Poster presented to the Student Research Conference in UHD.

1. Investigation of phylogenic relationships
of shrew populations using genetic markers
Juan Barrera, Suraj Basnet, Brigitte Goble and Richard Wrangpetch
Dr. Amy B. Baird, Dr. Yuanyuan Kang and Mr. Mehdi Esmaeiliyan, Research Mentors, Department of Natural Sciences, UHD
Acknowledgements
We would like to thank all the BIOL3103 students from Fall 2013 and Spring 2014 for all
their works. We express our sincere gratitude to American Society of Mammalogists and
Animal Diversity Web (University of Michigan) for the information and images.
Abstract
Polymorphic regions in the genome can be used as markers to study the
diversity of species in populations. In this study, DNA was isolated from tissue
samples from shrew species from different geographic regions. We conducted
Polymerase Chain Reaction (PCR) to amplify and sequence two mitochondrial
gene regions, cytochrome-c oxidase subunit 1 (COI) and cytochrome-b (cyt-b).
These genetic markers were then blasted for species identification. In
addition, a phylogenetic tree was constructed for all samples. Our study will
shed light on the diversity of shrews at the DNA level and reveal their
evolutionary relationships. This study was done as a collaborative project
between all students enrolled in BIOL3103 genetics lab in the Fall 2013 and
Spring 2014. Our data has been posted on Blackboard Learn as wiki pages and
this poster presentation is a summary of data from this website.
Introduction
Shrews are small mole like organisms that belong to family Soricidae. They
are among the smallest mammals, ranging from 2 ½ inches to 9 ½ inches
long. Shrew weight varies between 2 grams to 106 grams. Common features
include long pointed snouts, very small eyes, and short velvety fur. Previously,
researchers have relied on morphological traits to determine the phylogenetic
relationships between species. DNA barcoding, an advanced technique, is a
way to obtain unique DNA sequences for species identification and study of
phylogeny. Organisms that have more similarities in their DNA will be more
closely related on a phylogenetic tree. Through a collaboration project we have
constructed a phylogenetic tree to determine species relationships of various
species of shrews using a DNA barcoding and bioinformatics approach. We
used Cytochrome oxidase1(COI) and Cytochrome b genes for our analysis.
Department of Natural SciencesFigure 1: Images of Myosorex sp, Cryptotis parva and Cryptotis goodwini
Methods
DNA Extraction: The Qiagen DNEasy Blood and Tissue Kit was used to
complete DNA extraction.
PCR of COI and Cytochrome b: PCR beads (Fischer Scientific Inc.) and primer
cocktails (forward and reverse primers) for the targeted genes were used. After
completion of the reaction, quality of the amplified product was verified using
agarose gel electrophoresis. The samples containing ample amount of PCR
product were purified using Qiaquick PCR purification kit
Sequencing : The product from PCR was sent to external facilities for sequencing.
Sequencing alignment: The alignment of forward and reverse nucleotide
sequences was carried out using Geneious 6.0.5 software
Phylogeny: MEGA5.2 (Molecular Evolutionary Genetics Analysis) was used to
open and examine the quality of each sequences. Subsequently, all sequences were
aligned to construct a phylogenetic tree using neighbor joining where Armadillo
was used as an outgroup.
BLAST: The BLAST (Basic Local Alignment Search Tool) was used to compare
our nucleotide sequences with the sequences of previously identified species in the
NCBI Genbank.
Results and Discussions
•Cyt-b and COI had some similarities and some differences in the respective phylogenies.
•Overall, we found that Cyt-b is more accurate in species identification and analysis of phylogeny for shrews.
•Cryptotis goodwini is a paraphyletic group in both phylogenies. One lineage represents a possible new,
undescribed shrew species. More study of this is needed to confirm.
•We found some inconsistencies in BLAST results between COI and Cyt-b. This may be because the NCBI
Genbank does not have as much data for comparison with COI. The Cyt-b BLAST results often had a higher
percentage match to the expected genus and species of a particular sample.
•A few samples had both CO1 and Cyt-b BLAST results that did not match the expected genus and species by a
very high percentage. For example, a BLAST search on the nucleotide sequence of the CO1 region for several
samples of putative Myosorex species had an 83% match to Kerivoula minuta, a fruit bat in the Fall of 2013.
However, these same samples blasted as Myosorex species using cyt-b sequences in Spring 2014. This indicates
that BLAST search results resulted because Myosorex COI sequences were not available in Genbank, and BIOL
3103 students are the first to have sequenced it.
•Cyt-b sequences reduced the number of paraphyletic groups of the CO1 phylogeny. Cyt -b analysis revealed
Suncus murinus, Sorex articus and Blarina hylophaga to be their own monophyletic clade as opposed to COI
which placed them as a paraphyletic clade.
•One Suncus murinus grouped closely to Cryptotis goodwini on COI phylogeny. This may be due to
contamination of the sample. However, Cyt-b phylogenic tree establishes Suncus murinus as a monophyletic
clade.
•Cyt-b establishes Blarina hylophaga as a monophyletic clade. The COI sequences obtained for Blarina
hylophaga were not clear and could not be verified.
Figure 2: Cytochrome oxidase (COI) phylogeny for studied species subset
of family Soricidae conducted by students in BIOL 3103 in Fall 2013.
Figure 3: Cytochrome-b (Cyt-b) phylogeny for studied species
subset of family Soricidae conducted by BIOL 3103 students in
Spring 2014.