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Phylogeography of the Marine Toad Bufo marinus across the Eastern Extension of the Trans-Mexican Neovolcanic Belt


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Phylogeography of the Marine Toad Bufo marinus across the Eastern Extension of the Trans-Mexican Neovolcanic Belt
Benson Morrill

Department of Biology


Project Abstract
The Trans-Mexican Neovolcanic Belt (TMNB) of Mesoamerica has been shown to be a barrier that generally separates the Neotropical and Nearctic distributions of animal species (Perez-Higareda and Navarro, 1980). The Marine Toad (Bufo marinus) is among the few species that are exceptions to this generalization. Their natural distribution spans from the southern tip of Texas south through Central America and into the Amazon basin of South America (Conant and Collins, 1991). The purpose of this research project is to examine geographic patterns in the genomic diversity of this species---a phylogeographic study of genetic differentiation--- using populations sampled from either side of the biogeographic barrier of the TMNB. This study will be explicitly comparative as information derived from my study of B. marinus will be compared to previous work (Mulcahy and Mendelson, 2000) on other toads from the same region. The goal is to determine if the formation of the TMNB has affected the genetic history of these relatively unrelated toads in a similar fashion.
Literature Review

Prior to the year 2000 the belief was that the distribution of the Gulf Coast Toad (Bufo valliceps) also spanned the TMNB. To answer questions similar to those we will be asking about B. marinus, Mulcahy and Mendelson (2000) sequenced mitochondrial DNA from B. valliceps from areas north and south of the TMNB. From this data they were able to show the existence of two monophyletic clades within B. valliceps. One clade was found to be north of the TMNB, while the other was found to be south of the TMNB. Consequently, they named the northern clade Bufo nebulifer, and the southern clade Bufo valliceps. In addition, using a molecular clock they were able to provide convincing evidence that this speciation event occurred during the same time as the formation of the TMNB.


Bufo marinus has proved to be a species quite proficient in colonizing new areas and thriving there. These toads have caused many problems in areas they have been introduced. In 1965 a Dade County Florida official went so far as to propose a bounty be put on the heads of this species because of the nuisance they had become since their introduction there only the year before (Krakauer, 1970). Australians have also found this species, which they call the Cane Toad, difficult to get rid of since its introduction there (Hedgpeth, 1993). The resilient and durable ecology of this species leads to an interesting question that this research is hypothesized to answer: was B. marinus also affected by the formation of the TMNB, or due to its ecology was it able to overcome this geological barrier?
Research Objectives


  1. Collect DNA-sequences data (16S and cyt-b genes) from tissue samples of B. marinus collected by Mulcahy and Mendelson from areas north and south of the eastern extension of the TMNB to assess the amount of genetic variation between and within these two groups.

  2. Perform phylogenetic analysis of the resulting sequences to see if the genetic variation is significant.

  3. Use a molecular clock to estimate how long ago any major changes occurred, if significant genetic variation is found.


Research Methods


  1. Extract DNA from tissue samples using standard phenol/chloroform extraction methods (Maniatis et al., 1982).

  2. Amplify and PCR the DNA using the methods and primers described in Mulcahy and Mendelson (2000).

  3. Perform phylogenetic analysis and use a molecular clock as described in Mulcahy and Mendelson (2000).


Outcomes
We believe there to be three possible outcomes for this project:


  1. Our data will not show a significant difference in the amount of genetic variation between the northern and southern populations. This would lead us to believe that the TMNB did not form a barrier to the crossing of B. marinus.

  2. Our data will show a significant difference in the amount of genetic variation between the northern and southern populations, and additionally will show a similar change in time to that of B. valliceps. This would lead us to a conclusion that the TMNB also formed a barrier to the crossing of B. marinus.

  3. Our data will show a significant difference in the amount of genetic variation between the northern and southern populations, but this variation will not show a similar change in time to that of B. valliceps. This would lead us to conclude that an event other than the formation of the TMNB caused the difference in genetic variation between the two groups.


Public Presentation of Results
After the completion of this project I will publicly present our results at the following venues:


  1. USU Student Showcase here at Utah State University.

  2. 2004 annual International Herpetological Conference at the University of Oklahoma (May 26-31).



References
Conat, R., and J.T. Collins. 1991. “Reptiles and amphibians: Eastern/Central North America,” Houghton Mifflin Company. Boston.
Hedgpeth, J.W. 1993. Foreign invaders. Science, New Series, Vol. 261, No. 5117. (Jul. 2, 1993), pp. 34-35.

Krakauer, T. 1970. The invasion of the toads. The Florida Naturalist 12-14.


Maniatis, T., Fristch, E. F., and Sambrook, J. 1982. “Molecular Cloning: A Laboratory Manual,” Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Mulcahy, D. G., and Mendelson, J. R. 2000. Phylogeography and speciation of the morphologically variable, widespread species Bufo valliceps, based on molecular evidence from mtDNA. Molecular Phylogenetics and Evolution, Vol. 17, No. 2, November, pp. 173-189.
Perez-Higareda, G., and Navarro, L. D. 1980. The faunistic districts of the low plains of Veracruz, Mexico, based on reptilian and mammalian data. Bull. Maryland Herpetol. Soc. 16:54-69.

Budget
Total Budget Requested: $ 502.00
Amount Requested from URCO: $ 252.00
Matching Funds from Department of Biology: $ 250.00
Breakdown of total budget requested

Laboratory Expenses

I will use standard methods for extracting, amplifying, and sequencing of DNA from each sample. The detailed methodology may be found in Mulcahy and Mendelson (2000); I have used these techniques in our lab and had satisfactory results. I will generate sequence data for two genes from all samples currently in our lab. There are 30 samples of B. marinus, and an additional 12 samples of B. valliceps and B. nebulifer that are relevant to this project. (total: 110 gene sequences).

DNA extractions:


In total, I need to extract DNA from approximately 42 tissues samples. I estimate a success rate of 80%, based on preliminary work. Cost of Proteinase-K, phenol, and other reagents required for extraction in our lab is $2.00 /extraction.
DNA extractions (42 @ $2ea.)................................................................... $ 84.00

+ 20% repeats............................................................................................. $ 16.00



PCR amplification:


In total, I need to amplify and purify PCR products for approximately 42 samples. Based on experience in our lab, I estimate that only 1microliter reactions will be necessary for sequencing, with an 80% success rate, and at a cost of $2/reaction

PCR amplification & purification (42 @ $2/ea.)......................................... $ 84.00

+ 20% repeats.............................................................................................. $ 16.00

DNA sequencing:


In total, I need to sequence approximately 42 PCR products. Double-stranded DNA fragments will be sequenced in both directions to attain at least 80% overlap—representing 110 separate sequences. Based on experience with these taxa in our lab with our sequencer, I can generate these data at a cost of $3/sequence, including price of gels. I estimate an 80% success rate in these procedures. This cost is significantly less that that charged ($20/sequence) by the sequencing services of the USU Biotechnology Center.
DNA sequencing (84 @ $3/ea. proposed to be covered by URCO).........$ 252.00

+ 20% repeats..............................................................................................$ 50.00


TOTAL EXPENSES: $ 502.00



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