Application for Funding From the Colorado College
Venture Grant Program, Fall 2002
Biology Senior Thesis: Determining the Breeding Strategy of Male Flammulated Owls
I. Introduction
The Flammulated Owl (Otus flammeolus) is a small, poorly known raptor that inhabits montane forests throughout western North America (McCallum and Gehlbach, 1988). These owls are restricted to forests of commercially valuable tree species, such as mature ponderosa pine (Pinus ponderosa) and Douglas-fir (Psuedotsuga mensiesii) (Hayward and Verner, 1994). Since several studies found that densities of Flammulated Owls declined after tree removal (Groves et al., 1997), and because the owls require abandoned woodpecker holes for nesting, the Flammulated Owl is listed as a sensitive and vulnerable species by the United States (Hayward and Verner, 1994) and Canada (van Woudenberg, 1992). The legal status of the owls has resulted in the need for conservation plans, which require detailed information on life history traits such as reproduction and mating strategies, habitat requirements, and size of the breeding territory. Recent research by Dr. Brian Linkhart (2001) has elucidated information on a number of these life history characteristics; however, information about mating strategies is still needed.
Flammulated Owls are known to be socially monogamous, and each sex has a defined role in breeding (Linkhart, 2001). Females incubate the eggs and defend the nest, while the males are the primary foragers for females and their broods (Linkhart, 2001). The sex ratio for this bird is apparently male biased in the most intensely studied populations (Linkhart, 2001). As a result of this bias, only a small percentage of adult males breed annually. Males that are not involved in a pair bond during a given season, referred to as `bachelor’ males, typically inhabit territories without adequate nesting sites and contain a high proportion of dense, even-aged stands of young trees. In contrast, territories of nesting males usually contain optimal foraging sites that consist of open, mature forest (Linkhart et al., 1998). It therefore appears that females are selecting males each year on the basis of forest habitat (Linkhart, 2001). Most bachelor males, however, appear to occupy the same poor quality territories their entire lives, even when high quality territories occasionally become available. It is thus questioned why the bachelor males do not switch to a more optimal territory.
Linkhart (2001) has hypothesized that the answer to this question is that bachelor males are involved in a dual mating strategy. This hypothesis contains two main components. First, bachelor males return to poor quality territories for the possibility of establishing a pair-bond in areas where they have invested considerable time and energy learning the locations of important sites for feeding, sleeping, and escaping predators. Linkhart (2001) has already shown that most males, including bachelors, appear to return to original territories their entire lives and that even bachelor males occasionally may breed. Second, bachelor males perform extra-pair copulations in the meantime, which enable them to pass on their genes despite the lack of an established pair-bond. Casual observations of extra-pair copulation, as well as observations of unidentified owls in the vicinity of nesting females by Linkhart (2001), indicate that bachelor males may be involved in extra-pair copulation. Until recently, studies had not attempted to establish genetic evidence of extra-pair copulation.
Questions pertaining to the percentage of males participating in breeding annually are of great importance to conservation management plans. If extra-pair copulation is shown to occur regularly within this species, this may increase the effective population size, which is the number of breeding individuals within a population (Sugg and Chester, 1994). Loss of genetic variability is a concern when effective population size is small or declining, which can result from loss of breeding habitat, and can be exacerbated by uneven sex ratios (Primack, 1998). A diverse gene pool is essential so that the possibility for future adaptation, successful expansion, or reestablishment in natural populations is retained (Hedrick and Miller, 1992).
This past summer, with the aid of a Hughes Undergraduate Research Grant, Dr. Brian Linkhart, Ian Hopper (C.C. sophomore), Tyler McGrath (C.C. junior) and I performed field research and data collection at the 452ha Manitou Experimental Forest, Teller Co., Colorado; the site of Linkhart’s original Flammulated Owl study area. With the collection of blood samples obtained from 30 individuals this summer, the opportunity now exists to analyze the DNA within this blood for evidence of extra-pair copulation. This genetic analysis is the focus of my senior thesis.
II. Methods
In order to potentially exclude paternity from within family groups, genetic analysis of the Flammulated Owl breeding system will necessarily begin with the identification of DNA markers, called microsatellites. Microsatellite markers are elements within DNA that are composed of tandemly repeating one-, two-, or threenucleotide base (A, T, C, and G) sequences (Hartwell et al., 2000). These DNA markers are often used for breeding studies because they show a high level of polymorphism and are relatively stable within a few generations (Hartwell et al., 2000). Genetic research performed for the Flammulated Owl has been limited, and none of this research has specifically used microsatellite markers. As a result, the first main phase of this research will involve locating micro satellite markers and developing Flammulated Owl microsatellite primers. A primer is the section of DNA that flanks the microsatellite target region (Hartwell et al., 2000). Primers are necessary for a process called polymerase chain reaction (PCR), which makes multiple copies of a target region of DNA. Primers that have been developed for a particular species have frequently proven useful in amplifying the same locus for related taxonomic groups (Parker et al., 1998). Primers that have previously been developed for the Boreal Owl (Aegolius funereus) (M. Koopman, personal. communication, 23 Aug., 2002), Eagle Owl (Bubo bubo) (Isaksson and Tegelstrom, 2002), and the Spotted Owl (Strix occidentalis) (M. Koopman, personal communication, 23 Aug., 2002) will thus be tested with Flammulated Owl DNA during this first phase. Preliminary research on the feasibility of Boreal Owl primers amplifying Flammulated Owl microsatellites has shown that at least one of these primers will be highly polymorphic for the Flammulated Owl (M. Koopman, personal communication, 19 Aug., 2002).
Once primers are developed, the second phase of the project will focus on determining parentage. After manufacturing the multiple copies of the microsatellites through PCR, the PCR-amplified product is run through a process called gel electrophoresis. Through this process, the microsatellite fragments will travel at different rates depending on their size. After separating the micro satellite markers for individuals within a family group it is possible to exclude paternity. Specifically, the microsatellites from the 29 individuals will be amplified via PCR and the developed primers. The PCR product will be analyzed through polyacrylamide gel electrophoresis, which will be read by an automated DNA sequencer. This machine is an efficient way to analyze the polyacrylamide gels in that a laser beam automatically reads fluorescent labels attached to the PCR products in the gel (http://www.uwvo.edu/dbmcd/lab/msatintro.html). Maternity will be assumed for the female incubating the eggs within a given nest, thus providing the potential for paternity exclusion. After performing the polyacrylamide gel electrophoresis, the data will be analyzed using techniques developed by Dr. David McDonald at the University of Wyoming.
Research for this project will take place in the McDonald lab at the University of Wyoming, Laramie. Dr. McDonald’s lab is well equipped for research on avian microsatellite DNA genotyping. Current research in this lab includes population genetic research on the Boreal Owl and Black Rosy-Finches (Leucosticte atrata) (http://www.uwyo.edu/dbmcd/lab/lab.html). PhD student, Marni Koopman, developed the Boreal Owl microsatellite primers and is currently working with Dr. McDonald. She will show me how to perform these lab techniques. McDonald’s lab was chosen as the location to perform this research because the individuals involved in research there are experts in avian genetics, especially within the order Strigiformes. In working at this lab, I will be able to capitalize on the experience of these researchers, which will facilitate the processes of developing microsatellite primers and analyzing paternity data. Additionally, the use of this lab is more economical. I will pay only for the supplies that I use, rather than purchasing bulk amounts of supplies that are not necessary for this project.
This analysis of the mating strategy of Flammulated Owls is focus my senior thesis. First, second, and third blocks of this year have been designated solely for the completion of this research. Results from this project will be presented in written format as a Biology senior thesis and presented at the Biology Day colloquium seventh block. It is also intended that results from this project be published as a paper in a scientific journal, such as The Condor or Molecular Ecology Notes.
III. Budget
The total amount requested from the Venture Grant program is $850, which will be used to cover the cost of lab expenses at the University of Wyoming. Additional funding is requested from the Hughes Undergraduate Research Program, which includes for the semester a $1,500 undergraduate stipend and a $750 "supply and travel" budget for Dr. Brian Linkhart, my faculty sponsor. The Hughes Undergraduate Research Program provided funding for the summer 2002 field research and data collection.
Literature Cited
Groves, C. et al. 1997. Density, distribution, and habitat of Flammulated Owls in Idaho. Great Basin Naturalist 52(2): 116-123.
Hartwell et al., 2000. Genetics: From Genes to Genomes. McGraw Hill Companies, Inc., United States of America.
Hayward, G. D. and J. Verner (tech ed.). 1994. Flammulated, Boreal, and Great Gray Owls in the U.S.: A Technical Conservation Assessment. USDAFS RM- 253.
Hedrick, P.W. and P.S. Miller. 1992. Conservation Genetics: Techniques and Fundamentals. Ecological Applications 2(1): 30-46.
Isaksson, M. and H. Tegelstrom. 2002. Characterization
of polymorphic microsatellite markers in a captive population of the eagle owl (Bubo bubo) used for supportive breeding. Molecular Ecology Notes 2: 91-93.
Linkhart, B. D., R. Reynolds, and R. Ryder. 1998. Home range and breeding habitat of breeding Flammulated Owls in Colorado. Wilson Bulletin 110(3): 342-351.
Linkhart, B.D. 2001. Life history characteristics and habitat quality of Flammulated Owls in Colorado. PhD dissertation, University of Colorado, Boulder, Colorado. 206 pp.
McCallum, D.A., and F.R. Gehlbach. 1988. Nest-site preferences of Flammulated Owls in western New Mexico. The Condor 90:653-661.
McDonald, D.B. 1999. Microsatellite DNA: the half-page intro. Retrieved 27 August, 2002, from Dr. McDonald’s University of Wyoming web site: http://www.uwyo.edu/dbmcd/lab/msatintro.html.
McDonald, D.B. McDonald Lab: research focus. Retrieved 27 August, 2002, from Dr. McDonald’s University of Wyoming web site: http://www.uwvo.edu/dbmed/lab/lab.html.
Sugg, D.W. and R.K. Chesser. 1994. Effective population sizes with multiple paternity. Genetics 137: 1147-1155.
Parker et al., 1998. What molecular markers can tell us about populations: choosing and using a molecular marker. Ecology 79(2): 361-382.
Primack, R.B. 1998. Essential of Conservation Biology. Sinauer Associates Pub., Sunderland, Massachusets.
Van Woudenberg, A.M. 1992. Integrated management of Flammulated Owl breeding habitat and timber harvest in British Columbia. Masters thesis, Univ. British Columbia, Vancouver.