ASSESSING POPULATION STRUCTURE AND MIGRATORY PATTERNS OF WHITE-THROATED SPARROW (ZOSOTRICHIA ALBICOLLIS) BREEDING POPULATIONS IN WESTERN CANADA by Marcelo Mora B.Sc. Pontificia Universidad Catolica del Ecuador, 2007 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN NATURAL RESOURCES AND ENVIRONMENTAL STUDIES UNIVERSITY OF NORTHERN BRITISH COLUMBIA February 2012 © Marcelo Mora, 2012 1+1 Library and Archives Canada Bibliotheque et Archives Canada Published Heritage Branch Direction du Patrimoine de I'edition 395 Wellington Street Ottawa ON K1A0N4 Canada 395, rue Wellington Ottawa ON K1A 0N4 Canada Your file Votre reference ISBN: 978-0-494-87555-1 Our file Notre reference ISBN: 978-0-494-87555-1 NOTICE: AVIS: The author has granted a non­ exclusive license allowing Library and Archives Canada to reproduce, publish, archive, preserve, conserve, communicate to the public by telecommunication or on the Internet, loan, distrbute and sell theses worldwide, for commercial or non­ commercial purposes, in microform, paper, electronic and/or any other formats. L'auteur a accorde une licence non exclusive permettant a la Bibliotheque et Archives Canada de reproduire, publier, archiver, sauvegarder, conserver, transmettre au public par telecommunication ou par I'lnternet, preter, distribuer et vendre des theses partout dans le monde, a des fins commerciales ou autres, sur support microforme, papier, electronique et/ou autres formats. The author retains copyright ownership and moral rights in this thesis. Neither the thesis nor substantial extracts from it may be printed or otherwise reproduced without the author's permission. L'auteur conserve la propriete du droit d'auteur et des droits moraux qui protege cette these. Ni la these ni des extraits substantiels de celle-ci ne doivent etre imprimes ou autrement reproduits sans son autorisation. In compliance with the Canadian Privacy Act some supporting forms may have been removed from this thesis. Conformement a la loi canadienne sur la protection de la vie privee, quelques formulaires secondaires ont ete enleves de cette these. While these forms may be included in the document page count, their removal does not represent any loss of content from the thesis. Bien que ces formulaires aient inclus dans la pagination, il n'y aura aucun contenu manquant. Canada ABSTRACT The objective of the present study was to investigate the genetic and migratory differences among White-throated Sparrow populations in western Canada in order to locate the presence of a migratory divide. Deuterium isotopes and molecular markers were used to assess the migratory differences between sparrows west (Central Interior BC) and east (Peace Region BC) of the Continental Divide. Head feather isotopes showed that both populations are overwinter wintering in either the Pacific Coast, New Mexico/Arizona or Colorado/Kansas areas. Microsatellites and mitochondrial markers did not show genetic structure among populations, however tail feather isotopes were significantly different. Analysis of migratory samples is congruent with Peace region birds migrating east of the divide. The Central Interior birds were not detected in any migratory monitoring locations. Data of the present study is congruent with a migratory divide and an east/west migration pattern between Central Interior and the Peace Region populations. ii I would like to offer my thanks to NSERC for funding the project and to the Pacific Century Scholarship for the financial support that was provided to me. A very special thanks to my lovely wife, because without her technical and moral support as well her strong work ethic and wonderful skills in the field, this thesis would never be completed successfully. Another special thanks to Dr. Brent Murray, for all his guidance during this project, as well as the financial support that he has provided me. Also I would like to thank him for giving me the opportunity to be part of this project and for believing in international students. I would like to thank Dr. Ken Otter who has done much more for this project than what a committee member would normally do. A special thanks to him for his guidance and for all the training during the banding season, plus the technical support in the avian behaviour he gave me during this project. I would like to thank Thibault and Angelique Grava for all their help during my training in bird banding. To Dr. Chris Johnson and Ingebjorg Jean Mattson for their advice on how to prepare stable isotope samples. To Wendy Easton for her useful advices during my thesis proposal. To Timothy Meehan for providing me his deuterium stable isotope map GIS files and allowing me to use it in my analyses. To Roger Wheate for his assistance with GIS maps To Vi Lambie and all the personnel of Mugaha Marsh Bird Observatory, Tattlayoko Bird Observatory, Rocky Point Bird Observatory, Vaseux Lake Bird Observatory, Lesser Slave Lake Bird Observatory and Beaverhill Bird Observatory for all their hard work and time providing me the samples from migratory birds. Also, thanks to Scott Ramsay for providing me samples from Ontario and Prince George. A special thanks to Mark Thompson for all his help and advice during lab work, as well as, to Gayathri Samarasekera for her help during population structure data analysis. To the people of the Pouce Coupe Park for allowing us sampling within their facilities and to Dusty Walsh and Laura Kennedy for their assistance in collecting samples at Prince George. To my parents and my sister for all the support that they have provided me during all my life Very special thanks to Jacqueline Dockray, Linda Tackaberry, Hugues Massicotte and Juan Carlos Lopez for all your help and support. And finally, to my son Gabriel Adrian for giving me the inspiration during this final writing process. iv TABLE OF CONTENTS ABSTRACT II ACKNOWLEDGMENTS Ill TABLE OF CONTENTS V LIST OF TABLES VIII LIST OF FIGURES XII CHAPTER 1 GENERAL INTRODUCTION 1.1 MIGRATORY ROUTES AND CONNECTIVITY OF BIRD POPULATIONS 1 1.2 WHITE-THROATED SPARROW 7 1.3 OVERVIEW OF IMPORTANT MARKERS USED TO DELINEATE MIGRATORY PATHWAYS 11 1.4 MIGRATORY FLYWAYS AND BANDING RECOVERIES 15 1.5 BANDING STATIONS INFORMATION 16 1.6 RESEARCH OBJECTIVES AND THESIS ORGANIZATION 18 CHAPTER 2 DETERMINATION OF MIGRATORY CONNECTIVITY AND PATTERNS USED BY WHITE-THROATED SPARROWS IN WESTERN CANADA USING DEUTERIUM STABLE ISOTOPES 20 2.1 INTRODUCTION 20 2.2 METHODOLOGY 25 2.2.1 STUDY AREA AND SAMPLE COLLECTION 25 v 2.2.2 SAMPLE PREPARATION AND ANALYSIS 27 2.2.3 STATISTICAL ANALYSIS 30 2.3 RESULTS 31 2.3.1 DISTRIBUTION AND OUTLIERS 31 2.3.2 HEAD FEATHER SAMPLE ANALYSIS 34 2.3.3 TAIL FEATHER SAMPLE ANALYSIS 37 2.4 DISCUSSION 42 2.4.1 DETERMINATION OF WINTERING TERRITORIES BASED ON HEAD FEATHER DEUTERIUM ISOTOPES 42 2.4.2 DETERMINATION OF BREEDING TERRITORIES BASED ON TAIL FEATHER DEUTERIUM ISOTOPES 47 CHAPTER 3 GENETIC DIFFERENTIATION ANALYSIS OF WESTERN CANADA WHITETHROATED SPARROW POPULATIONS AND GENETIC ASSIGNMENT OF MIGRATORY INDIVIDUALS 3.1. INTRODUCTION 54 3.2 METHODOLOGY 58 3.2.1. STUDY AREA AND SAMPLE COLLECTION 58 3.2.2. GENOMIC DNA EXTRACTION 62 3.2.3. MICROSATELLITE AMPLIFICATION 62 3.2.4. MICROSATELLITE DATA ANALYSIS 63 3.2.5 MITOCHONDRIAL DNA AMPLIFICATION 65 3.2.6. MITOCHONDRIAL DATA ANALYSIS 66 vi 3.3. RESULTS 67 3.3.1 MICROSATELLITE BREEDING SAMPLES ANALYSIS 67 3.3.2 MICROSATELLITE MIGRATORY SAMPLES ANALYSIS 72 3.3.3 MITOCHONDRIAL DNA RESULTS 74 3.4. DISCUSSION 78 3.4.1 POPULATION HISTORY OF CENTRAL INTERIOR WHITETHROATED SPARROW POPULATIONS 78 3.4.2 POPULATION STRUCTURE AND GENETIC ASSIGNMENT OF WHITE-THROATED SPARROW POPULATIONS 81 CHAPTER 4 GENERAL DISCUSSION 4.1 MIGRATORY CONNECTIVITY BETWEEN BREEDING AND MIGRATORY POPULATIONS 84 4.2 UNIQUE NATURE OF CENTRAL INTERIOR 86 4.3 ENVIRONMENTAL/MANAGMENT IMPLICATIONS 88 4.4 IMPROVEMENTS TO THE TECHNIQUE 90 4.5 TECHNIQUE ASSESMENT 93 BIBLIOGRAPHY 97 APPENDIX 1: RAW DATA SUMMARY 107 APPENDIX 2: MITOCHONDRIAL DNA HAPLOTYPES 123 vii LIST OF TABLES Table 2.1 A) Five highest and lowest 5Df (%o) values of Central Interior tail feather samples. B) Five highest and lowest 8Df (%o) values of Peace River Region tail feather samples. C) Five highest and lowest 5Df (%o) values of head feather samples 32 Table 3.1.White-crowned sparrow (Zonotrichia leucophrys) microsatellite primers (5'-> 3') that showed polymorphism in White-throated Sparrow (Zonotrichia albicollis) (Poesel et al. 2009). All primers span a tetranucleotide repeat. The size range observed in White-crowned sparrows in base pairs (Poesel et al. 2009) is shown 64 Table 3.2 PCR primers and cocktail combinations (Wong and Hanner 2008) used in amplification of mitochondrial COI gene from White-throated Sparrows 66 Table 3.3 Amplification size (bp), number of alleles (Na), observed (Ho) and expected (He) heterozygosity, and number of private alleles found for all eight microsatellite loci in each region: Central Interior, Peace River Region and Ontario. Heterozygocity values in bold represent significant differences with Bonferroni corrections between Ho and He viii 69 Table 3.4 AMOVA from breeding population samples. Source of variation, degree of freedom (d.f), sum of squares and Percentage of Variation are shown. Distance method: 69 Sum of Squared size differences Rst; 1000 permutations Table 3.5 Pairwise Fst Population Comparison between Central Interior, Peace Region, and Ontario. Distance Method used: Sum of squared size differences (Rst). Significance (Pvalues) are shown in parenthesis next to Fst value 69 Table 3.6 Self-assignment of sampling locations during breeding season to their breeding regions using Geneclass2. The first number (outside the parenthesis) represents the number of individuals assigned with highest probability to each breeding region. In parenthesis is represented the number of individuals that were statistically rejected (P < 0.05) as being part of this location (using MonteCarlo resampling and Paetkau et al. 2004 simulation algorithm) 73 Table 3.7 Assignment of individuals from migratory sampling locations (Mugaha Marsh, Lesser Slave, Beaverhill, Rocky Point and Dawson Creek-migratory)to the three breeding regions (Central Interior, Peace River Region and Ontario) using Geneclass2. The first number (outside the parenthesis) represents the number of individuals assigned with highest probability to each breeding region. In parenthesis is represented the number of individuals that were statistically rejected (P < 0.05) as being part of this location (using MonteCarlo resampling and Paetkau et al. 2004 simulation algorithm) ix 73 Table 3.8 Number of mtDNA haplotypes per location and number of unique haplotypes as well as number of sequences 75 Table 3.9 Frequency of shared haplotypes per sampling location. Haplotypes name was stated with letters (A, B, C, and K). Central Interior, Peace River Region and Ontario populations were included 75 Table 3.10 AMOVA from mtDNA samples. Source of variation, degree of freedom (d.f), sum of squares and Percentage of Variation, and Fst are shown (1000 permutations) 77 Table 3.11 Pairwise Population Comparison of mtDNA haplotypes Fst between Central Interior, Peace River Region and Ontario. Statistical significant (P-value) are shown in parenthesis 77 Table 4.1 Summary of number of samples that were analyzed of each location for all the markers: Tail and Head feathers isotopes 5Df, Microsatellite, and Mitochondrial DNA x 95 Table Al.l Raw data of breeding individuals including: Alleles of eight neutral microsatellites, mtDNA Cytochrome Oxidase I (COI) haplotypes, deuterium Stable Isotopes 5Df (%o) for tail and head feather samples. GPS coordinates were taken per sampling location 107 Table A1.2 Raw data of migratory individuals including: Alleles of eight neutral microsatellites, deuterium Stable Isotopes 8Df (%o) of tail feather samples xi 119 LIST OF FIGURES Figure 1.1 Distribution of breeding (blue) and wintering (red) range of the White-throated Sparrow (Zonotrichia albicollis) in North America. The non-migratory range is shown in purple. The red dot indicates the Central Interior BC population which it not shown in the classical distribution map. Black arrows represent two potential wintering areas used by the Central Interior population. The distribution map was constructed with ArcMaps with layers of passerine distribution ranges obtained from NatureServe (Ridgely et al. 2007) 6 Figure 1.2 Banding recapture records obtained with the collaboration of the USGS Patuxent Wildlife Research Center Bird Banding Laboratory (http://www.pwrc.usgs.gov/bbl/). The map was constructed in Google Earth v.5.2 10 Figure 2.1 Map of sampling locations included in stable isotope analysis. Prince George (PG), Dawson Creek (DC), Sikanni (Sik), Mugaha Marsh (MBO), Rocky Point (RPBO), Vaseux Lake (VLBO), Mount Revelstoke (MRBO), Tattlayoko (TLBO), Lesser Slave Lake (LSLBO) and Beaverhill Bird Observatory (BBO) were included. Sampling locations west from the continental divide were grouped into the Central Interior population (in blue), and east from the divide were grouped as the Peace River Region population (in green). Sampling locations collected during fall migratory season in collaboration with banding station in BC and AB (in yellow) was also included. The number outside the parenthesis represents the amount of sparrows caught during the fall season of 2009 (if available).The xii first number inside parenthesis represent the amount of samples obtained from each location, Second and third numbers represent the amount of tail (T) and head (H) samples analyzed per location. The map was constructed in Google Earth v.5.2 26 Figure 2.2 Stable Isotope precipitation GIS map (6Dp) modified from Meehan et al. (2004). Isotope relief map contour areas from isotope environmental map were modified with adjustment factor of -30 %o to compensate the Hydrogen exchange of 8Df. A layer showing the White-throated Sparrow range was added; obtained from NatureServe (Ridgely et al. 2007). GIS map obtained with permission of Tim Meehan (December 20, 29 2010) Figure 2.3 Percentage of &Df(%o) from head samples (without outliers) binned based on the 8 D p (%o) relief contour areas from the modified Meehan et al. (2004) isotope environmental map (Figure 2.2). A) Central Interior, B) Peace River Region 35 Figure 2.4 Mean &Df (%o) values of head samples with Confidence Intervals 95% (95% CI). A) Central Interior, B) Peace River Region locations are shown xiii 36 Figure 2.5 Percentage of &Df (%o) from tail feather samples (outliers included) binned based on the &DP (%o) relief contour areas from the Meehan et al. (2004) isotope environmental map (modified with -30 %o adjustment). Locations: A) Central Interior, B) The Peace River Region, C) Sikanni River, D) Lesser Slave Lake bird observatory, E) Beaverhill bird observatory, F) Mackenzie bird observatory, and G) Rocky Point bird observatory are shown 39 Figure 2.6 Mean 8Df (%o) values of tail samples with Confidence Intervals 95% (95%CI). Breeding locations: A) Central Interior, B) Peace River Region, C) Sikanni as well as migratory locations: D) Mackenzie, E) Rocky Point, F) Lesser Slave Lake, G) Beaverhill are shown. Bold numbers indicate highly significant differences (P < 0.01) in pairwise comparisons (using Mann Whitney-U), while smaller font not-bold numbers indicate significant differences (P < 0.05) 42 Figure 2.7 Stable Isotope precipitation GIS map (6Dp ) modified from Meehan et al. (2004). Isotope relief patterns were modified with adjustment factor of -30 %o to compensate the hydrogen exchange of 5DF. A layer of showing the White-throated Sparrow range was obtained from NatureServe (Ridgely et al. 2007), and superimposed to the map. Blue circles show the probable wintering sites located east from the Rockies and the red circle shows the possible wintering area west from the Rockies. GIS map obtained with permission of Tim Meehan (December 20, 2010) 46 xiv Figure 3.1 Example of shrub-dominated habitat in Pouce Coupe (BC) where breeding and migratory birds were collected. Birds were attracted to the nest with seeds (autumn) or using a playback (summer) 59 Figure 3.2 Map of sampling locations from collected during breeding season. Prince George (PG), John Prince Research Forest (JPRF), MacLeod Lake (MacL), Tumbler Ridge (TR), Moberly Lake (ML), Dawson Creek (DC), and Sikanni (Sik) were included. Sampling locations west from the continental divide were grouped into the Central Interior population (in blue), and east from the divide were grouped as the Peace River Region population (in green). The first number in parenthesis represents the total amount of individuals collected per location; the second number represents the amount of individuals that were successfully genotyped for microsatellite analysis. Ontario samples were not included in map. Map was constructed with Google Earth v.5.2 60 Figure 3.3 Map of sampling locations collected during fall migratory season in collaboration with banding station in BC and AB. Mugaha Marsh (MBO), Rocky Point (RPBO), Tattlayoko (TLBO), Vaseux Lake (VLBO), Mount Revelstoke (MRBO), Dawson Creekmigratory (DC), Lesser Slave Lake (LSLBO) and Beaverhill Bird Observatory (BBO) were included. The number outside the parenthesis represents the amount of sparrows caught during the fall season of 2009 (if available). The first number inside parenthesis represents the total amount of individuals collected per location; the second number represents the xv amount of individuals that were successfully genotyped for microsatellite analysis. The map was constructed with Google Earth v.5.2 61 Figure 3.4 A) Example of one of the 10 runs of Structure Bar plot with K - 2. Color lines represent probability of regions (1. Central Interior, 2. Peace River Region, 3. Ontario) to be assigned to a population k. B) Example of Tess bar plot of one of the 100 runs with K - 2, Bars represent probability of a individual sample assign to a population. Green bars indicate that all individuals were assigned to the same population 70 Figure 3.5 A) Estimated Logarithm Probability of Data [LnP(D)] plotted against each estimated population (K), calculated with STRUCTURE. B) Deviance Information Criterion (DIC) plotted against each estimated population (K), calculated with TESS 71 Figure 3.6 Statistical Parsimony tree of mtDNA haplotypes based on 17 variable sites of a 461 bp COI gene. Circumference of circles is proportional with haplotype frequency. Colour represent the population where the haplotypes found: Central Interior (red), Peace River Region (Blue) and Ontario (White). Bars represent number of nucleotide changes between haplotypes 76 xvi Figure A2.1 Cytochrome Oxidase I (COI) fragment of the 19 different haplotypes of Whitethroated Sparrow sequences found during the present study at the breeding territories of Western Canada and Ontario 123 xvii CHAPTER 1 GENERAL INTRODUCTION 1.1 MIGRATORY ROUTES AND CONNECTIVITY OF BIRD POPULATIONS The study of migratory behaviour is important for determining the connectivity between breeding and wintering populations. Bird migration has been studied for many years, yet little is known about population specific migratory corridors and routes used by neotropical songbirds in North America. This lack of knowledge is of concern when human development is proposed along suspected migratory routes. In western Canada, increased interest in the development of wind energy is expanding in the northern Rocky Mountains (BC Hydro 2009). As this area also corresponds to the confluence of two migratory corridors, it is important to understand how human-made structures might impact migratory populations. In order to assess this impact, full understanding of the migratory routes and wintering areas used by breeding populations of migrant species is necessary. Once migratory connectivity is more fully understood, it will then be possible to assess the impact that a disruption on a migratory route could have on a specific breeding population. A number of migratory routes have been recognized in North-America (Lincoln 1998). Even though it represents an oversimplification of a more complex situation, migratory routes have been grouped into four general flyways: Pacific, Central, Mississippi and Atlantic. The delimitation of these four migratory flyways has been used as a general tool to understand migratory behaviour. It should be noted that several migratory routes crossover each other and the exact routes change according to species (Lincoln 1998). The Pacific flyway extends 1 through the west from Alaska, following the Pacific coastline of British Columbia, Washington, Oregon, and California. The eastern extension of the Pacific flyway follows the eastern foothills of the northern Rocky Mountains where it eventually turns west heading to Oregon or California through several passes, including the Columbia or the Snake River valleys (Wythe, 1938). The Central flyway extends from breeding locations in the Northwest Territories around the Mackenzie River watershed and then follows the eastern foothills of the Rocky Mountains, where it overlaps with the eastern Pacific flyway. Instead of turning west towards the Pacific coast however, birds continue on the eastern aspect of the Rockies straight through the Great Plains to overwintering locations in Texas and Mexico (Lincoln 1998). The Mississippi flyway also extends from the Northwest Territories, originating at the Mackenzie River delta and continuing unimpeded by mountains for more than 3000 miles to the Mississippi River delta (Lincoln 1998). The Atlantic flyway follows the Atlantic coast to Florida and South America. The Atlantic coast wintering area receives birds from three or four interior migration paths: coastal region south of Delaware Bay; Central Canada coming through the south-easterly path of the Great lakes; Ontario region, following the Ohio river valley or flying south-east crossing the mountains to the Atlantic coast (Lincoln 1998). Determining the migratory behaviour of an avian species can be very complex because birds can fly along the confluence of two or more migratory routes before heading to their final destination. In species' which utilize multiple migratory routes, these migratory overlaps complicate efforts to determine population-specific routes based solely on observational (i.e., banding station) information. However, it is essential to elucidate this complex network in 2 order to better infer which population(s) might be affected when human development is planned along known migratory pathways. A very important aspect of understanding migratory routes and behaviour of avian species is the study and delimitation of migratory divides. A migratory divide is a geographic boundary between two or more breeding populations that follow different migratory routes from each other (Irwin and Irwin 2005). In North America, the Rocky Mountains have long been considered to be an important migratory divide for some species. In northern British Columbia, breeding populations of several songbird species in the Peace River Region and in Central Interior British Columbia lie in a migratory divide (Dunn et al. 2006). Located in this area, however, there are two important passages through the Rocky Mountains (Pine Pass and Peace River) that are used by some migratory birds during fall and spring migrations. These passes may allow populations to breach the migratory divide or lead to areas of migratory overlap, i.e., between the Pacific and Central fly ways. In both cases, movement through a narrow mountain pass could be considered a migratory bottleneck. Migratory bottlenecks located in areas with abundant human structures (e.g. wind farm turbines, power lines) could represent a potential source of mortality for migratory species. Due to high wind speeds in the area, the eastern foothills are currently being developed for wind energy production (Environment Canada 2003). Previous studies have shown that wind farm facilities are a source of mortality for migratory birds. Wind farms in Buffalo Ridge (Minnesota) and Altamount Pass (California) showed an average of approximately 12 3 and 17.5 passerine casualties per year, respectively (Johnson et al. 2002; Smallwood and Thelander 2008). Although these numbers suggest a relatively low collision risk per wind farm, several of these facilities placed between nesting and wintering areas could act as a significant barrier to migration (Drewitt and Langston 2006). As birds will encounter several wind farms (plus several other man-made facilities) during migration, a barrier effect may be created due to these structures either compounding the collision risk, or by displacing migrants from their usual migratory route to a less efficient one, thereby altering fitness and increasing energy costs. In order to have a better understanding of the migratory populations of songbirds in western Canada, one can study a representative species that could be compared with species using similar migratory corridors. The White-throated Sparrow (Zonotrichia albicollis: Emberizidae) was chosen as a representative species because it is a common North American short distance migrant with a widespread distribution. It breeds mainly east of the Rocky Mountains (Campbell et al. 2001; Mazerolle and Hobson 2007), but a population located in the Central Interior of British Columbia (west from the Rockies), that is not included in most species breeding range maps also exists (e.g., Sibley 2000). The genetic relationship of the Central Interior population to those breeding east of the Rockies is unknown; however, preliminary data of song structure indicates some degree of differentiation (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.). There has been much speculation around the migratory behaviour of this population. It has been suggested by several authors (e.g., Campbell et al. 2001; Wythe 1938) that individuals from this population migrate to the disjunct south-western overwintering area of California and 4 Oregon instead of to the main overwintering area in the south-eastern United States (Figure 1.1). The migratory routes used by populations on both sides of the Rocky Mountains are still undetermined. It is also unknown if these birds cross the Rocky Mountains at any point through the several mountain passes under consideration for wind development. The main objective of this study was to examine the genetic relationship and migratory behaviour of the White-throated Sparrow populations in northern BC. By examining the genetic relationships of populations on either side of the presumed migratory divide the degree of spatial genetic structure was assessed. Differences in migratory routes were inferred through the analysis of population specific markers in birds collected at breeding locations and during the autumn migration. Deuterium stable isotopes were used to infer differences in migratory routes and to identify potential wintering areas while molecular markers were used to attempt to genetically assign migratory individuals. Understanding the spatial genetic variation and migratory behaviour of White-throated Sparrow populations will provide useful information for proactive conservation plans. These include: the amount of genetic differentiation that exists between populations, an understanding of how each population could be affected if a migratory route is disrupted or altered, and an indication of how this disruption could affect gene flow between populations. This information may lead to a better understanding of the implications of human development on species that use corridors that cross migratory divides. 5 Figure 1.1 Classical Distribution of breeding (blue) and wintering (red) range of the Whitethroated Sparrow (Zonotrichia albicollis) in North America. The non-migratory range is shown in purple. The red dot indicates the Central Interior BC population which it not shown in the classical distribution map. Black arrows represent two potential wintering areas used by the Central Interior population. The distribution map was constructed with ArcMaps with layers of passerine distribution ranges obtained from NatureServe (Ridgely et al. 2007). 6 1.2 WHITE-THROATED SPARROW The White-throated Sparrow (Zonotrichia albicollis: Emberizidae) is a common North American short distance migrant with a widespread distribution, mainly east of the Rocky Mountains (Campbell et al. 2001; Mazerolle and Hobson 2007). This species exhibits two distinct types of plumages or morphs, white and tan, which originate from a chromosomal polymorphism resulting from a pericentric inversion on the second chromosome (Tuttle 2003). White morph birds are usually heterozygous for the inversion, while tan morph birds are homozygous non-carriers (Tuttle 2003). Phenotypically, both morphs differ from each other based on the brightness in the median and superciliary crown stripes (Tuttle 2003; Campbell et al. 2001). Additionally, polymorphisms coincide with behavioural differences. For instance, white morph birds tend to be more aggressive with less parental care than tan morph birds (Tuttle 2003). Both morphs mate disassortatively (white males mate with tan females and vice versa). The maintenance of the morph polymorphism found within populations has been attributed to disassortative mating (Tuttle 2003; Campbell et al. 2001; Knapton et al.1984). Even though one of the two morphs has been reported to be in higher proportion in some populations, overall both morphs are equally represented through the entire breeding range (Falls and Kopachena 2010). Two hypotheses have been proposed to explain why this negative assortative mating has been maintained (Falls and Kopachena 2010). First, there is a possibility that homozygote individuals for the inversion are selected against as many deleterious mutations may have accumulated in the inverted region. Second, negative assortative mating could be maintained by a combination of different strategies. For instance, white birds tend to be more territorial while tan birds engage in higher levels of parental care. In this case, both strategies could 7 complement each other resulting in a higher fitness for mixed couples than for non-mixed couples that are very territorial but invest little in parental care or vice versa. White-throated Sparrow breeding populations are spread throughout large regions of Canada (Figure 1.1). Historically the distribution was believed to extend east from the slopes of the Rocky Mountains to the Atlantic coast, and north into large portions of the Yukon-Northwest Territories (Campbell et al. 2001). However, in 1919, breeding individuals were reported west of the Rocky Mountains in the Central Interior of British Columbia (Campbell et al. 2001). The origin of this breeding population is unknown, and it currently extends from the town of Mackenzie in the north, to Quesnel in the south, and to Houston, and possibly the Kispiox valley, in the west (Campbell et al. 2001; Wythe 1938). No studies have analyzed the genetic relationship of this population to those found east of the Rocky Mountains. An analysis of song however found population differentiation based on discriminant function analysis (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.). This differentiation was found to be higher when individuals were grouped per region (Central Interior BC, Peace River Region, Alberta) than as sample area. Additionally, misclassified individuals were assigned mostly to geographically close groups, suggesting higher contact between the Peace River Region and Central Interior than between Peace River Region and Alberta or Central Interior and Alberta (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.). 8 Wintering grounds of White-throated Sparrows have been reported mainly in the east and southeast United States, from Minnesota to Maine in the north, along the Gulf of Mexico from Texas to Florida in the south (Campbell et al. 2001). This wintering range has been extended according to observational and banding information that reported the presence of this species in California (Figure 1.1). Initially noted in the first records available in 1888 (Wythe 1938), these reports were first considered "accidental". However, with continued and increased reports in this area, authors like Whyte (1938) began to suspect that thousands of birds could be wintering along the Pacific coast. Reports of wintering sparrows have also been found in the coastal states north of California (i.e., Oregon and Washington). Wythe (1938) and Campbell et al. (2001) include all three states, as well as parts of southwestern British Columbia, as part of the White-throated Sparrow wintering range. Although the exact breeding location of these wintering coastal sparrows still needs to be determined, it has been speculated that the sparrows breeding in the Central Interior of British Columbia are the most likely source of sparrows wintering in this location (Wythe 1938). Compared with other wintering locations, White-throated Sparrows wintering in the Pacific coastal region have been reported to be rare but regular from Oregon to the Mexican border (Kucera 2008). Banding records of recaptures, obtained through literature searches and with the collaboration of the USGS Patuxent Wildlife Research Center Bird Banding Laboratory, as well as the110th Annual Christmas Bird Count, seem to indicate that these birds are found mainly in coastal areas, or centered around northern California (Kucera 2008; Garrison 2008; Wythe 1938; National Audubon Society 2010) (Figure 1.2). 9 vTSPta & rA.T=V 0 ATSPlS-jfl > -3 * Figure 1.2 Banding recapture records in California obtained with the collaboration of the USGS Patuxent Wildlife Research Center Bird Banding Laboratory (http://www.pwrc.usgs.gov/bbl/). The map was constructed in Google Earth v.5.2. 10 1.3 OVERVIEW OF IMPORTANT MARKERS USED TO DELINEATE MIGRATORY POPULATIONS Stable isotopes, molecular markers and banding recaptures are the most commonly used markers to study migratory connectivity between breeding and wintering populations. Each technique has its own pros and cons (Coiffait et al. 2009); however, none of them has been shown to be powerful enough to answer all the questions about bird migration. For this reason, several studies have used different combinations of techniques to understand migratory connectivity (e.g., Boulet and Gibbs 2006; Clegg et al. 2003; Mazerolle and Hobson 2007; Norris et al. 2006). Analysis of stable isotopes is a useful technique to study migratory connectivity of bird populations. The strength of this technique relies on the fact that feathers only grow during a short period of time, after which they become inert. This technique measures isotopes, ingested with food or from the environment, that accumulate in certain tissues (like feathers or claws). Isotope patterns are therefore signatures of the location where feather molting occurred, which generally happens in the breeding or wintering territories (Farmer et al. 2008). The most important disadvantages associated with stable isotopes (compared with other methodologies) are the effects of climatic variation and local environmental factors on the observed ratios. Hydrogen/deuterium presents a latitudinal gradient based on precipitation and altitude, so its utility as a marker depends also on the resolution of environmental isotope 11 maps available (Clegg et al. 2003). Isotope maps, however are based on long term averages. Year to year variation has been attributed to the amount of hydrogen exchanged between the feather and ambient water vapour in a given year (Mazerolle et al. 2005). To compensate for yearly variation, a discrimination factor is usually added to the predicted precipitation isotope ratios (Mazerolle et al. 2005). In addition to seasonal climatic changes in precipitation patterns, local anthropogenic factors, causing differences in moisture regimes, such as logging, can add variability to isotopes ratios (Coiffait et al. 2009), Other factors that can also increase local variability are the differences in proportions of isotopes absorbed, isotope fractionation, among organisms caused by different biological, physical or chemical processes (UGSS 2012). Isotopic fractionation can have great influence on the variability of isotope markers. For instance, differences in the diet (prey sources) between juveniles and adults have been suggested as a major cause of local variation in isotopic ratios of hydrogen (Langin et al. 2007). Another important tool for migratory connectivity studies are molecular markers. This diverse group of markers has proven to be very useful in differentiating east versus west populations that are normally located near a migratory divide (e.g., Zink 1994; Zink 2008; Lecomte et al. 2009; Perez-Tris et al. 2004). Nuclear markers such as microsatellites (e .g., Burg and Croxall 2001) and mitochondrial DNA sequences (e.g., Perez-Tris et al. 2004) are the two techniques most widely used for population genetic analysis. 12 Both of these techniques have their own advantages. On one hand, neutral microsatellites are codominant markers that can be very useful to determine population structure and genetic diversity due to their high mutation rate (Clegg et al. 2003). On the other hand, mitochondrial markers do not undergo recombination, and because of their relatively lower effective population size and maternal inheritance, can be very useful when studying populations that have differentiated recently in time (Zink 2008). The main disadvantage of genetic markers in connectivity studies is the dependence on finding a strong differentiation between populations. The degree of population structure observed depends on barriers to reproduction and on the time of divergence between populations. Genetic differentiation is an important prerequisite to effectively use a population assignment method to infer the breeding location of individuals collected along migratory routes or on wintering grounds. In order to find this differentiation, it may be necessary to develop a high number of markers, which can be difficult and time consuming in the case of non-model species. Additionally, finding population structure can be problematic in cases where avian species originate from a recent expansion from a single refugium (e.g., Davis et al. 2006). Other markers widely used in connectivity studies are leg/neck band recoveries. This is a cost-effective, widely-used method. Throughout North America, a large network of banding stations exists to aid in the study of bird migration and conservation. The biggest limitation with this technique is that, even in the most abundant species, low numbers of recaptures are 13 recovered (Mazerolle et al. 2005; Butler et al. 1996). However in spite of this limitation, several cases have shown that leg band recoveries provide useful information finding migratory differentiation when other techniques fail. For instance, Mazerolle et al.(2005), used hydrogen isotopes from tail and head feathers (collected in Delta Marsh banding station) in order to determine the breeding and wintering areas of White-throated Sparrows. In their study, stable isotope analysis was uninformative between estimated breeding and wintering areas (head and tail feather isotope ratios), while leg banding records showed that birds from breeding populations from western Canada (Alberta, Manitoba, Saskatchewan and British Columbia) wintered in more westerly locations than birds in central (Ontario and Quebec) and eastern (New Brunswick, Nova Scotia, Prince Edward Island, Newfoundland, and Labrador) Canada. In many cases, leg band recoveries are used to complement information from other markers. For example, Smith and colleagues (2003) used stable isotopes and leg band recoveries to study the spatial and temporal patterns of migration in sharp-shinned hawks. The stable isotope analysis data showed that early migrants passing through the migration site were from lower latitudes, while leg-band information indicated that these early migrants winter further south than birds passing later. These combined data suggest that sharp-shinned hawks use a chain migration pattern instead of leap-frog migration and illustrates the power of multiple markers to gain a full understanding of migratory behaviour. 14 1.4 MIGRATORY FLYWAYS AND BANDING RECOVERIES The migratory connectivity of White-throated Sparrows has been examined in only a few studies. Deuterium stable isotope samples from the Delta Marsh banding station in Manitoba, located in the Mississippi flyway, were used to determine the southeastern United States as the main wintering area for the birds passing through this migratory station (Mazerolle et al. 2005). This study was unable to find differentiation between estimated breeding (measured by tail feather isotope ratio) and wintering (measured by head feather isotope ratio) isotopic ratios. Banding recoveries, however, showed a slight east/west differentiation in wintering areas used by western (Northeastern British Columbia, Alberta and Saskatchewan) and the rest of the eastern breeding populations (Mazerolle et al. 2005). It is important, however, to state that this study, as well as others, has not been performed on birds breeding in the far western portions of the range, specifically the Central Interior population of BC. It has long been speculated that there is a link between the western wintering distribution (California and Texas) and the western breeding distribution (Central Interior British Columbia and western Alberta/Peace River Region). Wythe (1938) hypothesised that sparrows breeding at the western limit of the range (described until 1938, i.e. Alberta/Peace River Region) could be wintering in Texas (possibly using the Central flyway). Additionally, he suggested that birds breeding in the Central Interior could be using a Rocky Mountain passage to cross the Rocky Mountains and then head southwest from Alberta to California (using for instance the Columbia River flyway). No studies, however, have provided detailed information on migratory routes used by sparrows breeding at the western distribution of the species range that would allow an examination of these hypotheses. 15 The Yellowhead, Peace River and Pine Passes are described by Wythe (1938) as three possible passages used by White-throated Sparrows for fall migration. The Yellowhead Pass elevation is 975 m, the Peace River 600 m and the Pine Pass has an elevation of 869 m above sea level. Wythe (1938) suggested the Pine Pass/Peace River as possible passages used by the White-throated Sparrows that founded the first breeding population in Central Interior BC. In this hypothesis, White-throated Sparrows could have arrived when birds (normally breeding east of the Rockies) deflected from their usual migratory path, and followed the Peace River system or Pine Pass until they found suitable habitats in the Central Interior region (Wythe 1938). This hypothesis would predict a close genetic relationship between the populations on either side of the continental divide and a shared post-glacial history. It is also possible that birds in the Central Interior represent a much older population with an independent postglacial history, i.e., have separate refiigia. Although evidence of breeding was first described in 1919, this date corresponds with the major influx of European settlement in the area. Historic breeding populations may have simple gone unrecorded until this date. 1.5 BANDING STATION INFORMATION Another important source of information for understanding migratory behaviour is the banding records from banding stations across western Canada. From the banding stations that are located in BC and Alberta, eight collaborated with the present study. Five of these 16 stations arc located in British Columbia (Mackenzie, Tattlayoko, Revelstoke, Rocky Point, Vaseux Lake) and two in Alberta (Lesser Slave lake and Beaverhill). As expected from the species distribution maps, the Alberta banding stations reported the highest numbers of White-throated Sparrows during the Autumn migration. Beaverhill reports a total of 110 White-throated Sparrows (from 1997-2006) during the fall, and an average of 11 birds per fall (Priestley 2007), while Lesser Slave Lake reports a total of 161 White-throated Sparrows (from 2005-2008) during the fall, and an average of 40.25 birds per fall (Krikun 2005; 2006; 2007; 2008). White-throated Sparrows are regularly reported in these banding stations in the top 10 list of species captured (Krikun 2005; 2006; 2007; 2008; Priestley 2007). The Mackenzie Nature Observatory reported the highest numbers of White-throated Sparrows in British Columbia. From 1995 -2009 this station reported a total of 170 Whitethroated Sparrows, with an average of 11.33 birds per year each fall (note, reports from 1995-1997 come from a smaller number of nets used) (Mackenzie Nature Observatory 2009). Banding stations in the southern part of BC reported fewer numbers of sparrows. For instance, Rocky Point Bird Observatory reported only 38 banded/45 observed White-throated Sparrows from 1994 to 2009 (reports in 1998 and 2007 were not available) (Melcer and Nightingale 2009; David 2006; 2008; Jantunen 2003; 2004; Gibson 2002; Derbyshine 1999; 2000). Only 3.5 birds per year where observed during this range of time with most individuals banded between mid-September to late-October. 17 Other stations in the south part of the province, such as Tatlayoko, Revelstoke and Vaseux Lake Bird Observatories, rarely report captures of White-throated Sparrows. Tatlayoko, for instance, had an average of one bird banded (or observed) per year, from 2006 to 2009 (Ogle 2008; 2009a; 2009b). Vaseux Lake reports the same average from 2002 to 2009; however, their captures year-to-year are more inconsistent than Tatlayoko with a high of 3 sparrows captured in 2005 and a low of no sparrows captured in 2004, 2006, and 2007 (eBird Canada 2010). 1.6 RESEARCH OBJECTIVES AND THESIS ORGANIZATION The main objective of the present study was to investigate the genetic and migratory differences among White-throated Sparrow populations in western Canada. This information is needed to address hypotheses on the location of a migratory divide and on population specific migration routes that, in the context of ongoing development, can ultimately be used for proactive management. To do this, the differential use of migratory routes was inferred by analyzing the migratory connectivity between breeding populations and overwintering areas (Chapter 2), as well as the spatial genetic structure of breeding populations in Western Canada (Chapter 3). Chapter two compares natural ratios of deuterium in White-throated Sparrow head and tail feathers (5DF) with the latitudinal gradient displayed by deuterium isotope concentrations in precipitation (8D p) in order to identify wintering grounds used by breeding populations in 18 British Columbia on either side of the continental divide. Head feathers 5Dfwere used to infer the wintering grounds used by birds from known breeding populations, while tail feathers 6Df were used to infer breeding areas used by migratory sparrows in order to infer migratory routes used by those birds. Chapter three uses neutral microsatellite markers and mitochondrial DNA sequences to determine the amount of genetic differentiation among breeding populations of Whitethroated Sparrows in western Canada. This genetic structure was then used to attempt to assign migratory individuals captured with the collaboration of migratory banding stations across BC and Alberta. This technique could then allow us to establish routes of importance for many populations of White-throated Sparrows and help determine how different populations of these birds could be contributing to the mixed genetic stock found in each migratory corridor. Chapter four is a synthesis of the above information that indicates the utility of these techniques for proactive management of migratory bird species. This chapter also performs an assessment of the techniques used, describing their applicability to other species and how they can be improved. Additionally, this chapter shows how the present study contributed to the understanding of the migratory behaviour of White-throated Sparrows and what are the environmental and management implications that could be estimated based on the information obtained. 19 CHAPTER 2 DETERMINATION OF MIGRATORY CONNECTIVITY AND MIGRATORY PATTERNS USED BY WHITE-THROATED SPARROWS IN WESTERN CANADA USING DEUTERIUM STABLE ISOTOPES 2.1 INTRODUCTION Bird migration and the connectivity between breeding and wintering populations has been a central area of interest in ecological and evolutionary studies. Detailed knowledge of the use of migratory corridors and wintering areas by songbirds is important for understanding both their ecology as well as in the development of appropriate conservation plans for maintaining breeding populations. Such plans rely on an understanding of the connectivity between summer/winter grounds of individual populations and the potential impact that human-made structures may have as a result of altering or disrupting migratory routes. However, due to the inherent difficulty of tracking birds during migration, the study of migratory connectivity has been severely obstructed (Webster et al. 2002). The Peace River Region of British Columbia is one area of potential importance for bird migration and which is situated in a site of increasing human development (i.e., wind energy). The Peace River Region is located on the east side of the Continental Divide of the Americas, marked in this region by the presence of the Rocky Mountains. This region of the Continental Divide has also been associated with an important migratory divide for several avian species (Dunn et al. 2006). This region also has several passes that connect both sides 20 of the divide that are used by migratory birds. These passes are migratory bottlenecks for birds crossing both sides of the mountains. It is not known how human development in this area might affect migration. Depending on population specific migrations routes, bottlenecks could affect one or both of the populations on either side of the migratory divide. The White-throated Sparrow {Zonotrichia albicollis) is a common North American short distance migrant generally found east of the Continental Divide. Differences in song structure have been noted for a breeding population located west of the divide in the Central Interior of BC to those located east of the divide (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray B.). It has also been proposed that the Central Interior population has a different migratory route and overwintering area than those found east of the divide (Whyte 1938; Campbell et al. 2001). Details on population specific migratory routes, however, are lacking and hypotheses of migratory connectivity have yet to be tested. Understanding population specific migratory connectivity and routes on both sides of the Continental Divide (also possibly a migratory divide for this species) is crucial in order to identify how these populations could be affected by human development. Unlike to more traditional markers, such as leg/neck banding, data collection is relatively rapid and does not suffer from the limitations associated with the low number of band recoveries (Coiffait et al. 2009). Stable isotopes are variants of individual atomic elements, which differ in the number of neutrons and therefore have unique atomic masses (Coiffait et al. 2009). The relative proportions of isotope that accumulate in growing tissue vary 21 according to the environment and diet. In birds, the use of isotopes to identify wintering or breeding habitats has exploited the fact that specific body tissues, such as feathers or claws, incorporate different isotope signatures from the environment (e.g. rain or diet) during the period of their growth. These tissues then become metabolically inert, preventing the isotopic signatures from fluctuating over time (Mazerolle et al. 2005). These characteristics have been very useful for determining animal movements across a landscape, especially across latitudinal migratory patterns (e.g., Clegg et al. 2003; Smith et al. 2003; Wassenaar and Hobson 2001; Hobson and Wassenaar 1997). Hydrogen/Deuterium has been one of the most successful isotopes in bird studies, showing a clear latitudinal differentiation in several studies (e.g., Mazerolle et al. 2005; Clegg et al. 2003; Smith et al. 2003; Wassenaar and Hobson 2001; Hobson and Wassenaar 1997). Hydrogen isotopes (or deuterium 5D) have also been shown to be associated with rainfall patterns and other environmental variables. Levels of Deuterium seem to follow a latitudinal pattern decreasing at higher latitudes, elevations, and towards the continental interior (Clegg et al. 2003). However, temporal changes in climatic conditions and diet of birds can add variation to the observed isotope ratios (Wassenaar and Hobson 2006). The discrimination power of this element depends on the resolution of the environmental isotope map available (Clegg et al. 2003). These environmental maps are based on a 40-year average of both geographical and climatological isotope information available from the Global Network for Isotopes in Precipitation (GNIP) database. These models then use a 22 "kriging" statistical procedure to interpolate isotope data of unknown locations from known locations. In this method, a georeferenced isotope precipitation profile (5Dp) map is used to infer the location where tissue growth occurred based on the tissue, i.e., feather, isotope profile (&Df) (Meehan et al. 2004). Deuterium stable isotopes are particularly useful in White-throated Sparrows because these birds have two differential moults that happen during different parts of the season. The crown and tail feathers of this sparrow have different periods of growth that provide isotopic signatures of the breeding and winter grounds, respectively. In the first moult (on breeding grounds before fall migration) all feathers are replaced, while at the second moult (on wintering grounds before spring migration) only the body feathers in the head region are replaced (Mazerolle et al. 2005). Although this differential moult pattern is not present in all avian species, in the present study this characteristic was useful to determine wintering territories used by breeding individuals as well as to determine potential breeding areas used by migratory individuals. The first objective of this study was to use the head feather 5DF to establish the wintering areas used by western Canadian populations of White-throated Sparrows on both sides of the Continental Divide (i.e., Central Interior BC and Peace River Region). The second objective was to use the tail feather 5Df to estimate the most probable breeding grounds used by migratory individuals sampled at stopover sites and banding stations across British Columbia 23 and Alberta. These studies will provide evidence on the presence of a migratory divide and locations of migratory bottlenecks in northern British Columbia as well as the potential impact that human-made structures can have on the disruption of migratory routes of Whitethroated Sparrows. Elucidation of population's specific migratory routes and wintering areas used by White-throated Sparrows are vital in order to delineate future conservation projects in which important areas for bird migration could be conserved, not only for these songbirds, but also for other species using similar migratory corridors. 24 2.2 METHODOLOGY 2.2.1 STUDY AREA AND SAMPLE COLLECTION Standard mist-net technique was used to capture White-throated Sparrows from breeding and migratory locations (Figure 2.1). For most breeding birds captured (May2009- June 2010) in Prince George (n = 20) (19 tail and 18 head) and Dawson Creek (n = 27) (26 tail and 26 head) both tail and head feathers were analyzed. In addition, tail samples from three breeding individuals (collected May 2009) from Sikanni River (most northern location) were included for calibration purposes. Seven banding stations across British Columbia (BC) and Alberta (AB) collaborated to sample migratory White-throated Sparrows (July - Sept 2009). The frequency of White-throated Sparrows sampled varied according to the banding station; with no sparrows being captured at Vaseux Lake, Tatlayoko, and Revelstoke, three samples from Rocky Point, nine from Mugaha Marsh, nine from Lesser Slave Lake and seven from Beaverhill banding station. 25 Sik (3T) * "s „^, w fi: 2311 Google : ' image G 2011 TeTaMeif .cs ® .j,'« . •: 5.2011 Goes.'Spct image 52 29'32 40" N 117 01' 12 8?" O elev 1361 m w •wt : v'fcJbogle eat Ait OjO ! 200 74 km Figure 2.1 Map of sampling locations included in stable isotope analysis. Prince George (PG), Dawson Creek (DC), Sikanni (Sik), Mugaha Marsh (MBO), Rocky Point (RPBO), Vaseux Lake (VLBO), Mount Revelstoke (MRBO), Tattlayoko (TLBO), Lesser Slave Lake (LSLBO) and Beaverhill Bird Observatory (BBO) were included. Sampling locations west from the continental divide were grouped into the Central Interior population (in blue), and east from the divide were grouped as the Peace River Region population (in green). Sampling locations collected during fall migratory season in collaboration with banding station in BC and AB (in yellow) was also included. The number outside the parenthesis represents the amount of sparrows caught during the fall season of 2009 (if available).The first number inside parenthesis represent the number of samples obtained from each location, Second and third numbers represent the amount of tail (T) and head (H) samples analyzed per location. The map was constructed in Google Earth v.5.2. 26 2.2.2 SAMPLE PREPARATION AND ANALYSIS Tail and feather samples were subjected to three rounds of cleaning using a 2:1 chloroform:methanol solution, rinsed with MilliQ H 2 0, and air-dried for 24 hours under the fumehood (Wassenaar and Hobson 2006; Mazerolle et al. 2005). Entire head feathers and the terminal veins of the tail feathers were sub-sampled in the same location to avoid inconsistent periods of feather growth (Wassenaar and Hobson 2006). Subsamples were weighed until they reached an optimum weight (0.1 - 0.3 mg) and then packed into silver capsules (Isomass Scientific Inc). Silver capsules containing the feather samples were crushed into small balls and inserted in a 96-well PCR tray. Non-exchangable Hydrogen (5Df) of feathers was analyzed using a Hekatech HT Oxygen Analyzer interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK) at the University of California, Davis. The fraction of exchangeable hydrogen between feathers and environment was corrected using keratin standards as described by Wassenaar and Hobson (2003). Deuterium concentration was expressed in units per ml (%o) relative to the international standard V-SMOW (Vienna Standard Mean Ocean Water). To estimate the migratory areas used by White-throated Sparrows breeding populations, nonexchangeable feather deuterium isotope ratio values (6Df) were compared with the precipitation deuterium isotope ratio values (5D P ) for North America. However, &DF values are often different from 8D p values [because of different variables, such as, fractionation factors or the percentage of Hydrogen that is exchangeable with the environment (Farmer et al. 2008)]. 27 Forty per cent of the feather hydrogen is potentially exchangeable with the environment (Hobson and Wassenaar 1997). To adjust for amount of hydrogen exchanged with the environment, a correction factor was used to modify the mean growing season precipitation (5Dp) map of Meehan et al (2004) (Figure 2.2). This map is based on a 40-year average of geographical (sometimes altitudinal data) and climatologically isotope information available at the Global Network for Isotopes in Precipitation (GNIP) database. To estimate the correction factor to be used, &DF values of tail feathers from individuals captured at known breeding locations (Prince George, Dawson Creek and Sikanni) were compared with predicted precipitation isotopes for those locations. Based on these comparisons, an adjustment factor -30%o was used to calibrate &DP precipitation values. Predicted precipitation isotopes were obtained using the Online Isotopes in Precipitation Calculator OIPC v2.2 (Bowen G.J 2011). The modified Meehan et al. (2004) GIS map layer was combined with a White-throated Sparrow range layer available on the NatureServe web site (Ridgely et al. 2007) in order to estimate isotope values for breeding (tail feathers) and wintering (head feathers) areas. These values were compared to observed &DF ratio of the White-throated Sparrow feathers. 28 : Oto-54 -55 to-63 -6910-83 : -84 to-98 : -99to-114 Wintering Rang. 115 to-129 L301O-145 14610-160 : -16110-173 : -17410-209 Figure 2.2 Stable Isotope precipitation GIS map (5D p) modified from Meehan et al. (2004). Isotope relief map contour areas from isotope environmental map were modified with adjustment factor of -30 %o to compensate the Hydrogen exchange of 5Df. A layer showing the White-throated Sparrow range was added; obtained from NatureServe (Ridgely et al. 2007). GIS map obtained with permission of Tim Meehan (December 20, 2010). 29 2.2.3 STATISTICAL ANALYSIS All statistical analyses were done with SPSS v.18.0 with and without outliers. Outliers were determined using a stem-and-leaf plot and according to reported wintering and breeding ranges in Western Canada (>-130 %o 5Df for wintering samples and < -131 %o &Df for breeding samples). Once outliers were detected they were removed for all subsequent analysis. Descriptive statistics (Mean, Standard Deviation and Confidence Intervals) were calculated and plotted. Frequency distribution of tail and head feathers 5Df values (%o) were plotted in a bar graph against corrected 6D p ranges (based on Meehan et al. 2004 precipitation map). Normality of samples for each sampling location was assessed using a Kolmogorov-Smirnov test. Head feather isotope ratios were analyzed with parametric one­ way ANOVA and t-tests. To test statistical differences of tail feather isotope ratios, an initial comparison between all locations was done using Kruskal-Wallis tests. After this analysis, pairwise comparisons between Central Interior, Peace River Region and the banding stations were analyzed using non-parametric Mann-Whitney-U test. Additionally, in order to test if the date when birds were banded had any influence on the isotope ratios, ANCOVA analysis of banding stations data with date as a covariate was conducted. 30 2.3 RESULTS 2.3.1 DISTRIBUTION AND OUTLIERS Most stable isotopes data from head and tail samples fit a normal distribution (P>0.5) (Kolmogorov-Smirnov for one sample test). The only sample location that showed significant deviation from a normal distribution was the tail-feather samples from the Peace River Region (0.01> P <0.05). As a result of this deviation from normality, tail-feather samples were analyzed using non-parametric statistics while head-feather samples were analyzed with parametric (ANOVA) statistics. Analysis of tail samples showed three values of 5Df values (-85%o, -89%o and -130%o) as clear outliers (Table 2.1a, b). These values lie outside of the values noted for the breeding distribution of White-throated Sparrows in western North America. Head-feather analysis failed to show statistical outliers; however, as four of the lowest values (from -137.2%o to 145.9%o) clearly mapped outside of the wintering area, they were considered as outliers (Table 2.1c). Statistical analyses were done with and without outliers. However, as excluding outliers from analysis did not significantly change the outcome of the study all the analysis shown in the present study was done without outliers. 31 Table 2.1 A) Five highest and lowest &Df (%o) values of Central Interior tail feather samples. B) Five highest and lowest 5Df (%o) values of Peace River Region tail feather samples. C) Five highest and lowest 5Df (%o) values of head feather samples of all regions. A) Highest Lowest Relative Rank 1 2 3 4 5 5 4 3 2 1 Individual Zoal-jal74 Zoal-jal77 Zoal-jal76 Zoal-jal70 Zoal-jal71 Zoal-jal83 Zoal-jal73 Zoal-jal75 Zoal-jal88 Zoal-jal82 6D f (%0) Relative Rank 1 2 3 4 5 5 4 3 2 1 Individual Zoal-jdl57 Zoal-jdl47 Zoal-jdl45 Zoal-jdl56 Zoal-jdl50 Zoal-jdl55 Zoal-jdl54 Zoal-jdl37 Zoal-jdl38 Zoal-jdl60 8D f (%«) -89.8 -137.9 -138 -139.2 -139.6 -151 -151.4 -152.3 -156.7 -156.8 Included or Excluded Excluded Excluded Included Included Included Included Included Included Included Included B) Highest Lowest 32 -85.1 -130 -138.3 -147.5 -148.6 -162.2 -163.6 -164.3 -165.6 -167.6 Included or Excluded Excluded Excluded Included Included Included Included Included Included Included Included C) Highest Lowest Relative Rank 1 2 3 4 5 4 3 2 1 5 Individual Zoal-jdl41 Zoal-jdl53 Zoal-jdl60 Zoal-jdl56 Zoal-jal78 Zoal-jal73 Zoal-jdl48 Zoal-jd060 Zoal-jal88 Zoal-jdl46 33 6Df (%o) -54.3 -55.2 -58.7 -59.1 -60.4 -123.2 -137.2 -138 -138.7 -145.9 Included or Excluded Included Included Included Included Included Included Excluded Excluded Excluded Excluded 2.3.2 HEAD FEATHER SAMPLE ANALYSIS Head feather 5Df (%o) values for each sampling location were binned (Figure 2.3) according to the relief map countour lines (Figure 2.2). The Central Interior samples &Df (%o) values were mainly distributed (-40%) between -69 - -83 %o, while the remaining samples were equally distributed in a lower percentage across four other areas (Figure 2.3). The Peace River Region had 5Df (%o) values distributed across five different relief map contour areas, with higher percentages (-30%) between -69 - -83 %o and -99 - -114 %o (Figure 2.3). Headfeather samples showed a high standard deviation, which was twice as much as the standard deviation obtained from tail-feather samples. Central Interior head samples had a mean 5Df (%o) and standard deviation (-84.12±18.16 %o) lower than Peace River Region (-90.33±21.46 %o). No statistical differences were detected using ANOVA (F: 0.931, P: 0.341) or T-tests (F: 1.47, P: 0.233). Confidence intervals at 95% of head feather samples were also much greater than tail feather samples (Figure 2.4). 34 A) Central Interior Head samples 6D(%oVSMOW) Peace Region Head samples 50 . 40 j 6D(%oVSMOW) Figure 2.3 Percentage of &Df(%o) from head samples (without outliers) binned based on the 5DP (%0) relief contour areas from the modified Meehan et al. (2004) isotope environmental map (Figure 2.2). A) Central Interior, B) Peace River Region -70,00- -80.00" o •o c <3 2 -90.00- -100.00- -110.00- T A Location Error Bars: 95% CI Figure 2.4 Mean 5Df (%o) values of head samples with Confidence Intervals 95% (95% CI) bars. A) Central Interior and B) Peace River Region locations are shown. 36 2.3.3 TAIL FEATHER SAMPLE ANALYSIS To assess the distribution of data, tail feather 5Df (%o) values were also compared according to the &D P (%o) value relief areas (Figure 2.5) from the Meehan et al. (2004) isotope environmental map (Figure 2.2). All Central Interior sample &Df (%o) values were distributed between -130 %o - -160 %o zones (Figure 2.5A). In contrast the Peace River and Sikanni samples were distributed primarily in the -146 - -173 %o zones (Figure 2.5, B+C). Tail isotope values distribution from Lesser Slave Lake (LSLBO), Beaverhill (BBO) and Mackenzie (MBO) samples were mostly spread between -146 - -160%o, and -161 - -173%o while Rocky Point (RPBO) distributed in the -146 —160%o zone (Figure 2.5, D-G). 37 Breeding Locations Central Interior- Tail samples A) 80 £ 60 W £ 40 CL 20 ^ ID CO fO co in m lo ^ CO in o rn o> rH-f IN rf N O rH r-t 0 r-H r-H CsJ ^ . , oo a> u*> O m lO r-l r-»' M _i _-i 6D(%oVSMOW) B) Peace Region-Tail samples 80 c 60 QJ &40 20 0 ^ ,v V ^ 3 £>' <2>& J$> ^ y $ $ ?P >V 6D(%oVSMOW) 38 Migratory Locations 0) E) Lesser Slave Lake- Tail samples Beaverhill- Tail samples so so - 50 |40 ^ 20 0 v? j9> s?> & - «S» * II J? N# 60 1 8 40 ^ 20 0 -f J* J 1 J 1 cfDCwVSMOW) Mackenzie- Tail samples qj 80 Rocky Point- Tail samples iOO so . ~ 60 i 60 3 40 £ •$> •? ^ * * * #' «j£* cfDt^VSMOWj F) II , ll % ft. 40 •••: 20 i o ! * * * * *^N*- s>•,v*VVV <#> <£> <$> s \ v <*? ^ „<§> * * * * «r>V>VV' & #? tffi(*«VSMOW) dD 0.5) between banding stations isotope ratios with collection date as a covariant. 40 -140 00- 12345 •150 00- 16 36 a "O c rs jg -160 00" •170 004 h Breeding Locations Migratory Locations -180 00- "T 0 Location Error Bars: 95% CI Figure 2.6 Mean 5Df (%o) values of tail samples with Confidence Intervals 95% (95%CI). Breeding locations: A) Central Interior, B) Peace River Region, C) Sikanni, as well as migratory locations: D) Mackenzie, E) Rocky Point, F) Lesser Slave Lake, G) Beaverhill are shown. Bold numbers indicate highly significant differences (P <0.01) in pairwise comparisons (using Mann Whitney-U), while smaller font not-bold numbers indicate significant differences (P < 0.05). 41 2.4 DISCUSSION 2.4.1 DETERMINATION OF WINTERING TERRITORIES BASED ON HEAD FEATHER DEUTERIUM ISOTOPES Head feathers isotope values (6Df) presented a greater range of values compared with tail feather isotope values. This variability may reflect the higher complexity of the isotope 6DP relief patterns at the wintering range than at the breeding range and/or may be further influenced by the differential migration by males and females. Differential migration of both sexes, where females migrate longer (have more positive 8Df values) than males has been noted in White-throated Sparrows (Mazerolle and Hobson 2007) and could partially explain the wide distribution of head-feather samples. Differential migration could not be fully tested in this study because in many samples the sex of the bird could not be determined in the field. An alternative to avoid the difficulties of visual determination of sex in sparrows would be using molecular techniques for sex discrimination (such as the CHD gene amplification) for more detailed examination of sex specific migratory patterns (Dubiec and ZagalskaNeubauer 2006). However if present, the effect of sex specific migration patterns would appear minimal as when the small number of known males were tested statistically (not shown) no differences were observed with the overall mean. The timing of moults can also influence the range of values observed. If a proportion of the birds moult during migration then the range of values might not only reflect the isotopic signal of wintering sites but those of stopover locations. This effect, however, was not noted in previous studies of White-throated Sparrows, which have shown that less than 4% of 42 individuals had not completed their prealternate moult when banded at spring migration monitoring banding stations (e.g., Mazerolle et al. 2005). Additionally, head feather samples compared with claws and blood samples were reported as the most accurate and consistent samples used for isotopes studies (Mazerolle and Hobson 2005). Head feather stable isotope values did not show significant differences between the Peace River Region and Central Interior populations. Both populations, however, had higher isotopic signatures than what were expected if they would be wintering in the southeastern US (Figure 2.2). The southeastern US (east of Great Plains) has been described as the main wintering ground used by White-throated Sparrows (Campbell et al. 2001; Falls and Kopachena 2010). Stable isotope analysis has also identified this area as the main wintering ground (expected 5Df between 0 and ~68%o) used by migratory White-throated Sparrows captured at the centrally located Delta Marsh (Manitoba) banding station (Mazerolle et al. 2005). In contrast, the mean value of head feather 5Df observed in Central Interior and Peace River Region populations fit with the upper limit of the wintering range, limiting possible wintering areas used by these populations to three locations: The Pacific coast of the United States, New Mexico-Arizona and Colorado/Kansas (Figure 2.7). Previous studies have suggested that the Pacific coast is the most likely wintering area for breeding population of the Central Interior (Campbell et al. 2001; Wythe 1938). However, due to the overlap in expected isotope values we cannot differentiate among the Pacific coast 43 or the New Mexico-Arizona and Colorado/Kansas areas. In the Pacific wintering area, most White-throated Sparrows have been observed in the northern California coastal areas (e.g. San Francisco); although sparrows have been observed wintering in Washington and Oregon coasts and southeastern Vancouver Island (Kucera 2008; Garrison 2008; Wythe 1938; National Audubon Society 2010) (Figure 1.2). Very few birds have been observed wintering in the Sonora desert and at higher altitude in the Sierra Nevada Mountains (National Audubon Society 2010). This distribution predicts that the birds wintering along the Pacific coast should have a narrower isotope profile than birds wintering at the New Mexico/Arizona and Colorado/Kansas area (Figure 2.7). The Central Interior population does have a narrower isotope distribution than the Peace River Region, suggesting that this population could be wintering in the Pacific coastal region (Figure 2.3). The most parsimonious hypothesis for wintering areas used by the Peace River Region population would be that these birds are migrating to the New Mexico-Arizona and Colorado/Kansas region. Because of the proximity to the southern Rocky Mountains (altitude effect on 6Dp), these two regions present a wider range of isotope values (Figure 2.7), which seems to fit the pattern observed in the isotope distribution present at this population (Figure 2.3). Note also that if at least some of these birds are wintering in more southern locations (i.e. northwest Texas), it would also explain the higher variance in this sample location. White-throated Sparrows band recapture information suggests an east-west segregation at wintering grounds of White-throated Sparrows (Mazerolle et al. 2005). The 5DF values of 44 head feathers of western Canadian populations obtained in the present study (Figure 2.4), as well as those examined in Delta Marsh Manitoba (Mazerolle et al. 2005), are congruent with an east-west segregation on the wintering grounds, in which eastern birds migrate towards southeast US, and western birds (Central interior and Peace River Region) migrate to either the Pacific west coast or to New Mexico/Arizona and Colorado/Kansas areas. These data suggest a general trend of parallel migration in North America and is consistent with the connectivity of the Central Interior with the Pacific coast and the Peace River Region with the New Mexico/Arizona and Colorado/Kansas areas. 45 Figure 2.7 Stable Isotope precipitation GIS map (5DP) modified from Meehan et al. (2004). Isotope relief patterns were modified with adjustment factor of -30 %o to compensate the hydrogen exchange of 5Df. A layer of showing the White-throated Sparrow range was obtained from NatureServe (Ridgely et al. 2007), and superimposed to the map. Blue circles show the probable wintering sites located east from the Rockies and the red circle shows the possible wintering area west from the Rockies. GIS map obtained with permission of Tim Meehan (December 20, 2010) 46 2.4.2 DETERMINATION OF BREEDING TERRITORIES BASED ON TAIL FEATHER DEUTERIUM ISOTOPES Analysis of tail-feather isotopes showed significant statistical differences among breeding sample locations. These differences were maintained when outliers were included, showing that extreme values on the distribution did not affect the results. Notably, significant statistical differences were seen between the Peace River Region and Central Interior populations. This distribution is expected and is consistent with the predicted geographical distribution of isotope values for each sampling location (Figure 2.2), where the mean value in Central Interior (-146 ±6.22 %o SD) is 8%o higher than the Peace River Region (-154 ±7.01 %o SD) and 12%o higher than Sikanni River is (-158 ±2.87 % 0 SD) (Figure 2.6). The 95% confidence intervals of Central Interior and Peace River Regions do not overlap, reflecting a high degree of confidence in assigning samples to either of these locations. This lack of overlap in confidence intervals is unexpected as a high degree of variance associated with within-population estimates has been recorded for some species (Langin et al. 2007). For instance, Farmer and colleagues (2008) estimated that it is necessary to have a difference of 31%o in order to assign confidently two samples as coming from distinct latitudes. However, the confidence intervals at 95% values from Central Interior and Peace River Region showed that the 31%o value suggested by Farmer and colleagues (2008) might not apply for these populations. The banding stations in Alberta are important for the present study as they potentially capture White-throated Sparrow migrants using either the Central, Mississippi, or eastern Pacific 47 (Columbia River) flyways. Lesser Slave Lake Bird Observatory, located on the eastern shore of Lesser Slave Lake and, Beaverhill Bird Observatory, located 80 km southeast of Edmonton, is situated south of the sampled breeding locations, on the eastern side of the Continental Divide. Located at a convergence of the Mississippi, Central and eastern Pacific flyways (Krikun and Holroyd 2001) these banding stations have a high likelihood of capturing samples from the breeding locations tested if they are using a migratory path east of the Rocky Mountains. Tail feather isotopes of birds from Lesser Slave Lake and Beaverhill banding stations showed significant differences with the Central Interior suggesting that sparrows from this population are not being detected at these station (Figure 2.6). On the other hand, tail feather isotope ratio at these two banding stations showed no statistical differences with the Peace River Region. This is consistent with the Peace River Region birds are being detected at Beaverhill and Lesser Slave Lake. However, it is important to consider that other breeding locations (not sampled) will have similar isotope signatures and that the higher standard deviation (compared with the rest of locations) observed at both of these banding stations suggests that birds from more than one population are most likely being captured at these locations. The observed isotope signatures suggest that populations north of the Peace River Region are also being captured at these locations, since both banding stations sampled a number of birds from the -146 - -160 %o zone (Figure 2.2). 48 This data is consistent with the hypothesis that birds breeding in the Peace River region are using a Central migratory pathway to over wintering areas in New Mexico/Arizona and Colorado/Kansas areas. Although the use of the eastern Pacific flyway, crossing the Rockies at the Columbia River and proceeding to the Pacific coast area, cannot be rejected, the use of a northern Rocky Mountain pass seems unlikely. Data from Mugaha Marsh Banding Station also suggests that birds from the Peace River Region are not crossing through the Peace River Pass. Mugaha Marsh Banding Station is located 14km northwest of Mackenzie, BC, east of the Parsnip Reach of the Williston reservoir (Mackenzie Nature Observatory 2011) and may potentially capture birds using the two major mountain passes in this area, the Peace River and Pine Pass. The Williston reservoir is the impoundment of the Peace River watershed from the WAC Bennett Dam in the Peace Canyon, which cuts through the Rocky Mountains (600 m elevation), into the north and south arms west of the Rockies that extend along the former tributaries that flowed along in the Rocky Mountain trench. This banding station is also located 68 km from the entrance to the second major pass through the Rockies in this region, the Pine Pass. The Pine Pass (874 m elevation) has the distinction over other passages that it is the only one that cuts the Rockies in a transversal orientation (following the migratory direction). The Mugaha Marsh station has the highest amount of captures of White-throated Sparrows in British Columbia, with an average of 16.18 birds per year (1995 - 2011) and a high peak of 49 78 captures in the 2011 (Mackenzie Nature Observatory 2009). Because of being strategically located in the Rocky Mountains trench, Mugaha Marsh Banding Station is important for detecting birds that are using the Peace River Pass. Additionally this station is located near to the migratory divide for many bird species such as the Yellow-rumped Warbler (Dendroica coronata), Blackpoll Warbler (Dendroica striata), American Redstart (,Setophaga ruticilla), Northern Waterthrush (Seiurus novaboracensis) and Wilson's Warbler (Wilsoniapusilla) (Dunn et al. 2006). Tail feather isotopes samples from this banding station showed a different distribution from the rest of the banding stations. The distribution of samples at this station presented a high percentage of samples (-60%, 5/9) in the -161 - -173%o zone. This zone corresponds to the northern BC-Yukon border region (Figure 2.2). The high mean and distribution of &Df values at Mugaha Bird Observatory suggests that birds caught at this location could be either coming from a population north of the Peace River Region (east of the Rockies, e.g., Sikanni) or from a population north of Mackenzie (west of the Rockies, e.g., northern Williston Lake/Finlay River or even south-eastern Yukon following Liard river watershed). The lower standard error (CI 95%) (compared with the rest of banding locations) observed at this banding station may indicate that birds banded at this location come from a more narrow distribution than the Alberta banding stations. The range of values observed at Mugaha Marsh are significantly different from those observed in the Central Interior and Peace River breeding locations. Despite being located 50 on the western side of the Continental Divide, birds banded at this station have isotopic values that represent a higher latitudinal range than for both breeding locations. No evidence has been reported that the Central Interior population extends to such high latitudes along the west side of the Rocky Mountains. These data indicates that a third breeding population is potentially using the Peace River area during migration. The Rocky Point banding station also regularly captures White-throated Sparrows in British Columbia. With an average of 3 White-throated Sparrows each year (1994 to 2009), most banded between mid-September to late-October (Melcer and Nightingale 2009; David 2006, 2008; Jantunen 2003, 2004; Gibson 2002; Derbyshine 1999, 2000), the number of recaptures at this station is lower than Mugaha Marsh. Because of being located at the south end of Vancouver Island, this station represents an important Pacific flyway sampling location. The mean SDfratio of migrants banded at Rocky Point banding station (-151.77 ±3.6 %o SD, n = 3), as well as the lack of statistical differences with birds from Central Interior and Peace River Region suggest that birds from either Central Interior or Peace River Region breeding populations could be passing through this station and, as a result, using the Pacific Flyway. Given the small sample size, no definite conclusion can be drawn, although the low numbers of White-throated Sparrows annually banded at this station suggest that this is not a major migration route. The inclusion of other Pacific flyway banding stations (such as the Delta Marsh in Vancouver, not included in this study) may be more useful locations that could help in the understanding of the migratory behaviour of Central Interior and Peace River Region populations. 51 In summary, the results indicate that the populations breeding in the Central Interior and Peace River are overwintering in the western area of the wintering range. The results are also consistent with most parsimonious hypotheses of migration, i.e., that the Central Interior population overwinters in Pacific coast while the Peace River population overwinters in the New Mexico-Arizona and Colorado/Kansas area. Alternate hypotheses involving combinations of these breeding locations and wintering areas however could not be ruled out. Partial information on migratory routes was also obtained. Banding station data is consistent with birds from the Peace River region using the flyway east of the Continental Divide. At the same, time this data did not support use of this migratory corridor by the Central Interior population. A number of possible migratory pathways may be used by the Central Interior population. One possible migratory route would be that the sparrows are crossing through the Pine Pass and then following an undetected route, such as along the eastern foothills of the Rockies, before heading to their wintering destination. Another possibility is that Central Interior sparrows fly south following the Fraser River and then proceed down the coast. Other western inland routes seems to be unlikely as the Mount Revelstoke, Tattlayoko and Vaseux Lake banding stations rarely report White-throated Sparrow captures (eBird Canada 2010, Ogle 2008; 2009a; 2009b). Obtaining samples from additional banding stations in BC, e.g., Delta Marsh, and Alberta, e.g., Inglewood, as well as from strategically placed locations will be needed to provide the crucial evidence to support or refute these possible routes. It is also 52 recommended that both head and tail feathers be collected from all migratory samples so that both possible breeding and wintering locations can be linked to migratory samples. Based on the ability to successfully differentiate breeding populations on either side of the Continental Divide using tail-feather isotopes, one alternative study that would help understanding migratory behaviour would be to sample tail and head feathers from birds wintering along the Pacific coast or New Mexico-Arizona and Colorado/Kansas areas. These isotopic ratios could then be compared to the present study in order to infer breeding populations. A similar technique has been applied successfully before to other species, such as Swainson's Thrush (Catharus ustulatus) and has shown to be powerful enough to differentiate the origin of wintering individuals (Kelly et al. 2005) Elucidating the complexity of migratory routes in western Canada is crucial to understand the effects that local disruptions could have on White-throated Sparrow populations. These effects could be important as birds fly through migratory bottlenecks when they cross the Rocky Mountains. However, without detailed information on the migratory routes of western Canadian White-throated Sparrow populations, it would be very difficult to determine which population could be affected by a disruption on a migratory route and what would be the repercussions of these disruption events. 53 CHAPTER 3 GENETIC DIFFERENTIATION ANALYSIS OF WESTERN CANADA WHITETHROATED SPARROW POPULATIONS AND GENETIC ASSIGNMENT OF MIGRATORY INDIVIDUALS 3.1 INTRODUCTION Understanding genetic structure or differentiation of breeding populations along a migratory divide has been an important aspect of many migratory connectivity studies (i.e., Clegg et al. 2003, Kelly and Hutto 2005, Ruegg and Smith 2002, Davis et al. 2006). These studies have used genetic differences among breeding populations to identify discrete migratory groups, which can confirm the presence of a migratory divide. The amount of genetic divergence among populations however, is related to a number of factors including population/evolutionary history, the presence of barriers to gene flow, and the resolution of the genetic marker system used. Population history (response to glaciation and post-glaciation events) has a strong influence on the population structure of avian populations as well as the evolution of migratory routes. Many cases of migratory and genetic differentiation among populations appear to be a result of the independent evolution of populations that have originated from separate refugia (and then have come into secondary contact) after a process of post-glacial range expansion.(e.g., Sylvia atricapilla, Perez-Tris et al. 2004; Dendroica petechia, Boulet and Gibbs 2006). 54 Additionally, as in Blackcaps a population bottleneck prior to post-glaciation expansion can affect the genetic differentiation among populations (Perez-Tris et al. 2004). In contrast, a lack of differentiation, as found in Black-throated Blue Warbler {Dendroica caerulescens), is usually attributed to a recent expansion from a single refugium (Davis et al. 2006). Demographic events and population history can not entirely explain how differences in migratory behaviour are maintained. Genetic and migratory differences can promote reproductive isolation by pre-zygotic and post-zygotic mechanisms and restrict gene flow after populations meet in a secondary contact zone (Irwin and Irwin 2005; Toews and Irwin 2008). For example, the Greenish Warbler (Phylloscopus trochiloides) (Irwin and Irwin 2005) and Yellow-rumped Warbler {Dendroica coronata) (Brelsford and Irwin 2009) have shown differentiation enhanced by a selective pressure against hybrid individuals. In these cases, secondary contact between populations with different migratory strategies resulted in hybrid individuals that exhibit a sub-optimal migratory strategy with a lower fitness than either parental lineage. This lower fitness could be influenced by the presence of an ecological or geographical barrier complicating migration of hybrids, as is the case in the Greenish Warbler {Phylloscopus trochiloides) where the barrier is the Tibetan Plateau (Irwin and Irwin 2005). In many cases, genetic differentiation follows a geographical pattern. This pattern can be influenced by a geographical barrier or by the dispersal ability of the organism. Being able to differentiate between two or more populations along a geographical divide is important as 55 it may indicate that these populations use different migration corridors or wintering areas (e.g., Clegg et al. 2003; Kelly and Hutto 2005; Irwin and Irwin 2005). The Rocky Mountains, which form the main geographic feature of the Continental Divide, are an east/west genetic and migratory divide for breeding populations of many avian species (Boulet and Gibbs 2006; Kelly and Hutto 2005). The White-throated Sparrow (Zonotrichia albicollis) has breeding populations on either side of the Rocky Mountains. Differences in song structure (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.) and, possibly, migration strategies have been noted between populations west of the Rocky Mountains in the Central Interior of BC and those east of the Rockies in the Peace River region (Chapter 2). The degree of genetic differentiation between these populations is unknown. In spite of the fact that bird species tend to have lower levels of genetic differentiation than other vertebrates, probably because of their higher mobility and larger population sizes (Winker et al. 2000), many studies using a variety of molecular markers have found genetic differentiation among populations of the same species (e.g., Boulet and Gibbs 2006; Clegg et al. 2003; Lecomte et al. 2009; Helbig et al. 1995; Winker et al. 2000). Different markers have distinct strengths that, when combined, can provide a more complete picture of the spatial genetic variation within a species. For instance, mitochondrial DNA is a maternally inherited marker which does not undergo recombination and has a higher substitution rate than corresponding sequences in the nuclear genome, making it an effective marker to study populations that have recently diverged (Zink and Barrowclough 2008) and for phylogeographic studies (Avise et al. 1998). Microsatellites, on the other hand, are nuclear 56 codominant markers that usually produce unique individual genotypes that are used for a range of applications, from paternity or forensic analysis to studying population or species relationships. The primary objective of the present study was to determine the genetic differentiation of White-throated Sparrow breeding populations distributed on both sides of the likely migratory divide (the Rocky Mountains). Eight neutral rnicrosatellite markers as well as a partial sequence (500bp) of the mitochondrial Cytochrome Oxidase Subunit I (COT) gene were assessed. The combination of these markers is useful not only to determine the presence or absence of structure/differentiation between White-throated Sparrow populations, but also to help us infer historical demographic events (i.e., the use of a single refugium or multiple refugia) that could influence genetic differentiation and migratory behaviour. The second objective, if evidence of genetic differentiation is noted, is to directly study migratory behaviour through the genetic assignment of migratory individuals, banded along important migratory routes in western Canada, to their likely breeding grounds. 57 3.2 METHODOLOGY 3.2.1 STUDY AREA AND SAMPLE COLLECTION Based on White-throated Sparrow habitat preferences, sampling locations were located in shrub-dominated habitats (i.e., clearcuts, forest edges, oil and gas lines, and ATV trails) (Figure 3.1). One hundred and five feather samples from breeding birds were collected during the months of May-July of 2009-2010 across 3 regions in the Central Interior of British Columbia (BC), Canada; however, from these samples only one hundred and one samples were successfully used: Prince George (23), John Prince Research Forest (4) and MacLeod Lake (12), and 4 locations in Peace River Region of BC: Moberly Lake (11), Tumbler Ridge (11), Dawson Creek (37), and Sikanni River (3) (Figure 3.2). Additionally, 20 blood samples from Algonquin Provincial Park (Ontario) and 20 blood samples from Prince George (43 in total for this location) were obtained in collaboration with Dr. Scott Ramsay (collected between 2004-2009). Sixty-seven samples from fall migratory birds were obtained in August-September of 2009. Seven banding stations across British Columbia (BC) and Alberta (AB) collaborated to sample migratory White-throated Sparrows: Rocky Point Bird Observatory (3), Mugaha Marsh Bird Observatory (9), Lesser Slave Lake Bird Observatory (9), Beaverhill Bird Observatory (8), Tattlayoko Bird observatory (0), Vaseux Lake Bird Observatory (0) and Revclstoke Bird Observatory (0). Additionally (38) were collected in Dawson Creek in the month of September 2009 (Figure 3.3). 58 White-throated Sparrows from breeding populations (and from Dawson Creek in the fall) were captures using active mist-nest techniques. Birds were attracted to the nest with seeds (fall) or using a playback (summer). Migratory samples from banding stations were captured using passive mist-net technique. Two feather samples from external tail rectrices were obtained from each bird prior to their release. Feathers were transported in hermetic Ziploc bags that were labelled detailing: date, banding location, species, CWS number, and individual code. Figure 3.1 Example of shrub-dominated habitat in Pouce Coupe (BC) where breeding and migratory birds were collected. Birds were attracted to the nest with seeds (autumn) or using a playback (summer). 59 m HI • HHD wmmgmHHHIi MM l^nn||Mfp HH •H| mm••• nn ___ Figure 3.2 Map of sampling locations from collected during breeding season. Prince George (PG), John Prince Research Forest (JPRF), MacLeod Lake (MacL), Tumbler Ridge (TR), Moberly Lake (ML), Dawson Creek (DC), and Sikanni (Sik) were included. Sampling locations west from the continental divide were grouped into the Central Interior population (in blue), and east from the divide were grouped as the Peace River Region population (in green). The first number in parenthesis represents the total amount of individuals collected per location; the second number represents the amount of individuals that were successfully genotyped for microsatellite analysis. Ontario samples were not included in map. The map was constructed with Google Earth v.5.2. 60 J- LSLBO (9 9) ' g MBO 17(9 9) 30 0(0) *>.MRBd:0(0) 11 If 4 jt v^BO 0(0) , RPBO 4(3'.''3) 4 • |V, . .m. Farm Serv.ce Agency ' 2C1* ^ -t»s t-oo 0.05) with 1% of variation (Rst: 0.01031) explained among populations (Central Interior, Peace River Region, and Ontario) (Table 2.4). However, Pairwise Rst comparisons showed significant 67 differences (P < 0.05) between Central Interior and Peace River Regions (Rst: 0.02253), but non-significant (P > 0.05) between Central Interior and Ontario regions (Rst: -0.01452) (Table 3.5) and between Ontario and Peace River Region (Rst: -0.00255). Under a number of different models (e.g., Fst-like) the statistical results do not change even though the values change. Overall AMOVA still shows non-significant results (P > 0.05) but with a lower percentage of variation and Fst (Fst: 0.0028). Pairwise Fst comparisons showed similar results showing shallow non-significant results between Central Interior and Peace River Regions (Fst: 0.003) and all other comparisons. The symmetric proportion of samples assigned to each population showed that there is no structure in the samples analyzed (Pritchard et al.2007) with the program STRUCTURE which does not take in account geographical distances (Figure 3.4a). When the Estimated Logarithm Probability of Data [LnP(D)] was plotted against each estimated population (K), the curve does not plateau, also suggesting a lack of genetic structure (Figure 3.5a). This same pattern was observed in program TESS (which uses geographical distances) when the Deviance Information Criterion (DIC) was plotted against each K (Figure 3.4b, 3.5b). 68 Table 3.3. Amplification size (bp), number of alleles (Na), observed (Ho) and expected (He) heterozygosity, and number of private alleles (Pa) found for all eight microsatellite loci in each region: Central Interior, Peace River Region and Ontario. Heterozygosity values in bold represent significant differences after Bonferroni correction between Ho and He. Central Interior Peace River Region Ontario Marker Size (bp) Na Ho He Pa Na Ho He Pa Na Ho He Pa Zole-A08 252-286 9 0.71 0.78 1 9 0.64 0.72 1 8 0.75 0.77 2 Zole-BOl 252-304 17 0.95 0.88 0 20 0.92 0.91 4 12 1 0.9 0 Zole-B03 222-270 11 0.88 0.83 1 19 0.87 0.86 9 9 0.95 0.85 0 Zole-A02 160-204 12 0.88 0.87 0 19 0.79 0.9 6 8 0.75 0.85 1 Zole-C06 212-248 15 0.83 0.87 1 15 0.92 0.87 2 9 0.85 0.86 1 Zole-Cl 1 180-200 13 0.65 0.85 5 9 0.87 0.8 1 7 0.9 0.84 0 Zole-C07 192-280 23 0.61 0.95 3 24 0.79 0.95 2 21 0.85 0.96 5 205-291 15 0.87 4 14 0.72 0.86 4 10 0.8 0.88 15 129 29 84 Zole-Fl 1 0.81 115 Total 1 10 Table 3.4 AMOVA from breeding population samples. Source of variation, degree of freedom (d.f), sum of squares and Percentage of Variation are shown. Distance method: Sum of Squared size differences Rst; 1000 permutations. Source of Variation d.f. Sum of Squares Percentage of Variation Among populations 2 3555.133 1.03 Within populations 281 262410.628 98.97 Total 283 265965.761 RST: 0.01031 P-value: 0.1417 ± 0.0089 Table 3.5 Pairwise Fst Population Comparison between Central Interior (CI), Peace River Region (PR) and Ontario (ONT). Distance Method used: Sum of squared size differences (Rst). Significance (P-values) is shown in parenthesis next to Fst value. Central Interior Central Interior Peace River Region Ontario 0 Peace River Region 0.0225 (0.0107) 0 Ontario -0.0145 (0.9385) -0.0026(0.5615) 69 0 A.) Figure 3.4 A) Example of one of the 10 runs of Structure Bar plot with k - 2 . Color lines represent probability of regions (1. Central Interior, 2. Peace River Region, 3. Ontario) to be assigned to a population k. B) Example of Tess bar plot of one of the 100 runs with K = 2, Bars represent probability of an individual sample to be assigned to a population. Green bars indicate that all individuals were assigned to the same population. 70 A -) !.) Ln P(D) over k > 1 (• or estfcmrteil popuWOooi jit) B< ) DJC over k two A r. 7 Number of estimated populations (KJ Figure 3.5 A.) Estimated Logarithm Probability of Data (LnP(D)) plotted against each estimated population (K), calculated with STRUCTURE. B.) Deviance Information Criterion (DIC) plotted against each estimated population (K), calculated with TESS. 71 3.3.2. MICROSATELLITE MIGRATORY SAMPLES ANALYSIS The self-assigning test using Geneclass2 showed that 50.7% of samples were assigned correctly to their breeding population (Table 3.6). John Prince and McLeod Lake, in spite of being located within Central Interior were mostly assigned to the Peace River Region. Similarly, more samples from Algoquin Park (ON) were assigned to Central Interior and the Peace River Region than to Ontario. Assignment test of migratory locations to breeding populations showed that for all locations samples were assigned to all three breeding locations. More samples from Mugaha Marsh and Rocky Point were assigned to the Central Interior while more samples from Lesser Slave Lake and Dawson Creek (migratory) were assigned to the Peace River Region (Table 3.7). For few samples the probability of assignment to a population was rejected (P < 0.05) (Table 3.7). Notably, Mugaha Marsh showed the greatest amount of population rejection. Of interest, one breeding individual collected in Dawson Creek (Peace River Region) was recaptured the next year from a migratory flock captured at on the same location. 72 Table 3.6 Self-assignment of sampling locations during breeding season to their breeding regions using Geneclass2. The first number (outside the parenthesis) represents the number of individuals assigned with highest probability to each breeding region. In parenthesis is represented the number of individuals that were statistically rejected (P < 0.05) as being part of this location (using MonteCarlo resampling and Paetkau et al. 2004 simulation algorithm). Peace River Region Central Interior Prince John McLeod George Prince Lake Central Interior Peace River Region Ontario Excluded from all locations Moberlv Dawson Lake Creek Tumbler Ridge Sikanni River Ontario Algonquin Park 26(2) 0(0) 2(0) 4(0) 5(2) 4(1) 2(0) 7(1) 11(1) 4(0) 10(0) 6(0) 25(0) 6(0) 0(0) 7(0) 6(3) 0(0) 0(1) 1(1) 4(5) 1(0) 1(0) 5(1) 0 0 0 0 3 0 0 Table 3.7 Assignment of individuals from migratory sampling locations (Mugaha Marsh, Lesser Slave, Beaverhill, Rocky Point and Dawson Creek-migratory)to the three breeding regions (Central Interior, Peace River Region and Ontario) using Geneclass2. The first number (outside the parenthesis) represents the number of individuals assigned with highest probability to each breeding region. In parenthesis is represented the number of individuals that were statistically rejected (P < 0.05) as being part of this location (using MonteCarlo resampling and Paetkau et al. 2004 simulation algorithm). Mugaha Marsh Rocky Point Lesser Slave Beaverhill Dawson Creek (mig) Central Interior 4(2) 2(0) 3(0) 2(0) 7(2) Peace River Region 2(3) 1(0) 5(0) 2(0) 18(0) Ontario Excluded from all locations 2(2) 0(0) 1(0) 1(0) 6(0) 1 0 0 2 4 73 3.3.3 MITOCHONDRIAL DNA RESULTS One hundred and twenty nine COI sequences (461bp with 17 variable sites) from three regions, Central Interior (56), Peace River Region (54) and Ontario (19), were determined in the present study (Table 3.8). In total, 19 haplotypes were found with four haplotypes (A, B, C, and K) found more than once (Table 3.9). A statistical parsimony network tree showed a star-like arrangement with the most frequent haplotype in all locations, B (68-83%), found in the center. Most haplotypes (except for haplotype J and R) differ from B by only one mutation (Figure 3.8). Haplotype C was also present in all three locations while haplotype K was found in the Central Interior and Ontario and haplotype A found in the Central Interior and the Peace River Region (Table 3.9, Figure 3.6). AMOVA results for mtDNA haplotypes showed similar results to the nuclear microsatellite markers with non-significant differences among the three locations (Fst = 0.01, P > 0.05) (Table 3.10). Pairwise Fst comparisons also showed non-significant differences between all sampling locations (Table 3.11). 74 Table 3.8 Number of mtDNA haplotypes per location and number of unique haplotypes as well as number of sequences Central Interior Peace River Region Ontario Haplotype number 8 10 7 Unique haplotypes Number of sequences 4 7 4 54 53 19 Table 3.9 Frequency of shared haplotypes per sampling location. Haplotypes name was stated with letters (A, B, C, and K). Central Interior, Peace River Region and Ontario populations were included. Frequency of shared haplotypes Central Interior Peace River Region Ontario A 0.070 0.150 0 B 0.820 0.700 0.680 C 0.018 0.019 0.050 K 0.018 0 0.050 75 Figure 3.6 Statistical Parsimony tree of mtDNA haplotypes based on 17 variable sites of a 461 bp CO/gene. Circle circumference is proportional with haplotype frequency. Colour represents the population where haplotypes are found: Central Interior (red), Peace River Region (blue) and Ontario (white). Bars represent number of nucleotide changes between haplotypes. 76 Table 3.10 AMOVA from mtDNA samples. Source of variation, degree of freedom (d.f), sum of squares and Percentage of Variation, and Fst are shown (1000 permutations). Source of Variation d.f. Sum of Squares Percentage of Variation Among populations 2 0.596 1.01 Within populations 126 26.768 98.99 Total 128 27.364 FST: 0.0101 P-value : 0.1975 ±0.0121 Table 3.11 Pairwise Population Comparison of mtDNA haplotypes Fst between Central Interior, Peace River Region and Ontario. Statistical significance (P-value) are shown in parenthesis. Central Interior Central Interior Peace River Region Ontario 0 Peace River Region 0.0110(0.17) 0 Ontario 0.0157(0.14) 0.0039 (0.32) 77 0 3.4 DISCUSSION 3.4.1 POPULATION HISTORY OF CENTRAL INTERIOR WHITETHROATED SPARROW POPULATIONS Neutral nuclear markers as well as mitochondrial COI sequences showed little evidence for genetic structure among the sparrow breeding locations tested. Pairwise differences were noted between the Central Interior and the Peace River Region, but these values were small, (Rst < 0.0225). This lack of differentiation is reflected in the results from Bayesian assignment techniques (TESS and STRUCTURE) where no evidence of population structure was observed (Figure 3.4, 3.5). This low differentiation precludes the assignment of migratory individuals to breeding populations, but it provides important evidence on the historical and evolutionary history of Central Interior White-throated Sparrow population. The lack of differentiation and low Rst/Fst values found with microsatellite and mitochondrial data suggests either high gene flow among all populations or a recent range expansion into the Central Interior by eastern populations (although both events are not exclusive from each other). The paraphyletic star-like COI tree pattern (Figure 3.6) is also consistent with range expansion in which a mutation-drift equilibrium has not been reached (Beebee and Rowe 2008). This same pattern has been observed in other passerine species such as the Black-throated Blue Warbler where using both microsatellite and mitochondrial markers, no populations differences were observed despite migratory and phenotypic differences (Davis et al. 2006). Davis and colleagues suggested that the star like phylogeny and lack of genetic differences indicates that populations expanded from a single glacial 78 refugium in the Pleistocene, and further that migratory and phenotypic differences appeared more recently. Other birds, such as the prairie warbler (Dendroica discolor) and the chipping sparrow (Spizella passerina), also have similar star-like mtDNA trees (even though the prairie warbler showed a significant genetic differentiation between subspecies D. d. dicolor and D. d. paludicola) suggesting a rapid post-glacial expansion during Pleistocene followed by a subsequent differentiation of migratory behaviour (Buerkle 1999; Mila et al. 2006). Consistent with evidence of a single Pleistocene refugium, Whyte (1938) suggested that the Central Interior population arose from an expansion from populations along the eastern side of the Rocky Mountains, crossing west through lower altitude mountain passes. The Pine Pass and the Peace River have been suggested as the most probable routes of these passages, as birds probably were deflected from their migratory routes following the Peace River system until they found suitable territories west of the Rocky Mountains (Wythe 1938).The exact date of the establishment of the Central Interior population is unknown. The first historical records from the Central Interior are from around 1925 (Wythe 1938); however it is important to consider that banding information prior to the first influx of European settlers into the Central Interior in 1850's (Stevenson et al. 2011) is practically non-existent. Based on available information, a likely hypothesis would be that White-throated Sparrows have been breeding in the Central Interior in low numbers for at least a century, and that recently population numbers have increased as a result of the rise of the forest industry (around 1960's) which significantly modified the landscape creating more suitable habitat. 79 Despite evidence of westward range expansion, the presence of a geographical barrier such as the Rocky Mountains is expected to act as a barrier to gene flow between the Central Interior and Peace River Region. Interestingly, pairwise AMOVA of microsatellite results showed a statistical difference between the Peace River Region and Central Interior, but no differences between the former two and the Ontario population (Table 3.5). Although these results may indicate that the Rocky Mountains are acting as a barrier to gene flow between Central Interior and the Peace River Region, the overall genetic data do not follow what was expected according to an isolation-by-distance model. A range wide lack of isolation-bydistance would support the recent expansion hypothesis with high levels of gene flow. However, with only three sampling points conclusions are tentative and would require many more sampling locations in order to confirm a lack of isolation-by-distance among Whitethroated Sparrow populations. Weak population structure (at drift-dispersal equilibrium) is usually considered to be the result of high dispersal and gene flow (Lecomte et al. 2009). This lack of structure would be expected in many migratory bird species where dispersal and effective population sizes are higher than in other vertebrates (Winker et al. 2000). However differences in song structure (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.) the small, but significant, statistical genetic difference between Peace River Region and Central Interior and the possible migratory differences (Chapter2) suggest that populations on both sides of the Rocky Mountains could be in the early stages of differentiation. 80 3.4.2 POPULATION STRUCTURE AND GENETIC ASSIGNMENT OF WHITETHROATED SPARROW POPULATIONS To test confidence of the assignment technique used in the present study, breeding individuals were assigned back to the breeding populations sampled (Table 3.6). Consistent with the general lack of structure, this technique showed that only 40-50% of samples were assigned correctly to their breeding locations and that there were very few population exclusions. In spite of the relatively low percentage of samples assigned correctly in the present study, this percentage was slightly higher than what was expected under simulation studies (from 25 to 30%) for an overall Fst of 0.01 (with 10 loci and 30 samples per population) (Cornuet et al. 1999). The self-assignment test showed that individuals from breeding sample locations located east of the Continental Divide were mostly assigned to the Peace River Region. The only exceptions were Sikanni that was assigned mostly to the Central Interior (although it only has a sample size of 3 individuals) and Ontario that was mostly assigned to the Central Interior and the Peace River Region. Sampling locations west of the Continental Divide had two different scenarios: Prince George which was assigned mostly to the Central Interior, and the John Prince Research Forest and MacLeod Lake that were assigned mostly to the Peace River Region. However, it is hard to make any conclusions based on this test because of the low genetic structure and different sampling sizes among locations. 81 Samples originating from the different migratory stations, i.e., Mugaha Marsh, Lesser Slave Lake, Rocky Point and Beaverhill, did not show a clear preference of assignment of individuals to any of the sampled breeding populations (Table 3.6). The lack of genetic structure has confounded the assignment of migratory individuals. It is important to state that this likelihood technique only calculates a probability of a sample belonging to a reference population. Analysis of tail feather isotopes signatures (Chapter 2) suggests that at least some of the migratory samples at these locations have values outside the range observed in the breeding populations indicating that they could be breeding in populations not sampled. The Mugaha banding station, although west of the Rockies had a distinct isotope signature from the Central Interior breeding birds. These migratory samples also showed the greatest amount of exclusions for the breeding populations tested. The combined results support the hypothesis that this station is capturing migrants breeding outside, presumable north, of the breeding locations sampled in this study Migratory birds were also captured in Dawson Creek where 18 out of 35 migratory birds were assigned most likely to the Peace River Region breeding population (Table 3.7). This result was expected since this sampling location of migratory birds was also used during the breeding season and it is the closest to migratory stopover location sampled to the breeding locations included in this study. For this reason, it is very possible that at least part of the migratory flock sampled came from breeding territories near that sampled stopover site. This was confirmed by a recapture of an individual (code: Zoal-jdl63/Zoal-idl34) sampled two times in the same area, the first one with the migratory flock on the fall of 2008 and the second time in his breeding territory in the summer of 2009. 82 Lack of resolution in assignment tests caused by a poor population structure could be resolved by using additional markers, such as selective markers that are able to detect recent differentiation events at a genetic level. Selective markers have been used in other studies (i.e., Bredford and Irwin 2009) to detect hybrid zones in avian species and could provide enough resolution to find the genetic differentiation necessary in order to effectively use assignment tests to elucidate migratory behaviour of White-throated Sparrows. In conclusion, results from microsatellite and mitochondrial data of this study could not determine the presence or absence of a migratory divide in western Canadian White-throated Sparrow populations. However, they provide evidence that suggests that the breeding populations studied arose from an expansion from single glacial refugium. Additionally, shallow but significant genetic differences, plus differences in song structure (unpublished data, Mesias, V., Mora, M., Ramsay, S., and Murray, B, and Otter, K.) between the Central Interior and Peace River Region suggest that both populations could be in an early stage of differentiation. This early stage of differentiation implies that determining the migratory behaviour of western Canadian White-throated Sparrows using genetic markers will require additional (locally adaptive) markers. Further the results of the Mugaha banding station indicate that additional breeding populations are migrating through Northern BC and point for the need of sampling a wider range of breeding locations in Western Canada. 83 GENERAL DISCUSSION 4.1 MIGRATORY CONNECTIVITY BETWEEN BREEDING AND MIGRATORY POPULATIONS The aim of this study was to use genetic and stable isotope information in order to determine the migratory corridors and wintering areas used by western Canadian White-throated Sparrow populations. Elucidating the migratory connectivity of these populations will provide key information that could be applied to enhance conservation projects that aim to preserve ecological areas that are important for migratory species. Determining ecologically important areas of migratory birds is very important because if one of these areas is affected by human related activity, more than one breeding population could be threatened. One of the objectives of the present study was to elucidate wintering areas used by the Central Interior BC and Peace River Region populations. Deuterium stable isotopes did not completely elucidate wintering areas of these two populations; however, head feathers stable isotopes successfully narrowed down the tentative wintering areas to the south-western coast (California, Oregon, Washington) or to the northern isotopic limit of the eastern wintering range (New Mexico/Arizona or Colorado/Kansas) (Figure 2.7). Samples from head feathers isotopes did not show significant differences [but situated the 6Df mean value in the higher part of the wintering range (Figure 2.4 A, B)]. These values showed differences with other studies (e.g., Mazerolle et al. 2005) in which birds were wintering at the south-eastern part of the wintering range. 84 Another objective of this study was to investigate migratory differences among Whitethroated Sparrow populations located on either side of the continental divide in western Canada. It was not possible to determine exactly the migratory routes being used by each breeding population, but the significant differences between tail-feathers samples of Central Interior and all of the rest of banding stations imply that Central Interior sparrows are not using routes that correspond to the geographic location of any of those stations (except maybe Rocky Point). This suggests that sparrows from Central Interior might not be crossing the Rocky Mountains during fall migration (Figure 2.6). Still, another possibility is that Central Interior birds could be crossing the mountains, but using an undetected pathway; for this reason it would be important to extend the sampling in the future to other areas that could be important for migration. A lack of differences between Peace River Region and Beaverhill and Lesser Slave Lake banding stations suggest that birds from these breeding populations are not crossing the Rocky Mountains during fall migration (Figure 2.6). The most parsimonious explanation would be that these birds are using the Central flyway to a wintering destination close to New Mexico/Arizona or Colorado/Kansas area. However, there is still a possibility that those birds could be using another migratory route like the Columbia River route to California or the Mississippi flyway to Texas. However, most of the data used in this study is based on the information gathered during the fall migration, and data collected during spring migration might also help to determine the migratory behaviour of these sparrows. 85 The banding station with the highest number of White-throated Sparrow captures west of the Continental Divide is Mugaha Marsh. Even though it is located within the Central Interior, this banding station seems to be capturing birds from higher latitudes than any of the breeding populations sampled. This is evidenced by the northern isotopic distribution of the tail feather isotopes found in sparrows banded at this station (Figure 2.6). These isotopic ratios are statistically different from Central Interior and the Peace River Region, suggesting that sparrows from these breeding populations are not being detected at this banding station. This evidence suggests the importance of the Mackenzie area for migration, because instead of capturing birds from Central Interior or the Peace River Region during the fall, the banding station apparently receives sparrows from higher latitudes which then could be using low altitude passages (e.g., Pine Pass) to cross the Rocky Mountains to their wintering destinations. From there, they may follow more direct routes (e.g. the Fraser River) to the Pacific coast. As the area on the opposite side of the Continental Divide from Mackenzie is proposed for intense wind and other energy development, evidence suggesting this region may be a confluence of migratory corridors is important. 4.2 UNIQUE NATURE OF CENTRAL INTERIOR Molecular Genetic markers (Chapter 3) have been very useful to find genetic differentiation in migratory connectivity and behaviour studies (e.g., Boulet and Gibbs 2006; Clegg et al. 2003; Lecomte et al. 2009; Winker et al. 2000; Helbig et al. 1995). However, in the present 86 study no population structure was found using neutral microsatellites and the partial coding sequence of the mitochondrial Cytochrome Oxidase I gene (Table 3.4, 3.10, Figure 3.4). Mitochondrial DNA data showed a star-like haplotype tree that lacked reciprocal monophyly. This tree, as well as the lack of overall structure found in microsatellite data suggests that White-throated Sparrow populations originated from single glacial refugia, followed by a range expansion to the Central Interior from an Eastern population. Similar results have been obtained in other avian passerine species such as the Black-throated Blue Warbler, in which lack of differentiation was attributed to similar phenomenon (Davis et al. 2006). On the other hand, birds which were originated from two or more refugia (e.g., Yellow Warbler) showed clear genetic differentiation along an East/West axis (Boulet and Gibbs 2006). The question that remains unresolved is how recent is this vicariant event? The complexity of the haplotype tree and the number of rare alleles/haplotypes (Table 3.3, 3.7) obtained in the Central Interior seems to suggest that this population could be older than what was suggested from the first records of White-throated Sparrows in the province (Whyte 1938). Additionally, song analysis showed some degree of differentiation in song structure between western Canadian populations (unpublished data, Mesias, V., Otter, K., Mora, M., Ramsay, S., and Murray, B.) and these differences reflect that the vicariant event could be old enough for song differences to become established in these populations. 87 4.3 ENVIRONMENTAL/MANAGMENT LMPLICATIONS As wind power industry keeps growing as an important source of energy in many countries, concerns about the environmental impacts that this technology could have are increasing. Studies in birds have shown that in spite of the fact that wind farms have less impact in terms of bird casualties than other man-made structures like power lines (Johnson et al. 2002), a high number of birds can be affected within certain wind farm locations. For instance, significant numbers of Gryphon Vulture (Gyps fulvus) casualties have been reported at the Strait of Gibraltar, because installations seems to be located at a migratory bottleneck (e.g., De Lucas et al. 2008; Bildstein et al. 2009). Wind farms located at these migratory bottlenecks have affected raptors more than any other group of birds (e.g., Madders and Whitfield 2006), however, possibly in a lower degree, passerines have also shown to be affected by wind power structures during migration (Johnson et al. 2002). In the case of the present study, the effect that cumulative effect of multiple wind farm facilities located at a migratory bottleneck for the western White-throated Sparrow populations, as well as potentially other song birds could be important. Due to the high average wind speed in the area, there is increasing interest in the Peace River Region to develop wind power projects (BC Hydro 2009). Migrants crossing the Rocky Mountains could be funnelled into narrow passes, and this funnelling might concentrate movement to areas that overlap with wind farm development. Birds tend to lower their flying height during migration in locations like coast-lines and when crossing a ridge (Drewitt and Langston 2006), so migrants flying through the Pine Pass (or the Peace River) to eastern or western 88 wintering locations could be affected by potential wind power projects in the Peace River Region (plus several other wind farm projects in the United States). An extensive expansion of wind farm projects in Peace River Region could represent a significant impact on a disjunct population like the Central Interior. This impact would include a decrease on the gene flow between this population and the rest of the species' range (creating genetic isolation). However, it is hard to assess if Central Interior population would be affected by wind farms, because even though stable isotope evidence suggests that Central Interior birds might not be crossing the Rocky Mountains during the fall; this cannot be demonstrated with the data available so far. Additionally, Central Interior sparrows could still be funnelled into several wind farm projects while they cross the mountains in spring migration to northern breeding grounds. Estimating the impact that the cumulative effect of wind farm projects could have on migratory bird populations will be a difficult task, because as the White-throated Sparrow illustrate more than one population is likely to be affected. For instance, the Central Interior and Peace River Region are two likely populations to be affected by a wind farm project expansion. However, stable isotope results also suggest that Mugaha Marsh is banding birds from northern latitudes. These northern populations could be also affected by a sudden expansion in the wind power projects in the Peace River Region, because these birds could be funnelled into the area before or after crossing the mountains into the Mackenzie area during the fall (and possibly spring) migration. 89 4.4 IMPROVExMENTS TO THE TECHNIQUE In terms of efficiency, deuterium stable isotopes were effective in showing latitudinal discrimination. This is evident from feathers of White-throated Sparrows from the Central Interior and Peace River Region which indicate that these birds winter at latitudes with a higher isotopic signature than was obtained in previous studies (i.e., Mazerolle et al. 2005) for the rest of the distribution. However, deuterium isotopes were not able to discriminate wintering areas (Pacific South-west coast and New-Mexico/Arizona or Colorado/Kansas) at a longitudinal level. Other studies, such as Kelly and colleagues (2005), have used a combination of techniques to study connectivity between breeding and wintering areas at a longitudinal level (i.e., Coastal vs. Inland birds). These techniques include combining deuterium with sulphur isotopes and mitochondrial DNA data. Even though sulphur isotopes did not show differentiation by itself, it significantly increased the resolution of the technique when both isotopes were combined in a discriminant function analysis. Several other isotopes could be effectively used to complement the present study. Carbon isotopes have been effective in studying wintering habitat via reflecting the abundance of C3 versus C4 plants (Bearhop et al. 2004; Chamberlain et al. 2000; Pain et al. 2004; Yohannes et al. 2005). This element, as well as nitrogen, has been used successfully to find differentiation along a migratory divide between two subspecies of the Willow warbler (Phylloscopus trochiius trochilus and Phylloscopus trochilus acredula) (Chamberlain et al. 90 2000). Other studies such as Chambelain et al. (1997) have not only found that Carbon and Nitrogen are useful for discriminating breeding areas of migratory populations, but have also used strontium isotopes to find East/West differentiation. The latitudinal discrimination of tail feather isotopes on breeding populations could be used in order to infer breeding territories of migrants captured at their wintering areas. Other studies such as Kelly and colleagues (2005) have successfully used feathers from Swainson's Thrush (Catharus ustulatus) migrants in order to infer breeding territories using deuterium isotopes and mitochondrial haplotypes. A similar strategy could be employed to complement this study, in which feathers from wintering areas such as California, Oregon, New Mexico Arizona, Colorado and Kansas could be sampled, and tail feather isotopes could then be compared to the data from the present study. Another way to increase the efficiency of the technique used in the present study is to improve the resolution of the genetic markers. Increasing the resolution of genetic markers is very important in order to delineate the population structure of White-throated Sparrow breeding populations. Finding population differentiation with genetic markers could be a challenging task, as in many cases, it is necessary to develop high number of markers, which can be difficult and time consuming in the case of non-model species (as the White-throated Sparrow). Additionally, avian species that have originated from a single refugium and have recently separated or high levels of gene flow could present low levels of differentiation between populations {e.g., Davis et al. 2006). 91 Increasing the number of microsatellite markers may not increase the resolution significantly, Adaptive markers have been shown to work effectively in cases where other markers have failed to find genetic differentiation. For instance, Brelsford and Irwin (2009) found genetic differences in two possibly adaptive markers (one autosomal and one sex-linked) with fixed differences across a hybrid-zone in the Yellow-rumped warbler, when little differentiation previously was reported using mtDNA markers. Other markers such as Exon-Primed Intron-Crossing (EPIC) markers and Expressed Sequence Tags (EST)-linked microsatellites have become available thanks to the increasing amount of information on bird genomic projects. The main advantage of EPIC markers is that they are highly variable because they have target intronic regions which are flanked by conserved exonic regions (Thomson et al. 2010). A high number of these markers have been developed for bird population studies because of their advantages (e.g., Backstrom et al. 2008). For instance, besides been highly variable, they are also conserved at primer regions and can provide adaptive information via hitchhiking of close gene regions (Thomson et al. 2010). On the other hand, EST-linked microsatellites are markers that can be more useful than EPIC's for adaptive population studies; this is because the microsatellite allele variation of these markers can be located within the transcript region and have an important effect in the protein coding sequence. Additionally, a high number of these markers have been described 92 in passerine species, most of which have been successfully transferred between different avian species (Dawson et al. 2010; Karaiskou et al. 2008). 4.5 TECHNIQUE ASSESMENT Combining genetic data from mitochondrial and microsatellite markers with deuterium stable isotopes was useful for determining the population history and inferring the migratory routes in order to locate the presence of a migratory divide in White-throated Sparrows, however, the technique presented certain limitations that can be optimized to increase the resolution of this methodology. Several changes can be suggested for future projects in order to optimize these techniques including a fully integrated sampling strategy with additional sampling locations during both the breeding season, as well as, fall/spring migratory seasons to supplement banding station information. Lack of population structure in molecular genetic markers did not allow us to confidently estimate the origin of migratory individuals banded in western Canada. Adding more samples from other breeding populations would be very important to clarify if this lack of population structure is extended to all the breeding range or if there is a regional genetic structure that is not been detected. Understanding the genetic structure and the amount of gene flow between breeding populations of White-throated Sparrow can be very important to understand migratory behaviour since migration has a strong genetic component. 93 Besides the lack of structure obtained with genetic data, the significant differences observed on the tail feather isotopes ratios 5Df of Central Interior and the Peace River Region are promising, as they can be used in the future to estimate the breeding territories of migrants captured at their migratory and wintering grounds. Sampling birds at wintering and migratory grounds could be a great strategy to implement to optimize the techniques used in this study. This could be done by either choosing sampling sites that could complement migratory routes banded by banding stations (e.g., sampling birds in Quesnel that are flying down Fraser River or in Washington State (US) flying down the Columbia River drainage). Sampling birds from additional breeding locations could be also important to solve questions regarding genetic or demographic effects, such as isolation-by-distance, or bottleneck effects that could explain the genetic distribution. One alternative to study these effects would be sampling locations following one or two transects that cover both sites of the geographical divide. These two transects could be useful to determine if there is a general isolation-bydistance pattern across the species range or if there is a point which forms a genetic divide between the Central Interior and eastern populations. Another improvement to the study would be using an integrated sampling strategy in which head feather samples are collected not only from breeding populations but also from migratory birds. Unfortunately in the present study this was not the case as only head feather samples of breeding individuals were taken (Table 4.1). Results, such as the tail feather 94 isotope signal of Mugaha Marsh migrants, indicate the importance of sampling head feathers from migrants at banding stations in future studies. Table 4.1 Summary of number of samples that were analyzed from each location for all the markers: Tail and Head feathers isotopes 5Df, Microsatellite, and Mitochondrial DNA. Breeding Locations Prince George Central Interior John Prince Samples Collected Tail feather 5 Df Head feather 5 Df Microsat mtDNA 44 19 18 43 40 12 - - 61 19 37 Regions McLeod Lake CI Totals Peace River Region Dawson Creek Moberly Lake Tumbler Ridge Sikanni River PR Totals Ontario Ontario 4 4 18 12 59 10 54 26 26 37 32 12 - - 11 9 12 3 - - 3 - 11 3 10 2 64 29 26 62 53 20 - - 20 19 141 126 145 Totals Migratory Locations Mugaha Marsh Totals 5 9 9 Rocky Point 3 3 - Lesser Slave 9 9 - Beaverhill Dawson Creek 8 7 - 38 - - 67 9 3 9 - 7 - 35 - 63 95 - In conclusion, the strategy of the present study of combining genetic data from mitochondrial and microsatellite markers with deuterium stable isotopes showed that White-throated Sparrow has a good potential for being an indicator species in migratory behaviour studies. Results obtained were successful in providing evidence to determine the population history and migratory connectivity of breeding and wintering populations of White-throated Sparrows. While the genetic data provided evidence of a recent expansion from single glacial refugia, head feather isotopes suggested that Central Interior and the Peace River Region are not following migratory routes to south-eastern wintering grounds. I suggest that both populations could be following an east/west migration pattern where Central Interior population could be migrating to the south-west Pacific Coast and the sparrows from the Peace River Region to the New Mexico/Arizona or Colorado/Kansas area. Improving the resolution of the molecular and isotopic markers as well as optimizing the sampling strategy could be a very successful tool to study the connectivity between breeding and wintering populations of migratory species. Preferentially, species with and east/west distribution that originated from different glacial refugia are recommended if this technique is going to be implemented. If that is not the case, local adaptive markers and a fully integrated sampling strategy could be applied. In summary, this technique showed that White-throated Sparrow has potential to be a good indicator species for proactive conservation studies on migratory connectivity of avian species, but that more work needs to be done before applying this strategy in this and other species. 96 BIBLIOGRAPHY Avise, J. C., D. Walker, and G. C. Johns. 1998. Speciation durations and pleistocene effects on vertebrate phylogeography. Proceedings of the Royal Society of London. Series B: Biological Sciences 265: 1707-1712. Backstrom, N., S. Fagerberg, and H. Ellegren. 2008. Genomics of natural bird populations: a gene-based set of reference markers evenly spread across the avian genome. Molecular Ecology 17: 964-980. BC Hydro. 2009. BC Hydro Wind Data Study [Internet], [cited 2011 Nov 22], Available from http://www.bchydro.com/etc/medialib/internet/documents/environment/winddata/pdf/ winddata study_report_mayl_2009.Par.0001.File.bch_wind_data_study_mayl_09. pdf. Bearhop, S., G. M. Hilton, S. C. Votier, and S. Waldron. 2004. Stable isotope ratios indicate that body condition in migrating passerines is influenced by winter habitat. Proceedings of the Royal Society of London B 271: 215-218. Beebee, T., and G. Rowe. 2008. An Introduction to Molecular Ecology. Second Edition. Oxford University Press. Bello, N, O. Francino, and A Sanchez. 2001. Isolation of genomic DNA from feathers. Journal of Veterinary Diagnostic Investigation 13: 162-164. Bensch, S., G. Bengtsson, and S. Akesson. 2006. Patterns of stable isotope signatures in willow warbler Phylloscopus trochilus feathers collected in Africa. Journal of Avian Biology 37: 323-330. Bildstein, K., M. Bechard, C. Farmer, and L. Newcomb. 2009. Narrow sea crossings present major obstacles to migrating Griffon Vultures Gyps fulvus. Ibis 151: 382-391. Boulet, M., and L. Gibbs. 2006. Lineage origin and expansion of a Neotropical migrant songbird after recent glaciation events. Molecular Ecology 15: 2505-2525. Bowen, G. J. 2011. The Online Isotopes in Precipitation Calculator, version 2.2. [Internet], [cited 2011 Nov 18], Available from http://www.waterisotopes.org. Brelsford, A., and D. E. Irwin. 2009. Incipient speciation despite little assortative mating: The yellow-rumped warbler hybrid zone. Evolution 63: 3050-3060. Brown, M., Bomberger, and C. R. Brown. 2010. Blood Sampling Reduces Annual Survival in Cliff Swallows (Petrochelidon Pyrrhonota). The Auk 126: 853-861. 97 Buerkle, A. 1999. The Historical Pattern of Gene Flow among Migratory and Nonmigratory Populations of Prairie Warblers (Aves: Parulinae). Evolution 53: 1915-1924. Burg, T.M., and J.P. Croxall. 2001. Global relationships amongst black-browed and grey­ headed albatrosses : analysis of population structure using mitochondrial DNA and microsatellites. Molecular Ecology 10: 2647-2660. Buttler, R.W., F.S. Delgado, H. De la Cueva, V. Pulido, B.K. Sandercock. 1996. Migration Routes of the Western Sandpiper. The Wilson Bulletin 108: 662-672. Campbell, W., N. Dawe, I. McTaggart-Cowan, J. Cooper, G. Kaiser, A. Stewart, and M. McNall. 2001. The Birds of British Columbia. Vol. 4. Vancouver, BC, Canada: UBC Press. Chamberlain, C.P., J.D. Blum, R.T. Holmes, X. Feng, T.W. Sherry, and G.R. Graves. 1997. The use of isotope tracers for identifying populations of migratory birds. Oecologia 109:132-141. Chamberlain, C P, S Bensch, X Feng, S Akesson, and T Andersson. 2000. Stable isotopes examined across a migratory divide in Scandinavian willow warblers ( Phylloscopus trochilus trochilus and Phylloscopus trochilus acredula ) reflect their African winter quarters. Natural History 267: 43-48. Chen, C., E. Durand, F. Forbes, and O. Francois. 2007. Bayesian clustering algorithms ascertaining spatial population structure: a new computer program and a comparison study. Molecular Ecology Notes 7: 747-756. Clegg, S. M., J. F. Kelly, M. Kimura, and T. B. Smith. 2003. Combining genetic markers and stable isotopes to reveal population connectivity and migration patterns in a Neotropical migrant, Wilson's warbler (Wilsoniapusilla). Molecular Ecology 12: 819-830. Clement, M., D. Posada, and K. A. Crandall. 2000. TCS: a computer program to estimate gene genealogies. Molecular Ecology 9: 1657-1659. Coiffait, L., C. P F Redfern, R. M Bevan, J. Newton, and K. Wolff. 2009. The use of intrinsic markers to study bird migration. Environmental Research 24: 169-174. Cornuet, J.M., S. Piry, G. Luikart, A. Estoup, and M. Solignac. 1999. New Methods Employing Multilocus Genotypes to Select or Exclude Populations as Origins of Individuals. Genetics 153: 1989-2000. David, G.. 2006. Fall Migration at the Rocky Point Bird Observatory in 2005. [Internet], [cited 2011 Dec 16], Available from http://www.islandnet.com/~rpbo/finalreport05.pdf. 98 . 2008. Migration Monitoring at the Rocky Point Bird Observatory:Fall 2008. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~-rpbo/finalreport08.pdf. Davis, L., E. Roalson, K.L. Cornell, K. McClanahan, and M. S McWebster. 2006. Genetic divergence and migration patterns in a North American passerine bird: implications for evolution and conservation. Molecular Ecology 15: 2141-2152. Dawson, D., G. Horsburgh, C. Kupper, I. Stewart, A. Ball, K. Durrant, B. Hansson, et al. 2010. New methods to identify conserved microsatellite loci and develop primer sets of high cross-species utility - as demonstrated for birds. Molecular Ecology Resources 10: 475-494. Dawson, R. JG, H L. Gibbs, K.A. Hobson, and S. M Yezerinac. 1997. Isolation of microsatellite DNA markers from a passerine bird, Dendroicapetechia (the Yellow Warbler), and their use in population studies. Heredity 79: 506-514. De Lucas, M., G. F. E. Janss, D. P. Whitfield, and M. Ferrer. 2008. Collision fatality of raptors in wind farms does not depend on raptor abundance. Journal of Applied Ecology 45: 1695-1703. Derbyshine, D.. 1999. A report on migratory monitoring at Rocky Point: Fall 1999. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~rpbo/fmalrep99.html. — . 2000. A report on migratory monitoring at Rocky Point: Fall 2000. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~rpbo/fmalreportOO.html. Drewitt, A., and R. Langston. 2006. Assessing the impacts of wind farms on birds. Ibis 148: 29-42. Dunn, E., K.A. Hobson, L. I., Wassenaar, D. Hussell, and M. Allen. 2006. Identification of Summer Origins of Songbirds Migrating through Southern Canada in Autumn. Avian Conservation and Ecology - Ecologie et conservation des oiseaux 1:2:4. Dubiec, A., Zagalska-Neubauer, M. 2006. Molecular Techniques for Sex Identification in Birds. Biological Letters 43: 3-12. eBird Canada. 2010. Bird Observations Vaseux Lake Observatory. [Internet], [cited 2011 Dec 16]. Available from http://ebird.org/ebird/canada/. Falls, J. B., and J.G. Kopachena. 2010. White-throated Sparrow (Zonotrichiaalbicollis), The Birds of North America Online (A. Poole, Ed.). In Birds Of North America Online. Ithaca: Cornell Lab of Ornithology. [Internet], [cited 2011 Aug 12]. Available from http://bna.birds.cornell.edu/bna/species/128 doi: 10.2173/bna. 128. 99 Farmer, A., B. Cade, and J. Torres-Dowdall, 2008. Fundamental limits to the accuracy of deuterium isotopes for identifying the spatial origin of migratory animals. Oecologia 158: 183-192. Ganache, B., M. Raposo, M. Raymond, and C.F. Deschepper. 2001. M13-tailed primers improve the readability and usability of microsatellite analyses performed with two different allele-sizing methods. Biotechniques 31: 24-28. Garrison, B. 2008. Range map for White-throated Sparrow. California Wildlife Habitat Relationships (CWHR) Version 8.2. California Department of Fish and Game and California Interagency Wildlife Task Group.[Internet], [cited 2011 Jun 10]. Available from: http://nrm.dfg.ca.gov/ Gibson, G.. 2002. Migration Monitoring at Rocky Point Bird Observatory 2001. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~rpbo/finalreportO 1.html. Helbig, A. J., I. Seibold, J. Martens, and M. Wink. 1995. Genetic Differentiation and Phylogenetic Relationships of Bonelli's Warbler Phylloscopus bonelli and Green Warbler P. nitidus. Journal of Avian Biology 26: 139-153. Hobson, K. A., and L. I. Wassenaar. 1997. Linking Breeding and Wintering Grounds of Neotropical Migrant Songbirds Using Stable Hydrogen Isotopic Analysis of Feathers. Oecologia 109: 142-148. Irwin, D. E, and J. H Irwin. 2005. Siberian migratory divides: the role of seasonal migration in speciation. Pages 27-40 in Birds of Two Worlds: The Ecology and Evolution of Migration, Ed. R. Greenberg and P. P Marra. Johns Hopkins University Press. Jantunen, J. 2003. Migration monitoring at Rocky Point Bird Observatory in fall 2003. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~rpbo/RPB02003.htm. . 2004. Migration Monitoring at Rocky Point Bird Observatory in fall 2004. [Internet], [cited 2011 Dec 16]. Available from http://www.islandnet.com/~rpbo/finalreport04.html. Johnson, G. D., W. P. Erickson, M. D. Strickland, M. F. Shepherd, D. A. Shepherd, and S. A. Sarappo. 2002. Collision Mortality of Local and Migrant Birds at a Large-Scale Wind-Power Development on Buffalo Ridge, Minnesota. Wildlife Society Bulletin30: 879-887. Karaiskou, N., E. L. Buggiotti, and C. R. Primmer. 2008. High Degree of Transferability of 86 Newly Developed Zebra Finch EST-Linked Microsatellite Markers in 8 Bird 100 Species. Journal of Heredity 99: 688 -693. Kelly, J. F, and R. L Hutto. 2005. An East-West Comparison of Migration in North American Wood Warblers. The Condor 107: 197-211. Kelly, J. F., K. Ruegg, and T.B. Smith. 2005. Combining Isotopic and Genetic Markers to Identify Breeding Origins of Migrant Birds.Ecological Applications 15: 1487-1494. Knapton, R. W., R. V. Cartar, and J. B. Falls. 1984. A Comparison of Breeding Ecology and Reproductive Success between Morphs of the White-throated Sparrow. The Wilson Bulletin 96: 60-71. Krikun, R. 2008. Lesser Slave Lake Bird Observatory 2008 Annual Report.[Internet], [cited 2011 Dec 16] Lesser Slave, Alberta, Canada. Available from http://www.lslbo.org/research_library.asp . 2007. Lesser Slave Lake Bird Observatory 2007 Annual Report. [Internet], [cited 2011 Dec 16] Lesser Slave, Alberta, Canada. Available from http://www.lslbo.org/research_library.asp . 2006. Lesser Slave Lake Bird Observatory 2006 Annual Report. [Internet], [cited 2011 Dec 16] Lesser Slave, Alberta, Canada. Available from http://www.lslbo.org/research_library.asp . 2005. Lesser Slave Lake Bird Observatory 2005 Annual Report. [Internet], [cited 2011 Dec 16] Lesser Slave, Alberta, Canada. Available from http://www.lslbo.org/research_library.asp Krikun, R., and G. Holroyd. 2001. Beaverhill Lake Important Bird Area Conservation Plan. [Internet], [cited 2011 Dec 16]. Beaverhill Lake, Alberta, Canada. Available from http://www.lslbo.org/research_library.asp Kucera, T. 2008. Life history account for White-throated Sparrow. California Wildlife Habitat Relationships (CWHR) Version 8.2. California Department of Fish and Game and California Interagency Wildlife Task Group. [Internet], [cited 2011 Feb 20], Available from http://nrm.dfg.ca.gov/FileHandler.ashx?DocumentID:=2269. Langin, K. M, M. W. Reudink, P. P. Marra, E. R. Norris, T. K. Kyser, and L. M. Ratcliffe. 2007. Hydrogen isotopic variation in migratory bird tissues of known origin: implications for geographic assignment. Oecologia, 152: 449-457 Lecomte, N., G. Gauthier, J. F. Giroux, E. Milot, and L. Bernatchez. 2009. Tug of war between continental gene flow and rearing site philopatry in a migratory bird : the 101 sex-biased dispersal paradigm reconsidered. Molecular Ecology 18: 593-602. Lincoln, F. 1998. Revised by John Zimmerman. Migration of Birds circular 16. US Department of the Interior US Fish and Wildlife Service. Madders, M, and D. P. Whitfield. 2006. Upland raptors and the assessment of wind farm impacts. Ibis 148: 43-56. Mazerolle, D.F., K. A. Hobson, and L. Wassenaar. 2005. Stable Isotope and Band-Encounter Analyses Delineate Migratory Patterns and Catchment Areas of White-throated Sparrows at a Migration Monitoring Station. Oecologia 144: 541-549. Mazerolle, D. F., and K. A. Hobson. 2005. Estimating Origins of Short-Distance Migrant Songbirds in North America: Contrasting Inferences from Hydrogen Isotope Measurements of Feathers, Claws, and Blood. The Condor 107: 280-288. . 2007.Patterns of differential migration in White-throated Sparrows evaluated with isotopic measurements of feathers. Canadian Journal of Zoology 85: 413-420. Meehan, T. D, J. T. Giermakowski, and P. M. Cryan. 2004. GIS-based model of stable hydrogen isotope ratios in North American growing-season precipitation for use in animal movement studies. Isotopes in Environmental and Health Studies 40: 291 300. Melcer R., and A. Nightingale. 2009. Rocky Point Observatory Fall landbird migratory monitoring: 2009 data summary. [Internet], [cited 2011 Dec 16]. Available fromhttp://www.islandnet.com/~rpbo/finalreport09.pdf Mila, B., T.B. Smith, and R. K. Wayne. 2006. Postglacial Population Expansion Drives The Evolution Of Long-Distance Migration In A Songbird. Evolution 60: 2403-2409. Mugaha Marsh Nature Observatory. 2009. Mugaha Marsh Nature Observatory 2009 Annual Report. [Internet], [cited 2010 Nov 11]. Mackenzie, British Columbia, Canada. Available from http://www.mackenzienatureobservatory.ca/documents/Annual%20Reports.htm . 2011. Mugaha Marsh Nature Observatory 2011 Annual Report. [Internet], [cited 2012 Jan 08], Mackenzie, British Columbia, Canada. Available from http://www.mackenzienatureobservatory.ca/documents/Annual%20Reports.htm National Audubon Society. 2010. 110th Annual Christmas Bird Count. [Internet], [cited 2011 Jul 06]. Available from http://www.christmasbirdcount.org. 102 Norris, D R., M. B. Wunder, M. Boulet, and B Wunder. 2006. Perspectives on Migratory Connectivity. Ornithological Monographs 61: 79-88. Ogle, S.. 2008. Annual Report for2006 and 2007. [Internet], [cited 2011 Dec 16]. Available from http://www.natureconservancy.ca/site/DocServer/TLBO_Annual_Rreport_2006_and _2007.pdf?docID=4721. . 2009a. Tatlayoko Lake Bird Observatory Annual Report 2008 -Draft. [Internet], [cited 2011 Dec 16]. Available from http://www.natureconservancy.ca/site/DocServer/TLBO_Annual_Rreport_2008.pdf? docID=4741. . 2009b. 2009 Annual Report Tatlayoko Bird Observatory. [Internet], [cited 2011 Dec 16], Available from http://www.natureconservancy.ca/site/DocServer/TLBOreport09_SOgle.pdf7docID5441. Osborn, R. G., C. D. Dieter, K. F. Higgins, and R. E. Usgaard. 2011. Bird Flight Characteristics Near Wind Turbines in Minnesota. The American Midland Naturalist 139: 29-38. Paetkau D, R Slade, M. Burden, and A. Estoup. 2004. Direct, real-time estimation of migration rateusing assignment methods: a simulation-based exploration of accuracy and power. Molecular Ecology 13:55-65. Pain, D. J, R. E Green, B. GieBing, A. Kozulin, U. Ottosson, M. Flade, and G. M Hilton. 2004. Using stable isotopes to investigate migratory connectivity of the globally threatened aquatic warbler Acrocephalus paludicola. Ecology 138: 168-174. Perez-Tris, J., S. Bensch, R. Carbonell, A. J Helbig, and J. L. Tellerfa. 2004. Historical Diversification of Migration Patterns in a Passerine Bird. Evolution 58: 1819-1832. Petren, K. 1998. Microsatellite primers from Geospiza fortis and cross-species amplification in Darwin's finches. Molecular Ecology 7: 1782. Piry, S., A. Alapetite, J.-M. Cornuet, D. Paetkau, L. Baudouin, and A. Estoup. 2004. GENECLASS2: A Software for Genetic Assignment and First-Generation Migrant Detection. Journal of Heredity 95: 536 -539. Poesel, A., H. L. Gibbs, and D. A. Nelson. 2009. Twenty-one novel microsatellite DNA loci isolated from the Puget Sound white-crowned sparrow, Zonotrichia leucophrys pugetensis. Molecular Ecology Resources 9: 795-798. Pritchard, J., W. Xiaoquan, and D., Falush. 2007. Documentation for structure software: 103 Version 2.2. [Internet], [cited 2011 March 01]. Available from http://pritch.bsd.uchicago.edu/software. Priestley, L. 2007. Beaverhill Bird Observatory 1987 to 2006 - 20 Years of Monitoring. [Internet], [cited 201 IDec 16]. Beaverhill Bird Observatory, Edmonton, AB. Available from http://beaverhillbirds.com/docs/BB0 20_years.pdf Rannala, B., and J. L. Mountain. 1997. Detecting immigration by using multilocusgenotypes. Proceedings of the National Academy of Sciences of the United States of America 94: 9197 -9201. Ridgely, R.S., T.F. Allnutt, T. Brooks, D.K. McNicol, D.W. Mehlman, B.E. Young, and J.R. Zook. 2007. NatureServe: Digital Distribution Maps of the Birds of the Western Hemisphere Version 3.0. [Internet], [cited 2010 Nov 18]. Available from http://www.natureserve.org/getData/birdMaps.jsp. Rodriguez, J. P.. 2002. Range Contraction in Declining North American Bird Populations. Ecological Applications 12: 238-248. Rozen, S., and H. Skaletsky. 2000. Primer3 on the WWW for general users and for biologist programmers. In Krawetz S, Misener S (eds) Bioinformatics Methods and Protocols: Methods in Molecular Biology, 365-386. Totowa, Nj: Humana Press. Ruegg, K., and T. Smith. 2002. Not as the Crow Flies: A Historical Explanation for Circuitous Migration in Swainson's Thrush (Catharus ustulatus). Proceedings of the Royal Society: Biological Sciences 269: 1375-1381 Segelbacher, G. 2002. Noninvasive genetic analysis in birds: testing reliability of feather samples. Molecular Ecology Notes 2: 367-369. Sibley, D., C. Elphick, and J. Dunning. 2001. Sibley Guide to Bird Life and Behavior. First Edition. Alfred A. Knopf. Sibley, D. 2000. The Sibley Guide of Birds. First Edition. Alfred A. Knopf. Smith, R. B, T. D Meehan, and B. O. Wolf. 2003. Assessing migration patterns of sharpshinned hawks Accipiter striatus using stable-isotope and band encounter analysis. Journal of Avian Biology 34: 387-392. Smallwood, K.S., and C. Thelander. 2008. Bird mortality in the Altamont Pass Wind Area, California. Journal of Wildlife Management 72: 215-223. Stevenson, S., H. Armleder, A. Arsenault, D. Coxson, C. Delong , and M., Jull. 2011. British Columbia's Inland Rainforest: Ecology, Conservation and Management. Vancouver, BC, Canada: UBC Press. 104 Tamura, K., J. Dudley, M. Nei, and S. Kumar. 2007. MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0. Molecular Biology and Evolution 24: 1596-1599. Toews, D., and D. E. Irwin. 2008. Cryptic speciation in a Holarctic passerine revealed by genetic and bioacoustic analyses. Molecular Ecology 17: 2691-2705. Thomson, R., I. J. Wang, and J. Johnson. 2010. Genome-enabled development of DNA markers for ecology, evolution and conservation. Molecular Ecology 19: 2184-2195. Tuttle, E. 2003. Alternative reproductive strategies in the White-throated Sparrow: behavioral and genetic evidence. Behavioral Ecology 14: 425 -432. USGS. 2012. Resources on Isotopes. [Internet], [cited 2012 March 09], Available from: http://wwwrcamnl.wr.usgs.gov/isoig/res/fiinda.html Wassenaar, L I, and K A Hobson. 2003. Comparative equilibration and online technique for determination of non-exchangeable hydrogen of keratins for use in animal migration studies. Isotopes In Environmental And Health Studies 39: 211-217. . 2006. Stable-hydrogen isotope heterogeneity in keratinous materials : mass spectrometry and migratory wildlife tissue subsampling strategies. Rapid Communications in Mass Spectrometry 20: 2505-2510. . 2001. A stable-isotope approach to delineate geographical catchment areas of avian migration monitoring stations in North America. Environmental Science & Technology 35: 1845-1850. Webster, M. S., P.P. Marra, S.M. Haig, S. Bensch, and R. T. Holmes. 2002. "Links between worlds: unraveling migratory connectivity".Trends in Ecology & Evolution 17: 7683. Winker, K., G. R. Graves, and M. J. Braun. 2000. Genetic differentiation among populations of a migratory songbird : Limnothlypis swainsonii. Journal of Avian Biology 31: 319328. Wong, E, and R. Hanner. 2008. DNA barcoding detects market substitution in North American seafood. Food Research International 41: 828-837. Wythe, M. W. 1938. The White-throated Sparrow in Western North America. The Condor 40: 110-117. Yohannes, E., K. A. Hobson, D. J. Pearson, and L. I. Wassenaar. 2005. Stable isotope analyses of feathers help identify autumn stopover sites of three long-distance migrants in northeastern Africa. Journal of Avian Biology 36: 235-241. 105 Zink, R. 2008. Microsatellite and mitochondrial DNA differentiation in the fox sparrow. The Condor 110: 482-492. Zink, R., and G. Barrowclough. 2008. Mitochondrial DNA under siege in avian phylogeography. Molecular Ecology 17: 2107-2121. Zink, R. M. 1994. Geography of mitochondrial DNA variation, population structure, hybridization and species limits in the fox sparrow (Passerella iliaca). Evolution 48: 319-328. 106 APPENDIX 1 RAW DATA SUMMARY Table Al.l Raw data of breeding individuals including: Alleles of eight neutral microsatellites, mtDNA Cytochrome Oxidase I haplotypes, deuterium stable isotopes 5Df (%o) for tail and head feather samples. GPS coordinates were taken per sampling location. Additional Microsatellites MtDNA Stable isotopes Tail 6D, Sample ID Location Prince George UTM Collection Date - A08 B01 B03 A02 C06 Cll C07 Fll 272 284 254 182 232 180 224 261 4-01 Haplotype Head 8 <%•> (%o) 7 7 B Prince George - 272 294 262 190 236 188 224 269 264 272 246 178 224 182 256 254 4-02 Prince George - 294 262 194 252 182 256 265 268 268 258 190 232 190 240 249 4-03 7 Prince George - 280 262 190 236 194 240 257 268 280 254 174 224 188 224 291 4-04 - 7 7 - 7 7 B Prince George - 268 294 258 194 224 188 248 291 268 284 258 178 232 194 256 249 4-09 - 7 7 B Prince George - 280 288 258 186 236 194 256 253 272 252 254 182 216 180 216 253 4-10 - 7 7 B Prince George - 272 282 262 190 232 194 260 261 268 256 246 174 224 184 216 249 4-11 - 7 7 B Prince George - 272 294 254 174 236 194 260 249 276 268 250 190 224 180 220 253 4-12 - 7 7 B Prince George - 284 284 258 198 228 196 220 277 272 288 250 170 224 180 276 245 272 294 258 170 232 184 276 249 276 252 250 174 236 188 232 241 4-17 - * - A Prince George - 4-18 - 7 7 B Prince George - 276 288 254 194 240 194 232 257 272 276 246 182 216 194 236 245 4-19 276 276 254 186 107 224 200 240 261 - 7 B Song recorded 7 B 280 ( - B 280 Info d 7 Table A 1.1 Continued Additional Microsatellites Sample ID Location Prince George UTM - MtDNA Collection Date AOS B01 B03 A02 C06 Cll C07 Fll - 272 280 246 174 216 180 224 253 4-20 Stable Isotopes Haplotype Info Tail 5 D, Head 80 f Song <%•) (%o) recorded ? ? A Prince George - 272 294 258 182 232 196 224 269 276 280 242 186 220 188 248 253 284 284 254 198 224 194 248 253 272 264 254 182 206 188 248 253 1 - ? ? - - - 4-21 Prince George - - 4-26 A Prince George - - 276 268 262 186 206 192 276 253 264 272 254 174 232 180 236 245 4-27 ? -> B Prince George - 276 288 258 182 240 188 276 249 272 280 246 188 228 190 256 261 4-33 - - - B Prince George - - 272 294 250 194 232 194 276 265 276 288 250 186 224 180 205 249 4-34 - 7 ? B Prince George - - 280 290 258 194 232 194 224 261 268 284 258 190 232 184 225 257 272 294 266 194 236 184 232 257 260 260 254 182 216 184 208 249 4-35 - ? ? B Prince George - - 4-46 - ? ? B Prince George - - 260 294 258 190 228 184 234 257 260 268 258 182 228 180 232 249 4-52 - ? ? B Prince George 10U 0512078 E 26-May-09 280 294 266 190 228 180 248 253 272 264 250 174 216 194 240 245 Zoal-ia008 - ? ? B 5971972 N Prince George 10U 0512002 E 27-May-09 276 280 258 186 232 196 240 261 276 268 254 174 232 180 260 265 Zoal-ia009 - ? ? B 5971738N Prince George 10U 0512078 E 26-May-09 276 294 262 182 232 188 264 273 268 264 246 178 224 194 252 253 Zoal-iaOlO - ? -> B 5971972 N Zoal-jal64 - Prince George " - 280 288 258 186 240 196 252 261 276 280 242 182 216 184 232 269 276 294 250 194 232 184 252 273 108 B - -148.7 -77.6 - Table A 1.1 Continued Additional Microsatellites Sample ID Location UTM Prince George MtDNA Collection Date AOS 801 B03 A02 C06 Cll C07 Fll . 272 276 258 182 232 172 240 265 276 298 266 198 232 196 240 269 264 284 254 178 220 180 ? 253 Haplotype Stable Isotopes Head &D f Song (%.) (%.) recorded -150.9 -110.6 ? Zoal-jal6S Prince George . Zoal-jal66 - -142.8 -107.6 B Prince George 272 294 258 186 228 180 ? 261 268 280 254 190 220 180 228 249 276 294 270 190 224 194 234 261 272 252 254 190 212 180 208 249 Zoal-jal67 - -140.1 -91.5 . B Prince George . Zoal-jal68 -141.9 -65.2 B Prince George 280 294 262 194 236 194 236 269 280 284 232 166 232 180 232 245 Zoal-jal69 - -148.2 -95.6 B Prince George 284 294 258 182 236 180 252 253 272 284 250 186 224 184 232 249 284 294 250 190 232 184 252 273 276 272 246 182 228 168 228 ? Zoal-jal70 - -139.2 -79.3 B Prince George Zoal-jal71 - -139.6 -77.2 B Prince George 276 290 254 186 240 168 228 ? 264 260 254 186 212 184 232 241 Zoal-jal72 Info Tail 6 O r - _ _ -151.4 -123.2 B Prince George . 272 294 254 190 220 184 244 245 268 288 250 178 216 180 216 205 Zoal-jal73 B Prince George 10 U 0511144 E 30-May-10 276 294 254 194 236 188 248 205 268 264 242 182 232 180 228 253 Zoal-jal74 . -89.8 -73.6 B 5971689 N Prince George 10 U 0511144 E 30-May-10 272 294 258 194 240 180 228 253 272 294 242 178 228 180 216 241 Zoal-jal75 . -152.3 -64 B 5971689 N Prince George 10 U 0511144 E 31-May-10 272 294 262 186 228 180 252 253 7 268 258 178 228 178 188 261 Zoal-jal76 -138 K 5971689 N ? 294 266 178 109 228 178 272 265 . -76.7 . Table A 1.1 Continued Additional Microsatellites MtDNA Sample ID Location UTM Collection Date A08 B01 B03 A02 C06 Cll C07 Fll Prince George 10U0511144E 30-May-10 272 280 242 178 224 180 228 241 272 294 258 182 228 184 228 249 272 280 242 186 232 180 248 249 Zoal-jal77 Prince George 10 U 0511144 E 31-May-10 Zoal-jal78 HeadSDi Song <%•) <%o) recorded -137.9 -102.2 - -142.1 -60.4 B 5971689 N Prince George 10 U 0511144 E 31-May-10 276 290 250 198 232 180 264 261 268 276 258 182 228 180 188 249 Zoal-jal79 - -142.7 7 D 5971689 N Prince George 10U0511144E 31-May-10 280 276 262 186 232 180 220 257 276 264 258 198 236 192 7 249 Zoal-jal80 Info Tail8Dr O 5971689 N - -148.5 -65.7 7 -81.3 N 5971689 N Prince George 10 U 0511144 E 31-May-10 276 290 262 202 236 192 253 268 290 246 182 220 7 257 Zoal-jal81 B 5971689 N Prince George 10 U 0511144 E 30-May-10 276 294 258 186 228 7 272 272 254 182 220 188 - 257 253 -156.8 ? -151 -78.3 7 Zoal-jal82 5971689 N Prince George 10 U 0511144 E 30-May-10 276 280 262 198 228 192 7 272 272 262 178 228 7 7 257 249 7 Zoal-jal83 5971689 N Prince George 10 U 0511144 E 30-May-10 280 290 266 180 232 7 7 273 7 7 ? 7 7 7 7 7 ? ? 7 7 7 7 7 7 272 272 246 182 214 184 236 249 - -156.7 -138.7 - - 7 Zoal-jal88 5971689 N John Prince Forest 10U 0409749 E 17-Jun-09 Zoal-ib025 B 6055679 N John Prince Forest 10U 0416594 E 19-Jun-09 286 280 254 190 224 192 236 261 280 264 254 178 232 184 220 245 280 272 258 194 248 188 248 261 280 280 246 194 224 188 212 249 Zoal-ib028 - - - B 6052975 N John Prince Forest 10U 0416594 E 19-Jun-09 Zoal-ib045 - Yes B 6052975 N Zoal-ib055 Haplotype Stable Isotopes John Prince Forest 10U 0416594 E 6052975 N 19-Jun-09 284 294 254 198 236 192 244 261 268 280 254 186 232 188 268 249 280 284 254 186 236 192 268 261 110 C - Table Al.l Continued Additional Mierosatellites Sample ID Location UTM Collection Date MacLeod Lake 10U 0513725 E 26-Jun-09 Zoal-ih019 10U 0513725 E MacLeod Lake 10U 0498878 E Zoal-ih020 MacLeod Lake Zoal-ih032 10U 0500490 E MacLeod Lake 10U 0500490 E 10U 0500490 E MacLeod Lake 10U 0498878 E Zoal-ih034 182 228 180 220 237 228 192 234 261 272 284 257 174 210 184 224 249 276 294 262 182 236 192 224 257 268 276 254 186 228 180 188 249 272 300 254 194 236 192 252 261 268 276 238 178 240 180 224 253 282 294 266 190 245 196 240 269 268 284 246 182 216 188 260 253 276 296 250 182 224 192 260 253 272 252 250 170 212 192 264 249 272 292 254 178 236 192 268 261 272 268 254 180 228 180 220 257 276 294 254 198 240 192 240 265 272 264 250 178 220 188 212 245 280 280 254 186 228 192 240 245 28-Jun-09 272 268 254 178 228 192 248 257 276 276 254 194 236 192 248 265 26-Jun-09 276 292 254 174 216 188 264 253 280 294 262 182 220 192 270 253 276 260 250 182 220 176 212 249 280 294 258 190 232 188 212 287 272 260 238 190 208 176 204 249 272 284 254 194 228 192 244 253 26-Jun-09 26 Jun-09 29-Jun-09 29-Jun-09 29-Jun-09 29 Jun-09 26-Jun-09 6108321 N MacLeod Lake Zoal-ih047 10U 0508224 E 6078477 N MacLeod Lake Zoal-ih052 10U 0513725 E 6067293 N MacLeod Lake Zoal-ih053 10U 0498993 E 26-Jun-09 6108354 N MacLeod Lake 10U 0498878 E 6108321 N Fll 186 6114644 N Zoal-ih046 C07 266 6114644 N MacLeod Lake Cll 254 6114644 N Zoal-ih033 C06 264 6114644 N MacLeod Lake Zoal-ih054 10U 0500490 E A02 294 6108321N Zoal ih031 B03 272 6067293 N Zoal-ih029 BOl 280 6067293 N MacLeod Lake AOS MtPNA 28-Jun-09 111 Haplotype Stable Isotopes Info Tail SD, Head 6 D, Song <%o) (%») recorded - - Yes B - - - - B B - - - - - B ? Yes - - Yes B - - A Yes - - - - - - - - - - B B Yes B B ? - Yes Table A 1.1 Continued Additional MtONA Microsatellites Sample ID Location Moberly Lake UTM 10U 0588005 E Collection Oate 31-May-09 A08 BOX B03 A02 C06 Cll C07 Fll 272 294 262 182 228 180 240 249 272 296 262 198 232 188 240 253 264 288 250 186 224 184 248 241 Haplotype Stable Isotopes Head 6 0, Song (%„) (%.) recorded _ A Zoal-ic012 6181234 N Moberly Lake 10U 0571140 E 31-May-09 Info Tail 8D, Yes - ? Zoal-ic013 6182770N Moberly Lake 10U 0588005 E 01-Jun-09 276 288 254 198 228 192 252 249 280 264 254 182 224 184 224 249 Zoal-ic014 - H 6181234 N Moberly Lake 10U 0571140 E 30-May-09 280 294 266 186 236 192 236 253 272 268 254 186 224 176 248 249 ZoaMc018 - _ B 6182770N Moberly Lake 10U 0571140 E 30-May-09 280 298 268 194 236 192 280 249 276 280 246 182 220 180 248 241 Zoal-ic039 _ B 6182770 N Moberly Lake 10U 0571140 E 31-May-09 276 294 256 190 232 188 248 261 280 268 254 174 228 176 248 213 Zoal-ic040 - _ B 6182770 N Moberly Lake 10U 0571140E 30-May-09 280 268 262 182 228 192 268 213 272 280 246 170 224 180 240 245 Zoal-ic048 _ B 6182770 N Moberly Lake 10U 0571140 E 30-May-09 280 294 250 186 232 192 240 269 272 280 258 182 236 192 228 257 Zoal-ic049 Yes _ B 6182770 N Moberly Lake 10U 0571140 E 30-May-09 272 284 266 190 248 192 228 261 276 272 246 170 232 188 232 237 Zoal-icOSO . _ B 6182770N Moberly Lake 10U 0571140 E 31-May-09 280 298 254 204 236 188 244 257 272 272 250 182 212 192 248 257 Zoal-ic058 Yes _ B 6182770 N Moberly Lake 10U 0588005 E 29 May-09 276 276 254 186 248 192 272 261 272 264 250 190 228 188 208 249 Zoal-ic065 . _ _ . 7 6181234 N Dawson Creek 10U 0680870 E 02-Jun-09 272 284 262 198 228 192 260 249 272 280 262 170 228 164 244 233 Zoal-id007 _ B 6176372 N 280 294 262 194 112 232 192 256 253 _ Table Al.l Continued Additional Microsatellites MtDNA Collection Sample ID Location Dawson Creek UTM 10U 0680708 E Date 03-Jun-09 A08 B01 803 A02 C06 Cll C07 Fll 276 268 ? 178 224 192 200 245 276 284 ? 180 228 196 228 261 272 272 246 178 216 184 244 249 Zoal-id015 Stable Isotopes Tail §Dr Head 8Dt Song e <%.) (%o) recorded _ B 6177208 N Dawson Creek 10U 0668852 E 04-Jun-09 Zoal-ie017 Info Haplotyp - _ A 6176114 N Dawson Creek 10U 0668852 E 04-Jun-09 272 290 250 178 224 192 276 253 286 280 246 186 232 180 252 239 Zoal-ie027 _ _ B 6176114 N Dawson Creek 10U 0668852 E 04-Jun-09 288 290 254 198 248 196 268 239 280 272 254 166 216 188 228 261 Zoal-ie030 . - _ B 6176114 N Dawson Creek 10U 0680870 E 02-Jun-09 280 304 262 178 236 192 236 261 272 268 254 190 232 188 252 249 Yes -153.5 ? _ _ _ _ ? Zoal-id038 Dawson Creek 6176372 N 06-Jun-10 276 272 254 194 236 192 268 253 10U 0668852 E 04-Jun-09 264 280 246 178 220 184 220 257 Zoal-ie044 B 6176114N Dawson Creek 10U 0668852 E 04-Jun-09 280 294 258 186 228 188 236 257 272 280 254 178 224 180 224 253 Zoal-ie051 B 6176114N Dawson Creek 10U 0668852 E 04-Jun-09 272 294 262 186 228 180 240 269 264 252 250 182 220 188 228 249 Zoal-ie060 - -151.9 -138 _ _ L Dawson Creek 6176114 N 05-Jun-10 272 276 274 190 236 192 232 269 10U 0680809 E 05-Jun-09 272 252 246 194 224 192 272 245 Zoal-id062 B 6176912 N Dawson Creek 10U 0668852 E 04-Jun-09 280 260 258 198 228 196 272 253 272 250 254 186 232 184 236 257 Zoal-ie064 . _ _ B 6176114N Dawson Creek 10U 0680721E 07-Sep-09 276 284 254 190 236 192 272 257 268 276 256 194 224 184 244 241 Zoal-iel34/Zoal-jdl63 _ B Dawson Creek 6177627 N 05-Jun-10 272 294 270 198 232 188 276 249 10U 0680721E 05-Jun-10 272 272 250 186 224 188 224 245 276 276 254 190 228 200 256 253 Zoal-jel36 6177627 N 113 R . -154.3 -116.8 Table Al.l Continued Additional Microsatellites MtPNA Stable Isotopes Sample ID Location UTM Collection Date A08 B01 B03 A02 C06 Cll C07 Fll Dawson Creek 10U 0680721E 05-Jun-10 272 288 236 178 216 176 252 253 280 290 250 194 240 192 264 253 272 280 250 162 228 188 234 241 Haplotype Song (%.) (%„) recorded -164.3 -82.6 ? Zoal-jel37 6177627 N Dawson Creek 10U 0680721 £ 05-Jun-10 - -165.6 -117.1 B Zoal-jel38 6177627 N Dawson Creek 10U 0680721E 05-Jun-10 272 284 266 162 248 192 252 241 268 284 242 182 216 184 220 245 276 294 246 186 244 188 264 249 264 260 ? 182 220 188 232 225 272 272 ? 294 244 192 248 273 272 268 226 170 232 180 240 249 Zoal-jel39 info Head 80, Tail 8D, - ? -81.9 -150.3 -101.4 B 6177627 N Dawson Creek 10U 0668826 E 0S-Jun-10 Zoal-jel40 B 6176112 N Dawson Creek 10U 0668826 E 05-Jun-10 Zoal-jel41 - -161.5 -54.3 A 6176112 N Dawson Creek 10U 0680833 E 06-Jun-10 272 294 254 194 236 184 240 265 268 268 246 182 224 192 240 241 Zoal-jdl42 - -160.3 -105.5 M 6176357 N Dawson Creek 10U 0680833 E 06-Jun-10 268 272 246 182 232 196 248 249 276 292 230 182 228 180 244 245 Zoal-jdl43 - -149.9 -102.7 B 6176357 N Dawson Creek 10U 0680833 E 06-Jun-10 276 294 254 194 232 192 244 261 276 260 230 182 220 184 240 249 280 280 238 190 224 188 264 253 272 264 246 178 224 184 224 257 Zoa!-jel44 - -150.6 -111.6 A 6176357 N Dawson Creek 10U 0680833 E 06-Jun-10 Zoal-jel45 - -138.3 -68.7 B 6176357 N Dawson Creek 10U 0668675 E 07-Jun-10 280 280 262 190 232 188 228 257 272 268 234 162 232 196 256 253 Zoal-jel46 - -148.7 -145.9 ? 6175712 N Dawson Creek 10U 0668675 E 07-Jun-10 276 280 254 190 240 200 256 253 276 268 246 162 224 188 228 249 Zoal-jel47 - -130 -109.6 ? 6175712 N Dawson Creek 10U 0668675 E 07-Jun-10 284 284 254 166 224 196 260 257 272 280 222 194 214 188 207 ? Zoal-jel48 -148.9 C 6175712 N 272 280 226 202 114 232 192 216 ? - -137.2 - Table Al.l Continued Additional Microsatellites MtDNA Stable Isotopes Sample ID Location UTM Collection Date A08 B01 B03 A02 C06 Cll C07 Fll Dawson Creek 10U 0668675 E 07-Jun-10 268 268 246 177 224 188 252 253 272 282 250 177 236 192 252 253 276 282 254 170 228 188 232 245 Zoal-jel49 Haplotype Song (%.) (%„) recorded -154.2 -89.9 S 6175712 N Dawson Creek 10U 0668675 E 07-Jun-10 Zoal-jel50 - -148.6 -104 B 6175712 N Dawson Creek 10U 0665804 E 07-Jun-10 284 282 280 288 280 294 268 276 254 258 186 232 192 272 262 194 228 176 212 253 262 194 232 188 228 253 170 228 184 ? 245 257 249 Info Head 50< Tail 6Dr - 253 Zoal-jel51 -156.1 -75 -156.8 -106.8 B 6173116 N Dawson Creek 10U 0665804 E 07-Jun-10 Zoal-jelS2 B 6173116 N Dawson Creek 10U 0665804 E 07-Jun-10 272 288 278 178 244 192 ? 272 276 254 ? 224 188 200 - -157.7 -55.2 ? Zoal-jel53 6173116N Dawson Creek 10U 0665804 E 07-Jun-10 284 280 258 ? 240 200 244 257 272 272 254 180 224 184 192 245 Zoal-jel54 - -163.6 -121.1 B 6173116N Dawson Creek 10U 0665804 E 07-Jun-10 276 294 270 188 232 188 252 245 272 256 254 180 230 188 264 241 Zoa[-jel55 - -162.2 -81.6 B 6173116 N Dawson Creek 10U 0665804 E 272 08-Jun-10 294 258 180 244 196 272 . 261 272 272 254 166 210 180 240 249 276 294 258 202 232 196 244 253 272 264 242 194 228 180 240 253 Zoal-jel56 -147.5 -59.1 B 6173116 N Dawson Creek 10U 0665804 E 08-Jun-10 Zoal-jel57 - -85.1 -103.9 A 6173116 N Dawson Creek 10U 0665804 E 08-Jun-10 272 294 258 194 232 188 244 261 264 268 252 178 210 184 224 249 272 294 270 190 240 188 252 253 276 280 254 172 202 192 260 249 Zoal-jel58 . -153.6 -97.9 B 6173116 N Dawson Creek 10U 0665804 E 08-Jun-10 Zoal-jel59 - -149.4 -72.3 A 6173116 N Dawson Creek 10U 0665804 E 08-Jun-10 276 288 258 172 248 196 260 257 264 272 262 186 220 184 220 241 Zoal-jel60 -167.6 B 6173116 N 272 294 262 186 115 236 188 220 249 - -58.7 Table A 1.1 Continued Additional IVIicrosatellites Sample ID Location UTM Collection Date Sikanni River 10U 0523139 E 08-Jun-09 MtDNA AOS B01 B03 A02 C06 Cll C07 Fll 264 256 250 182 216 188 244 257 264 294 258 198 236 188 276 265 272 276 250 178 228 180 258 261 Haplotype Stable Isotopes Head 6 D( Song (Xo) |%°) recorded A Zoal-if016 6327484 N Sikanni River 10U 0523139 E 09-Jun-09 Zoal-if021 - _ Yes 8 6327484 N Sikanni River 10U 0523139 E 07-Jun-09 272 294 254 178 232 192 268 261 268 268 250 178 232 180 249 257 _ ? Zoal-if043 6327484 N Tumbler Ridge 10U 0631034 E 12-Jun-09 272 294 262 178 236 188 256 261 272 264 270 162 232 ? 246 261 Zoal-ig006 - - 0 6106110 N Tumbler Ridge 10U 0625243 E 13-Jun-09 276 264 274 194 232 ? 252 261 272 256 254 182 228 188 224 253 Yes _ ? Zoal-ig022 6109316 N Tumbler Ridge 10U 0625243 E 13-Jun-09 280 266 266 190 252 188 224 257 272 284 254 182 224 180 260 257 Zoal-ig023 - _ B 6109316 N Tumbler Ridge 10U 0626343 E 13-Jun-09 272 294 254 186 224 196 272 261 276 276 254 182 240 180 220 253 Zoal-ig024 - _ B 6110423 N Tumbler Ridge 10U 0625243 E 13-Jun-09 280 294 258 190 244 196 268 253 272 272 254 174 216 188 232 245 Zoal-ig026 Yes _ Q 6109316 N Tumbler Ridge 10U 0631034 E 12-Jun-09 276 280 262 182 220 192 232 261 272 268 258 178 224 192 232 ? Zoal-ig042 - _ A 6106110N Tumbler Ridge 10U 0631034 E 12-Jun-09 276 292 262 178 232 200 256 ? 264 280 246 174 220 180 236 233 Zoal-ig056 - _ _ B 6106110N Tumbler Ridge 10U 0631034 E 12-Jun-09 276 290 258 194 224 188 264 249 272 280 254 186 236 180 224 253 Zoal-ig057 Yes _ B 6106110 N Tumbler Ridge 10U 0614788 E 10-Jun-09 280 286 258 186 240 192 272 261 260 264 254 160 216 188 236 249 6126559 N 272 276 258 186 116 232 188 252 253 _ Yes _ ? Zoal-ig059 Info Tail &Df Table Al.l Continued Additional Microsatellites Sample ID MtDNA Location UTM Collection Date A08 B01 B03 A02 C06 Cll C07 Fll Tumbler Ridge 10U 0614788 E 10-Jun-09 272 264 246 168 216 180 268 253 Zoal-ig061 Haplotype Stable Isotopes Head 60, Song (%.) (%•) recorded _ B 6126559 N Tumbler Ridge 10U 0625243 E 13-Jun-09 272 294 250 180 228 192 280 261 272 268 254 182 224 172 224 245 Zoal-ig063 - _ Yes B 6109316 N Tumbler Ridge 10U 0625243 E 13 Jun-09 280 288 256 186 228 192 228 253 272 284 250 182 220 192 232 245 Zoal-ig066 _ B 6109316N Ontario - - 280 288 274 194 224 192 248 249 272 260 258 182 220 176 192 261 5-24 Info Tail 5D, - _ B Ontario - - 284 290 262 194 228 196 252 265 252 284 258 186 220 188 256 253 7-06 _ G Ontario - 280 288 262 190 220 196 268 257 280 276 242 174 224 180 256 245 8-52 - _ F Ontario _ _ 280 294 250 186 224 196 268 253 272 276 242 190 232 184 236 241 276 294 262 194 236 184 252 257 268 288 258 186 232 192 220 253 8-55 . B Ontario - _ 9-05 _ B Ontario _ _ 276 290 262 186 240 196 276 257 272 272 254 182 208 180 200 245 9-81 _ B Ontario - _ 280 284 258 194 240 192 218 257 272 284 254 178 224 188 232 257 9-82 - _ C Ontario - 276 288 258 178 224 188 252 273 276 280 250 178 224 184 254 261 9-84 _ B Ontario - 280 284 266 182 228 188 276 261 272 252 254 178 228 192 224 257 9-85 _ B 276 268 258 186 117 232 196 272 265 . Table Al.l Continued Additional MtDNA Microsatellites Location UTM Collection Date A08 B01 B03 A02 C06 Cll C07 Fll Ontario - - 272 288 246 178 224 188 200 249 111 292 258 178 232 196 200 249 268 252 250 182 228 184 236 249 272 290 254 194 236 188 252 249 272 276 250 174 220 188 228 245 9-87 Haplotype Stable Isotopes Head 5 D, Song (%„) (%o) recorded - - - - - - - - - - - - - - - - - - - - - - B Ontario - - 9-89 B Ontario - - 9-90 B Ontario - - 272 294 258 174 232 192 264 261 256 276 254 182 220 180 242 245 9-91 J Ontario - - 268 294 266 194 228 188 242 249 268 272 254 186 216 176 192 245 9-93 K Ontario - - 272 294 268 186 232 188 236 249 272 268 262 182 234 180 228 221 276 294 268 184 240 188 236 253 9-94 Ontario - - 268 280 246 178 224 176 258 253 272 294 250 186 240 180 264 257 272 284 245 174 224 184 240 245 9-95 rt B Ontario - - 9-96 B Ontario - - 272 288 258 190 232 196 276 257 268 276 258 186 234 172 226 269 272 284 268 190 240 188 278 269 272 276 258 182 228 180 248 249 9-99 B Ontario - - 9-100 ? Ontario - - 276 300 262 198 232 184 256 261 264 276 254 182 232 184 220 221 9-101 B 264 294 254 186 240 192 118 220 249 Info Tail 5 D, Table A1.2 Raw data of migratory individuals including: Alleles of eight neutral microsatellites, Deuterium Stable Isotopes 5Df (%0) of tail feather samples. Tail 6 D, Collection Sample ID Location Zoal-ij097 Mugaha UTM Date 29-Aug-09 Marsh Zoal-ij098 Mugaha 29-Aug-09 Marsh Zoal-ij099 Mugaha 29-Aug-09 Marsh Zoal-ij 100 Mugaha 01-Sep-09 Marsh Zoal-ijlOl Mugaha 09-Sep-09 Marsh Zoal-ijl02 Mugaha 13-Sep-09 Marsh Zoal-ijl03 Mugaha 14-Sep-09 Marsh Zoal-ijl04 Mugaha 16-Sep-09 Marsh Zoal-ijlOS Mugaha 18-Sep-09 Marsh Zoal-iol06 Lesser 16-Sep-09 Slave Lake Zoal-iol07 Lesser 22-Aug-09 Slave Lake Zoal-iol08 Lesser 24-Aug-09 Slave Lake Zoal-iol09 Lesser 28-Aug-09 Slave Lake Zoal-iollO Lesser 16-Sep-09 Slave Lake Zoal-iolll Lesser 16-Sep-09 Slave Lake Zoal-ioll2 Lesser Slave Lake 16-Sep-09 AOS B01 B03 A02 C06 Cll C07 Fll 268 272 250 186 232 188 248 249 276 272 254 194 232 192 260 265 268 264 270 186 232 188 248 253 272 294 274 190 240 192 272 257 272 292 258 190 228 188 228 249 276 294 266 194 228 192 240 265 272 284 250 182 214 180 234 276 288 270 194 220 190 254 ? 260 284 254 170 228 196 254 261 264 288 242 174 228 196 266 265 272 268 258 194 228 180 228 249 276 290 258 194 232 192 240 257 268 284 250 178 220 180 242 237 280 288 260 186 228 186 266 261 276 264 238 182 216 176 244 241 280 268 250 194 224 196 272 245 272 268 258 ? 232 194 250 243 280 290 262 ? 240 194 282 251 280 268 254 182 232 188 ? 261 284 298 258 186 236 188 ? 269 272 268 254 170 216 184 236 249 280 284 266 182 224 192 248 261 276 254 254 174 228 184 240 257 280 294 262 190 228 192 272 257 272 288 258 186 232 188 236 245 272 294 258 194 236 192 236 253 272 284 254 174 244 180 ? 249 280 292 254 174 244 184 ? 261 268 288 258 182 228 180 220 253 272 294 258 182 252 184 232 261 268 280 262 178 240 188 212 249 280 294 274 186 252 188 260 261 119 (X.) -161.2 -160.8 -168.2 -163 -153.8 -154 -162.4 -157.5 -158.7 -142.8 -163.9 -151.1 -148.8 -149 -154 -148.7 Table A1.2 Continued Stable Microsatellite Isotopes Tail &D[ Collection Sample ID Location UTM Zoal-ioll3 Lesser - Date A08 B01 B03 A02 C06 Cll C07 Fll <%•) 16-Sep-09 268 280 238 170 220 188 208 257 -173.2 272 284 254 186 224 192 232 273 276 288 262 182 232 180 228 245 280 294 266 198 236 190 228 249 268 276 254 174 232 172 228 245 272 294 258 194 240 192 252 249 272 284 258 182 216 188 216 249 272 288 270 186 228 196 252 257 Slave Lake Zoal-ioll4 Lesser - 12-5ep-09 - 16-Sep-09 - 16-Sep-09 -160.6 Slave Lake Zoal-ikll5 Beaverhill Zoal-ikll6 Beaverhill Zoal-ikll7 Zoal-ikllS Beaverhill Beaverhill - - 16-Sep-09 16-Sep-09 274 258 248 194 220 184 ? 253 274 288 252 202 236 192 ? 253 280 284 254 184 228 192 216 249 280 284 258 194 232 192 264 265 260 288 246 182 228 184 232 253 Zoal-ikl20 Beaverhill - 21-Sep-09 268 292 262 186 228 188 240 265 Zoal-ikl21 Beaverhill - 21-Sep-09 268 260 258 194 228 192 240 245 268 264 262 194 232 192 272 249 Zoal-ikl22 Beaverhill - 21-Sep-09 276 254 268 178 232 188 204 253 280 254 288 202 236 192 276 253 276 252 254 178 220 190 236 249 276 268 258 202 224 190 260 257 272 290 234 198 220 184 236 233 280 294 254 198 232 188 240 261 272 272 246 186 216 180 222 249 280 294 250 198 224 190 252 253 268 288 254 182 214 182 254 261 276 294 258 194 220 190 266 265 276 294 254 186 224 172 272 249 280 304 258 190 232 188 272 261 272 276 238 178 224 176 224 245 280 290 250 194 224 184 244 269 272 280 258 168 224 176 244 245 272 282 262 194 236 188 260 253 264 276 254 178 232 172 228 249 276 294 254 186 232 192 240 249 Zoal-iml2B Rocky - 01-0ct-09 -149.5 -158.7 -175.7 -152.2 -163.2 -161.2 -150.2 -151.5 Point Zoal-iml24 Rocky - 30-Sep-09 - 26-Sep-09 -148.3 Point Zoal-iml25 Rocky -155.5 Point Zoal-id068 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181 N Zoal-id070 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181N Zoal-id071 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181 N Zoal-id072 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181 N Zoal-id073 Dawson 10U 0680970 E 08-Sep-09 Creek 6177181 N 120 - Table A 1.2 Continued Stable Microsateilite Isotopes Tail 6Df Collection Sample ID Location UTM Date AOS B01 B03 A02 coe Cll C07 Fll (%.) Zoal-id074 Dawson 10U 0680970 E 08-Sep-09 266 234 250 190 198 176 256 253 - 274 240 254 194 228 192 256 269 264 276 254 186 222 188 248 249 276 294 254 190 236 198 248 249 272 268 254 178 224 176 224 249 276 294 266 198 228 192 236 249 264 276 254 182 228 196 192 249 276 294 254 198 232 200 248 253 272 284 250 178 226 196 228 245 6177181N 272 284 262 186 232 200 240 257 10U 0680970 E ? ? 256 ? ? 188 252 249 ? ? 256 ? ? 188 256 249 264 264 250 194 220 176 240 257 276 276 264 202 240 188 248 261 268 280 258 182 232 180 244 249 276 294 262 194 232 184 252 265 272 280 246 178 232 188 266 249 284 294 258 182 244 196 272 257 09-Sep-09 272 276 262 178 232 192 250 245 272 288 274 182 232 196 284 269 09-Sep-09 256 232 254 194 232 184 252 241 272 268 266 198 256 184 260 265 272 280 256 ? 224 180 252 261 276 294 266 1 232 188 266 265 276 272 250 176 220 192 202 249 280 280 270 196 228 198 202 257 260 268 250 178 220 180 232 249 272 300 254 194 230 192 242 257 272 294 250 182 228 180 228 253 272 294 254 198 240 192 248 257 272 284 254 182 232 180 236 261 276 288 266 194 232 190 260 265 Creek 6177181N Zoal-id075 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181N Zoal-id076 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181N Zoal id077 Dawson 10U 0680970 E 08-Sep-09 - Creek 6177181N Zoal-id079 Dawson 10U 0680970 E 09-Sep-09 - Creek Zoal-id080 Dawson 09-Sep-09 - Creek 6177181N Zoal-id081 Dawson 10U 0680970 E 09-Sep-09 - Creek 6177181N Zoal-id082 Dawson 10U 0680970 E 09-Sep-09 - Creek 6177181N Zoal-id083 Dawson 10U 0680970 E 09-Sep-09 _ Creek 6177181N Zoal-id084 Dawson 10U 0680970 E - Creek 6177181N Zoal-id085 Dawson 10U 0680970 E _ Creek 6177181N Zoal-id086 Dawson 10U 0680970 E 10-Sep-09 Creek 6177181N Zoal-id087 Dawson 10U 0680970 E 10-Sep-09 - _ Creek 6177181N Zoal-id088 Dawson 10U 0680970 E 10-Sep-09 _ Creek 6177181N Zoal-id089 Dawson 10U 0680970 E 10-Sep-09 - Creek 6177181N Zoal-id090 Dawson 10U 0680970 E 10-Sep-09 Creek 6177181N 121 _ Table A1.2 Continued Stable Microsatellite Isotopes Tail Sof Collection Sample ID Location UTM Date A08 B01 B03 A02 C06 Cll C07 Fll (%.) Zoal-id091 Dawson 10U 0680970 E 10-Sep-09 276 276 258 182 220 184 214 245 - 276 288 266 190 228 188 224 253 272 276 250 178 232 176 216 245 276 298 254 182 236 192 272 265 272 284 254 186 220 180 214 253 272 294 254 186 228 188 214 261 272 268 254 174 232 192 228 249 280 268 254 182 236 192 228 265 272 276 246 178 216 192 212 249 272 288 260 194 224 196 264 253 272 284 262 182 224 180 234 253 284 294 262 186 236 192 236 257 268 260 250 178 224 176 236 249 284 290 258 186 240 188 248 253 272 276 246 178 216 192 212 249 272 288 260 194 224 196 266 253 272 268 256 178 220 192 236 249 280 288 260 186 224 192 260 269 272 280 254 186 232 180 224 245 276 294 262 190 232 192 252 249 264 284 254 178 224 188 200 241 268 294 266 182 228 192 260 249 266 272 266 190 228 188 ? 257 276 276 274 198 240 188 ? 265 ? 276 250 186 224 188 248 253 ? 276 254 206 228 196 242 257 276 268 254 182 220 180 224 249 276 294 258 190 220 192 260 253 Creek 6177181N Zoal-id092 Dawson 10U 0680970 E 10-Sep-09 _ Creek 6177181N 2oal-id093 Dawson 10U 0680970 E 10-Sep-09 - Creek 6177181N Zoal-id095 Dawson 10U 0680970 E 10-Sep-09 - Creek 6177181N Zoal-id096 Dawson 10U 0680970 E 10-Sep-09 - Creek 6177181 N ZoaMdl26 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl27 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl28 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl29 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl30 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181N Zoal-idl31 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl32 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl33 Dawson 10U 0680970 E 07-Sep-09 - Creek 6177181 N Zoal-idl3S Dawson 10U 0680970 E 07-Sep-09 Creek 6177181 N 122 _ APPENDIX 2 MITOCHONDRIAL DNA HAPLOTYPES >•!» i f,e, i r , T i T T f . T U T T a T f , f i ( T , f f , i f i r , i f T f , f , <*, r, f. a T i f. f. i f, T i r, r, i f, f, r T f. C T f i i T T i f t A A f T f , i T r i T i f f. vpc B napfcx.ps - c i - ; a i tj j « . t i ^ - i a o c . i c «. i w - ^ < Ha plot, pe C pe P Ha pbtv pe G 1 la plot, pe 11 ra pfcrrv pe 1 Ha pbtv pe J Itepbti pe K Ha pbt> pe I Hapfcvt, pfc M Ha pbtv p* ri Ha pbtv pe O l"Wt yLrtvpe pHa pbtv pe G. Ha pfet> pc r* rcptot/pe 1 < T 5 T T C i . 6 C C T 4 T O C C « i C i C < T ( 5 C T T C f i i i C < 3 i 3 T i O i 6 a f i i T < j C T A < 3 6 i 6 < > A O i i 4 < j i A i i O A T < S i < 5 < i « ATT<3AOOAOATA Mpbtv|>« I Hapbtvp* F haobtvoe 0 Ha plot* pe H MapL-ut* l J Hapbt, pc \ : Fbpbty p« L Knplrvtvp* M Hapbtvpe N nspioTv pe U MapluU j* F 1 toploty pe O. Ha pbt, pc r* Kiplntv'p* s t ( c hC» p Ir.r •|V I a i • i I I J. I I I (• <2 < I R LhduM.'iw D us p few .• p* k htaplo^.-pc F Ra pfc»t - pa F„ HaplOTv'pa M Haptotrpc 1 Ha pbt,-pc J Hapto-t.-pc I: i tapk>t. pe I I tap lot , pe r.i ltopb*> pe I-J 1 topbti pe © 1 tapbtv pc P 1 topbt. pe o. f fcpkM . pe X rk4pLriv-|« 5 Figure A2.1 Cytochrome Oxidase I (COI) fragment of the 19 different haplotypes of White-throated Sparrow sequences found during the present study at the breeding territories of Western Canada and Ontario. 125