume 1682, M. Nielsen, P. Johansen, O. Fogh Olsen, J. Weickert (Eds.).
11. Kerckhove, M. 1999b. A Shape Metric for Blum Ribbons, Journal of Mathematical Imaging and Vision 11( 2):
12. Boggiano*, M.K., M. DeSantis*, and M. Kerckhove. In press. The Set of Hemispheres containing a Closed Curve on the Sphere. Journal of Undergraduate Mathematics.
13. Radice, G.P., M.K. Boggiano*, M. DeSantis*, P. Larson*, P. Oppong*, M. Smetanick*, T. Stevens*, J. Tripp*, R. Weber*, M. Kerckhove, and R.O. de Sá. In Press. Three-dimensional reconstructions of tadpole chondrocrania from histological sections. Journal of the Virginia Academy of Sciences.
14. d'Heursel_, A. and R. O. de Sá. 1999. Cranial and postcranial ossification sequence in Hyla geographican and Hyla semilineata (Anura: Hylidae). Cuadernos de Herpetologia. Accepted.
15. Smetanick*, M., R.O. de Sá, and G.P. Radice. 1999. Does myogenesis correlate with life history modes in anurans? Submitted, In review. (Journal of Herpetology).
16. Fan*, S.-Y., R.O. de Sá, and G.P. Radice. 1999. A common pattern of somite cell rotation in three species of pipids. Submitted, In review. (Journal of Herpetology).

B. What have you presented as a result of this work?
* indicates undergraduate student
_ indicates graduate student

Presentations at UR.
1. Weingard*, R. and M. DeSantis*. 1996. Our Summer Research Experience. Math and CS Dept. Colloquium, University of Richmond.
2. Hill*, S. and R.O. de Sá. 1996. The skull anatomy and skeletogenesis of a poison arrow frog (Dendrobates auratus). Poster presentation at Undergraduate Research Symposium, University of Richmond.
3. Oppong*, J. 1998 Quantification of Shape Difference using SL(2,R). Math and CS Dept. Colloquium, University of Richmond.
4. DeSantis*, M. and M. Kerckhove. 1997. Descriptors of Reconstructed Cartilage Elements. 3D Quantitation Workshop, University of Richmond.
5. de Sá, R. An Overview of the CRUI Project and chondrocranial anatomy 1997. 3D Quantitation Workshop, University of Richmond
6. Swart, C_. 1997. The chondrocranial anatomy of a primitive frog: Rhinophrynus dorsalis (Anura: Rhinophrynidae). 3D Quantitation Workshop, University of Richmond
7. Radice, G. 1997. Some Technical Issues in Preparing Serial Sections. 3D Quantitation Workshop, University of Richmond
8. Smetanick*, M. 1997. Heterochronies in Muscle Development. 3D Quantitation Workshop, University of Richmond.
9. Kerckhove, M. 1997. Describing the Muscular Process of the Palatoquadrate. 3D Quantitation Workshop, University of Richmond
10. Wilburn*, R., M. DeSantis*, and M. Kerckhove. 1997. Math and the Tadpole Chrondrocranium. Poster at Undergraduate Research Symposium, University of Richmond.
11. Boggiano*, M.K. and M. DeSantis*. 1997. Our Summer Vacation: a long drive on a sphere. Math and CS Dept Colloquium, University of Richmond.
12. Schwartz*, G., T. Stevens*, R. O. de Sá. 1997. Chondrocranial anatomy, skeletogenesis, and internal oral morphology of three species of Ptychohyla (Amphibia: Anura: Hylidae). Poster presentation at Undergraduate Research Symposium, University of Richmond.
13. Weber*, R. and R. O. de Sá. 1997. Diagnostic characteristics of Hypopachus tadpoles (Anura: Hylidae). Poster presentation at Undergraduate Research Symposium, University of Richmond.
14. Larson*, P. and R. O. de Sá. 1997. Chondrocranial morphology and skeletogenesis in the genus Leptodactylus (Amphibia: Anura: Hylidae). Oral Presentation at Undergraduate Research Symposium, University of Richmond.
15. Smetanick*, M. and G. Radice. 1997. Patterns of Musculoskeletal Development in Direct and Indirect Developing Anurans. Oral Presentation at Undergraduate Research Symposium, University of Richmond.
16. Tripp*, J. 1998. Stable Medialness Measures in the Presence of Boundary Noise. Poster at Undergraduate Research Symposium, University of Richmond.
17. Tripp, J. 1998. Height Ridges and Medial Loci for Image Analysis. Math and CS Dept. Colloquium, University of Richmond.
18. Tripp*, J. Height Ridges and Medial Loci for Image Analysis. Math and Computer Science Departmental Colloquium, University of Richmond. September, 1998.
19. Oppong*, J. M. Jr.. Quantification of Shape Difference using SL(2,R). Math and Computer Science Departmental Colloquium, University of Richmond. September, 1998.
20. Smetanick*, M. 1998. Patterns of Musculoskeletal Development in Direct and Indirect Developing Anurans. University of Richmond 13th Annual Undergraduate Research Symposium.
21. Spear*, S. and R.O. de Sá. 1999. The chondrocranial anatomy of Hyla chrysocelis. Poster presentation at Undergraduate Research Symposium, University of Richmond.
22. Pingelski*, E. and L. Smith. 1999. Comparison of Muscular Processes for Tadpoles within the Family Hylidae. Poster presentation at Undergraduate Research Symposium, University of Richmond.
23. Smith*, L. 1999. Comparison of Skeletonization and Distance Map Algorithms in NIH-Image and IP Toolkit. Math and CS Dept Colloquium, University of Richmond.
24. Tripp*, J. 1999. The Perona-Malik Model in Computer Vision. Math and CS Dept. Colloquium, University of Richmond.

Presentations at Professional Meetings.
1. Weingard*, R. 1996. Shape; the unsolved mystery. PME Student Presentation, MAA MathFest, Seattle, WA
2. Hill, S*. and R. O. de Sá. 1996. Chondrocranial anatomy and ossification sequence of Dendrobates auratus (Anura: Dendrobatidae). Poster Presentation 39th Annual Meeting Society for the Study of Amphibians and Reptiles, July 1996.
3. de Sá, R.O. and E. O. Lavilla. 1996. Descripción de la larva de Pseudis minuta (Anura: Pseudidae) y de su morfología oral interna. IV Latinamerican Congress of Herpetology, Santiago de Chile, Chile, October 1996
4. Arrieta*, D. and R. O. de Sá. 1996. Características condrocraneales de bufonidos del género Melanophryniscus (Anura: Bufonidae). IV Jornadas de Zoología del Uruguay. Montevideo, September 1996
5. de Sá, R. and E. Hines*. 1997. Anatomical characteristics of Phyllomedusinae tadpoles. Third World Congress of Herpetology. Abstract and Oral Presentation. August, 1997.
6. Larson*, P. and R. O. de Sá. Chondrocranial morphology and skeletogenesis in the genus Leptodactylus (Amphibia: Anura: Hylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
7. Swart_, C. and R. O. de Sá. The chondrocranium of Rhinophrynus dorsalis. Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
8. Schwartz*, G., T. Stevens*, R. O. de Sá. Chondrocranial anatomy, skeletogenesis, and internal oral morphology of three species of Ptychohyla (Amphibia: Anura: Hylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
9. Weber*, R. and R. O. de Sá. Diagnostic characteristics of Hypopachus tadpoles (Anura: Hylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
10. Swart_, C. and R. O. de Sá. Development of the suprarostral plate in pipoid frogs. Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Guelph, Canada. July 1998.
11. Riley*, T.D. and R. O. de Sá. Chondrocranial anatomy and skeletogenesis in two species of neotropical Hyla. Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
12. Hines*, E. and R. O. de Sá. Internal oral anatomy, a comparative analysis of the Phyllomedusinae (Anura: Hylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
13. Smetanick*,M. G. P. Radice, and R. O. de Sá. Patterns of musculoskeletal development in the direct and indirect developing anurans. Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Seattle Washington, June 1997.
14. Tripp*, J. 1998. Height Ridges and Medial Loci for Image Analysis. PME Student Presentation, MAA MathFest, Toronto, Canada.
15. Oppong*, J. 1998 Quantification of Shape Difference using SL(2,R). PME Student Presentation, MAA MathFest, Toronto, Canada.
16. Turner_, W. H. and R. O. de Sá. Chondrocranial morphology and skeletogenesis in three species of Physalaemus (Anura: Leptodactylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Guelph, Canada. July 1998.
17. Spear *, S. F. and R. O. de Sá. Chondrocranial and internal oral anatomy of Hyla chrysoselis (Anura: Hylidae). Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Guelph, Canada. July 1998.
18. Smetanick*, M. R. O. de Sá, and G. P. Radice. The timing and pattern of myogenesis in Hymenochirus boettgeri. Combined meetings American Society Ichthyologists and Herpetologist, Herpetological League, and Society for the Study of Amphibians and Reptiles. Guelph, Canada. July 1998.
19. de Sá, R. O. The monophyly of anurans in relation to the development of the tadpoles' rostral region. Fifth Symposium of the Herpetological Association of Africa, Stellembosch, South Africa, September 1998.
20. Tripp*, J. 1999. Pullback Metrics and Medial Loci. MAA Student Poster Session. San Antonio, TX
21. Swart_, C. R. O. de Sá, and A. Lathrop_. Internal oral anatomy of the larvae of seven species of megophryids (Anura: Megophryidae). Joint Meeting of the American Society of Ichthyologists and Herpetologists, American Elasmobranch Society, the Herpetologists' League, and the Society for the Study of Amphibians and Reptiles. State College, Penn State University, June 1999.
22. Smith*, L. 1999. Comparison of Skeletonization and Distance Map Algorithms in NIH-Image and IP Toolkit. MAA Student Presentation. MAA MathFest, Providence, RI.
23. Tripp*, J. 1999. The Perona-Malik Model in Computer Vision. PME Student Presentation. MAA MathFest, Providence, RI
24. Kerckhove, M. 1999. Use of Pullback Metrics in Core Extraction. Imaging Group Colloquium, University of North Carolina.
25. Kerckhove, M. 1999. Computation of Ridges via Pullback Metrics from Scale Space. Conference on Scale-Space Theories in Computer Vision, Corfu, Greece
26. Turner_, W.H. and R. O. de Sá. Larval chondrocranial morphology of Limnomedusa macroglossa (Anura: Leptodactylidae: Leptodactylinae). Joint Meeting of the American Society of Ichthyologists and Herpetologists, American Elasmobranch Society, the Herpetologists' League, and the Society for the Study of Amphibians and Reptiles. State College, Penn State University, June 1999.
27. Swart_, C., S. Spear*, and R. O. de Sá. Variation in larval chondrocranial morphology among three species of Xenopus. Joint Meeting of the American Society of Ichthyologists and Herpetologists, American Elasmobranch Society, the Herpetologists' League, and the Society for the Study of Amphibians and Reptiles. State College, Penn State University, June 1999.
28. Larson, P*. and C. Swart_. 1999. Chondrocranial morphology of Rana larvae (Anura: Ranidae): Insights into the phylogenetics relationships of New World Rana. Joint Meeting of the American Society of Ichthyologists and Herpetologists, American Elasmobranch Society, the Herpetologists' League, and the Society for the Study of Amphibians and Reptiles. State College, Penn State University, June 1999.
29. de Sá, R. O. and P. Larson*. 1999. Anatomy condrocraneal, phyllomedusines, y filogenia. V Latinamerican Congress of Herpetology, Montevideo, Uruguay, December 1999. Accepted for presentation.
30. Swart, C_. 1999. Evolucion de la morfologia condrocraneal de anuros pipoides. V Latinamerican Congress of Herpetology, Montevideo, Uruguay, December 1999. Accepted for presentation.

C. What Web site or other Internet site have you created?

We created a web site with the purpose of further disseminate the research done under the NSF-CRUI. This web site is for public access at: http://www.science.richmond.edu/~biology/rdesa.www/grant.html

D. Other anticipated contributions.

We anticipate that additional manuscripts and presentations will continue to be produced from work initiated under the NSF-CRUI. For example, Dr. Kerckhove's group is preparing a manuscript on the comparison of skeletonization algorithms, this manuscript will be submission to the Journal of Computer-Assisted Microscopy. In addition , as mentioned above, Dr. Kerckhove will be given a presentation at the upcoming Joint Meetings of the American Mathematical Society and the Mathematical Association of America, January 2000 , "Undergraduate Research in Math and CS at the University of Richmond." Also, Ms. Lee-Ann Smith will be presenting a poster entitled "Benchmarks for Skeletonization Algorithms" at the UR Undergraduate Research Symposium, Spring 2000.
An additional two presentations already schedule will be given by Dr. de Sá, Mr. C. Swart, and Mr. Peter Larson in the upcoming Latinamerican Congress of Herpetology in December 1999.
Dr. Radice and Dr. de Sá are actively trying to seek sources of live specimens of frogs of the genus Pipa to extend the myogenesis and somitogenesis analysis to this genus and to test the patterns predicted for Pipa based on the results obtained during the CRUI. In December 1999, Dr. de Sá is starting a experiment of manipulation of hormonal level to try to reconstruct the ancestral pattern of coloration in the genus Pseudis. This line of research could determine which of the two processes, either acceleration or hypermorphosis, played a role in Pseudis speciation.
Manuscripts already in preparation relate to chondrocranial morphology of Bufonid frogs (Lavilla and de Sá), Phyllomedusine frogs (de Sá, Larson, and Hines), and Melanophryniscus (de Sá, Arrieta, and Larson). We also anticipate a few additional presentations at National meetings based on the work initiated under the NSF-CRUI.

4. Contributions:
How has the project contributed?

A. To the development of your own discipline(s)?

The mathematical analyses of shape are significant contributions to newly developed field of Scale Space Theory
Myogenetic events and patterns of myogenesis have not been previously study in an evolutionary framework. Our work show that myogenesis is phylogenetically useful.
Analyses of chondrocranial variation showed the need for interspecific comparisons if chondrocranial characters are going to be used in phylogenetic analyses.
The data obtained from the study of the formation of the suprarostral plate in pipoid frogs are conclusive about the homology of the suprarostral plate to the cartilages observed in the rostral area of non-pipoid frogs. These results should end the long standing controversy regarding a possible diphyletic origin of Anurans.

B. To other disciplines of science or engineering?

The image analysis community consists of computer scientists, engineers, and mathematicians. As mentioned above, Dr. Kerckhove publications have contributed to the field by introducing and applying novel ideas from differential geometry. One of the reviewers of those papers wrote "We ought to encourage more differential geometers to work in this area."

C. To education and development of human resources?
Students assistants working on the project gained valuable experience in
how to become professional scientists. First, they learned to participate in a research team, on a project separate from that required by normal class work. They learned that they were responsible for completing certain tasks for the good of the project, not for themselves. Several of the students worked with us for more than a year, so these students also gained experience working on a project for an extended time. Both of these experiences were valuable introductions to working as professional scientists. Second, several of our students wrote and illustrated publications based on their work, and gave presentations about their work to local and national audiences. Again, this was excellent training, especially for those who want to go on in science or medicine, but also for those headed for other fields that require written and oral project reports.

D. To physical, institutional, and information resources for
science and technology?

Our work highlighted a need for more rapid computer processing power, larger memory storage, and a need for a way to archive digital images. Our 3 dimensional reconstructions were of relatively small size, a few tens of megabytes, and they still took several minutes to render on the fastest available desktop computers. For the techniques to become widespread and routine will probably require another 10 folds increase in computer processing speed. To reconstruct larger images will require larger storage capacity and faster methods of transmitting files between computers or from computers to backup devices. Archiving techniques are also a big concern. For example, the removable storage devices we used at the beginning of the project are now obsolete. We will need to transfer those images to other media for long term storage, but there is no guarantee that the next media will be useful 10, 20, or 50 years from now.