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