Investigation of phylogenic relationships of shrew populations using genetic markers

Juan Barrera, Suraj Basnet, Brigitte Goble and Richard WrangpetchDr. Amy B. Baird, Dr. Yuanyuan Kang and Mr. Mehdi Esmaeiliyan, Research Mentors, Department of Natural Sciences, UHD

AcknowledgementsWe 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.

AbstractPolymorphic 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.

IntroductionShrews 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 Sciences Figure 1: Images of Myosorex sp, Cryptotis parva and Cryptotis goodwini

MethodsDNA 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 kitSequencing : 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 softwarePhylogeny: 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.