Genetic evolution within the genus Liolaemus
Meghann Strain1, Dr. James A. Schulte II2
Department of Biology
Until recent years, the field of phylogenetics has been rooted in comparing species based on
physical characteristics and geographic location. With new developments in genetic research, these
relationships have been put under further scrutiny. Through gene sequencing, it is possible to determine
the genetic similarities between species on a smaller, more exact scale.
The purpose of this research is to compile genetic data from one hundred species of iguanas from
the genus Liolaemus. These data will then be used to infer a phylogenetic hypothesis based on genetic
similarities between species. This research is based on the assumption that the more similar the genetic
code, the more closely related the species.
The DNA used in these procedures is nuclear DNA rather than mitochondrial DNA. Nuclear
DNA is a better option for this type of research as it has a lower rate of substitution and mutation than
mitochondrial DNA, and because it is inherited from both parents.
The gene isolated in these procedures is the KCNA2 gene. It is a protein-coding gene in the
shaker subfamily. It encodes a voltage-gated channel protein in the plasma membranes of cells. The
channel permits or denies entry of ions into the cell depending on the electrochemical gradient across the
membrane. This gene has only recently been isolated and sequenced in other species, and has not been
previously sequenced in iguanas.
Before the DNA can be sequenced, it needs to be amplified to ensure that enough of the gene is
present in the sample to observe. This is done through polymerase chain reactions, or PCRs. Each
specimen of DNA is prepared with a cocktail of water, buffer, magnesium chloride,
deoxyribonucleotides (dNTPs), taq polymerase enzyme, and two primers, one that matches the DNA in a
forward direction, and another that matches it in reverse. The use of two primers ensures that the gene is
fully prepared. A control is also used during this step, which consists of all of the components of the
cocktail without the DNA. This will ensure that the chemicals used are not contaminated.
After the amplification process is completed, the DNA needs to be purified. Purification involves
the removal of all extraneous substances, leaving just the isolated DNA suspended in water. This is done
by first mixing the amplified solution with a solution of suspended magnetic particles in an eppindorf
tube. Due to the negative nature of DNA, the magnetic particles adhere to it. The solution is then placed
on a magnetic plate. The plate attracts the magnetic particles, forming a ring around the edge of the tube.
The remaining solution is then removed from the tube, leaving the magnetic bead ring intact. The ring is
then cleaned with a wash of ethanol to remove any impurities. After the ethanol is removed, the tube is
1
2
Class of 2007. Biology. Honors Program. Oral Presentation
Associate Professor. Clarkson University. Dept. of Biology
dried. Water is then added to the tube, and mixed until the solution is homogenous, causing the water to
adhere to the DNA. The solution of water and DNA is then removed from the tube, and the magnetic
beads are discarded.
Following the purification process, the DNA is prepared in a series of single primer sequencing
reactions in a 96 well plate. The plate is then sent to the University of Wisconsin, Madison
Bioinformatics Center, to be run on their automated sequencer. Individual sequencing reactions are made
available electronically. Each reaction represents a single-stranded segment of the nucleotide sequence.
For each species, these segments are compiled into one long strand by aligning them in a consensus
sequence called a contig, and examined for errors. The computer programs used in this process are
SeqMan, FinchTV, and PAUP.
After each sequence has been assembled, they are put into a text file in the PAUP program that
allows side-by-side comparisons of each sequence. This text file is then executed in the program to infer
a phylogenetic tree. The tree is generated using parsimony, maximum likelihood, and distance methods.
After this work has been completed, the phylogeny determined from these data will be compared
to the phylogenetic hypothesis of Liolaemus based on mitochondrial DNA sequences. Finally, we intend
to evaluate the evolution of body size in this genus in a phylogenetic context to understand how this
ecologically important character has changed through time.
1
2
Class of 2007. Biology. Honors Program. Oral Presentation
Associate Professor. Clarkson University. Dept. of Biology
Tropidurus.spinulosusTS116
Leiocephalus personatus602
Sceloporus.consobrinusJAS194
Crotaphytus.collarisJAS195
Gambelia wislizenii201275
Liolaemus.kriegiJAS32
Liolaemus.plateiJPV150
Liolaemus.isabelaeISAB
Liolaemus.lemniscatus3721
Liolaemus.nitidusJPV147
Liolaemus.sp.nigromaculatusJPV116
Liolaemus.josephorumPlat
Liolaemus.curisJPV152
Liolaemus.tenuispunctJPV180
Liolaemus.melanopsFBC45
Liolaemus.rothi.hermannuneziJAS66
Liolaemus.xanthoviridisFBC2
Liolaemus.reichei2148
Liolaemus.fitzingeriiFML
Liolaemus.audituvelatus2162
Liolaemus.aymararumSDSU4014
Liolaemus.pulcherrimus15ornat
Liolaemus.stolzmanniSTOLTZ
Liolaemus.torresiPhrynosp
Liolaemus.molinaeO81
Liolaemus.canqueliPT4811
Liolaemus.famatinaeREE193
0.005 substitutions/site
Figure 1. Phylogenetic relationships among Liolaemus lizards based on maximum likelihood using the
GTR+I+G model (log likelihood – 3743.60817). Branches represent relative lengths among species
sampled.
1
2
Class of 2007. Biology. Honors Program. Oral Presentation
Associate Professor. Clarkson University. Dept. of Biology