1. Spotted owl KG6 B2 H2 644 749 KG2 KG1 480 M2 KG5 136 H1 M4 B1 M1 H4 B3 M3 382 249 71 150 120 262 189 837 Introduction The northern saw-whet owl, Aegolius acadicus, is a small predatory bird that migrates south in the fall and is caught and banded at owl banding stations in many areas across the United States. One of the big questions about northern saw-whet owls is how they migrate. Are there several geographical populations of saw-whet owls, each with its own migration route, or are there multiple migration routes within the different geographical populations? In order to study this, one must first determine if there are different, genetically distinct geographical populations of northern saw-whet owls. This question can be explored using a phylogeographic study. Phylogeography is the study of the genetic similarity between organisms from different geographical populations and generally involves sequencing DNA for comparison. In past phylogeographic studies, mitochondrial DNA has been sequenced as opposed to nucleic DNA. This is due to mitochondrial DNA’s rapid evolution and maternal mode of inheritance (Li et al. 2003). Uniparental inheritance means that siblings have identical mitochondrial DNA and makes it so that mitochondrial DNA variation is solely due to mutation (Ruokonen 2001). The d-loop is a noncoding control region located between the cytochrome b and 12S regions of mitochondrial DNA (see Figure 2) and is considered the most variable region of mitochondrial DNA because of its high mutation rate (Ruokonen 2001). Sequencing a variable region of DNA is helpful in phylogeographic studies because it is more likely to reveal genetic differences between organisms from the same species. Phylogeographic Analysis of Migrating Northern Saw-Whet Owls Carrie Shaw Department of Biological Sciences, York College of Pennsylvania Acknowledgements Thank you Dr. Karl Kleiner and Dr. Jeffrey Thompson for your guidance and help! Thank you Scott Weidensaul, Project Coordinator for central Pennsylvania owl banding project! Thank you Ned Smith Center for Nature and Art, supporter of the central Pennsylvania owl banding project! Thank you York College Biology Department for the supplies, lab space and the joy! Methods A)Primers Isolated a 147 base pair fragment of the mtDNA d- loop using primers from Sorenson (1999). Objective and Hypotheses The objective of this project is to assess the genetic similarity of northern saw-whet owls from different geographical locations. I hypothesize: 1) That we will find that sibling northern saw-whet owls travel together 2) That owls banded at the same location will be more closely related on a cladogram (see Figure 1) B) Feather Samples Feathers were taken from the breasts of hatch year owls from: 1) King’s Gap Environmental Education Center in Cumberland Co., PA *KG 1 & 2—same net check *KG 5 & 6—same night 2) Hidden Valley Banding Station in Schuylkill Co.,PA *H 1&2—same night *H4—separate night 3) Choteau, Montana *M1&2—same net check *M4—same night as M1&2 *M3—separate night 4) Bainbridge Island, Washington *all birds on different nights C) Sample Processing D) Phylogenetic Analysis All 21 sequences were aligned using Clustal X software (Gibson et al. 2001). A cladogram based on the sequences was constructed using Clustal X and Tree View software (Page 1996). Conclusion The alignment suggests that sibling owls may migrate together, however the genome sequence traces were ambiguous at the few base positions that separate these potential siblings. The analysis indicates no genetically distinct geographic subpopulations of northern saw-whet owls based on the region of the mtDNA d-loop sequenced. This suggests that there may be substantial gene flow among breeding populations of northern saw- whet owls in North America. Dr. Kleiner and saw-whet owl! Literature Cited 1.Gibson, T., Higgins, D., Thompson, J. and Jeanmougin, F. 2001 June 18. Clustal X software. Available from: http://www-igbmc.u-strasbg.fr/BioInfo/ClustalX/Top.html. Accessed 3 April 2005. 2. Li, Ming, Wei, Fuwen, Groves, Pamela, Feng, Zoujian, and Junchu Hu 2002. Genetic structure and phylogeography of the takin (Budorcas taxicolor) as inferred from mitochondrial DNA sequences. Available from NRC Research Press at http://cjz.nrc.ca. Accessed 21 January 2005. 3. Marshall, H.D. and Baker, A.J. 1997. Structural Conservation and Variation in the Mitochondrial Control Region of Fringilline Finches (Fringilla spp.) and the Greenfinch (Carduelis chloris). Molecular Biology and Evolution 14 (2): 173-184. 4. Page, R. D. M. 1996. TREEVIEW: An application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12: 357-358. Software version 1.6.6., 2001 September 3 available from: http://taxonomy.zoology.gla.ac.uk/rod/treeview.html. Accessed 3 April 2005. 5. Ruokonen, Minna 2001. Phylogeography and conservation genetics of the lesser white fronted goose (Anser erythropus). Department of Biology, University of Oulu, Finland. Available from: http://herkules.oulu.fi/isbn9514259483/. Accessed 21 January 2005. 6. Sorenson, Michael D., Ast, Jennifer C., Dimcheff, Derek E., Yuri, Tamaki, and David P. Mindell 1999. Primers for a PCR-based approach to mitochondrial genome sequencing in birds and other vertebrates. Molecular Phylogenetics and Evolution 12: 105-114. Figure 2. Chicken (Gallus gallus) mtDNA displays location of d-loop (Sorenson 1999). Figure 2 Figure 1. Predicted results for cladogram Results  The alignment (see Figure 3) revealed no 100% identical sequences; however, KG1 & KG2 only differ by 1 base and M1 & M2 differ by only 2 bases. The genome sequence traces at these positions were indistinct.  Owls are not grouped by geographical location on the cladogram (see Figure 4) Figure 5. Diagram of vertebrate mitochondrial control region from Ruokonen (2001). Domain II begins at the 400 th base and ends at the 873 rd base (Marshall and Baker 1997). KG=King’s Gap, PA H=Hidden Valley, PA M=Choteau, Montana B=Bainbridge Island, WA Figure 3. Alignment of owl mtDNA d-loop fragment sequences Figure 3 Figure 5 Figure 1 The region of the mtDNA d-loop studied may also be more conserved than other regions, as indicated by Marshall and Baker (1997) and by Ruokonen (2001) (see Figure 5). Our 147 base pair fragment is located from the 390th base to the 537th base (Sorenson et al. 2001), which is mostly located in the conserved domain II. Further research using different primers to compare different sequences may clarify these results. Figure 4. Cladogram of saw-whet owl mtDNA sequences created using Clustal X and Tree View software.