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Aquatic and terrestrial movements of tailed frogs (ascaphus truei) in relation to timber harvest in Coastal… Wahbe, Tanya Rhoda 2003

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A Q U A T I C A N D T E R R E S T R I A L M O V E M E N T S O F T A I L E D F R O G S {ASCAPHUS TRUEI) IN R E L A T I O N T O T I M B E R H A R V E S T IN C O A S T A L BRITISH C O L U M B I A  by  TANYA RHODA WAHBE  B . S c , California State Polytechnic University, 1993 M . S c , The University of British Columbia, 1996  A T H E S I S S U B M I T T E D IN PARTIAL F U L F I L M E N T O F THE REQUIREMENTS FOR THE D E G R E E OF  DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Department of Forest Science; Centre for Applied Conservation Research)  W e accept this thesis as conforming to the required standard  T H E UNIVERSITY O F BRITISH C O L U M B I A July 2003 © Tanya Rhoda Wahbe, 2003  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada  ABSTRACT  ii  ABSTRACT  In British Columbia, Oregon, and California, coastal Ascaphus populations are designated 'at risk.' Local extirpation is a concern because recolonization may be slow, even if post-logging habitats recover quickly. In coastal BC streams flowing through clearcuts, second growth, and old growth, I investigated movements of larval Ascaphus and associations with stream parameters using area-constrained stream surveys. In old growth, larvae moved seven times farther than in clearcuts. Embedded logs, abundant in clearcut streams, may explain shorter larval movements. Using pitfall traps, I examined terrestrial movements of Ascaphus in clearcuts and old growth. More juveniles were trapped in clearcuts but more mature adults were trapped in old growth, suggesting fewer effective migrants in clearcuts. Many frogs moved at least 100 m from streams, and exhibited weaker stream affinity compared to inland Ascaphus that moved at least 12 m. In fall, I trapped frogs farther from streams in old growth than in clearcuts, and more frogs were trapped within 25 m of streams in clearcuts. Long distance overland movements appear more likely where forested stands are present. Using RAPDs, I examined population genetic structure of Ascaphus in an old growth and clearcut stream. In the clearcut, larvae were less diverse than in the old growth and exhibited no relationship between physical distance and genetic relatedness. In the old growth, larvae decreased in genetic similarity with increasing physical distance. Lower heterozygosity in the clearcut suggests that Ascaphus may be less able to adapt to environmental fluctuations and more susceptible to disease than larvae in the old growth. Maintaining viable populations throughout the range of Ascaphus is an underlying assumption of my thesis. My results suggest reduced recolonization potential and lower genetic variation where forest cover is absent. Aggregations of Ascaphus at individual streams may not represent distinct populations, and should not be managed as distinct units. Connectivity between multiple streams within a watershed will be a more meaningful unit of management than individual streams with forested buffers. Conservation measures more likely to promote long-term population persistence should be considered, such as the retention of a partial forest matrix between streams.  TABLE OF CONTENTS  iii  TABLE OF CONTENTS  Abstract  ii  Table of Contents  iii  List of Tables  vi  List of Figures  vii  >  Acknowledgements  *  C H A P T E R O N E : General Introduction  1  C H A P T E R T W O : Movements of Larval Ascaphus  6  INTRODUCTION STUDY A R E A METHODS  6 •  7 7  Stream Surveys and Parameters  7  Mark-Recapture  9  Statistical Analyses  9  RESULTS  10  Mortality Due to Marking  10  Recapture Rates  10  Movement Rates  12  DISCUSSION  14  Stream Gradient, Logjams, and Movement Rates  14  Algal Productivity and Movement Rates  15  C H A P T E R T H R E E : Terrestrial Movements of Juvenile and Adult Ascaphus INTRODUCTION  16 ••• 16  STUDY A R E A  17  METHODS  18  Pitfall Trap and Drift Fence Arrays  18  Mark-Recapture  19  Statistical Analyses  20  RESULTS  21  Catch Per Unit Effort  21  Recapture Rate  22  TABLE OF CONTENTS  iv  Movement Patterns  25  Distance from Stream  25  Movement Rates and Directions  29  Body Size and Condition DISCUSSION  30 30  Catch Per Unit Effort  30  Recapture Rate  31  Movement Patterns  31  Distance from Stream  31  Movement Rates and Directions  32  Body Size and Condition  C H A P T E R F O U R : Population Genetic Structure of Ascaphus at the Watershed Scale  35  36  INTRODUCTION  36  STUDY AREA  38  METHODS  39  Tissue Sampling Design  39  Isolation of DNA  40  Assay for R A P D Markers  40  Screening of Primers  41  Scoring of Diploid R A P D Markers  42  Statistical Analyses  42  RESULTS  43  Polymorphic Loci and Heterozygosity  43  Frequency of Dominant Alleles  45  Population Differentiation  45  Genetic Relatedness Along Stream Transects  48  D I S C U S S I O N ....  52  Heterozygosity  52  Population Differentiation  53  Geographic Distance and Genetic Relatedness  55  TABLE OF CONTENTS C H A P T E R FIVE: Summary and Conservation Implications  v 56  INTRODUCTION  56  S U M M A R Y OF RESULTS  57  IMPLICATIONS O F M Y R E S U L T S T O ASCAPHUS C O N S E R V A T I O N G O A L S  58  L I T E R A T U R E CITED  63  APPENDIX I Table A-1.1 Small mammal mortalities during field seasons  81  Table A-l.2 Number of amphibians encountered, trapped, and recaptured  82  Table A-l.3 Relative abundance of amphibians trapped  82  Figure A-1.1 Relationship between soil moisture and distance from stream  83  A P P E N D I X II Table A-l 1.1 R A P D s data and loci scored Table A-II.2 Gel schemes for electrophoresis  84 102  A P P E N D I X III Figures A-lll. Photo plates  104  Squamish and Mamquam watersheds  104  Adult female and male Ascaphus  105  Upland trapping array and second-growth stream  106  Streamside trapping array and old-growth stream  107  Green and red visual implant fluorescent elastomers  108  LIST OF TABLES  vi  LIST O F T A B L E S  Table 2.1. Larval Ascaphus movements (m/day) and stream parameters in southwestern British Columbia. 1995-1996 and 1999. Means are in parentheses. Logjams are ranked as 1 = low, 2 = medium, and 3 = high  11  Table 3.1. Number of nights of trapping and number of Ascaphus recorded (trapped and encountered. Southwestern British Columbia. 1998-2000  22  Table 3.2. Total captures, catch per unit effort ( C P U E : number of frogs per 100 trap nights), mean (+ Sx)  distance from stream, mean snout-vent length, individual mass, and total mass by  site for juvenile and adult Ascaphus at six sites. 1998-2000  24  Table 4.1. Site locations and stream attributes of Ascaphus truei tissue collection sites. Sample sizes are given at left of table  39  Table 4.2. Mean heterozygosity (H) and percentage of polymorphic loci at each stream reach station for old growth and clearcut. Mean values for old growth and clearcut were calculated using estimates obtained for each of the 61 loci; these are not based on means for each stream reach station. The total number of samples used to calculate estimates is given in the last row of the Average n column  44  Table 4.3. Genetic differentiation between old growth and clearcut ("population"), among stream reaches ("subpopulations") pooled within old growth and clearcut, and among stream reaches analyzed separately within old growth and within clearcut, as given by F j values S  47  Table 4.4. Hierarchical analysis of molecular variance: between old growth and clearcut ("among populations") and among stream reaches ("within populations") pooled within old growth and clearcut, as given by ® T values S  47  LIST OF TABLES  vii  Table 4.5. Similarity of R A P D bands within and among stream reach stations in a) clearcut and b) old growth. Reach 0 m is the top of the stream sampling area, and 180 m is the bottom. Values in the first row indicate similarities among individuals within each station. The rows that follow indicate similarities between stations of different distances from one another. For example, row four of the 8 0 - m station in the clearcut indicates that individuals sampled at the 8 0 - m and 140-m stations have a band-sharing frequency of 0.925  49  Table 4.6. Mean frequency of R A P D band sharing among individuals sampled within the same stream reach station (0 m) and among individuals sampled at increasing distances from one another (20-m apart, 4 0 - m apart, etc.). Categories indicate distances between stream reach stations. Standard errors for the parameter estimates ( S E ) were obtained with bootstrapping in E  which 100 repeated random samples were selected from the data and the model is estimated from each one (Norusis 1993)  50  Table A-1.1. Small mammal mortalities: forest cover type, watershed, year, month, and distance from stream (m). All shrew mortalities occurred in traps where escape ropes were not properly secured (either not touching bottom of trap, or rope missing due to bear disturbances)  81  Table A-l.2. The number of amphibians trapped, encountered, and recaptured during the Ascaphus field season. Southwestern British Columbia. 1998-2000  82  Table A-l.3. The relative abundance (number per 100 trap nights) of amphibians trapped during the Ascaphus field season. Southwestern British Columbia. 1998-2000  82  Table A-11.1. Six primers, and 61 loci scored with high confidence. Lab sample number, tadpole DNA sample number, treatment (TR: O G = Old Growth, C C = Clearcut), and distance (stream sampling station, in meters)  84  Table A-II.2. Gel scheme used for all samples. Gel scheme for samples 141-158 provided skipped wells for the repeats necessary when samples gave no reaction or bands showed weak signals  102  LIST OF FIGURES  viii  LIST O F F I G U R E S  Figure 1.1. Stream flowing through old-growth forest. Photo: T. Wahbe  3  Figure 1.2. Larval and adult male Ascaphus. Photo: T. Wahbe  3  Figure 2.1. Sampling scheme: Initial marking occurred in three 5 - m reaches (shaded) separated by 25 m; sampling for recaptures included two additional 10-m reaches (hatched). In the text, recaptures noted for reaches 2 and 3 include the contiguous 1 0 - m reaches. Arrows indicate direction of stream flow  8  Figure 2.2. Logjams and larval movements, a) Logjams estimated in each forest cover type: old growth, second growth, and clearcut, b) Larval movements per day (m) in streams with low, medium, and high levels of logjams. Values of 2.5 (between medium and high rankings) were lumped with values of 3 because movement values were similar. Error bars represent standard error of the mean  13  Figure 2.3. Individual movements of larvae in relation to stream gradient (some points overlap). Solid line above extreme movements represents a potential factor ceiling sensu Thompson et al. (1996)  14  Figure 3.1. Schematic map of study area showing relative positions of the four river basins, and the three replicates of each treatment: old growth (OG) and clearcut (CC)  17  Figure 3.2. A trapping grid (representing one experimental unit) showing arrays of streamside and upland pitfall traps and drift fences. Not to scale  18  Figure 3.3. Number of juvenile and adult Ascaphus trapped: a) differences among three watersheds, b) differences among three years  23  Figure 3.4. Number of Ascaphus trapped during each two-week period: a) juveniles and adults, b) females and males (juveniles and adults combined)  27  LIST OF FIGURES  IX  Figure 3.5. Number of Ascaphus trapped at each distance from stream: a) juveniles and adults, b) females and males (juveniles and adults combined). Values within bars represent sample sizes  28  Figure 3.6. Relative proportion of Ascaphus moving in four directions relative to stream: upstream, downstream, downslope (towards stream), and upslope (away from stream). Legend shows 5% scale for each direction. Fall data only  29  Figure 4.1. Map showing location of study area in south coastal British Columbia and schematic map showing relative positions of river basins near collection sites of Ascaphus truei. The streams flowing through the clearcut (CC) and old-growth (OG) sites are tributaries of the Mamquam River. Sites are approximately 1.6 km apart  38  Figure 4.2. Schematic showing 10 stream reaches (vertical bars) sampled for larval Ascaphus. Arrows indicate direction of stream flow. The 0 m reach is at the head of the stream sampling area, and the 180 m reach is closest to the Mamquam River. This sampling scheme was established in both the clearcut and the old growth  39  Figure 4.3. R A P D pattern obtained with primer 213. Lanes labelled "L" are size marker DNA fragments. The brightest middle band is 600 base pairs in molecular weight. Lane " N C " is the negative control, and " P C " is the positive control. Samples, sites ( C C = clearcut, O G = old growth), and sampling distances from left to right are: A) 96 = C C - 0 m; 9 7 - 9 9 = C C - 2 0 m; 100 = O G - 6 0 m; 101-103 = C C - 6 0 m; 104-107 = C C - 8 0 m, B) 108-109 = C C - 8 0 m; 110-115 = C C - 1 0 0 m; 116 = O G - 1 2 0 m  41  Figure 4.4. Frequency distributions of the dominant allele among the 61 R A P D loci scored: a) old growth, and b) clearcut  46  Figure 4.5. Scatterplot of genetic relatedness (frequency of R A P D band sharing) and physical distance (distance between stream sampling stations; meters apart) for individuals sampled from the clearcut and old-growth site  51  Figure A-1.1. Relative soil moisture at four distances from stream in old growth (OG) and clearcut (CC): 0 m, 25 m, 50 m, and 100 m. a) summer 1999, b) fall 1999, and c) fall 2000... 83 Figures A-lll. Photo plates of Ascaphus, trapping arrays, rivers, and marking technique  104  ACKNOWLEDGEMENTS  x  ACKNOWLEDGEMENTS My undergraduate advisors, Dr. Laszlo Szijj and Dr. Keith Arnold, encouraged me to come to the University of British Columbia to obtain my doctoral degree, and I am forever grateful. Many people have supported me throughout my doctoral research with their contributions of technical, academic, and personal guidance. I am most grateful to my co-supervisors, Dr. Fred Bunnell and Dr. R. Bruce Bury, but also to my current and past committee members, Dr. Antal Kozak and Dr. Alton Harestad, for their invaluable guidance. All their teachings will be treasured. This project could not have been completed without tremendous help from volunteers and field assistants. Jeff Arsenault, Mark Brooks, Leigh Burrows, Jennie Christensen, Michelle Eiswerth, Debbie Higgins, Nancy Holling, Isabelle Houde, Nancy Job, Eduardo Jovel, Brian Kreowski, Katherine Maxcy, Randy Morris, Yuka Ota, Jeffrey Peterson, Judy Rodrigues, Keri Sadowski, Peter Sandiford, Mark van Kleunen, and Mae Wahbe were all enthusiastic, dedicated field assistants. My gratitude is extended to the Vanderhoefs of Glacier Valley Farm for providing the use of their cabins and bunkhouse for field crews in Brackendale. Dave Guilbride of International Forest Products and Stu MacDonald of the Ministry of Forests assisted with site location and road access. I thank the Squamish Nation for access to their traditional territory. Discussions and reviews of previous drafts of the thesis or individual chapters were provided by numerous individuals outside of my committee. Dr. Mike Adams, Dr. Glenn Sutherland, Don Major, Ralph Wells, and Mark Boyland provided feedback and helpful discussions. Dr. XinSheng Hu provided informative discussions in AMOVA techniques for genetic analyses. Dr. Kyle Young and Dr. Ann Chan-McLeod reviewed an early draft of the thesis. Dr. David Huggard reviewed a draft of Chapter 3. Dr. Carol Ritland, Dr. Kermit Ritland, Charles Chen, and Jodi Krakowski reviewed drafts of Chapter 4 and provided helpful discussions in population genetics. Several anonymous reviewers provided critical reviews of Chapters 2 and 3. I am very grateful to those who assisted with molecular lab work. DNA isolation, PCR, and RAPD techniques in the Genetic Data Centre could not have been successful without patient training from Dr. Carol Ritland. Washington Gapare, Cherdsak Liewlaksaneeyanawin, Allyson Miscampbell, and Dr. Andreas Hamann also provided invaluable assistance in the lab. Outside my committee, I received additional support in statistical analyses from Dr. Val Lemay and Dr. Peter Marshall. Dr. Kermit Ritland wrote the band sharing frequency program and assisted with analyses.of genetic relatedness. Cherdsak Liewlaksaneeyanawin provided a tutorial in RAPDs scoring techniques, and provided assistance with the program WINAMOVA. Critical funding for my research came in the form of grants to Dr. Fred Bunnell from Forest Renewal British Columbia, Natural Sciences and Engineering Research Council of Canada, Western Forest Products, Weyerhaeuser, and Wildlife Habitat Canada. Also, I am grateful for two fellowship awards: a British Columbia Environmental Research Scholarship from the Ministry of Water, Land and Air Protection, and a California State University Forgivable Loan/Doctoral Incentive Program award. Special thanks to my friends and colleagues at the Centre for Applied Conservation Research for their support and encouragement throughout my dissertation research. I am most grateful to my soulmate and life partner, Eduardo Jovel, who encouraged and inspired me, and whose undying enthusiasm made my experience at UBC a delight. Special thanks goes out to my family for the tremendous encouragement, understanding, and support provided throughout my dissertation research. Thank you all. I dedicate this thesis to my parents, Samia Sabra Al Awar and Badih Salim Wahbe.  CHAPTER ONE: General Introduction  1  CHAPTER  ONE  G e n e r a l Introduction  Forests are structurally complex ecosystems that tend to contain greater vertebrate species richness than other terrestrial biomes (Wilson 1988; Bunnell and Kremsater 1990). Timber harvest can significantly alter the structure and function of forests, and in the temperate zone, habitat loss and forest fragmentation are major threats to species (Soule 1991; Caughley and Gunn 1996). Amphibians are particularly susceptible to removal of forest canopy because of their physiological requirements for cool, moist conditions. In many forests, amphibians are the most abundant vertebrate group (Burton and Likens 1975a; Crisafulli and Hawkins 1998), and some workers argue they may play a key role in ecosystem dynamics (deMaynadier and Hunter 1995). Although little experimental evidence for the role of amphibians in forest ecosystems exists, indirect evidence suggests amphibians may play a significant role in nutrient cycling (Burton and Likens 19755; Hairston 1987) and food web dynamics (Jaeger 1972; Burton and Likens 1975b; Fraser 1976). Thus, forest management practices that modify the density and distribution of amphibians may have important implications for forest function and productivity (Bormann and Likens 1979). Early signs of ecosystem dysfunction usually appear at the level of populations, affecting species with narrow ecological tolerances (Odum 1992). Numerous correlative studies have shown that the abundance of terrestrial and aquatic amphibian species is lower in harvested forests compared to mature forests in both western (Bury 1983; Bury and Corn 1988a; Dupuis and Bunnell 1999; Grialou etal. 2000) and eastern (Po.ugh etal. 1987; Ash 1988; Waldick 1997) North America. Because of their biphasic life histories (water and land), specific habitat requirements (Blaustein etal. 1994), longevity, and strong site fidelity compared with most vertebrates, amphibians are especially sensitive to environmental changes in ecosystems and may serve as valuable indicators of ecosystem dysfunction. There is limited knowledge about the ecological role, population ecology and life history of amphibians in Pacific Northwest forest systems. For some species, the effects of forest management have begun to be addressed, but for many species with complex life histories, we know little about them and the potential effects of timber management. For my  CHAPTER ONE: General Introduction  2  research, I selected the tailed frog {Ascaphus truei Stejneger) as the focal species for three reasons. 1) Ascaphus  truei has specialized habitat requirements and is of considerable  conservation interest. They are considered "vulnerable" in coastal British Columbia (BC Conservation Data Centre 2001), Oregon (Oregon Natural Heritage Program 2001), and California (California Natural Heritage Program 2001). 2) Current recommendations for protection of the species with riparian forest buffers is based on larval habitat needs, but data on the effects of timber harvest on larval population abundance and density are contradictory or dependent on geographic location. Also, available evidence from inland populations indicates that adults are long lived and have strong site fidelity, remaining within a narrow range of 0 20 m around streams (Daugherty and Sheldon 1982a). Movement potential appears low (Daugherty and Sheldon 1982a; Bury and Corn 1988a). Research on post-metamorphic Ascaphus  is sparse, and management strategies fail to  incorporate half of the life history. Thus, management efforts to protect  Ascaphus  populations in areas subject to timber harvest may not be effective. 3) Species richness in riparian areas of small streams is usually higher than upslope areas (Bunnell and Dupuis 1994), hence other species could benefit from protection of Ascaphus survival of Ascaphus Ascaphus  habitat were that found to be important to continued populations.  is endemic to the Pacific Northwest of North America. It is the only  representative of the family Ascaphidae (Nussbaum era/. 1983), and is a highly distinct lineage, perhaps a sister group to other anurans (Ford and Cannatella 1993; Jamieson ef al. 1993). It is believed to be the most primitive frog living due to the retention of many primitive morphological features that were lost in most extant anuran lineages (Ford and Cannatella 1993). Primitive features include the possession of ribs, short arms on sternum, tailwagging muscles, and nine vertebrae in front of the sacrum. The only other living frogs as primitive as Ascaphus  are three species in the genus Leiopelma (family Leiopelmatidae,  terrestrial-breeding frogs) of New Zealand (Green and Cannatella 1993), but the two groups may not be closely related (Green ef al. 1989; Green and Campbell 1992). Ascaphus  range from British Columbia (BC) to western Montana, between the  Pacific Coast and the Rocky Mountains, and southward to northern California (Corkran and Thorns 1996). Ascaphus  are found in mountainous, coniferous forests in cool, fast-flowing,  perennial streams that usually lack fish (e.g., Figure 1.1). The larvae (Figure 1.2) have a  CHAPTER ONE: General Introduction  large suctorial mouth to cling to rocks, an adaptation for their stream environment (Bury 1988; Welsh 1990). Ascaphus range in elevation from near sea level to above 2,140 m (Nussbaum era/. 1983; Leonard era/. 1993; Corkran and Thorns 1996) but, in coastal BC, Ascaphus occurs from near sea level to subalpine zones (30-1,100 m; Sutherland 2000). Habitat structure is reported to influence the occurrence and abundance of terrestrial (deMaynadier and Hunter 1995; Aubry 1997) and stream amphibians (Welsh and Ollivier 1998; Sutherland and Bunnell 2001). Several researchers have documented negative effects of forest harvesting activities on population density and relative abundance of Ascaphus larvae (Bury 1983; Bury etal. 1991; Corn and Bury 1989; Welsh and Lind 1991; Dupuis and Steventon 1999). Ascaphus are believed to be sensitive to habitat alteration because of their lengthy larval period of 1-5 years (Wahbe 1996; Wallace and Diller 1998; Bury and Adams 1999), small clutch size of 30-70 eggs (Metter 1967; Leonard et al. 1993), biennial reproduction at high elevation, inland sites (Rocky Mountains, Metter 1967; reproduction may be annual elsewhere, Bury era/. 2001), low recolonization potential (with the exception of Crisafulli and Hawkins 1998), and specialized characteristics of their habitats (Bury and Corn 1988a; Hawkins et al. 1988). They also have a low desiccation tolerance (Claussen 1973) and one of the lowest and narrowest temperature tolerances among anurans (Brattstrom 1963; de Vlaming and Bury 1970). Also, recent metamorphs are more vulnerable to desiccation and predation than adults (Jones and Raphael 1998).  Figure 1.1. Stream flowing through oldgrowth forest. Photo: T. Wahbe.  Figure 1.2. Larval and adult male Ascaphus. Photo: T. Wahbe.  Ascaphus in the Rocky Mountains may live 15 to 20 years (Daugherty and Sheldon 1982b) but do not reach reproductive maturity until they are seven or eight years of age (Daugherty and Sheldon 1982b; Brown 1990). Ascaphus breed in the fall (Metter 1964a;  CHAPTER ONE: General Introduction  4  Nussbaum era/. 1983). However, unlike most North American frogs, breeding migrations of Ascaphus (i.e., from stream to stream, or upland to stream) have not been reported. Ascaphus differ from most Pacific Northwest frogs in that fertilization is internal. Male Ascaphus possess an extended cloaca (misnamed the tail; Figure 1.2), which allows internal fertilization of the eggs. No other anuran is known to engage in copulation (Stebbins and Cohen 1995). Females (Appendix III) can retain sperm through winter (Metter 1964b). In mid-summer (after spring runoff), female Ascaphus attach strings of eggs to the undersides of rocky substrate (Brown 1975; Adams 1993; Karraker and Beyersdorf 1997; Bury et al. 2001). Ascaphus eggs are slow to develop, averaging six weeks to hatching (Brown 1975). Free-swimming larvae emerge from late August to September (Metter 1964a; Adams 1993), often overwinter at the nest site (Metter 1964a), and feed on the yolk sac (Brown 1990) until their first spring when a suctorial mouth fully develops (Metter 1964a). In general, time to metamorphosis varies with geographic location (Bury and Adams 1999) and elevation. Larvae in most populations metamorphose after 2 - 3 years (Metter 1967), but may take 4 - 5 years in high elevation or northern locales (Brown 1990; Wahbe 1996), or only one year in coastal areas in the southernmost portion of their range (Wallace and Diller 1998; Bury and Adams 1999). These life history traits suggest that Ascaphus are slow to reach maturity and best persist in relatively stable habitats (i.e., old-growth forests). Many amphibian species live in habitats that are altered or fragmented by human activities. Because of their permeable skin, most amphibians move relatively short distances and have limited dispersal capabilities compared to other vertebrates (Sinsch 1990). Some authors (e.g., Welsh 1990) have argued that recolonization of logged sites is critical to sustaining productive amphibian populations, but movement may be impeded in altered habitats. Blaustein era/. (1994) suggested that recolonization of sites following local extinction may be difficult for amphibians because: 1) physiological constraints limit amphibians to cool, moist habitats, 2) many amphibians move only short distances, and 3) many amphibian species show extreme site fidelity. Thus, when local amphibian populations become extinct, they may be less likely to recover than are other tetrapods. Movement patterns can indicate the dispersal ability of individuals (Daugherty and Sheldon 1982a), and the potential for recolonization of disturbed areas (Kramer et al. 1993). Information on the movement rates of organisms also is critical for predicting extinction thresholds (Diffendorfer 1998; Fahrig 2001). In partially harvested watersheds, recolonization by Ascaphus will be important for continued regional persistence. However, habitat fragmentation may create barriers to their dispersal. Molecular studies suggest that  CHAPTER ONE: General Introduction  gene flow among Ascaphus  5  populations is low (Pauken and Metter 1971; Ritland et al.  2000; Neilson et al. 2001), but processes governing genetic structure render interpretations of small spatial scales from available genetic results uncertain (Wahbe ef al. 2001). Direct measures of movement could be more revealing. Understanding the movement patterns and dispersal abilities of Ascaphus  could lead to improved conservation actions for this and  other riparian obligates, especially in managed forest landscapes. In the following chapters, I use three different approaches to examine the recolonization potential of Ascaphus  in managed and unmanaged forests. In Chapter 2, I  examine the instream movements of larval Ascaphus. larvae may contribute to the dispersal of Ascaphus.  It is unknown how stream-dwelling However, downstream movements  would be energy-efficient, because they can be passive with the stream current. I ask how movement rates of larvae differ between managed and unmanaged forests, and explore the relationship between stream and site parameters and dispersal ability among three treatments: old-growth forests, mature second-growth stands, and clearcuts. Ascaphus  movements important in dispersal may occur laterally and overland. In  Chapter 3,1 examine terrestrial movements of juvenile and adult Ascaphus.  I begin by  addressing differences in abundance and proportions of each developmental stage in clearcuts and old growth. I ask how stream affinity and patterns of movement differ between clearcuts and old growth, compare findings with evidence from inland populations, and explore differences in movement patterns among developmental stages and sexes. Habitat fragmentation can play a significant role in limiting dispersal and gene flow. In Chapter 4, I use R A P D s to examine population genetic structure of Ascaphus streams within a single watershed. I use larval Ascaphus  in two  tissue to compare genetic  variation in streams flowing through a clearcut and an old-growth forest. Also, I examine the relationship between genetic relatedness and physical distances within each stream. Maintaining viable populations throughout the range of Ascaphus  is an underlying  assumption of my thesis. The general context for my research is the issue of fragmentation via timber harvesting and whether it reduces recolonization potential of Ascaphus.  In the  final chapter, I synthesize the components of my thesis and summarize my major findings. Based on available data, and recognizing that uncertainties exist, I discuss some implications to conservation, provide habitat protection options that may better protect Ascaphus  populations arid other species dependent on headwater stream habitats, and  recommend directions for future research.  CHAPTER TWO: Movements of Larval Ascaphus  6  CHAPTER TWO Movements of Larval Ascaphus  INTRODUCTION Recolonization of habitats vacated by local extinction is problematic for amphibians because of their physiological constraints and movement behaviors (Blaustein et al. 1994). Many factors can influence the recolonization potential of amphibians. For example, the mountain yellow-legged frog {Rana muscosa) has become extinct at many high elevation sites in the Sierra Nevada Mountains of California. Bradford (1991) suggested that recolonization might never occur because streams connecting extant populations are inhabited by introduced fish that eat tadpoles. Forest practices (e.g., clearcut harvest) often have negative impacts on adult and larval Ascaphus (Metter 1968; Bury 1988; Welsh 1990) due to a reduction in soil moisture and increases in temperature, sedimentation, and solar radiation. Amphibians require moist, cool conditions to avoid desiccation, thus the removal of forest cover may lead to a reduction in the recolonization potential of disturbed habitats for some species. Corn and Bury (1989) reported no detection or low numbers of Ascaphus in streams 14-40 years after clearcut harvest. At inland sites, Daugherty and Sheldon (1982a) found that 50% of reproductively mature Ascaphus remained in the same 20-m area of their previous capture and concluded that adult Ascaphus exhibit extreme site fidelity. Given the long larval stage of Ascaphus, movements by larvae may be particularly important to recolonization of managed forests. After clearcutting, however, small streams often contain logging debris and sedimentation that could impede movement and recolonization by Ascaphus larvae. Few laboratory studies have examined the movements of larval amphibians. Anholt et al. (2000) reported reduced larval ranid frog activity with increased predator density and food availability, and greater activity among larger animals. In stream-breeding salamander larvae, Sih era/. (2000) reported ineffective anti-predator behavior (high emergence rate from refugia, and high activity while out of refuge) and high predation by sunfish. I found no published studies addressing movements of larval amphibians in streams in western North America. In this chapter, I investigate larval Ascaphus movement rates and assess influences of stream parameters on instream movements in managed and unmanaged forests. I test hypotheses based on three predictions: (1) I predicted that larval movement rates in recently harvested forests are lower than in unmanaged forests; (2) Because stream gradient, which is related to flow rate, may influence larval movements, I predicted that larval movements are less in streams where logjams are abundant; and (3) Post-harvest volumes of woody debris in  CHAPTER TWO: Movements of Larval Ascaphus  7  streams have been reported to increase three times over pre-harvest levels (Baillie ef al. 1999). Because embedded logs may influence larval movement rates, I predicted that larval movements are greater in high gradient streams.  STUDY AREA I conducted my study near Squamish in southwestern British Columbia (49° N, 122° W) within forests dominated by western hemlock (Tsuga heterophylla).  Sites were distributed  within four basins that drain into Howe Sound (Ashlu, Elaho, Mamquam, and Squamish Rivers; see Appendix III), and included reaches transecting old-growth forest (250+ years old), mature second-growth forest (60-80 years since logging) and recent clearcuts (~5 years since logging). These sites are hereafter referred to in this thesis as old-growth streams, second-growth streams, and clearcut streams. In reaches designated as 'old growth,' the predominant forest type in the watershed upstream was old-growth forest. In reaches designated as 'second growth,' the predominant forest type in the watershed upstream was mature second-growth forest. And, in reaches designated as 'clearcut,' the predominant forest type in the watershed upstream was clearcut ( 5 - 1 0 years since logging). Reaches within each basin were located on three separate tributaries, with predominant forest cover type being old growth, mature second growth, or clearcut. In 1996, the Squamish clearcut site was replaced with the Ashlu clearcut site due to loss of road access. Three replicates of each forest cover type were selected. The nine streams, approximately 3 m in wetted width, were selected on the basis of larval presence, and were chosen upstream (except Ashlu and Mamquam clearcut sites) from logging roads.  METHODS Stream Surveys and Parameters I determined larval movement patterns of Ascaphus  using area-constrained stream  surveys (adapted from Bury and Corn 1991 and Shaffer ef al. 1994) together with markrecapture techniques. Three 5 - m sections (reaches) per stream were selected 25 m apart. Location of the first downstream reach was randomly selected. Larvae were marked in these three reaches. To maximize number of recaptures, two additional 1 0 - m reaches were added directly below the second and third 5 - m reaches (Figure 2.1). A 1 0 - m reach was not added below reach 1 because I sought to maintain a distance of 50 m from logging roads or clearcut edges where appropriate. Area-constrained searches were conducted in all stream reaches by scanning the stream for surface-active larvae, then slowly moving up the stream turning and brushing undersides of rocks and capturing larvae with dipnets as they became dislodged. Stream searches were conducted between 0700-2200 hrs, taking three people roughly 3 hrs per stream, with an additional 3 hrs to process larvae and record stream parameters.  CHAPTER TWO: Movements of Larval Ascaphus  8  Initial stream surveys were conducted in June and early July of 1995 and 1996. Each stream was resurveyed once in July and once in August (approximately 20 days apart), and yielded information on the extent of larval movements. Three sizes of dipnets each with 1-mm mesh were used for sampling; appropriate net size (width of net: small, medium, large) was based on dominant stream substrate size. No seines were placed at the lower end of stream reaches.  1m Reach 3  5m  15m  30m Reach 2  35m  45m  60m Reach 1  65m  Figure 2.1. Sampling scheme: Initial marking occurred in three 5-m reaches (shaded) separated by 25 m; sampling for recaptures included two additional 10-m reaches (hatched). In the text, recaptures noted for reaches 2 and 3 include the contiguous 10-m reaches. Arrows indicate direction of stream flow.  In July of 1995, 1996, and 1999, I measured stream parameters I thought could influence the movement of larvae: stream gradient (using a clinometer), logs embedded in the stream (logjams; visually estimated and ranked low, medium or high), stream wetted width (meter tape), and canopy closure (visually estimated as percent cover).  CHAPTER TWO: Movements of Larval Ascaphus  9  Mark-Recapture  Larvae captured in the three 5 - m reaches were marked by fin clipping with a code unique-to-reach (e.g., two notches for individuals captured in reach two). Taking special care not to cut into the central axis of caudal muscle, the dorsal fin of the tail was marked with "V"shaped notches following Turner (1960). Notches were visible for at least one month and allowed for identification of recaptures by reach in subsequent stream surveys within that period. After a reach had been searched, I marked larvae, recorded the number of notches given, measured the distance from the upstream end of the reach, replaced disturbed rocks, and released larvae less than one meter upstream from the reach of capture. I kept 25 marked larvae (three notches each) in a lab enclosure to evaluate potential impacts of marking on larval survival. Lab conditions closely mimicked field conditions: 6-hr light: 18-hr dark cycle (using timer and fluorescent lamp). An aquarium of fresh stream water and algae-covered rocks, collected weekly from known Ascaphus  streams, was maintained at 5  °C and oxygenated with an air pump.  Statistical A n a l y s e s  Each of the nine streams was treated as an experimental unit for statistical analyses. I used the statistics package SPSS® for Windows® ( S P S S Inc., Chicago, Illinois, U.S.A.) to run analyses on mean values per experimental unit (i.e., nine treatment means) for the 737 Ascaphus  larvae captured during three years. I evaluated potential patterns in initial capture  data using Kendall's coefficient of concordance, where a coefficient of 1.0 indicates complete concordance among ranks. I used a two-way analysis of variance (ANOVA) to test differences in distance moved by larvae among old-growth, second-growth, and clearcut streams. The randomized complete block design included three rivers (as blocks; 2 degrees of freedom; Elaho, Squamish, Mamquam), and three forest cover types (2 degrees of freedom; old growth, mature second growth, clearcut). The design is unbalanced because of two missing observations (no recapture data in Squamish and Elaho watersheds), leaving a total of 2 degrees of freedom for experimental error. These data, however, could be evaluated by the General Linear Model Factorial Procedure (Norusis 1993). To explain variation in larval movement rates and recapture rates, I used the forward selection method for multiple linear regression analyses. To evaluate the effect of logjams, I used Spearman's rank correlation procedure (Spearman's rho; Zar 1984). All statistics were tested against a preset significance level of a = 0.05. The + values reported indicate the standard error of the mean.  C H A P T E R T W O : M o v e m e n t s o f L a r v a l Ascaphus  10  :  RESULTS Mortality Due to Marking During the first six-week period, no mortality w a s o b s e r v e d in larvae given three tailfin notches a n d kept in the laboratory. In w e e k s e v e n , three larvae (12%) w e r e found d e a d in the a q u a r i u m . T h e r e m a i n i n g 22 larvae lived for another five w e e k s .  I c o u l d not determine whether  their death w a s related to tailfin notching, handling stress, d i s e a s e or unnatural living conditions. H o w e v e r , time to a n y mortality a p p e a r e d long e n o u g h that marking itself w o u l d not h a v e influenced recapture e s t i m a t e s .  Recapture Rates During the initial s e a r c h e s in J u n e a n d July, I c a p t u r e d a n d m a r k e d 7 3 7 larvae.  During  July a n d A u g u s t , I r e c a p t u r e d 52 larvae (7% recapture rate). A l t h o u g h recapture n u m b e r s w e r e low, larval m o v e m e n t s differed by forest c o v e r type with greater m o v e m e n t rates in old growth than in clearcuts ( T a b l e 2.1).  O n l y d i s t a n c e s of 0 to 15 m, 30 to 4 5 m or 60 to 65 m could be  detected b e c a u s e of the two stretches (15 m e a c h ) of u n s a m p l e d s t r e a m b e t w e e n r e a c h e s (Figure 2.1). C a p t u r e s of larvae w e r e u n e v e n l y distributed a c r o s s s t r e a m r e a c h e s .  T h i s h a d the  potential to bias results if, for e x a m p l e , most larvae in clearcut s t r e a m s w e r e initially captured in r e a c h 1.  H o w e v e r , within a n y forest c o v e r type, I failed to detect a significant difference in the  proportion of initial c a p t u r e s a c r o s s r e a c h e s 1 through 3 (Kendall's coefficient of c o n c o r d a n c e = 0.86; asymptotic significance = 0.051). C o n s i s t e n t with m o v e m e n t from u p p e r to lower r e a c h e s , rates of recapture differed a m o n g r e a c h e s (Kendall's coefficient of c o n c o r d a n c e = 0.149; asymptotic significance = 0.719).  In s t r e a m s flowing through old growth a n d s e c o n d growth,  recapture rates in the two lower r e a c h e s c o m b i n e d (1 a n d 2 of F i g u r e 2.1) w e r e 12.6% a n d 10.7%, respectively; recapture rates in r e a c h 3 were 2.5% a n d 2.4%, respectively.  Conversely,  in clearcuts w h e r e m o v e m e n t s w e r e shorter, the recapture rate in r e a c h 3 w a s 10.5% but only 3.7% for the lower two r e a c h e s (consistent with less drift from the u p p e r r e a c h e s ) .  C H A P T E R T W O : Aquatic Movements of Larval  co  rTJ  TJ  e  p  co co  CD  co  csi  Ascaphus  CNJ  CN  ,-  CD  CN  LO  O  CN  d  CN  CN  co  CD  ^ o >* CL O  c ro  o  CD  o  LO CO  LO CNJ  O  o  CD  LO  00  o  LO OJ  O  o  o  or? CNJ  CN  T —  ~~  C/J  CO  o  o  O  csi  CN  q  CD  LO  LO  o  LO  CO  CN  CN  co  CN  E ro co  CL  E O co  105  d  o o  LO  d  oo d  LO  T —  00  LO  cq  d  oo 00  CD CO  co  CO  co  CD* d  oo"  00  o  " — •  E  LO LO  X  CD  CL  -»—  1  CO  oo  TT  o o  LO  CD  o  LO  1^  LO CD  o  E  c  IOT  TJ  p  CD  CO  5  o CD  CD  o d  o d  T —  c/j c co Q  E  o  CO  ^r d  d  CNJ  LO  d  ,^  LO  LO  od  o d  c  co  CD  ro  TJ  d  o  O  LO  CN LO  CD CO  d  d  d  d  o d  co •<-  co o  csi  ^ d  JZ  i 5  LO  co"  ro ro CT CO  oo  co LO  o D  d  p^  JZ CD >  ST  d  CD  CN  co  CD  CN  a)  o u-  o  v  cn  0  O  CN  CNj  E > o  CN  CNT d_  CN  T —  ro ro  oo  , , CD d  CD  CD  1^  o  CD  T —  d  •<-;  E ro n cr  LU  CD CD CD O O O  co  CD  O  JC  C/J  'E  ro D CT  CO  o JC  _ro LU  co d  d  E ro  D CT  E  ro  CD CD CD  CO  CD  CO CO  o d  o  <  JZ  ro LU  LO T —  d  E ro  ZJ CT  E  ro  o o o o o o  CHAPTER TWO: Aquatic Movements of Larval Ascaphus  12  Movement Rates Although larvae in old-growth streams moved about 7.1 times as far as larvae in streams flowing through clearcuts (Table 2.1), distances moved did not differ significantly among forest cover types (F ,2) - 1 -935; P = 0.341). Maximum movement rates also reflected forest cover {2  type associations. Over the average 2 0 - d a y period, larvae moved up to 3.76 m/day in oldgrowth streams, up to 1.94 m/day in mature second-growth streams, and up to 0.30 m/day in clearcut streams. My sampling design could not detect upstream movements <15 m. However, I did observe short upstream movements of 1 5 - 3 0 cm during area-constrained searches. Neither movements per day nor rate of recapture showed any relation with larval densities, which were highly variable (Table 2.1). I examined the potential influence of logjams on movement rates because all clearcuts contained abundant logjams that could have constrained movements. A multiple linear regression analysis was conducted with four stream variables (stream gradient, logjams, stream wetted width, percent canopy cover). Using the forward selection method, the only variable that was selected and w a s significant was logjams, which explained 1 3 % of the variation in movement rates (P = 0.009). None of the other stream variables added significantly to the amount of variation in larval movement rates that was explained by logjams. Abundance of logjams was ranked higher in clearcuts than in old-growth streams. Large amounts of logs in streams found mainly in clearcuts (Figure 2.2a) were associated with lower rates of movement (Spearman's rho (r ) = -0.507; P = 0.001: Figure 2.2b). s  CHAPTER TWO: Aquatic Movements of Larval Ascaphus  13  1.25 •  0.75 4  0.50 -  <  0.25 •  o.oo •  Old Growth  S e c o n d Growth  Clearcut  Low Logs  Medium Logs  High Logs  Figure 2.2. Logjams and larval movements, a) Logjams estimated in each forest cover type: old growth, second growth, and clearcut, b) Larval movements per day (m) in streams with low, medium, and high levels of logjams. Values of 2.5 (between medium and high rankings) were lumped with values of 3 because movement values were similar. Error bars represent standard error of the mean.  Because stream gradients tended to vary among treatments (Table 2.1), I tested for the effect of gradient on movement rate. Assuming a linear relationship, there was no effect of stream gradient on distance moved per day (r = 0.01, P = 0.643), and the extremes of 2  movement tended to be higher at lower gradients (Figure 2.3). Given the possibility of a factorceiling distribution sensu Thomson et al. (1996), there was relatively little power to test the relationship between movement per day and gradient. This inability may be due to insufficient sampling at the upper threshold rather than the result of weak relationships. Wider streams (i.e., large wetted width) were correlated with higher rates of movement (Spearman's rho, r = s  0.420; P = 0.002). There was no relationship between percent canopy closure and larval movement rates.  C H A P T E R T W O : A q u a t i c M o v e m e n t s of L a r v a l  70  Ascaphus  14  -I  60 •  50 •  40 •  -  30 •  CD T3  Is  20 •  c  10 •  0 . 10  12  14  16  18  20  22  Stream Gradient! ) 0  Figure 2.3. Individual movements of larvae in relation to stream gradient (some points overlap). Solid line above extreme movements represents a potential factor ceiling sensu Thompson et al. (1996).  DISCUSSION Stream Gradient, Logjams, and Movement Rates  I detected considerable downstream movements by Ascaphus larvae. Larval movements were associated with forest age class, stream wetted width, gradient, and logjams. Logjams explained 13% of the variation in movement rates. Though not statistically significant, I found larval movement to be much greater in old-growth streams regardless of gradient over a range of 4 to 44%. On the basis of stream gradient, second-growth streams should have shown the greatest movements per day, followed by old growth, then clearcuts. That pattern was not apparent, and maximum movement rates tended to be inversely, but not significantly, related to gradient. These results suggest that movements by Ascaphus larvae are active movements rather than passive drift.  CHAPTER TWO: Aquatic Movements of Larval Ascaphus  15  The sampled movement rates could be low because larvae moved outside the 65-m range of distances sampled. I believe that is unlikely for three reasons. First, the recapture rate is not uncommonly low (see review of dispersal studies of Sutherland et al. 2000). Second, at least some amphibians are documented to show strong site fidelity (Martof 1953; Stebbins 1954; Bellis 1965; Kleeberger and Werner 1982; Ovaska 1988). Third, the relative movement rates among forest cover types suggest that other factors are involved. Clearcut streams all contained abundant debris and logjams, sufficiently embedded in the stream that they could have constrained movements by serving as dispersal barriers. I found a negative relationship between distance moved and amount of logjams estimated in a stream. In some studies, postharvest volumes of woody debris have been reported to increase three-fold on average over pre-harvest levels (Baillie et al. 1999). Logjams may serve to reduce drift rates and possibly the recolonization potential of Ascaphus larvae.  Algal Productivity and Movement Rates  Alternatively, greater biomass of algae in clearcut streams may influence rates of larval movement. Ascaphus are commonly found in oligotrophic streams (Brown 1990; Rosenfeld 1997), and some data suggest that larvae are food limited (Kiffney and Richardson 2001). Primary production is often directly related to stream gradient and incident radiation (Mclntyre 1966). The greater influx of solar radiation in clearcut streams generally increases biomass of periphyton (see Kiffney and Bull 2000). A superior food supply in clearcut streams could result in lower movement rates of larvae. The extreme movements (potential factor ceiling of Figure 2.3) are consistent with the pattern expected from associations of primary production with gradient (shorter movements with steeper gradients and potentially greater food supply). Hawkins etal. (1983) suggested that the higher autotrophic production following canopy removal (or in naturally open stream sections) is responsible for higher abundances of invertebrates and stream vertebrates. When larval anurans are more active, they encounter both more food and more predators, thus activity rates mediate a trade-off between growth rates and predation risk (Anholt et al. 2000). Shorter movements per day in clearcuts, where incident radiation was higher and periphyton likely more abundant, also are consistent with the concept of lower food supply encouraging movement. My analyses indicate that larval movements were shorter in streams transecting clearcuts, and that these streams contained more woody debris. However, algal productivity in headwater streams transecting clearcuts and old-growth forests requires investigation.  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  16  CHAPTER THREE Terrestrial M o v e m e n t s of J u v e n i l e a n d A d u l t  Ascaphus  INTRODUCTION Available data on the effects of forest practices on larval population abundance and density are contradictory or vary by geographic location. While most studies report negative influences of forest harvest on larval Ascaphus, others have shown no effect or increases in abundance in some areas (e.g., in maritime locations). Still, relatively little is known about the larval ecology of Ascaphus, and even less is known about the transformed frogs, especially in terrestrial habitats. To determine influences of habitat disturbance and to better conserve Ascaphus populations, an understanding of both aquatic and terrestrial life stages is necessary. Patterns of Ascaphus movements may vary among regions due to inherent geographic differences. Rainfall is plentiful and desiccation risks for Ascaphus are less in coastal forests of the Pacific Northwest with maritime climates than for drier, inland locales. In coastal areas, some juvenile and adult Ascaphus have been found >100 m from streams during wet weather (e.g., Welsh and Reynolds 1986; Bury and C o m 1988a), but movements after metamorphosis are poorly documented. Ascaphus are afforded some protection in B C , but guidelines are based on larval habitat requirements (Ministry of Forests and Ministry of Environment, Lands and Parks 1999). Understanding the movement patterns of metamorphosed Ascaphus will help in the development of effective conservation actions, especially in managed forest landscapes. In a species like Ascaphus, which may have low vagility or dispersal controlled by abiotic factors (e.g., climate, wind or water currents), source-sink dynamics are expected to be common (Diffendorfer 1998). Despite the linear nature of streams and the ease with which movements can occur near water, Ascaphus may disperse laterally and overland. Larval movements downstream would have to be followed by extensive movements upstream to arrive at stream reaches that are cooler and less disturbed than lower reaches. However, these movements would represent a downstream drift - upstream movement cycle, not dispersal. Thus, to arrive in new habitats (streams) and introduce new members to the gene pool, terrestrial movements by Ascaphus may be critical to population persistence. My goal in this chapter was to examine the colonization potential of juvenile and adult Ascaphus in clearcuts and old-growth forests. I tested hypotheses based on three predictions: (1) To avoid desiccation, amphibians require moist habitats, so I predicted that frogs in clearcuts remain closer to streams than frogs in old-growth forest; (2) Some researchers (Bury and Corn 1987, 1988b) recorded juveniles moving at least 75 m from streams during fall. Others reported  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  17  reduced movement at the onset of reproductive maturity (Daugherty and Sheldon 1982a), so I predicted that juveniles undertake most upland movements, while adults move along streams; and (3) Precipitation and temperature strongly influence amphibian activity. Coastal populations should experience less thermal stress than inland populations, and I predicted that Ascaphus in coastal regions move greater distances than in inland regions.  STUDY AREA I conducted research near Squamish in southwestern B C (49° N, 122° W), 60 km N of Vancouver in Coastal Western Hemlock (CWH) and lower portions of Mountain Hemlock (MH) biogeoclimatic zones (Meidinger and Pojar 1991). Both zones are relatively moist with annual precipitation ranging 1000-4400 mm in C W H and 1700-5000 mm in MH (Meidinger and Pojar 1991). Sites were located in four adjacent river basins (Figure 3.1) that flow into Howe Sound.  Ashlu River  Figure 3.1. Schematic map of study area showing relative positions of the four river basins, and the three replicates of each treatment: old growth (OG) and clearcut (CC).  In the upper headwaters of watersheds, I selected rocky streams in old-growth forests (250+ years old; n = 3) and recent clearcuts (five years since logging; n = 3) using 20-min stream searches to establish presence of larval Ascaphus. All streams were 1-3 m wide and fishless. Each site covered about 2.25 hectares (Figure 3.2), adjacent to which was a surrounding 'buffer' of at least 50 m of contiguous forest. For example, a trapping grid in old growth was surrounded by at least 50 m of old-growth forest, beyond which a clearcut, road, or different forest cover type may be present. Mamquam and Elaho clearcuts were 200 and 500 m  C H A P T E R T H R E E : Terrestrial Movements of Juvenile and Adult Ascaphus  18  downstream from old growth, respectively, while the Ashlu clearcut was 300 m downstream from a rocky, unforested ridge. Selected streams were 200-400 m from any adjacent streams. Vegetation cover in clearcuts consisted mainly of an open canopy of sparsely distributed shrubs and small trees (< 1 m tall). About 1/3 of the Mamquam and Elaho clearcuts contained a closed canopy of shrubs and small trees (< 2 m tall).  METHODS  Pitfall Trap and Drift Fence Arrays To evaluate frog movements, I used streamside and upland arrays of pitfall traps with clear, plastic drift fences, adapted from prior designs (Corn and Bury 1990; Corn 1994). Arrays each consisted of four pitfall traps and one or five drift fences (Figure 3.2). Traps were constructed of white, smooth-walled polyvinyl chloride (PVC) sewer pipe, 15 cm in diameter and 40 cm deep. Streamside arrays were designed to detect movements up and down the stream bank. Each array was constructed with a 5-m long drift fence with two traps at each end, installed perpendicular to the stream as close to the water's edge as possible (Appendix III). Upland arrays detected movements towards and away from streams, and included five 5 - m long drift fences installed in zigzag formation with one trap at each elbow (adapted from suggestions by R.B. Bury; Appendix III). Arrays were 20 m in length and parallel to the stream.  -5m-  •25m-  -*-50rrr  -MOOrn  25m  o = trap < = drift fences  25m  Figure 3.2. A trapping grid (representing one experimental unit) showing arrays of streamside and upland pitfall traps and drift fences. Not to scale.  CHAPTER THREE: Terrestrial Movements of Juvenile arid Adult Ascaphus  19  At each site, I installed 48 pitfall traps and 48 m of drift fence (total = 288 traps and 240 m of fence for all sites). Selection of the first streamside array was random, while all others were systematic. The minimum distance of the first array from the nearest logging road was 50 m. In most cases, the first array was upstream from a logging road. In the Ashlu and Mamquam clearcuts, the first arrays were established downstream from logging roads because upstream forests were not the appropriate size or forest cover type. I placed a bottomless margarine container at the trap opening, forming a funnel, to keep frogs from hopping or climbing out and checked traps every 2 - 3 days for 1-4 week periods. To reduce small mammal mortality, I facilitated escape by placing sisal rope in traps (see Appendix III), securing it in soil beside the trap (adapted from Wind 1996). Insertion of ropes significantly reduces small mammal mortality (Karraker 2001) while Ascaphus are retained in traps. Small mammal mortality during three field seasons is given in Table A-1.1 (Appendix I). I inclined a cedar shingle against the fence over each trap opening (see Appendix III) to protect animals from direct sunlight, predation, and rainfall that can flood traps. The cover also may serve to attract frogs. To prevent desiccation of animals, I placed wet moss in traps, and added fresh water on every visit to maintain moisture. During rainy periods (mid-September through November), when captures were expected to be high (Bury and Corn 1987), grids were operated almost continuously and traps were visited frequently. I operated traps Sep 18-30 and Oct 4-13 in 1998, Jul 18-25, Aug 8-17, and Sep 10-Nov 1 in 1999, and Sep 1-Nov 9 in 2000. Unlike fall trapping, grids were not operated continuously in the summer of 1999: 2 - 3 times during Jul (eight days total), Aug (10 days), and early Sep (seven days). At 0 m and 100 m from stream, soil temperature at 15 cm depth (digital thermometer: + 0.1 °C) and air temperature in the shade 15 cm above the soil surface (mercury thermometer: + 0.5°C), and a relative measure of soil moisture at 15 cm depth (conductivity meter: + 0.5 units) were recorded on each site visit. Soil moisture also was recorded at each capture location. My conductivity meter had a scale from zero (dry) to 10 (wet), and Thomson et al. (1996) reported that this device provides stable, reproducible readings.  Mark-Recapture I measured, individually marked, and released frogs on the opposite side of the drift fence where they were captured, assuming that was their movement direction. Frogs were classified into two developmental stages based on snout-vent length (SVL) and morphological features: "juveniles," most with residual tail present (16.3-27.9 mm SVL) and "adults," most with secondary sexual characteristics present (28.0-50.1 mm SVL).  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  20  I marked frogs using Visual Implant Fluorescent Elastomer Tags (VIE; Northwest Marine Technologies 2002; see Appendix III). VIE has no reported effects on mortality or behavior of amphibians (Anholt et al. 1998; Jung era/. 2000; Davis and Ovaska 2001) and is preferable to toe clipping (Clarke 1972). To avoid potentially harmful chemical anaesthetics (e.g., tricaine methanosulfonate, aka M S - 2 2 2 ) , hyperactive frogs were submerged briefly in ice water to reduce their activity. This likely caused minimal or no stress as some species of terrestrially hibernating frogs endure freezing of extra-cellular body fluids (Churchill and Storey 1993), and Ascaphus are cold-adapted frogs. Directions of frog movement were categorized as upstream, downstream, upslope, or downslope. Upstream movements were parallel to the stream, against stream flow, and within 5 m of the edge of stream. Downstream movements were parallel to stream, in the direction of stream flow. Upslope movements away from stream were roughly perpendicular to stream and within 100 m of stream edge. Downslope movements towards the stream were roughly perpendicular. All animals were cared for in accordance with the principles and guidelines of the Canadian Council on Animal Care. I held the required certificate from the University of British Columbia Committee on Animal Care (#A0-0110), and the British Columbia provincial permit (#C088692) necessary for fieldwork on a "blue^listed" species.  Statistical Analyses Each trapping grid was treated as an experimental unit for statistical analyses. I installed one trapping grid at each of the six sites (three old growth, three clearcut), for a total of six experimental units. I used the statistics package SPSS® for Windows® ( S P S S Inc., Chicago, Illinois, U.S.A.) to run one-way analyses of variance (ANOVAs) on mean values per experimental unit (i.e., six treatment means) for the 254 Ascaphus captured during three years (1998-2000). In a separate analysis, interactions among the two treatments, three watersheds, and three years were tested using an A N O V A based on a randomized complete block design with a split-plot year effect. In this mixed-model A N O V A , year and forest cover were fixed factors and watershed was a random factor. Because interactions among forest cover, year, and watershed were not significant when analyzing mean distances, I pooled watershed and year interaction degrees of freedom into experimental error degrees of freedom to obtain a more precise estimate of variance (Hicks 1982). When differences among multiple means were statistically significant, I used a multiple comparison test (Bonferroni) to evaluate which means differed. All reported P-values were obtained using statistics tested against a preset significance level of a = 0.05. The + values reported indicate the standard error of the mean. When analyzing distance  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  21  categories, tests for independence (chi-square, x ) were performed on numbers of frogs 2  captured. Cochran-corrected tests for independence (Xc ; Zar 1984) were used for 2x2 2  contingency tables. Numbers of frogs captured were converted to numbers of frogs per 100 trap nights (TN), or catch per unit effort (CPUE), to address unequal trapping effort. Although C P U E are effectively percentage data, C P U E values are presented only in text and tables, thus arc sine transformation was not employed. A trap night is the number of traps in operation * the number of nights in operation. Traps full of water, pushed above ground (due to rising water table), or broken were deemed inoperable and deleted from trap night totals. To assess body condition of individual frogs in old growth and clearcuts, I used a body condition index (BCI). I calculated BCI using observed mass divided by expected mass. Expected mass (predicted Y) was based on residuals derived from a regression equation of mass (Y) against snout-vent length (X). This calculation incorporates length-corrected mass and has been used in studies of whiptail lizards (Dickinson and Fa 2000), terrestrial salamanders (Dupuis etal. 1995), and bears (Cattet et al. 2002). BCI calculations were restricted to males and non-gravid females to avoid bias created by differences in reproductive status (Stamps 1983).  RESULTS Catch Per Unit Effort  I recorded 281 Ascaphus  from 1998-2000: 254 captured, 27 incidental encounters  (Table 3.1). Other amphibians that were encountered, trapped, and recaptured are summarized in Tables A-l.2 and A - l . 3 (Appendix I). Of 254 Ascaphus  captured, 142 were in clearcuts and  112 were in old growth. Catch per unit effort (CPUE) was 1.3 times greater in clearcuts than in old-growth forests (Table 3.2) but was not statistically significant. Distributions of developmental stages differed significantly between clearcuts and old-growth forests (x<3 = 52.30; P < 0.001) 2  with 2.9 times more juveniles in clearcuts than in old growth, and 2.3 times more adults in old growth than in clearcuts (Table 3.2). Juvenile and adult captures also were unevenly distributed across the three watersheds and across the three years. The Mamquam watershed yielded three times more adult captures than the Ashlu watershed, and nearly twice as many juveniles were captured in the Elaho watershed compared with Ashlu and Mamquam watersheds (Figure 3.3a). The most adults and juveniles were captured in 1999, and the least were captured in 1998 (Figure 3.3b). It later becomes more apparent that relations with forest cover, watershed, and year result from different distributions in developmental stages captured among watersheds and years (Figure 3.3).  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  22  Table 3.1. Number of nights of trapping and number of Ascaphus recorded (trapped and encountered. Southwestern British Columbia. 1998-2000.  Year  1998 1999 2000  Number of F r o g s  #of ..... Nights  _ . Trapped  21 63 70  32 128 94  Incidental  Total  Encounters  Recorded  13 8 6  45 136 100  Recapture Rate  In three years, I recaptured only 17 frogs (6.7%) of the 254 marked animals. None were recaptured in 1998. Recapture rates in 1999 and 2000 were 6.3% and 9.6%, respectively. Recaptured frogs included 10 juveniles and seven adult frogs captured 4-400 days after initial capture. In old growth, recaptures included four adults (three males, one female) and one juvenile female. Recaptures in clearcuts included one adult male, two adult females, and nine juvenile males. Recapture rates were 4.5% in old growth and 8.5% in clearcuts. Adult males in old growth were recaptured 25-70 m from their initial capture site, about one year after initial capture (345-420 days; n = 3). One adult male was recaptured in a clearcut 330 days after initial capture, 85 m from the initial capture site. Two adult females were recaptured in a clearcut after five and 360 days, 90 m and 100 m from initial capture sites, while one adult female was recaptured in old growth after five days, 50 m from initial capture site. Nine juvenile males were recaptured in clearcuts after 5-14 days, in the same trap or up to 50 m away from initial capture site. One juvenile female was recaptured in old growth after four days, 30 cm from initial capture site.  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  120  100  20 • Q I Elaho  Ashlu  Adult I Juvenile  Mamquam  3 Adult Juvenile 1998  1999  2000  Figure 3.3. Number of juvenile and adult Ascaphus trapped: a) differences among three watersheds, b) differences among three years.  C H A P T E R T H R E E : Overland Movements of Juvenile and Adult  o  CM  CN  o o 5  o  o 3 U  .29 + 6.  m o  CN  ro  CT) O  O  Q_  O CN CD  CO o d n  57 + 9.  00  o  CN  CN CN  si  oo  CN  o  ro  +1 CD CN CO  II  CL ih" o  O  m  oo  CN  +1  CN O  CN  II  00 oo  a.  in  m  CN  II  00  CM CD  a.  iri  +1  CN  II  o d  m co  II  T  i_  G>  a ro  IO  0. .C  o  ro O  £. t o 3: UJ  0_  CD N-  +1 co  LD d  oq  CM CN  00 00  CN CD  II  CD  II  a.  CD  o d n  +1 00  00  d  CN CM CM d  n  a.  +1  m  oo  o  CD o csi  CD  il  oo  II  +1  +1 CO  00  b  I  ^1-  CT) c c CO a» O  E  CO  >  w  11  d II  CM  CD  +1 00  o CN  § 3  co o  6o~  CT) u c ro  II  CT) a.  6o"  co i^Lf)  CD CD 00 d  m  o  LO  65  n  II  CD CD iri CO  CO  0. oq d  o d  CN CT)  CD  o o  CD d n  CN  CD CN  CM  o o +  CD 00  c  co  +1  CN  O  CO  CN O  +1  CN  CD  o  00 CD  LO oq d  d n  +1  CN  CO  co +1  II  II  CL 00  CD CD CN  o o  00  m o  CM 00  CD CD  iri  CD CN  CD O  ET  00  d  Csi  +1  uT  +1  O  co  oo  CD CD d  00"  00 oq  T—  a.  m  FT  in  II  +1  o  csi  00 CN  m  +1  CD CM CM d  ih"  +1 00  LT  +1  CD  0)  3 O  d  CT) CN  CN  1  l3  CN O  n  0)  o 03 o c  CD CD  +1  o  o  +1  oo CT)  O  ro  II  iri CN  % o  ,"5 1^  m 00 o  24  m  o  CN  5  Ascaphus  CO CO  ro  ro o  II  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  25  Movement Patterns Distance from Stream I quantified colonization potential of Ascaphus by evaluating their mean distances from streams in old-growth forests and clearcut sites. I also evaluated distribution of captures across distance categories. I recognized two trapping periods: summer (Jul-Aug) and fall (Sep-Nov). Summer trapping occurred in 1999 and yielded 13 frogs. During the three years of fall trapping, no frogs were captured in November during snowfall. I captured the most frogs (n = 241) between Sep 2 6 - O c t 23. In summer, I captured six frogs in streamside arrays and seven in upland arrays; in fall, 134 were streamside, 107 were in upland arrays. I found a significant difference in the proportion of frog captures across distance categories among old growth and clearcut (x<3> = 23.16; P<0.001). The number of frogs 2  captured within 25 m of the stream was not independent of forest cover type (Xcci  8.04; P  2 =  <0.005) with more frogs captured within this distance in clearcuts. However, the proportion of frogs captured at distance categories 2 5 - 1 0 0 (x 2) = 2.69; P >0.250) and 5 0 - 1 0 0 (Xc i) = 1-70; 2  2  (  (  P >0.100) were independent of forest cover type. I failed to detect a difference when examining mean distances from streams in oldgrowth forests and clearcuts (mixed-model A N O V A : F<1,2)= 1.810; P= 0.311). However, I detected a watershed (mixed-model A N O V A : F , ) = 8.440; P = 0.011) and year (F , ) = 7.270; (2  8  (2  8  P = 0.016) effect on distance from stream. Mean distances from streams in the Ashlu (15.15 + 4.80 m) and Mamquam (31.53 + 9.13 m) watersheds were 2 - 4 times greater than in the Elaho watershed (7.72 + 6.13 m), and the difference between Mamquam and Elaho watersheds was statistically significant (P = 0.013). During fall, mean distances from streams in 1999 (19.56 + 9.12 m) and 2000 (28.22 + 8.59 m) were 3 - 4 times greater than in 1998 (6.62 + 4.16 m), and the difference between 1998 and 2000 was statistically significant (P = 0.019). Potential relations with forest cover may have been obscured by variation in spatial and temporal conditions. Site variables, however, did not appear different among forest cover types. Mean elevation of old-growth sites (717 m) was similar to that of clearcuts (752 m). Mean gradient (hillslope relative to stream) in old-growth sites (12°) was equal to that in clearcuts (12°). Mean aspect of streams in old-growth sites (235°) was also similar to that in clearcuts (272°). Relative soil moisture at increasing distances from stream also was not informative for explaining patterns of frog movements (Figure A-1.1, Appendix I). Mean distances from streams differed in summer and fall. During summer, frogs were captured in only one clearcut and two old-growth sites, but were 8.8 times farther from the clearcut stream (55.00 m versus 6.25 + 4.09 m). In fall, frogs in old growth (21.59 + 9.15 m; n = 3) were captured 1.3 times farther from streams than in clearcuts (17.00 + 3.93 m; n = 3).  CHAPTER THREE: Terrestrial Movements of Juveniie and Adult Ascaphus  26  Different trapping periods between years also sampled different developmental stages of frogs. For example, a greater proportion of juveniles were captured in fall (Figure 3.4a) and these were found farther from streams in old growth (Table 3.2). Because the uneven distribution of developmental stages had a dominating influence on relations with forest cover (Table 3.2), I examined differences in distance from stream separately for juveniles and adults (Figure 3.5a). On average, adult distance from stream (28.91 + 6.65 m) was farther than juvenile distance from stream (17.19 + 4.48 m; F io) = 2.296; P - 0.029). Juveniles were captured 1.4 times (12  farther from streams in old growth (20.01 + 9.12 m) than in clearcuts (14.36 + 3.05 m; F , = (1  4)  0.346; P = 0.588). Adults were captured 1.8 times farther from streams in clearcuts (37.25 + 7.22 m) than in old growth (20.57 + 9.98 m; F  (1i4)  = 1.835; P = 0.247). When distance from  stream was examined separately by forest cover type, I found no difference between mean juvenile distance (20.01 + 9.12 m) and adult distance (20.57 + 9.98 m) in old growth (F  (1|4)  =  0.002; P = 0.969). However, I found a significant difference between mean juvenile distance (14.36 + 3.05 m) and adult distance (37.25 + 7.22 m) in clearcuts ( F  (14)  = 8.527; P - 0.043).  I captured 72 females and 180 males during the three years. Mean distances from streams were 23.29 + 7.80 m for females and 16.81 + 3.90 m for males. I found that 29% of mature females and 61% of mature males were captured within 25 m of the stream (Figure 3.5b). At streamside, I captured 4.5 times more males than females. Males and females were differentially represented in fall when I captured significantly more males than females (Figure 3.4b). Captures of females during the coldest two-week interval (Oct 24-Nov 6) were similar to captures of females during the driest months.  CHAPTER THREE: Terrestrial Movements of Juveniie and Adult Ascaphus  27  100-  80co  CO  o  60-  CD  40 H  20  H ] Adult I  %  %  \  \  \  %  \ >\ >\  v  °<v *  %  *a  ^  I Juvenile  o  %  ^  ^  100  80  O  60  0)  E  40  -I  20 4  •'•'I  I  r.  \  o>  ^  .  \  /  X  .  /<~.  V •,A  \ 7  X /a  \ VP  >rfo  \  ^ 0  X v> O  |  I Male  I  I Female  >, ^7 \  \  ,  •'V  Figure 3.4. Number of Ascaphus trapped during each two-week period: a) juveniles and adults, b) females and males (juveniles and adults combined).  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  28  120  100 98 80 O  60 H E 40 H  42 : 35 27  20  1c 10 0m  25 m  13  11  50 m  •  Juveniles Adults  100 m  Distance from Stream 120  i K1-14  100 -\  80  o 60 E 40 •] , 30 20  25  22  23 Female  14  11  ' 13 |~|Male  0m  25 m  50 m  100 m  Distance from Stream  Figure 3.5. Number of Ascaphus trapped at each distance from stream: a) juveniles and adults, b) females and males (juveniles and adults combined). Values within bars represent sample sizes.  C H A P T E R T H R E E : Terrestrial Movements of Juvenile and Adult  Ascaphus  29  Movement Rates and Directions Although sample size was small, I estimated movement rates based on distances between initial and subsequent capture. Frogs recaptured in old growth moved an average 2.09 m/day (n = 5), and frogs in clearcuts moved 3.36 m/day (n = 12). Nine of 12 frogs recaptured in clearcuts were juveniles. Also, more males than females were recaptured in old growth, and males were captured nearer streams, on average. There appeared to be no difference in direction of movement between sexes. Although almost no downstream movement was recorded for females, they were responsible for most downslope movement. For all years combined, directions of movement in the fall tended to differ between juvenile and adult Ascaphus in old-growth forest and clearcut sites (Figure 3.6). Adult movement appeared little affected by forest cover type. However, juveniles exhibited stronger stream affinity in clearcuts. For both forest cover types combined, upstream movements constituted 57% of all juvenile movement, and 28% of all adult movement. Fifteen percent of all juvenile movement and 4 2 % of all adult movement was downslope. The apparent differences between forest cover types results primarily from the proportion of developmental stages captured in each forest cover type.  upstream  Adult downstream Proportion (%): H 0 5  Juvenile  O l d Growth  Clearcut  Figure 3.6. Relative proportion of Ascaphus moving in four directions relative to stream: upstream, downstream, downslope (towards stream), and upslope (away from stream). Legend shows 5% scale for each direction. Fall data only.  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  30  Body Size and Condition I evaluated variation in frog body size between old growth and clearcuts. Differences were small and none were statistically significant (Table 3.2). When stages were analyzed separately, juvenile and adult frogs both tended to be longer and weighed more in old growth (Table 3.2). Total mass of frogs captured in clearcuts (294 g; n = 142 frogs) appeared lower than in old-growth forests (396 g; n = 112), primarily because there were more adults in old growth (Table 3.2). Evaluating stages separately, mean total mass of juveniles in clearcuts (n = 109) was 2.5 times greater than in old growth (n = 37). Mean total mass of adults in old growth (n = 75) was 1.7 times greater than in clearcuts (n = 33). Juvenile BCI did not differ between clearcuts (0.999 + 0.001) and old growth (1.040 + 0.020; F , = 4.064; P = 0.114). Adult body (1  4)  condition in clearcuts (0.999 + 0.002) also did not differ from old growth (1.001 + 0.001; F  (1|4)  =  1.143; P = 0.345). DISCUSSION Catch Per Unit Effort Research on the effects of timber harvesting on Ascaphus habitat has focused on impacts to streams and riparian zones, and often failed to distinguish different responses by developmental stage (Corn and Bury 1989; Welsh 1990; Dupuis and Steventon 1999; Welsh and Lind 2002). Welsh and Lind (2002) reported more Ascaphus in streams in late serai forest compared with streams in younger forests. In Oregon, Biek ef al. (2002) reported Ascaphus densities (primarily adults) of 0.11/m in streams transecting clearcuts and 0.21/m in old 2  2  growth. In contrast, CPUE of frogs in clearcuts tended to be greater than in old growth for my study. However, I also captured 2.9 times more juveniles in clearcuts than in old growth, and 2.3 times more adult frogs in old growth than in clearcuts (Table 3.2). The capture of more juveniles in clearcuts may be linked to greater primary productivity (Murphy and Hall 1981; Hawkins et al. 1983), related to increased solar radiation reaching streams in clearcuts (reviewed by Beschta etal. 1987). Wahbe (1996) and Kim (1999) reported larger (length and weight) Ascaphus larvae in stream reaches through clearcuts compared with those through old growth in southwestern BC. Greater in-stream productivity and warmer temperatures could increase larval growth rates and, in turn, survival, which appears low (Sutherland 2000). However, if stream temperature rises too high, Ascaphus may be excluded as eggs die above 18.5°C (Brown 1975). At least two southwestern BC datasets revealed greater densities of larvae in streams flowing through clearcuts (Richardson and Neill 1998; Wahbe unpubl. data). Furthermore, data from within my study area suggest faster larval growth and earlier metamorphosis in clearcuts (3-4 year larval period) compared to a 4 - 5 year larval period in old-growth streams (Wahbe 1996). Therefore, Ascaphus in clearcut streams may exist  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  3J.  at higher densities and have a shorter larval period, which together can lead to the emergence of more metamorphic juveniles compared with streams in old-growth forests. I captured significantly more juveniles in clearcuts than in old growth, and 94% of those captures were new metamorphs. Thus, there would be an increased number of juvenile frogs and catch per unit effort in clearcuts. However, I captured fewer adult frogs in clearcuts, which may suggest lower survival of young frogs. Also, larval movement tended to be greater in old growth than in clearcut streams (Chapter 2). This may result in a more densely distributed larval population closer to the headwaters in clearcut streams, which could translate into the emergence of more metamorphs from a given area, compared with old growth where larvae are likely more spread along the length of the stream. In old growth, some metamorphs may be missed in my trapping efforts if the older individuals (which also take longer to reach metamorphic stage compared with larvae in clearcuts) have moved further downstream. However, this possibility is difficult to assess without sampling lower sections of the stream.  Recapture Rate My recapture rates were low (4.5-8.5%) compared to other studies. Daugherty and Sheldon (1982a) reported rates up to 33% for juveniles and up to 73% for adult Ascaphus captured during summer at inland sites. Landreth and Ferguson (1967) reported a 37.5% recapture rate, also during summer at inland sites. No developmental stages were reported for either study. These higher rates of recapture may result from summer sampling when Ascaphus aggregate near streams, increasing captures. I operated traps mainly during fall, and away from streamside into adjacent forest where frogs are less abundant. Among studies, there were also different sampling designs and intensities of effort. My lower recapture rates may reflect capturing mostly young frogs, which have a higher rate of mortality (Jones and Raphael 1998), or high vagility in moist, coastal areas. Furthermore, compared with old growth, recapture rate tended to be greater in clearcuts where I captured more juveniles than adults. Juveniles were found closer to streams than adults and were shorter distances between captures.  Movement Patterns Distance from Stream My results were consistent with the prediction that frogs in clearcuts would remain closer to streams than frogs in old growth. I found that there were more frogs captured within 25 m of streams in clearcuts than in old growth. This result may suggest stronger stream affinity of frogs in clearcuts where the most favorable microclimatic conditions are likely near streams. I captured frogs 1.3 times farther from streams in old growth compared with clearcut sites,  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  32  although no differences were significant when using mean distances (Table 3.2). In a radiotelemetry study of Pacific giant salamanders  (Dicamptodon tenebrosus) in B C , stream affinity,  longer refuge duration, and smaller home ranges were reported in clearcuts compared with those in old-growth forests (Johnston and Frid 2002). Developmental stage was not reported, however mean distance from streams was four times greater in old-growth forests, where refuge duration was two days shorter, and home range size was 10 times greater than that in clearcuts. Being less restricted by microclimate constraints, stream-breeding amphibians in old-growth forests may move greater distances from streams for foraging purposes or dispersal to adjacent streams. Although number of  Ascaphus captured at distance categories of 25 m or greater was  independent of forest cover type, influences of forest cover on distance from stream varied with developmental stage, and, to a lesser extent, sex of the frogs. Adult frogs were captured 2.6 times farther from streams than juveniles in clearcuts. Because desiccation is more likely, juveniles may remain closer to streams in clearcuts. In old growth, I recorded juveniles 1.4 times farther from streams than in clearcuts. Although patterns are consistent with forest cover reducing anticipated adverse microclimate effects, my measurements of microclimate showed little difference with distance from the streams. Other site variables measured (elevation, gradient, aspect) did not explain differences in distances frogs moved away from streams.  Movement Rates and Directions Rates of movement estimated by successive recaptures are difficult to interpret because frogs likely did not move consistently in the same direction, but these data provide an index of relative vagility. Other difficulties in interpretation stem from the low recapture rate, a small sample size, and a large variation in time between captures ( 4 - 4 0 0 days) that may represent daily movement or in some cases, dispersal. I observed movement rates that were slightly higher in clearcuts (3.36 m/day) than in old growth (2.09 m/day). The majority of recaptures in clearcuts were juveniles, which may be a more exploratory life stage than adults. There also tended to be more male recaptures than females in old growth (although sample size is small), and males tended to be found shorter distances from streams, suggesting less movement, than for females. Frogs may move more often (but shorter distances from streams) in clearcuts because of increased physiological stress. Maxcy (2000) reported greater mean daily movement rates for  Ascaphus in forested sites (12.27 + 3.48 m/day) compared with buffered  sites (8.53 + 5.01 m/day) in southwestern B C . During summer in western Montana (mean annual precipitation = 635 mm; B C : - 2 5 0 0 mm), Daugherty and Sheldon (1982a) evaluated streamside movements and reported mean distances between successive captures of 0.34 m/day (within years) and 0.75 m/day (between years). I did not recapture any frogs during  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  33  summer, but during fall, I recorded a mean distance between successive captures of 3.0 m/day. Daugherty and Sheldon (1982a) reported a maximum streamside movement of 360 m by an immature female over 12.5 months, a movement rate of 0.96 m/day. My results were inconsistent with predictions that younger developmental stages would perform most overland movements and dispersal, while adults would move primarily along streams (having left the natal area when younger). Juveniles were found to move predominantly upstream (Figure 3.6), and adults were twice as far from streams as juveniles (Table 3.2, Figure 3.5a). In contrast, Bury and Corn (1987, 1988b) captured many recently metamorphosed Ascaphus in fall > 75 m from streams in pitfall traps set in forested stands. Also, in summer, Daugherty and Sheldon (1982a) showed a reduction in movement at the onset of reproductive maturity, with greater movements in pre-reproductive frogs and extreme site fidelity in reproductively mature frogs. Considerable downstream movements by Ascaphus larvae occurred over distances up to 64 m in old-growth forests and 3 m in clearcut sites within a few weeks (Chapter 2). Frogs may compensate for these movements by moving predominantly upstream following metamorphosis. I found that directional movements in clearcut sites differed from those in oldgrowth forests. While most frogs in clearcut sites moved upstream, most frogs in old-growth forests moved towards streams (downslope). The closest adjacent stream was 200 m, and movement patterns of frogs in old-growth forests may represent dispersal from adjacent streams. In both old-growth forests and clearcut sites, upstream movements constituted 57% of all juvenile movements, and 28% of all adult movements. Juveniles clearly showed stronger stream affinity in clearcut sites, but adults appeared little affected by forest cover type. Metter (1964a) observed fewer frogs during summer sampling and speculated that frogs moved upstream for "protection," presumably in more shaded stream reaches. In clearcut sites, there may be a tendency to move upstream because higher elevation sites or steeper gradients in upper portions of streams are often less disturbed due to historical patterns of logging starting at valley bottoms. These upstream movements may also represent movements towards breeding or oviposition sites. Within-stream movements by adults are believed by some to be critical for survival in clearcut sites (Adams and Frissell 2001). Movements toward streams may be dispersal events, breeding migrations, movements associated with foraging, or searches for oviposition, overwintering or over summering sites. Breeding migrations in Ascaphus are unreported but hypothesized (Landreth and Ferguson 1967; Brown 1975; Wahbe et al. 2001). Males do not vocalize (Schmidt 1970), and aggressive or territorial behavior in Ascaphus has not been detected. Frogs aggregate near streams during summer or fall for breeding. During summer, marked Ascaphus adults in Montana did not demonstrate any movement associated with breeding or oviposition (Daugherty and Sheldon  CHAPTER THREE: Terrestrial Movements of Juvenile and Adult Ascaphus  34  1982a). However, my data (Figure 3.6) indicate upstream movements and movements towards streams by adult frogs during the breeding season, which supports speculations by Brown (1975) that frogs move upstream to breed in headwaters. Some authors suggest that frogs move downstream to mate (Landreth and Ferguson 1967) or overwinter (Adams and Frissell 2001), but these may be condition-specific responses (e.g., behavioral thermoregulation as suggested by Adams and Frissell 2001). The variation in distance from streams and movement directions between males and females may be explained by migrations during the breeding season. I observed stream affinity and upstream movements in mature males during breeding season and believe these frogs aggregated in search of mature females. I recorded almost no downstream movement for females, but females were responsible for most of the downslope movement. While some females may remain beside streams to breed or move towards streams to deposit eggs (some were gravid), others may leave the site and disperse to breed or locate suitable oviposition sites in neighboring streams. All three gravid females I captured along streams were moving upstream. Of 15 gravid females captured upslope, 7 3 % moved towards streams. These data may provide the first evidence suggesting breeding migrations (stream to stream or upland to stream movements) in Ascaphus. However, some females may not breed every year (Metter 1964a). Because females may store sperm for a year (Metter 1964b), they may move through forest uplands and later return to the same stream for oviposition. Amphibian capture rates vary within and among years because patterns of precipitation and temperature strongly influence surface activity and likelihood of detection (Bury and Corn 1987; deMaynadier and Hunter 1998; Aubry 2000). I expected movement patterns to be similarly influenced, and hypothesized that Ascaphus in wet regions (coastal BC) would show longer movements than frogs in drier regions (e.g., Idaho). My results were consistent with this prediction. Ascaphus in coastal B C moved at least 100 m from streams in both old growth and clearcuts. Few studies of inland populations have sampled Ascaphus away from streams. In southeastern Washington and northern Idaho, Ascaphus moved at least 12 m from streams (Metter 1964a). Sampling took place during wet and dry seasons for both studies. However, it is difficult to make a valid comparison because although 100 m was the maximum trapping distance for my study, the area sampled was not reported for the inland study. For inland populations, high site fidelity in Ascaphus has been attributed to low summer precipitation and warm conditions of interior regions that restrict movements of amphibians, and suggests low recolonization potential for inland populations (Daugherty and Sheldon 1982a). Ascaphus should experience less thermal stress on the coast than in the interior Rocky Mountains (Diller and Wallace 1999; Welsh and Lind 2002). Climatic conditions likely favorable for Ascaphus along the coast (e.g., high humidity, extended periods of rain) may enable adults  C H A P T E R T H R E E : Terrestrial Movements of Juvenile and Adult  Ascaphus  35  to occupy larger home ranges or move longer distances than in inland populations. I recorded fall movement rates that were 8.8 times greater than those reported by Daugherty and Sheldon (1982a) in summer. Available data suggest that affinity for streams is much greater in dry regions compared to wet regions. Although coastal conditions are not as restrictive to movements, a greater proportion  of Ascaphus in clearcuts were captured within 25 m of streams  compared to Ascaphus in old growth, which suggests greater stream affinity in clearcuts.  Body Size and Condition  Despite evidence of larger-sized larvae in stream reaches flowing through clearcuts (Wahbe 1996; Kim 1999), in this study juveniles and adults in clearcuts did not differ in size from those in old growth. Size at metamorphosis and length of larval period are important to amphibians because they influence larval and juvenile survival and the potential level of adult fecundity (Travis 1984). A shorter larval period is expected when there is more food early in larval development, while less food at this stage will lengthen the larval period (Wilbur and Collins 1973). A smaller size at metamorphosis may lead to reduced survivorship, because body size has a major influence on an animal's energetic requirements, its potential for resource exploitation and its susceptibility to natural enemies (Werner and Gilliam 1984). Body size in amphibians is also directly related to their rehydration and desiccation rates (Ray 1958; Spotila 1972) and to their ability to withstand food deprivation. I found more juvenile frogs in clearcuts, but both juveniles and adults were in slightly reduced condition in clearcuts compared with old growth. My findings suggest that while the aquatic environment in clearcut sites initially may be beneficial to larvae, the terrestrial habitat may not be conducive to long-distance movements by juveniles. Some alternatives may be that clearcuts provide poor habitat cover and reduced foraging opportunities, increased competition, or shorter active seasons due to the increases in temperature and decline in relative humidity.  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  36  CHAPTER FOUR P o p u l a t i o n G e n e t i c Structure of Ascaphus  at the W a t e r s h e d S c a l e  INTRODUCTION Timber harvesting may reduce habitat patch size and habitat connectivity, thus reducing dispersal among fragments and increasing the probability of local extinction (e.g., Sjogren 1991; Bunnell etal. 1992; Fahrig and Merriam 1994). Connectivity between habitat patches is believed to be key to metapopulation persistence (Sjogren 1991) because it allows dispersal between populations (Taylor 1990; Hanski and Gilpin 1991). Ascaphus populations may exist in a metapopulation structure (Metter and Pauken 1969; Daugherty and Sheldon 1982a; Ritland et al. 2000). Because direct estimates of dispersal can be problematic, Vos et al. (2001) recommended using genetic techniques to determine influences of landscape connectivity on animal dispersal. A primary goal of conservation genetics is to estimate the level and distribution of genetic variation within and among populations of rare and endangered taxa (Fritsch and Rieseberg 1996). Research examining gene flow among Ascaphus populations has focused on genetic differences among watersheds across portions of the species' range (i.e., genetic comparisons based on geographically extensive samples). Having estimated relationships among populations and inferring evolutionary processes, Nielson era/. (2001) suggested recognition of inland populations of B C , Idaho, Montana, Washington, and Oregon as a distinct species, Ascaphus montanus (following the epithet of Mittleman and Myers 1949). However, Ritland et al. (2000) reported that although inland populations in British Columbia were distinct, their genetic distance from other groups (north coast, mid coast, and south coast) was equal to that expected from isolation by geographic distance alone, as opposed to taxonomic differentiation. Nonetheless, both studies report strong genetic differences (i.e., low gene flow) among Ascaphus populations, suggesting a complex history of restrictions to geographic refugia and range expansions. Genetic structure among Ascaphus populations at large (range-wide) spatial scales is now better understood. However, we know little about possible metapopulation genetic structure. Gene flow among populations is necessary for long-term persistence. This information is critical to our understanding of the effects of forest management on Ascaphus populations.  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  37  Population genetic structure can be inferred using a variety of genetic markers. Isozyme electrophoresis is traditionally the most popular and cost-effective method (Fritsch and Rieseberg 1996). Other techniques include microsatellites, sequencing of variable regions of mitochondrial DNA, and randomly amplified polymorphic DNAs (RAPDs). All of these techniques have been used to examine population genetic structure in amphibians (e.g., Daugherty 1979; Green ef al. 1989; Rowe ef al. 1999; Ritland ef al. 2000; Neilson ef al. 2001). DNA sequencing is useful for studies at the species level, but it is not relevant for studies of populations at a small spatial scale. Although microsatellite markers are useful for population studies, they are not yet developed for Ascaphus.  Isozymes require a large sample size and a  large amount of tissue, which were not possible for this study. Isozymes also provide highly biased genomic sampling and generate too few loci (a maximum of 40 loci). I selected R A P D markers to examine population genetic structure in  Ascaphus.  For conservation genetics studies, R A P D s provide robust data for estimating levels and patterns of genetic variation (Fritsch and Rieseberg 1996). Compared with other techniques, the R A P D technique can generate essentially unlimited numbers of loci, provides a more random sample of the genome, requires only a small amount of genomic DNA, is economical, and requires simple and relatively fast procedures. Because Ascaphus  populations are at risk  and protected from destructive sampling in British Columbia, I was limited by the amount of tissue I could collect. I was also limited by the number of larvae available for tissue sampling in each stream. Therefore, the R A P D technique was well suited for my analysis of Ascaphus population genetic structure. My goal in this chapter was to examine the population genetic structure of Ascaphus  in  one clearcut stream and one old-growth stream in a single watershed. Also, I examine patterns of spatial distribution among larvae within each stream along a 1 8 0 - m transect. In Chapter 2, I recorded larval movement rates that were lower in clearcut streams than in old-growth streams. In Chapter 3,1 reported stronger stream affinity, suggesting lower colonization potential, of juvenile frogs in clearcuts. The purpose of this study was to determine whether I could infer fragmentation impacts from differences in genetic structure. In this chapter, I test hypotheses based on two predictions: (1) I captured twice as many breeding adults in old-growth forests as in clearcuts so I predicted that Ascaphus  larvae in the clearcut stream would be less diverse  than larvae in the old-growth stream; and (2) Female Ascaphus  deposit eggs in the upper  headwaters of streams and I recorded considerable downstream movements by larvae, so I predicted that larvae would exhibit lower genetic relatedness with increasing physical distance along a 1 8 0 - m stream transect.  C H A P T E R F O U R : Population Genetic Structure of Ascaphus at the Watershed Scale  38  STUDY A R E A  Ascaphus tissue samples were collected from two streams within the Mamquam River drainage in the south coast of British Columbia's Coast Mountains, near the city of Squamish (Figure 4.1). I selected one stream flowing through a clearcut site and a second stream flowing through old-growth forest. These sites were about 1.6 km apart. Aside from forest age and stream aspect, stream and site attributes did not differ substantially between the two sites (Table 4.1).  Figure 4.1. Map showing location of study area in south coastal British Columbia and schematic map showing relative positions of river basins near collection sites of Ascaphus truei. The streams flowing through the clearcut (CC) and old-growth (OG) sites are tributaries of the Mamquam River. Sites are approximately 1.6 km apart.  C H A P T E R F O U R : Population Genetic Structure of  Ascaphus  39  at the Watershed Scale  Table 4.1. Site locations and stream attributes of Ascaphus truei tissue collection sites. Sample sizes are given at left of table.  Forest Cover T y p e  Site Locations  Forest  Stream Attributes Site  Age  Latitude  (years)  Longitude  Elevation  10  49° 3 8 ' 0 1 " 123° 0 3 ' 5 8 "  (meters) 840  250 +  49° 3 7 ' 5 0 " 123° 0 3 ' 5 0 "  935  Clearcut  Gradient  . Width Aspect . . .  (meters)  (°) 9  NW  1.6  6  SE  2.7  (n = 87) Old Growth  (n = 63)  METHODS T i s s u e Sampling Design  Ten reaches were established within each stream for tissue sampling positioned 20 m apart starting at 0 m moving downstream to 180 m (Figure 4.2). I attempted to sample a minimum of 100 Ascaphus larvae per stream, but fewer were found. In all, I collected 63 individuals from the old-growth forest and 87 individuals from the clearcut. Tissue was collected by clipping two or three 2 - m m notches (approximately 5 mg) from the tails of larval Ascaphus. Samples were preserved in 95% ethanol and stored at -70°C until D N A extraction. I used all 150 individual Ascaphus larvae from two streams residing in one watershed for subsequent DNA assays.  —> —I  0 m  1  20 m  1  40 m  1  60 m  1  80 m  1  100 m  1  120 m  1  140 m  1  160 m  1  180 m  >  Figure 4.2. Schematic showing 10 stream reaches (vertical bars) sampled for larval Ascaphus. Arrows indicate direction of stream flow. The 0 m reach is at the head of the stream sampling area, and the 180 m reach is closest to the Mamquam River. This sampling scheme was established in both the clearcut and the old growth.  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  40  Isolation of D N A  DNA extraction from clippings of larval Ascaphus tailfins followed Johnson et al. (1994). Clippings were air-dried briefly before being subjected to proteinase-K treatment. Samples were then incubated in 360 p\ S T E buffer (pH 8.0), 40 p\ of 1 0 % S D S , and 4 p\ of 20 mg//vl proteinase K at 55° C for 1 6 - 2 4 hours. To increase volumes, 100 p\ of S T E buffer was then added to samples. Samples were extracted twice with phenol - chloroform - isoamyl alcohol (25:24:1). D N A was precipitated by the addition of 0.15 M N a C l (< 7.5 p\) and two volumes of 20°C absolute (100%) ethanol, and was pelleted at 10 000 x g in a microcentrifuge. Pelleted DNA was frozen for a minimum of two hours at -20° C and centrifuged at 10 000 x g for 15 min at 4° C. The pellet was then washed twice with -20° C 7 0 % ethanol to remove excess salt. D N A was recovered by centrifugation, dried with a speed vacuum, and resuspended in 50 p\ distilled water for a minimum of 48 hours at 4° C. DNA was quantified and yield was estimated using a spectrophotometer (Pharmacia Biotech, Ultrospec® 3000) and electrophoresed on a 0 . 8 % agarose gel. On average, 10 mg of tail clippings yielded 100 ng of DNA.  A s s a y for R A P D Markers  An assay for R A P D markers (Williams et al. 1990) was performed using 10.0 ng DNA in the presence of reaction buffer (50 mM Tris pH 7.6, 2.1 mM MgCI , 10 mM KCI; Roche 2  Diagnostics, Germany), 0.2 m M of each dNTP (Invitrogen Life Technologies, Canada), 0.3 pM decamer R A P D primer (Nucleic Acids and Protein Service Unit, U B C , Canada), and 0.13 U Taq DNA polymerase (Roche Diagnostics, Germany). The polymerase chain reaction (PCR) cycling conditions for the R A P D reactions were: two minutes at 94° C, followed by 45 cycles of one minute at 94° C, one minute at 36° C, and one minute at 72° C. A final extension of 10 min at 72° C was performed to ensure complete amplification. All P C R reactions were performed on the same Programmable Thermal Controller (PTC 100, MJ Research, Inc., USA). Following P C R , the products were electrophoresed on 2 % agarose gel in 1x T B E buffer at 140 V for 3.5 hours. Gels were then stained with ethidium bromide and photographed using ultraviolet light. Images were electronically saved, and light levels were standardized across all images using Photoshop (Version 6.0) prior to scoring. R A P D bands were manually sized and scored as presence versus absence of banded phenotypes. I performed all assays at the University of British Columbia (UBC) Genetic Data Centre. To ensure consistency, I scored all gels. Two gel images demonstrating a R A P D pattern obtained with primer 213 are presented in Figure 4.3. All data used in analyses of six primers and 61 loci are given in Table A-11.1 (Appendix II). I established gel schemes that would evenly sample individuals from both clearcut and old-growth sites, and from multiple sampling distances (see Table A-l!.2, Appendix  C H A P T E R F O U R : P o p u l a t i o n G e n e t i c S t r u c t u r e of Ascaphus  at the W a t e r s h e d  Scale  41  II). However, an equal number of individuals from both clearcut and old-growth sites were not always possible because sample size was slightly smaller for clearcuts.  Screening of Primers Among 16 primers screened against a subset of individuals, six primers were used for the full analysis ( U B C primers 211,213, 221, 268, 352, and 400). The sequences are viewable at http://www.biotech.ubc.ca/frameset.html  (Services, Nucleic Acids and Protein Service Unit,  Primer Kits, Primers.pdf). Overall, the six primers yielded 97 zones of R A P D band activity.  NC i  10  m  00 O)  Ci  o T-  O  o  ro o  o  IO O  <Q O  f~ O •  *»•-*  u  600 bp  m  ' mm  mmt  mm  u  M  HV  •sw  mm M  Ii fcaH iNMi  M  *•"-" *™ mm  A.  c\j o  L  L  co o  cn o  O  i  -  C M CO  ^  LO  CD  m  w  E5  II i i •Ml  t  ** «*  mm mm  ii  '•" 1  PC L  U U B  H I Mil  Lug £u •H ^"1 91 1 1  M  •MM  ***** * ** • ,  ,  S; ;  H  —  600 bp  B.  Figure 4.3. R A P D pattern obtained with primer 213. Lanes labelled "L" are size marker DNA fragments. The brightest middle band is 600 base pairs in molecular weight. Lane " N C " is the negative control, and " P C " is the positive control. Samples, sites ( C C = clearcut, O G = old growth), and sampling distances from left to right are: A) 96 = C C - 0 m; 97-99 = C C - 2 0 m; 100 = O G - 6 0 m; 101-103 = C C - 6 0 m; 104-107 = C C - 8 0 m, B) 108-109 = C C - 8 0 m; 110-115 = C C - 1 0 0 m; 116 = O G - 1 2 0 m.  C H A P T E R F O U R : Population Genetic Structure  of Ascaphus  at the Watershed Scale  42  Scoring of Diploid RAPD Markers To score R A P D markers as presence and absence data, I made the following assumptions (following Lynch and Milligan 1994): 1) Each scorable band per primer (different molecular weights) represented an independent locus and was treated as a two-allele system; 2) Marker alleles from different loci did not comigrate to the same position on a gel; and 3) The interpretation of banding patterns on gels was accomplished in a completely unambiguous manner (i.e., the investigator was unbiased and accurate in matching bands from different lanes within and among gels). I visually scored monomorphic and polymorphic R A P D markers once from the electronically saved gel images. A blind scoring method was used, in which I was unaware of the identity or origin of the individuals (e.g., old growth, 60 m). Regardless of intensity, if any bands were present in negative control lanes, those loci were omitted from the dataset. Only relatively easy to score bands (i.e., usually bright and discrete) were considered. Presence of a band was scored as a one and absence as a zero. Images were grouped by primer, and all possible markers for one primer were scored prior to scoring markers generated from a different primer.  Statistical Analyses Allele frequencies of loci scored in the old growth and clearcut were calculated using the program T F P G A (Tools For Population Genetic Analysis; Miller 1997). Genetic distances between stream reaches were calculated using Nei's genetic distance (Nei 1978) included in the program T F P G A . A one-way A N O V A was used to test differences in heterozygosity estimates obtained from the old growth and clearcut. All reported P-values were obtained using statistics tested against a preset level of significance, a = 0.05. F  S T  values, which measure the degree of population divergence, were obtained using the  program T F P G A by applying Weir and Kockerham's (1984) methods of calculating Wright's (1931) F-statistics. Assumptions of Hardy-Weinberg (H-W) equilibrium are made by T F P G A when calculating F T values. Standard errors of F T estimates were calculated using jackknifing S  S  where replicates were obtained from sequential elimination of loci. Confidence intervals (95%) of FST estimates were determined by the bootstrap method, where individuals within populations were resampled to create replicated data sets (1000 iterations). Such genetic equilibrium assumed when using F-statistics can only occur under conditions of zero selection and mutation, no emigration or immigration, random mating, and a large population size. Ascaphus  populations are probably not at equilibrium, because of the  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  43  relatively recent recolonization, and colonization of northern habitats that may still be occurring (Ritland era/. 2000). Therefore, when using dominant markers, OsWphis-r).  a  n  analog to  F T S  that does not rely on H-W equilibrium, may be an appropriate alternative. I used the program WINAMOVA (Excoffier 1996) to perform an analysis of molecular variance (AMOVA; Excoffier etal. 1992) and calculate 0 T - ^ST, like its analog, F , indicates genetic differentiation. ST  S  AMOVA is essentially a multivariate analysis of variance (MANOVA), but it uses the pairwise distance matrix as input, rather than the raw Y-vectors themselves (Smouse et al.  2001).  The  program AMOVA-PREP (Miller 1998) was used to prepare the group, distance, and population input files for use with WINAMOVA. The band-sharing coefficient is the main index of similarity used to describe DNA fingerprints from two individuals (Bruford and Beaumont  1998).  It gives the probability that a  band will be shared for a certain molecular weight range within the fingerprint. Band sharing coefficients were calculated using a program written in Fortran by Kermit Ritland. Shared bands are those that have comigrated (within 0.5 mm) and have no more than two-fold intensity difference (i.e., the difference between a homozygote and heterozygote; Bruford and Beaumont 1998).  The coefficient describes behavior, or how the phenotypic difference changes with  distance (behavior in spatial pattern), and calculates the frequency of shared dominant phenotypes. Standard errors of the estimates were obtained by bootstrapping, where 100 repeated random samples were selected from the data and the model is estimated from each one (Norusis  1993).  The relationships between genetic relatedness and physical distance were  analyzed using Pearson correlation coefficients (Norusis  1993).  RESULTS  Polymorphic Loci and Heterozygosity I scored a total of 97 RAPD loci. However, loci that were faint or not present on all gels were omitted. The total number of RAPD loci scored with high confidence was 61, and these loci were used in subsequent analyses. No loci were unique to differentiate between Ascaphus larvae from either sampling location (old growth or clearcut), but streams were too close for this to be possible. However, there were 61% (37/61)  in clearcut (Table  4.2).  72% (44/61)  polymorphic loci present in old growth, and  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  44  Table 4.2. Mean heterozygosity (H) and percentage of polymorphic loci at each stream reach station for old growth and clearcut. Mean values for old growth and clearcut were calculated using estimates obtained for each of the 61 loci; these are not based on means for each stream reach station. The total number of samples used to calculate estimates is given in the last row of the Average n column.  H  % Polymorphic Loci  Average n  0.0263  49.18 31.15 24.59 37.70 0.00 9.84 57.38 57.38 47.54 40.98 72.13  9.80 10.00 6.80 10.00 1.00 1.97 5.90 6.66 4.41 4.00 60.54  0.0259  13.11 14.75 16.39 36.07 36.07 34.43 44.26 26.23 27.87 42.62 60.66  10.00 9.67 3.62 9.64 9.97 9.98 10.00 5.00 10.00 7.30 85.18  1  2  Old Growth Om 20m 40m 60m 80m 100m 120m 140m 160m 180m Mean  0.2396 0.1493 0.1169 0.1818 0.0000 0.0486 0.2620 0.2578 0.1994 0.1908 0.3109  Clearcut 0m 20m 40m 60m 80m 100m 120m 140m 160m 180m Mean 1  0.0575 0.0673 0.0849 0.1643 0.1781 0.1679 0.2130 0.1288 0.1323 0.1965 0.2275  Nei's (1978) unbiased heterozygosity Estimates based on 9 5 % criterion (gives percentage of loci that are not fixed for one allele)  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  45  Mean heterozygosity that is expected under random mating (H) for old growth and clearcut is given in Table 4.2. In old growth, the estimate at 80 m is zero because only one Ascaphus larva was available for the calculation. Based on mean heterozygosity, larvae in the clearcut were genetically less diverse, or more closely related old growth  ( 0 . 3 1 0 9 + 0.0263;  F  = 4.259;  P=  ( 0 . 2 2 7 5 + 0.0259)  than larvae in  0.041).  Frequency of Dominant Alleles  Allele frequencies were estimated based on Lynch and Milligan's (1994) Taylor expansion estimate, which takes into account the dominance of RAPD markers and the statistical bias introduced by small sample size. The distribution of RAPD marker allele frequencies (Figure 4.4) showed the frequency of the dominant allele ranged from 0.01 to 1.00 (fixed). Fewer loci were nearly fixed for the dominant allele in the old growth (15) compared with the clearcut (22). These loci contribute little information about population structure and genetic differentiation, as none are specific to either site. Furthermore, most loci were not sufficiently variable to be informative. Population Differentiation Genetic differentiation estimates between "populations" (forest cover types) and among "subpopulations" (stream reaches), based on F T values, are provided in Table 4.3. The degree S  of differentiation was high among stream reaches forest cover types  (F  S T  = 0 . 0 6 7 7 + 0.0169).  (F  S T  = 0 . 3 2 2 8 + 0.0211),  but low between  Forest cover types also were analyzed separately,  revealing that the level of differentiation among stream reaches in the clearcut appeared to be lower  (F  0.0232;  S T  = 0 . 2 2 8 5 + 0.0185)  Table  (97.11%)  4.3).  than among stream reaches in the old growth  (F  S T  = 0.3136 +  The AMOVA (Table 4.4) confirmed that most of the genetic diversity  was found among reaches within each stream ("within population"). Genetic variation  between the two populations (old growth and clearcut: "among populations") was small  (2.89%).  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  46  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  47  Table 4.3. Genetic differentiation between old growth and clearcut ("population"), among stream reaches ("subpopulations") pooled within old growth and clearcut, and among stream reaches analyzed separately within old growth and within clearcut, as given by F values. ST  Unit  FST  SE  E  95% Confidence Intervals Lower Upper  2  1  Population  0.0677  0.0169  0.0383  0.1034  Subpopulation  0.3228  0.0211  0.2835  0.3658  Old Growth  0.3136  0.0232  0.2689  0.3613  Clearcut  0.2285  0.0185  0.1945  0.2632  1  2  Based on jackknifing over loci Based on bootstrapping over loci (1000 iterations)  Table 4.4. Hierarchical analysis of molecular variance: between old growth and clearcut ("among populations") and among stream reaches ("within populations") pooled within old growth and clearcut, as given by 0 values. S  Variance Component  T  Observed Partition Variance  % Total  P  Among Populations  0.123  2.89  0.024  Within Populations  4.135  97.11  0.008  1  Probability of having a more extreme variance component and 0 observed values by chance alone  *ST  0.029  1  S  T  than the  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  48  Genetic Relatedness A l o n g Stream Transects  Similarity of RAPD bands within and among stream reach stations are given in Table 4.5; the smaller the value, the less band sharing (i.e., lower genetic similarity). For both forest cover types, larvae sampled from the furthest upstream reach (0 m) were more genetically similar than larvae sampled from furthest downstream reach (180 m). Frequency of RAPD band sharing was averaged for individuals at increasing distances from one another and summarized in Table 4.6. These values are for distance categories (e.g., 0-m apart, 20-m apart) and do not represent stations. On average, frequency of band sharing was greater for larvae sampled in the clearcut stream compared with the old-growth stream, regardless of their physical distance (Table 4.6). In the old-growth stream, larvae sampled within one reach station had a high band similarity frequency, while larvae sampled at stations of increasing distances from one another showed a low band similarity frequency. This pattern was not seen in the clearcut stream.  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  49  Table 4.5. Similarity of R A P D bands within and among stream reach stations in a) clearcut and b) old growth. Reach 0 m is the top of the stream sampling area, and 180 m is the bottom. Values in the first row indicate similarities among individuals within each station. The rows that follow indicate similarities between stations of different distances from one another. For example, row four of the 8 0 - m station in the clearcut indicates that individuals sampled at the 8 0 - m and 1 4 0 - m stations have a band-sharing frequency of 0.925.  Clearcut Site Station  0m  20 m  40 m  60 m  80 m 100 m 120 m 140 m 160 m 180 m  0.921 0.940 0.927 0.938 0.948 0.946 0.940 0.963 0.968 0.968  0.934 0.953 0.946 0.948 0.956 0.954 0.947 0.964 0.978  0.928 0.944 0.927 0.937 0.948 0.945 0.934 0.957  0.917 0.929 0.920 0.924 0.929 0.930 0.929  0.916 0.931 0.922 0.925 0.933 0.927  0m  20 m  40 m  60 m  80 m 100 m 120 m 140 m 160 m 180 m  0.876 0.836 0.835 0.862 0.875 0.890 0.914 0.928 0.922 0.902  0.892 0.843 0.848 0.870 0.902 0.918 0.935 0.952 0.945  0.905 0.851 0.859 0.883 0.911 0.930 0.942 0.954  0.890 0.850 0.853 0.863 0.908 0.920 0.926  0.885 0.842 0.856 0.841 0.915  0.917 0.936 0.923 0.926 0.933  0.912 0.924 0.918 0.914  0.918 0.927 0.919  0.922 0.914 0.934  Old-Growth Site Station  0.887 0.815 0.858 0.851 0.931  0.852 0.823 0.812 0.855  0.835 0.817 0.821  0.830 0.810  0.856  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  CO  o  CD  TJ  C CD  Si ^  .52 o  E ®  r-co CO  g .E c  O  CO  -<= 8,  00  u ro  J=> JC  E ° ro T CD o  ,  E o  -2  ro  O CD CD  E £ E oI CD O E ^ 5 J= CD  £  -—- T J  ^ S.co I co  'J=  co  ^ f  0  CD CM CO i-i-  E ~  1° TJ  CD CD C  •— C CO O  II  co  to co  co •§  S  E  CD  0  E £ 2 TJ ro cc Q . 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The between-individual mean band sharing frequency was from 0.921 + 0.023 to 0.937 + 0.017 in the clearcut, and 0.861 + 0.018 to 0.889 + 0.017 in the old-growth site (Table 4.6). Overall, larvae were more genetically similar in the clearcut but less similar in old growth (Figure 4.5). In the clearcut, larvae 0-m to 160-m apart showed no decrease in genetic relatedness (Figure 4.5). In old growth, however, larvae 0-m to 140-m apart decreased in genetic similarity (Figure 4.5). Values at 160-m and 180-m stream reaches in old growth, and at the 180-m reach in the clearcut (Figure 4.5) have high standard errors of the estimate (Table 4.6) and may represent outliers. Pearson correlations revealed a low negative correlation between genetic relatedness and physical distance of individuals in the clearcut site (r= -0.475, P = 0.165), but a high negative correlation between genetic relatedness and physical distance in old growth (r= 0.786, P = 0.007; Figure 4.5).  .950  Clearcut  n  •52 .930 •  r= -0.475 P> 0.05  O" CD  .910 • to CD  c  T3  CD  ro  Old Growth  -890 •  CD  r= -0.786 P< 0. 05  cr  CJ  0 c  CD  •  •  .870-^  CD  .850 -20  0  20  40  60  80  100  120  140  160  180  200  Physical Distance (metres) Figure 4.5. Scatterplot of genetic relatedness (frequency of RAPD band sharing) and physical distance (distance between stream sampling stations; meters apart) for individuals sampled from the clearcut and old-growth site.  CHAPTER FOUR: Population Genetic Structure  of Ascaphus  at the Watershed Scale  52  DISCUSSION Heterozygosity In my RAPD study, Ascaphus  had lower genetic variation in the clearcut (H = 0.23 +  0.03) than in the old growth (H - 0.31 + 0.03), based on mean heterozygosity and percentage of polymorphic loci (72% and 61%, respectively). Using RAPD data, Ritland etal. (2000) reported a mean heterozygosity of 0.21 + 0.02 for Ascaphus  in a clearcut stream. Ascaphus  tissue  samples were collected from the same clearcut site in each study, and their estimate corresponds closely with that obtained in my RAPDs study. I found no studies examining differences in genetic structure between populations in old growth and clearcuts using RAPD techniques. However, some results based on allozyme data are noteworthy. Green et al. (1989) reported a mean heterozygosity of 0.096 for Ascaphus  (six  frogs; coastal Oregon). Hitchings and Beebee (1997) reported lower genetic variation (H = 0.06; 42% polymorphic loci) in urban populations compared with rural populations (H = 0.07; 50% polymorphic loci) of Rana temporaria,  a common pond-breeding frog. Nevo and Beiles  (1991) reported decreases in genetic diversity with decreasing ecological heterogeneity, from terrestrial and arboreal to aquatic and subterranean habitats (i.e., with decreasing niche breadth, or with increasing ecological stability and predictability). However, the terrestrial genus Leiopelma  generally had the lowest levels of heterozygosity (0.01-0.09) among aquatic,  terrestrial, and arboreal frogs (Green et al. 1989; Nevo and Beiles 1991). Reported heterozygosity of other amphibian taxa included 0.05 for tree frogs (Hylidae), 0.08 for true frogs (Ranidae), and 0.11 for true toads (Bufonidae; Nevo and Beiles 1991). Because these data are : based on allozymes, they cannot be directly compared with results from RAPD assays. However, trends in genetic variation among anurans can be evaluated and may provide the basis for predictions when using other molecular markers. For some species, heterozygosity is positively correlated with fitness components such as survival, growth rates, and fertility (Mitton and Grant 1984; Reed and Frankham 2003). If this is true for Ascaphus  my data may suggest that individuals in the clearcut are less capable of  adapting to environmental fluctuations (e.g., increases in temperature and ultra-violet radiation). Equally important is their increased susceptibility to disease (e.g., pathogenic fungi such as chytrids or Saprolegnia Ascaphus  water molds). My results suggest lower population persistence for  in the clearcut.  A number of factors could be responsible for the observed pattern of lower genetic variation in the clearcut than in the old growth. Harvested 10 years ago, the clearcut may now have few individuals entering the population. Dispersal may be limited in clearcuts due to this organism's physiological constraints. Recall that Ascaphus  have extremely low desiccation  tolerance (Claussen 1973), and one of the lowest and narrowest ranges of temperature  CHAPTER FOUR; Population Genetic Structure of Ascaphus at the Watershed Scale  53  tolerance among anurans (Brattstrom 1963; de Vlaming and Bury 1970). Although it is possible that genetic variation may have been low originally (i.e., few founding members prior to logging), it is unlikely as the heterozygosity estimate for the nearby old growth is higher. Another factor that may affect estimates of genetic variation is the number of mating individuals. Over three years, I captured 33 mature adults in clearcuts and 75 mature adults in old-growth forests (Chapter 3). During this time, I captured an equal number of gravid females in both forest cover types across the three years. However, with less than half the number of reproductively mature adults contributing to the gene pool in clearcuts, estimates of genetic variation were expected to be lower in clearcuts than in old growth. Populations in harsher habitats (e.g., clearcuts, urban settings) are expected to show reduced genetic diversity due to lower effective population sizes and temporary contractions of population size (Ritland et al. 2000).  Population Differentiation I estimated large F  S T  among stream reaches ( F  = 0.32), indicating a high degree of  S T  genetic differentiation among stream reaches (old growth and clearcut combined). When sites were analyzed separately, the degree of genetic differentiation among reaches within old growth was still high (F T = 0.31). However, F T in the clearcut was low (0.23). The lower genetic S  S  differentiation among reaches in the clearcut and lower heterozygosity are consistent with a smaller effective population size. These results suggest that the larval population in the clearcut stream is less stable than that in the old-growth stream. I estimated a small F T value between S  old growth and clearcut (F T = 0.07), indicating a low degree of genetic differentiation among S  streams. Prior to forest harvesting activities in that portion of the watershed (10 years ago), it may be possible that adults moved between the two streams, which are 1.6 km apart. This could explain the currently small degree of differentiation between the two streams.  Ascaphus  in the clearcut stream may have reduced in numbers since logging (recall the capture of fewer mature frogs in clearcuts than in old growth; Chapter 3), resulting in a less stable population genetic structure than in the old growth. Using R A P D s , Ritland et al. (2000) reported an F  S T  of 0.04 among five populations in the  south coast of B C , near the city of Squamish. Samples in their study were collected from sites too distant from one another for gene flow to occur. However, the authors suggested that the low values for F T and H, and a lack of association between genetic and geographic distance, S  were the result of more recent colonization. Based on allozymes, genetic differentiation among populations for other anurans include: Bufo japonicus Rana temporaria  (F  S T  = 0.24), Bufo bufo (F T = 0.49), and S  (F T = 0.32; summarized by Hitchings and Beebee 1997). Using R A P D s , a S  high degree of within-population differentiation among Rana pipiens  also was reported by  Kimberling era/. (1996). Based on allozyme data, Hitchings and Beebee (1997) reported a high  CHAPTER FOUR: Population Genetic Structure of Ascaphus at the Watershed Scale  degree of genetic differentiation ( F  ST  54  = 0.35) among urban populations of Rana temporaria,  and  a low degree of differentiation (F j = 0.11) among rural populations. The authors attributed the S  high degree of urban subpopulation differentiation to the increasingly inhospitable terrain of the urban environment. Because F-statistics rely on assumptions of H-W equilibrium, I thought it appropriate to use an alternative estimator of population differentiation that did not rely on this assumption. There were a number of reasons why I chose to explore alternative statistics. H-W equilibrium is rare and probably does not characterize Ascaphus  populations. For example, in the fall,  reproductively mature adults congregate at the upper headwaters of streams, and there is evidence that adults have strong site fidelity (Daugherty and Sheldon 1982a). In my study, of 17 total recaptures, five reproductively mature adults (29% of recaptures) were recaptured at the same stream in consecutive years (Chapter 3) suggesting some adults may return to their natal stream. Therefore, random mating may not occur. Also, Ascaphus  population size is  unknown; however there is evidence that it is small. I captured only 254 frogs (many were metamorphs) at six sites over three years (Chapter 3). Furthermore, because of the relatively recent and continuing colonization of northern habitats suggested by Ritland et al. (2000), some degree of immigration and emigration likely exists. Based on A M O V A , estimates confirmed genetic differentiation among old-growth and clearcut streams was small (2.89%; 0  S  T  = 0.03), and most genetic differentiation (97%) was  found within streams. A major disadvantage to using the program W I N A M O V A is that it cannot analyze a dataset containing missing observations, thus 26 samples (17 from old growth and nine from clearcut) were omitted. Using a small sample size when calculating estimates for genetic differentiation means that some rare alleles may be missed, which may under- or overestimate F T values (and d> ), which are directly related to estimates of heterozygosity. S  ST  However, the reduction in sample sizes due to omission of those data is probably not critical (C. Ritland pers com). In another study of Ascaphus,  A M O V A indicated that both coastal (88.7%)  and inland populations (82.7%) exhibited significant genetic differentiation (Neilson etal. 2001). Significant differentiation also occurred within a group encompassing 14 populations (® T = S  0.571; Neilson ef al. 2001). The long generation time in Ascaphus  ( 6 - 8 years) and low  metabolic rate may result in a slow evolutionary rate, which can reduce genetic divergence (Martin and Palumbi 1993).  CHAPTER FOUR: Population Genetic Structure  of Ascaphus  at the Watershed Scale  55  Geographic Distance and Genetic Relatedness  Based on band-sharing frequencies, individuals in the clearcut were more related to one another than were individuals in the old growth. This is consistent with heterozygosity and F T S  estimates. Both streams had more genetically similar individuals at the top of the stream than at the bottom. This observation suggests that larval drift from upper reaches influenced the patterns of genetic relatedness as larvae move further downstream. The difference was greater in old growth. Results are not surprising, as siblings are more likely to be found within close proximity of one another at the upper reaches, closer to where egg masses are deposited. However, in the clearcut, there was a high degree of genetic relatedness among individuals, and geographic distance did not appear to have any influence on relatedness of larvae up to 160 meters away. Individuals in old growth, however, showed a decrease in genetic relatedness with increasing geographic distance between sampled reaches. These findings may suggest that more movement of larvae across reaches occurs in the clearcut stream. However, that would be inconsistent with results of Chapter 2, in which larval Ascaphus in clearcut streams moved shorter distances (up to 3 m) compared with larvae found in old-growth streams (up to 65 m). Stream and site parameters have the potential to influence larval movement within streams. I measured stream gradient, wetted width, and site aspect (Table 4.1), however none appear significantly different to have caused a response in larval movement patterns (also see Chapter 2). An alternative to more movement is the possibility that many larvae in the clearcut are siblings. The entire sample may be offspring from as few as two mating pairs, or from closely related parents. This is a plausible explanation for the inconsistency in results. Mating pairs that were restricted to the clearcut stream since logging may explain the greater genetic similarity of larvae in the clearcut stream compared with those in the old-growth stream.  CHAPTER FIVE: Summary and Conservation Implications  56  C H A P T E R FIVE S u m m a r y a n d C o n s e r v a t i o n Implications  INTRODUCTION Timber harvesting reduces habitat patch size, increases habitat fragmentation (increasing probabilities of local extinction), and removes habitat connectivity, thus reducing dispersal among patches (e.g., Sjogren 1991; Bunnell etal. 1992; Fahrig and Merriam 1994). Welsh and Lind (2002) believed Ascaphus  populations would continue to decline in the Pacific  Northwest in response to anthropogenic disturbance regimes. If Ascaphus  movements through  upland habitats are restricted in recently harvested forests, connectivity among populations will be lower and recolonization of streams from which they have been extirpated may be more difficult (Aubry 2000). A metapopulation is a collection of smaller subpopulations occupying habitat patches, connected by dispersal, and balanced by extinction and colonization (Levins 1969; Hanski and Simberloff 1997). The subpopulations can each be characterized in terms of genetic variation and demographic parameters (Galbraith 1997). In many respects, amphibian spatial dynamics resemble classical metapopulation models. This is particularly evident where subpopulations in breeding ponds blink in and out of existence overtime, and extinction and colonization rates are functions of pond spatial arrangement (Marsh and Trenham 2001). The extent of individual movement is related to the potential for gene flow among populations (Lande 1988). Breeding ponds form discrete habitat patches for many anurans, but the connected nature of  Ascaphus  streams may not result in discrete populations. Also, aggregations of amphibians at individual streams or ponds may not represent distinct populations. Many amphibian species regularly disperse between ponds (Marsh and Trenham 2001), and the same may be true of streambreeding amphibians. I investigated the role of forest cover in the movement patterns and colonization potential of Ascaphus.  I examined differences in movement patterns and dispersal abilities of larval,  juvenile, and adult Ascaphus,  and explored genetic variation in Ascaphus  populations in  managed and unmanaged forests. I reviewed reasons for concern about the likelihood of Ascaphus  to be extirpated locally and then recolonize managed forests, and indicated existing  information gaps in Chapter 1. In Chapter 2, I evaluated larval movements of Ascaphus  and  discussed associations with stream and site parameters. In Chapter 3, I examined terrestrial movements of juvenile and adult Ascaphus,  and the multiple factors that can confound  responses to timber harvesting. I conducted a preliminary examination of the population genetic  CHAPTER FIVE: Summary and Conservation Implications  structure (using R A P D s ) of Ascaphus  57  in two streams within a single watershed in Chapter 4.  Here, I present a synthesis of results among investigations. I discuss the implications of those results to Ascaphus  conservation goals, and I propose options to develop more effective  management for Ascaphus  and other species that rely on forested headwater streams.  SUMMARY OF RESULTS Maximum daily movement rates of larvae in streams flowing through old-growth forests (3.8 m/day) appeared to be greater than rates in clearcuts (0.3 m/day) and mature secondgrowth forests (1.9 m/day). Stream gradient differed among forest cover types (ranging from 4 to 44%), and larval movement rates had a tendency to be greater in low gradient streams, suggesting active movement versus passive drift. Higher rates of larval movement were observed in streams with larger wetted widths. Neither larval density nor percent canopy closure influenced larval movement rates. Logjams had a significant role and explained 1 3 % of the variation in larval movement rates. Logs embedded in the stream are features that can create physical barriers to larval movement. All clearcut sites contained abundant logjams, and larval movement rates were greater in streams with lower levels of logjams. Alternatively, lower movement rates in clearcuts may be a response to greater instream primary productivity (related to less canopy and more solar insolation), which could encourage faster growth and reduced movement of larvae. More juvenile and adult frogs were captured in clearcuts (0.91/100 TN) than in oldgrowth forests (0.72/100 TN). Developmental stages were differentially represented with 3X more juveniles in clearcuts (0.70/100 T N ; 9 1 % of these were metamorphs) than in old growth (0.24/100 TN), but 2X more adults in old growth (0.48/100 TN) than in clearcuts (0.21/100 TN). There appeared to be more recaptures (8.5%) and higher movement rates (3.36 m/day; n = 12) in clearcuts than in old-growth forests (4.5%; 2.09 m/day; n = 5), but sample sizes were small. Many (n = 24) of these coastal Ascaphus  moved at least 100 m from streams compared to at  least 12 m from streams reported for inland populations (Metter 1964a; Landreth and Ferguson 1967). However, in the fall, frogs were captured 1.3X farther from streams in old-growth forests (21.59 + 9.15 m) than in clearcuts (17.00 + 3.93 m), and there were more frogs captured within 25 m of streams in clearcuts. Adults were captured farther (2X) from streams than were juveniles. Females were captured twice as far as males, and movement patterns appear to suggest breeding migrations (upland to streams). Reproductively mature frogs were captured twice as far from streams in old growth compared with clearcuts. Most frogs in clearcuts were moving upstream, and most frogs in old-growth forests were moving towards streams. Adult movement directions appeared little affected by forest cover type. Juveniles, however, exhibited stronger stream affinity in clearcuts than in old-growth forests, and moved mainly upstream.  CHAPTER FIVE: Summary and Conservation Implications  58  Juveniles and adults in clearcuts tended to be slightly smaller and had a lower body condition index (BCI) than frogs in old-growth forests. In both the old growth and clearcut, there were no unique loci to differentiate between larvae from either forest cover type. Fewer loci were nearly fixed for the dominant allele in the old growth (n = 15) than in the clearcut (n = 22). Forty-four (72%) polymorphic loci were present in the old growth, and 37 (61%) polymorphic loci were present in the clearcut. Based on mean heterozygosity, larvae in the clearcut were less diverse, or more closely related (H = 0.23 + 0.03), than larvae in the old growth (H = 0.31 + 0.03). The degree of genetic differentiation was high among stream reaches ( F  S T  = 0.32 + 0.02), but low between forest cover types (F T - 0.07 S  + 0.02). When forest cover types were analyzed separately, the level of differentiation among stream reaches in clearcut was lower ( F growth ( F  S T  S T  = 0.23 + 0.02) than among stream reaches in old  = 0.31 + 0.02). AMOVA confirmed that most of the genetic diversity (97%) was  found within streams, and genetic variation between the old-growth and the clearcut site was small (3%). For both the old growth and the clearcut, larvae sampled from the furthest upstream reach (0 m) were more genetically similar than larvae sampled from furthest downstream reach (180 m). Frequency of band sharing (genetic relatedness) was greater for larvae sampled in the clearcut compared with the old growth, regardless of their physical distance. In the old growth, larvae sampled within the same stream reach had high band similarity (high genetic relatedness), and larvae sampled at stream reaches of increasing distances from one another had lower band similarity. Thus, geographic distance and genetic similarity were negatively correlated in the old growth (r= -0.786, P = 0.007). Larvae in the clearcut showed no pattern in genetic relatedness with increasing geographic distance (r= 0.475, P = 0.165).  IMPLICATIONS OF MY RESULTS TO ASCAPHUS CONSERVATION GOALS Conservation has long been an important part of wildlife management. One goal of conservation biologists is to maintain natural levels of biodiversity and reduce factors driving species to extinction (Caughley and Gunn 1996). Ascaphus  is a taxonomically unique endemic  of the Pacific Northwest of North America, and is believed to be the most primitive frog in the world (Ford and Cannatella 1993). Maintaining representative viable populations throughout the range of Ascaphus on Ascaphus  is an underlying assumption of my thesis. Here, I outline how my research  contributes to the overall conservation goal. I also provide some management  recommendations and directions for future research.  CHAPTER FIVE: Summary and Conservation Implications  59  1. My research revealed a tendency for shorter larval movements in streams containing many embedded logjams. Embedded logs were abundant in clearcut streams and may reduce the recolonization potential of Ascaphus  larvae. Stream ecologists use the term  'drift' to describe the downstream movement of stream organisms either passively with floods or actively as in behavioral drift. Muller (1974) hypothesized that drift would eventually wash entire populations out of streams unless organisms actively moved upstream to compensate for drift. He coined the term "colonization cycle" to describe the maintenance of stream populations through a dynamic interplay between downstream drift and upstream movement.  It appears that Ascaphus  may follow a colonization cycle. Larvae exhibit downstream  movements, and my terrestrial data suggest that many post-metamorphic  Ascaphus  move upstream, compensating for larval movements. Reduced larval movements in clearcuts may be problematic in maintaining the colonization cycle. Logging activities resulting in a major increase in the density or embeddedness of large wood in stream channels should be avoided. Falling and yarding away from streams will be necessary to maintain slash-free channels (Chapter 2; Dupuis and Steventon 1999).  My recommendations for future research on larval Ascaphus  include: 1) estimation of  algal production among headwater streams transecting clearcuts and old growth; 2) assessment of larval movements along a continuous stream sampling area (e.g., 3 0 50 m in length), more regular sampling (e.g., every 2 - 3 days), and the placement of a weir at the base of the lower sampling reach to increase recapture rates; and 3) sampling of larvae along an elevational gradient to determine dispersion of larvae in streams transecting clearcuts and old-growth forests.  2. My research suggests that long distance overland movement is more likely when forested stands are present. Terrestrial habitat use by Ascaphus  (particularly juvenile  frogs) during fall appears more spatially reduced in clearcuts than in old-growth forests. Recent metamorphic wood frogs (Rana sylvatica) and Hunter 1999), and I captured juvenile Ascaphus  prefer closed canopies (deMaynadier farther from streams in old growth  than in clearcuts. Adult frogs are considered the most evolutionarily effective migrants (sensu MacArthur and Wilson 1967) because their reproductive value is high, especially for gravid females. I recorded adult Ascaphus  undertaking more long  distance overland movements than juveniles. Also, in old growth, I captured twice as many reproductively mature frogs as in clearcuts. Therefore it appears there are fewer  CHAPTER FIVE: Summary and Conservation Implications  60  evolutionarily effective migrants available to disperse through clearcuts to adjacent streams. The capture of fewer mature frogs in clearcuts may result from poor survival ofjuveniles. These results have implications to population persistence of Ascaphus.  Limited  movement might lead to increased population subdivision and isolation (Nijhuis and Kaplan 1998) due to a reduction in gene flow, and increase the chance of local extinction by demographic or environmental stochasticity. Amphibians are known to benefit from retention of riparian habitats during logging (Gomez and Anthony 1996; Dupuis and Steventon 1999; Maxcy 2000). Large riparian buffers may ensure moist and less variable microclimatic conditions, particularly in times of drought. Despite reports of strong stream fidelity for inland populations of Ascaphus,  I found that BC  frogs could move at least 100 m from streams into the upland forest in rainy weather. Maintaining some gene flow between populations is important and  Ascaphus  movement is more likely where forest cover is present. A forest buffer on both sides of streams appears to be a useful approach to maintaining Ascaphus  populations (Welsh  and Lind 2002). However, an effective width is not clear from my results and will require further investigation and monitoring. Adjusting zone widths depending on local conditions (e.g., stream wetted width, gradient, intensity of adjacent forest harvest) may be an appropriate option (deMaynadier and Hunter 1995). Windthrow damage is estimated to amount to 4% of the provincial annual allowable cut (Mitchell 1995), thus buffer integrity should be maintained by ensuring that harvested edges of buffers are windfirm.  My recommendations for future research include: 1) investigation of daily movements ofjuveniles and adults using passive integrated transponders (PITs) or radio transmitters for tracking individuals among clearcuts and old growth; 2) long-term markrecapture studies to reveal stream-to-stream movements where different buffer widths are maintained; 3) summer assessment of juvenile and adult abundance (CPUE) along an elevational gradient to determine dispersion along clearcut and old-growth streams; and 4) daily tracking of gravid females (using PITs or radio transmitters) in the summer and fall to evaluate possible breeding migrations, reproductive cycle (e.g., number of clutches per year), and determine oviposition site selection.  CHAPTER FIVE: Summary and Conservation Implications  61  3. My results revealed lower heterozygosity (genetic variation) for larvae captured in the clearcut site. In some species (including amphibians), heterozygosity is positively correlated with fitness components such as survival, growth rates, and fertility (Mitton and Grant 1984; Reed and Frankham 2003). If this is true for Ascaphus,  then my data  may suggest that individuals in the clearcut site will be less capable of adapting to environmental fluctuations and will be more susceptible to factors such as disease. The capture of fewer breeding adults and the lower genetic variation recorded in the clearcut is consistent with a smaller effective population size, which may mean clearcut populations are less stable (i.e., lower population persistence) than in old growth. Small effective population size and a lack of dispersal between sites may limit mate choice, which leads to breeding with close relatives. Results also suggest Ascaphus  populations exist in a metapopulation structure, but  further investigation is required. Through monitoring of genetic diversity within and among Ascaphus  populations, metapopulation dynamics may be revealed. Riparian  buffers likely serve as movement corridors for juvenile and adult Ascaphus,  but large  openings in adjacent forests may prevent frogs from reaching nearby streams via overland movements. A partial forest matrix between streams could be retained within each watershed to provide habitat pathways for dispersing frogs. When connectivity between streams cannot be maintained, habitat conservation strategies for  Ascaphus  could be improved by including riparian management areas on multiple adjacent headwaters in areas favoring Ascaphus.  Reducing distances between mature forest  patches would improve the chances of Ascaphus  moving through harvested  watersheds. To encourage gene flow among populations, I recommend monitoring Ascaphus  populations where a partial forest matrix is retained between streams.  Studies could focus on varying the level of harvest within the matrix. My RAPD study in two Ascaphus  streams provides some understanding of how habitat fragmentation  can influence population genetic structure. The examination is incomplete, but signals the need for further research on this topic.  My recommendations for future research include: 1) increasing studies to include at least three replicates and larger sample sizes (e.g., >30 larvae/sampling location; >100 larvae/stream); 2) evaluating population genetic structure in Ascaphus  using additional  molecular markers (e.g., microsatellites when these become available for  Ascaphus);  3) analysis of genetic relatedness among mature frogs and larvae in streams transecting clearcuts and old growth to examine site fidelity in Ascaphus;  4)  CHAPTER FIVE: Summary and Conservation Implications  62  examination of population genetic structure and possible metapopulation structuring of Ascaphus  populations within multiple adjacent watersheds and multiple streams within  each watershed; and 5) molecular monitoring of genetic diversity within and among Ascaphus  populations where a partial forest matrix is retained between streams.  My results, though not all significant statistically, are internally consistent and suggest reduced recolonization potential and lower genetic variation in Ascaphus forest cover has been removed. Aggregations of Ascaphus  populations where  at individual streams may not  represent distinct populations, and should not be managed as distinct units. Ascaphus  may  regularly disperse between streams. Thus, connectivity between multiple streams within a watershed will be a more meaningful unit of management than individual streams with forested buffers. Promoting some connectivity between streams may be the single most important issue to address when defining Ascaphus  conservation strategies. Riparian buffers alone may not be  effective for promoting long-term persistence of Ascaphus  populations, but when no other  habitat protection is provided, buffers should be retained along streams as a first step towards Ascaphus  protection. Conservation measures that are more likely to promote long-term  population persistence should be considered, such as the retention of a partial forest matrix between streams. Whitlock and McCauley (1999) suggest that movement of individuals is usually more relevant to our understanding of dispersal than gene flow alone. However, direct estimates of dispersal are difficult (Vos era/. 2001), and there are additional limitations to ecological studies such as confounding factors (e.g., watershed, year, developmental stage; see Chapter 3). Using ecological or molecular techniques alone to conduct monitoring of Ascaphus  populations  can be problematic because each technique has limitations. I recommend that any future Ascaphus  research include both ecological and molecular tools. By coupling ecological and  molecular tools, conservation biologists and forest managers may be better able to answer questions about species at risk, and with reduced uncertainty. For example, long-term markrecapture studies (or when technologies are improved, tracking individuals using radio transmitters to evaluate long-distance overland movements) and molecular monitoring of genetic diversity over at least one generation would constitute an effective monitoring program for Ascaphus.  Monitoring populations within different management scenarios and evaluating  the success of management will allow us to determine if we are indeed achieving our conservation goals.  LITERATURE CITED  63 LITERATURE CITED  Adams, M.J. 1993. Summer nests of the tailed frog (Ascaphus truei) from the Oregon coast range. Northwestern Naturalist. 74:15-18. Adams, S.B. and C A . Frissell. 2001. Thermal habitat use and evidence of seasonal migration by Rocky Mountain tailed frogs, Ascaphus montanus, in Montana. Canadian Field-Naturalist. 115(2):251-256. Anholt, B.R., S. Negovetic, and C. Som. 1998. Methods for anaesthetizing and marking larval anurans. 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The ontogenetic niche and species interactions in sizestructured populations. Annual Review of Ecology and Systematics. 15:393-425.  Whitlock, M . C and D.E. McCauley. 1999. Indirect measures of gene flow and migration: F  S T  ^  1/(4/V +1). Heredity. 82(2):117-125. m  Wilbur, H.M. and J . P . Collins. 1973. Ecological aspects of amphibian metamorphosis. Science. 182:1305-1314.  LITERATURE CITED  80  Williams, J.G.K., A . R . Kubelik, K.J. Livak, J.A. Rafalski, and S.V. Tingey. 1990. D N A polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Research. 18:6531-6535.  Wilson, E.O. 1988. The current state of biological diversity. In: Wilson, E.O. and F.M. Peter (eds.). Biodiversity. National Academy Press. Washington, D.C. Pp. 3-18.  Wind, E. 1996. Habitat associations of wood frogs (Rana sylvatica), and effects of fragmentation, in boreal mixedwood forests. M.Sc. Thesis. Department of Forest Sciences. The University of British Columbia. British Columbia. Vancouver, British Columbia. 69 p.  Wright, S . 1931. Evolution in Mendelian populations. Genetics. 16:97-159.  Zar, J . H . 1984. Biostatistical Analysis. Second Edition. Prentice-Hall, Inc. Englewood Cliffs, N J . 718 p.  APPENDIX I  81  Table A-1.1. Small mammal mortalities: forest cover type, watershed, year, month, and distance from stream (m). All shrew mortalities occurred in traps where escape ropes were not properly secured (either not touching bottom of trap, or rope missing due to bear disturbances).  Small Mammal  1  shrew shrew mole mole mole mole mole mole mole mole shrew shrew mole mole shrew shrew shrew shrew shrew shrew shrew  1  Forest Cover Watershed Clearcut Clearcut Old Growth Old Growth Old Growth Old Growth Old Growth Clearcut Old Growth Old Growth Old Growth Old Growth Old Growth Old Growth Clearcut Clearcut Clearcut Clearcut Clearcut Clearcut Old Growth  Mamquam Mamquam Ashlu Ashlu Ashlu Ashlu Ashlu Mamquam Ashlu Mamquam Ashlu Mamquam Ashlu Ashlu Elaho Elaho Mamquam Ashlu Ashlu Ashlu Mamquam  Year  Month  1998 1998 1999 1999 1999 1999 1999 1999 1999 1999 1999 1999 2000 2000 2000 2000 2000 2000 2000 2000 2000  August August July August August August . August September October October September September September October September September September October October October October  Distance from Stream (m) 5 5 5 100 0 0 0 25 25 . 50 100 25 0 25 25 25 25 50 50 50 50  Moles (shrew-mole, Neurotrichus gibbsii) were identified by Dr. Kevin Campbell, U B C . Shrews {Sorex spp.) were not identified to species.  APPENDIX I  82  Table A-1.2. The number of amphibians trapped, encountered, and recaptured during the Ascaphus field season. Southwestern British Columbia. 1998-2000.  Year  Month  1998  July August September October July August September October September October November  1999  2000  1  Numbers Trapped" (Encountered) 0  1  AMGR  AM MA  ENES  PLVE  BUBO  RAAU  0(0) 0(0) 8(2) 13(0) 2 (0) 6(0) 4 (0) 22 (0) 28 (0) 22 (0) 4(0)  0(0) 0(0) 8(2) 10 (0) 0(0) 2(0) 4(0) 18 (0) 11 (0) 9(0) 2(0)  0 (0) 0 (0) 5 (0) 4 (0) 0 (0) 2 (0) 2 (0) 6 (0) 4 (0) 4 (0) 2 (0)  0(0) 0(1) 4(1) 6(2) 0(0) 1 (0) 4(0) 5(0) 6 (0) 4(0) 3 (0)  0(0) 0(0) 4(0) 4(1) 11 (0) 10 (0) 9(0) 8(0) 43 (2) 13 (0) 0(0)  0(0) 0(1) 0(0) 0(0) ' 0(0) 0(0) 0(0) 2(0) 1 (0) 0(0) 0(0)  1  3  3  1  1  1  1  1  HYRE  0(0) 0(0) 0(2) 0(0) 0(1)  0(0) 0(2) 29 (7) 37 (3) 13 (1) 21 (1) 23 (0) 61 (1) 93 (3) 52 (0) 11 (0) 1  1  0(1) 0(0) 0(1)  4  0(1) 0(0) 0(0)  4  1  1  Recaptures were included in counts, and reported as superscripts; A M G R = Ambystoma  gracile, A M M A = Ambystoma macrodactylum, E N E S = Ensatina eschscholtzi, P L V E = Plethodon vehiculum, B U B O = Bufo boreas, R A A U = Rana aurora, H Y R E = Hyla regilla.  Table A-l.3. The relative abundance (number per 100 trap nights) of amphibians trapped during the Ascaphus field season. Southwestern British Columbia. 1998-2000.  Year  Month  1998  September October July August September October September October November  1999  2000  1  AMGR  AMMA  0.31 0.52 0.08 0.35 0.08 0.22 0.37 0.30 0.28  0.31 0.40 0.00 0.12 0.08 0.20 0.16 0.12 0.14  Relative Abundance (#/100 TN) ENES PLVE BUBO  0.19 0.16 0.00 0.12 0.04 0.07 0.06 0.05 0.14  0.15 0.24 0.00 0.06 0.08 0.06 0.07 0.05 0.14  0.15 0.16 0.84 0.53 0.18 0.09 0.63 0.16 0.00  1  RAAU  HYRE  0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00  Incidental encounters and recaptures not included; A M G R = Ambystoma gracile, A M M A =  Ambystoma macrodactylum, E N E S = Ensatina eschscholtzi, P L V E = Plethodon vehiculum, B U B O = Bufo boreas, R A A U = Rana aurora, H Y R E = Hyla regilla.  83  APPENDIX I  Summer 1999  -OG -CC  0m  25m  50m  100m  Distance from Stream  Fall 1999 -—. 3 CO  o o  CO o >  ro cu  CC  10.0 8.0  • OG  6.0  -CC  4.0 2.0 0.0  0m  25m  50m  100m  Distance from Stream  Fall 2000 cu i_  3 *J CO  o  o  00  >  cu  or  10.0 8.0  -OG  6.0  CC  4.0 2.0 0.0  0m  25m  50m  100m  Distance from Stream  Figure A-1.1. Relative soil moisture at four distances from stream in old growth (OG) and clearcut (CC): 0 m, 25 m, 50 m, and 100 m. a) summer 1999, b) fall 1999, and c) fall 2000.  APPENDIX II  84  Table A-11.1. Six primers, and 61 loci scored with high confidence. Lab sample number, tadpole DNA sample number, treatment (TR: O G = Old Growth, C C = Clearcut), and distance (stream sampling station, in meters).  Primer 211 Sample  DNA  #  #  1  36  2  37  3  38  4  31  5  32  6  33  7  40  8  46  9  47  10  192  11  193  12  194  13  50  14  141  15  142  16  202  17  203  18  204  19  147  20  148  21  149  22  207  23  208  24  209  25  157  26  158  27  159  28  237  29  238  30  239  31  167  32  168  33  169  34  247  35  248  36  249  37  174  38  175  39  176  40  250  41  251  42  252  43  11  44  12  45  13  46  39  47  34  48  35  49  189  50  48  TR  OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC CC OG  L o c i (bp)  Distance (m)  0 0 0 0 0 0 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 120 120 120 120 120 120 140 140 140 140 140 140 160 160 160 160 160 160 180 180 180 0 0 0 0 20  1250  1050  975  925  875  840  825  780  650  525  500  1 1  1 0 0 0 0 0 1 0 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 0 0 0 0 1 1  1 1 1 1  0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 X 0 0 0 0 0 0 0 0 0 0 0 X 0 0 1  0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 X 0 0 0 0 0 0 0 0 0 0 0 X 0 0 0  1 1 1  1 1 1  0 0 1 0 1 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0  1 0 0 0 0 0 0 1 1 1 1 1 0 0 1 1 0 1 0 1 1 1 1 1 0 0 0 0 0 1 1 1 1 1 X 0 1 1 1 1 1  1 1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1  1  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1  1 1 1 1 1 1  1 1 1  1 1 1  1 1  1  1  1  1 1 1 1 X 0 0 1  1 1 1 1 X 0 0 0  1 1 1 1 0 X 0 0 1  1  1  1  1  1  1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 0 X 0 0 0  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0  1  1 1 ' 1 0 0 0 1 1 1 1 1 0 1  X 0 1 1 0 0 0 0 1 0 0 0 X 0 0 0  1 1  1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1  1  1 1 1 1 1 1 1  1 1 1 0 X 0 1 0  0 0 0 0 X 0 0 0  A P P E N D I X II  T a b l e A-II.1. C o n t i n u e d .  Primer 211  Loci (bp)  Sample # 51  DNA # 49  OG  Distance (m) 20  52  195  CC  20  53  196  CC  54  197  55  TR  1250  1050  975  925  875  840  825  780  650  525  500  1  1  0  0  0  0  1  1  0  0  0  1  1  0  0  0  0  1  1  0  0  0  20  1  0  1  0  0  0  0  1  0  0  0  CC  20  1  1  0  0  0  0  1  1  0  0  0  1  0  0  0  0  1  0  0  0 1 0  0 0 0  0  0 0 1  0 0  1 1  0 0 0  0 0  1  1 0 0  143 144  OG  40  56 57 58 59  OG  40  145 205  OG CC  40 40  206  CC  40  1  0  0  0  1  60 61  150 151  OG OG  60 60  0 0  0 0  1 0  0 0  0 0  62  152  OG  60  0  0  0  0  63 64  210 211  CC  60  0  65  212  60 60  0 0  0  CC CC  0 1 1  0  0 0  0 0  66 67  154  OG  80  1  0  0  0  OG  80  68  155 217  CC  80  1 1  0 0  0 0  0 0  69  218  CC  80  1  1  0  70 71  219 156  CC OG  80 100  1  1  1  0  0 0  72  226 227 228  CC  100 100 100  1 1 1  0  CC CC  160 161 162  OG  120 120  1  1  1 1  1 1  73 74 75 76  OG OG CC  77 78 79  240 241  CC  120  80  242  CC  120  1  81  170  OG  140  1  120 120  0 1  X  0 0 0 0 1 0 X  1 1  0  0 0 0 1  1  0  0 0  0 0  0 0  0 1  0  0  0  0  0  0 0 0  0 1  0  0  1  0 1  0  0  1  0 0 0  0  0  0 0  0 0  1 1  0 1  0  0  0  1  0  0  1  1  0  0 0  0  1  0  1  0 0 0  0  1 1 1  1 1 1  0  0 1  1  0  1 1  0 0  1 1 1  0 0  0 0  0  0  0  0 0  0 0  0 0 0 0  0 1 0 1 0 0 0  0 1 0 0 0 0 0 1 1  0  1  0 1  0 0  0  1  0  0  0 1  0 1  0 0 1  1 1 1  0  0 0  0 1  1  X  1 X  0 0 X  X  X  X  0 X  X  0 X  X 1  X  X  X  0  1  1  1  1  1  X  X  X  0  0  X 1  1  0  X 1  0 X  1  0  1  X 1  0  1  0  0  1  1  0  1  1  X 1  X X  1  X  82  171  OG  140  1  X 1  83  172  OG  140  1  1  84  260  CC  140  1  1  1  1  0  0  1  1  0  1  1  85  177  OG  160  1  1  1  1  0  0  1  1  1  1  1  86  178  OG  160  OG  160  1 1  1 1  0 0  0  179  1 1  0  87  0  1 1  1 1  0 1  0 1  1 1  88  253  CC  160  1  1  0  0  1  1  0  1  1  254  160  1  1  1  0 X  1  0 X  1  1  255 190  0 X  1  90 91  CC CC  0 1 X  1  89  X  1  1  X 1  92  1  1  93  1  1 1  0  CC  160 0  1  1  1  1  0  0  X 1  198  CC  20  1  1  1  1  0  0  1  1  0 1  199  CC  20  1  1  1  1  0  1  1  0  94  200  CC  20  1  1  1  1  0  0  1  1  0  1  95  153  OG  60  1  X  1  X  X  1  1  1  X  96  213  CC  60  1  1  1  1  0  1  1  1  0  1  1  97  214  CC  60  1  1  1  1  0  0  1  1  1  1  1  98  215  CC  60  1  1  1  1  0  0  1  1  0  1  1  99  220  CC  80  1  1  1  1  0  0  1  1  0  1  1  100  221  CC  80  0  0  0  1 1  1  80  1 1  0  CC  1 1  0  222  1 1  1  101  1 1  0  1  1 1  1  0  APPENDIX II  86  Table A-11.1. Continued.  Primer 211 Sample # 102  DNA # 223  103 104  224 225  105  229  106  230  107 108 109  231 232 233  110  235  111  163  112  164  113  165  114  243  115 116 117  244 245  118 119  180  120 121  182 256  122  257  123  14  173 181  124  15  125  184  126 127  185  128 129  16 17  130  18  186  131  19  132  20  133  261  134  262  135  263  136  191  137  201  138  146  139  216  140  234  141  236  142  166  143  246  144  183  145  258  146  259  147  187  148  188  149  264  150  265  Loci (bp)  TR  Distance (m)  CC CC CC CC CC CC CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC OG OG OG OG OG CC CC CC CC CC CC CC CC CC CC OG CC CC CC OG CC OG CC CC OG OG CC CC  80 80 80 100 100 100 100 100 100 120 120 120 120 120 120 140 160 160 160 160 160 180 180 180 180 180 180 180 180 180 180 180 180 180 0 20 40 60 80 100 120 120 160 160 160 180 180 180 180  1250  1 1 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1050  1 1 1 1 1 1 1. 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  975  925  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 •1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 1 0 0 0 1 0 0 1 0 0 0 0 0 0  875  840  0 0 0 0 1 0 1 0 0 0 0 .0 0 0 0 0 0 0 0 0 0 0 0 . 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 1 0 0 0 0 0 0 1 0 0 0 1 0 1 0 0 1 0 0 0 0 0 0 0 0 0  825  1 1 1 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  780  650  525  500  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0 1 1 1 0 1 0 0 0 0 0 0 1 0 1 0 0 0 1 0 0 0 0 0 1 0 1 1 0 0 0 0 0 1 1 0 0 0 0 0 1 0 0 0 0 1 0 0 1  0 1 0 .1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1  1 1 1  1 1 1 1 1 1 1 1  1 1  APPENDIX II  Table A-11.1. Continued. Primer 213 Sample # 1 2  DNA # 36 37  3 4  38 31  5  32  6 7  33 40 46  8 9 10 11  47 192 193  12  194  13  50 141  14 15 16  142 202  17 18 19 20 21  203 204 147  22 23 24  207  148 149  25  208 209 157  26  158  27  159  28  237  29  238  30  239 167  31 32  168  33  169  34  247  35  248  36  249 174  37 38  175  39  176  40  250  41  251  42  252  43  11  44  12  45  13  46  39  47  34  48 49  35 189  50  48  Loci (bp)  TR  Distance (m)  OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC CC OG  0 0 0 0 0 X 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 120 120 120 120 120 120 140 140 140 140 140 140 160 160 160 160 160 160 180 180 180 0 0 0 0 20  1500  1000  850  775  710  675  500  400  340  1 1 1 1 1 X 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 X 0 0 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1  1 1 0 0 1 X 0 0 0 1 0 0 0 0 0 1 1 1 1 1 0 1 1 1 0 0 1 0 0 0 1 1 0 1 0 1 1 1 1 1 1 1 1 1 1 0 X 0 0 0  1 1 1 1 1 X 0 0 1 1 1 1  1 1 1 0 1 X 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 X 0 0 0  1 1 1 1 1 1 1 1 1 1 1  1  1 1 1 1 1  1 1 1 1 1  1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 X 0 0 0  1 1 1 1 1 1 1 1 1 X 1 0 0  X 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0 X 0 1 1  0 X 0 0 0  APPENDIX II  88  Table A-11.1. Continued. Loci (bp)  Primer 213 Sample DNA Distance TR 1500 (m) # # 1 49 OG 20 51 1 195 CC 20 52 1 196 CC 20 53 1 197 CC 20 54 143 OG 40 55 1 144 OG 40 56 145 OG 40 57 1 205 CC 40 58 1 206 CC 40 59 1 150 OG 60 60 1 151 OG 60 61 1 152 OG 60 62 1 60 210 CC 63 1 211 CC 60 64 1 212 CC 60 65 1 154 OG 80 66 80 155 OG 67 1 217 CC 80 68 1 218 CC 80 69 1 219 CC 80 70 1 156 OG 100 71 1 100 72 226 CC 1 227 CC 100 73 1 100 74 228 CC 1 160 OG 120 75 1 161 OG 120 76 1 162 OG 120 77 1 240 CC 120 78 1 241 CC 120 79 1 242 CC 120 80 1 170 OG 140 81 1 171 OG 140 82 1 172 OG 140 83 1 140 84 260 CC 1 177 OG 160 85 1 178 OG 160 86 179 OG 160 87 1 160 88 253 CC 254 CC 160 X 89 X 255 CC 160 90 1 0 190 CC 91 1 20 92 198 CC 1 20 199 CC 93 1 20 94 200 CC 1 153 OG 60 95 1 213 CC 60 96 1 CC 60 214 97 1 215 CC 60 98 1 80 220 CC 99 1 221 CC 80 100 222 CC 1 80 101  1000  850  775  710  675  500  400  340  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0 0 0 0 0 0 0 0 0 1 1 1 1 1  0 0 0 0 1 0 0 0 0 1 1 0 1 0 1 1 1 0 0 1 1 1 0 0 1 1 1 0 0 0 1 1 1  1 1 1 1 1 1  1 1 1 1 1 1  0 0 0 0 0 0 0 0 0 1 1 1 0 0 0 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X X 1 1 1 1 1 1 1 1 11 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 0 0 0 0 0 0 1 0 1 1 0 0 0 1 1 1 1  1 1 1 1 1 1 1 1 X X 1 1 1 1  0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 1 0 0 0 0 0 0 0 0 1 1 0 0 0 0 1 1 1 1 1 1 1 1 X X 1 1 1 1  0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 0 0 0 0 0 0 0 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 X 1 1 1 1 1 1 1 1 1 1 1  1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  89  A P P E N D I X II  Table A-II.1. Continued. P r i m e r 213 Sample # 102 103 104  DNA # 223 224 225  105 106  229  107 108 109  231 232  110  235  111  163  112  164  113 114  165 243 244  115  230  233  116 117 118  245  119  181  120 121  182 256 257  122 123 124  173 180  14  125  15 184  126  185  127  186  128  16  129  17  130 131  18  132  20  133  261  134  262  135  263  19  136  191  137  201  138  146  139  216  140  234  141  236  142  166  143  246  144  183  145  258  146  259  147  187  148 149  264  150  265  188  Loci bp)  TR  Distance (m)  CC CC CC CC CC CC CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC OG OG OG OG OG CC CC CC CC CC CC CC CC CC CC OG CC CC CC OG CC OG CC CC OG OG CC CC  80 80 80 100 100 100 100 100 100 120 120 120 120 120 120 140 160 160 160 160 160 180 180 180 180 180 180 180 180 180 180 180 180 180 0 20 40 60 80 100 120 120 160 160 160 180 180 180 180  1500  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1000  1 1 1 1. .1 1 1 1 '1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1. 1 1 1 1 1 1 1  850  775  710  1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 •1 1 1  1  1 1 1 1 1 1  1 1 1 ' 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1  1 1  1 1 1 1 1  1 1 1 1  1 1  1 1  675  1 1 1 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  500  400  340  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  APPENDIX II  90  Table A-11.1. Continued.  1 r i m Br 221 3  Sample #  1 2 3 4 5 6 7 8 9 10 11 12  DNA  # 36 37  15  38 31 32 33 40 46 47 192 193 194 50 141 142  16 17 18 19 20 21 22 23 24 25 26 27 28  202 203 204 147 148 149 207 208 209 157 158 159 237  29  43  238 239 167 168 169 247 248 249 174 175 176 250 251 252 11  44  12  13 14  30 31 32 33 34 35 36 37 38 39 40 41 42  45  13  46  39  47  34  48 49 50  35 189 48  TR  OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC CC OG  L o c i (bp)  Distance (m)  0 0 0 0 0 0 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 120 120 120 120 120 120 140 140 140 140 140 140 160 160 160 160 160 160 180 180 180 0 0 0 0 20  1380  1020  975  810  775  650  1 1 1 1 . 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1  1 1 1 1 1  1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1  1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0  480  420  360  325  1 1 0 0 0 0 0 0 0 1 1 1 0 0 0 1 1 1 X 1  1 1 -I -I 1  -| 1 •j -|  1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0  1  1 1 1 1 1 1 1 1 1 1 1 1  -j "  -| 1 1 1 1 1 -| 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1  1 1 1  -j  1 1 -| 1 -| 1 1 1 •j 1 1 1 1 •) 1 1 1 1 1 1 1 1 1 -) 1 1. 1 1 1 1 1 1 1 1 1 1 1 1 1  APPENDIX II Table A-11.1. Continued.  Primer 221 DNA Sample # # 49 51 52 195 53 196 54 197 55 143 144 56 57 145 58 205 59 206 60 150 61 151 152 62 63 210 64 211 212 65 154 66 67 155 217 68 69 218 219 70 71 156 72 226 73 227 74 228 75 160 76 161 77 162 240 78 79 241 80 242 81 170 82 171 83 172 84 260 177 85 86 178 87 179 88 253 89 254 255 90 91 190 92 198 93 199 94 200 95 153 96 213 97 214 98 215 99 220 100 221 101 222  TR OG CC CC CC OG OG OG CC CC OG OG OG CC CC CC OG OG CC CC CC OG CC CC CC OG OG OG CC CC CC OG OG OG CC OG OG OG CC CC CC CC CC CC CC OG CC CC CC CC CC CC  Distance 1380 (m) 20 1 20 1 20 1 20 1 1 40 40 1 40 1 40 1 40 1 60 1 60 1 60 1 60 1 1 60 60 1 1 80 80 1 1 80 1 80 80 1 1 100 1 100 100 1 100 1 120 1 1 120 120 1 1 120 1 120 120 1 140 1 140 1 140 1 140 1 1 160 160 1 160 1 160 1 160 1 160 1 0 1 20 1 20 1 20 1 60 1 60 1 60 1 60 1 80 1 80 1 1 80  Loci (bp) 1020  975  810  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  775 650 480 420 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  0 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 0 0 0 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  -|  1 1 1 1 1 1  1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  360  325  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  -| 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1  APPENDIX II  92  Table A-11.1. Continued.  Primer 221 Sample #  DNA  #  102  223  103  224  104  225  105  229  106 107  230  108 109  231 232 233  110  235  111  163  112  164  113  165  114  243  115  244  116 117  245  118  173 180  119  181  120 121  182 256  122  257  123  14  124  15  125  184  126  185  127  186  128  16  129  17  130 131  18  132  20  133  261  134  262  135 136  263 191  137  201  19  138  146  139  216  140  234  141  236  142  166  143  246  144  183  145  258  146  259  147  187  148 149  188 264  150  265  Loci (bp)  TR  Distance (m)  1380  CC CC cc cc cc cc cc cc cc OG OG OG cc CC CC OG OG OG OG CC CC OG OG OG OG OG CC CC CC CC CC CC CC CC CC CC OG CC CC CC OG CC OG CC CC OG OG CC CC  80 80 80 100 100 100 100 100 100 120 120 120 120 120 120 140 160 160 160 160 160 180 180 180 180 180 180 180 180 180 180 180 180 180 0 20 40 60 80 100 120 120 160 160 160 180 180 180 180  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1020  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 .1 .  975  810  775  650  480  420  360  325  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 .1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1  1 1 1 1 1 1 1 1  1 1  1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  APPENDIX  II  93  Table A-II.1. Continued. Primer 268 Samp # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50  DNA # 36 37 38 31 32 33 40 46 47 192 193 194 50 141 142 202 203 204 147 148 149 207 208 209 157 158 159 237 238 239 167 168 169 247 248 249 174 175 176 250 251 252 11 12 13 39 34 35 189 48  Loci (bp)  TR Distance 1500 1350 1250 1050 1000 875 760 640 610 590 510 490 400 350 (m) OG 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 0 1 1 1 1 1 1 1 1 1 1. 1 1 1 1 OG 0 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 CC 0 1 1 1 1 1 1 1 1 1 1 1 1 1 0 CC 0 X 1 1 1 1 1 1 X X X X X X X OG 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 20 1 1 1 1 1 1 1 1 0 OG 20 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 20 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 20 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 40 1 1 1 1 1 1 1 1 1 0 OG 40 1 1 1 1 1 1 1 1 1 1 1 1 0 OG 40 1 1 1 1 1 1 1 1 1 1 1 1 1 0 CC 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 40 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 60 1 X 1 1 X X X X X X OG 60 1 1 1 1 1 1 1 1 1 1 1 1 1 • .1 OG 60 1 1 1 1 1 1 1 1 1 1 1 1 CC 60 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 60 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 60 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 120 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 120 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 120 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 120 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 120 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 120 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 140 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 140 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 140 1 1 1 1 1 1 1 1 1 1 1 1 CC 140 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 140 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 140 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 160 1 1 1 1 1 1 1 1 1 1 1 1 OG 160 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 160 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 160 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 160 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 160 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 180 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 180 1 1 1 1 1 1 1 1 1 1 1 1 OG 180 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 0 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CC 0 1 1 1 1 1 1 1 1 1 1 1 1 "I 1 CC 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 OG 20 X X X X X X 1 1 1 X X 1 X X  94  A P P E N D I X II  Table A-l 1.1. Continued. Loci (bp)  Primer 268 Samp DNA Distance TR 1500 (m) # # 49 O G 20 X 51 1 195 C C 20 52 1 196 C C 2 0 53 1 197 C C 20 54 1 143 O G 40 55 1 144 O G 40 56 1 145 O G 40 57 1 205 C C 40 58 1 40 206 C C 59 1 150 O G 60 60 151 O G 60 61 152 O G 60 62 1 210 C C 60 63 1 211 C C 60 64 1 212 C C 60 65 1 154 O G 8 0 66 1 155 O G 80 67 1 217 C C 80 68 1 218 C C 80 69 1 219 C C 80 70 1 156 O G 100 71 1 226 C C 100 72 1 227 C C 100 73 1 228 C C 100 74 1 120 160 O G 75 1 120 161 O G 76 1 162 O G 120 77 1 240 C C 120 78 1 241 C C 120 79 1 242 C C 120 80 1 170 O G 140 81 1 171 O G 140 82 1 172 O G 140 83 1 260 C C 140 84 1 177 O G 1 6 0 85 1 178 O G 160 86 1 179 O G 160 87 1 253 C C 160 88 1 254 C C 160 89 1 255 C C 160 90 1 190 C C 0 91 1 198 C C 20 92 1 199 C C 2 0 93 1 20 94 200 C C 1 153 O G 60 95 1 60 213 C C 96 1 214 C C 60 97 1 215 C C 60 98 1 220 C C 80 99 1 221 C C 80 100 222 C C 80 1 101  1350  1250 1050 1000 875  760  640 610 590 510 490 400 350  1  1  X  X  1  1  X  X  X  X  X  1  X  1  1  1  1  1  1  1  1  1 .  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  . 1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  0  1  0  1  1  1  1  1  0  0  1  X  1  X  1  1  X  X  1  X  1  X  1  1  1  1  1  1  X  X  1  1  1  1  1  1  1  1  1  1  1  0  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  . 1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  X  1  X  X  1  1  1  1  1  X  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  1  95  APPENDIX II  Table A-11.1. Continued. Loci (bp)  Primer 268 Samp DNA # # 223 102 224 103 225 104 229 105 230 106 231 107 232 108 233 109 235 110 163 111 164 112 165 113 243 114 244 115 245 116 173 117 180 118 181 119 182 120 256 121 257 122 14 123 15 124 184 125 185 126 186 127 16 128 17 129 18 130 19 131 20 132 261 133 262 134 263 135 191 136 201 137 146 138 216 139 234 140 236 141 166 142 246 143 183 144 258 145 259 146 187 147 188 148 264 149 265 150  Distance 1500 TR (m) 1 80 CC 1 80 CC 1 CC 80 1 100 CC 1 100 CC 1 100 CC 1 100 CC 1 100 CC 1 CC 100 1 120 OG 1 120 OG 1 OG 120 1 120 CC 1 CC 120 1 CC 120 1 140 OG 1 160 OG 1 160 OG 1 OG 160 1 160 CC 1 160 CC 1 180 OG 1 180 OG 1 OG 180 1 180 OG 1 180 OG 1 180 CC 1 CC 180 1 180 CC 1 180 CC 1 CC 180 1 180 CC 1 180 CC 1 180 CC 1 0 CC 1 CC 20 OG 40 1 60 CC CC 80 1 100 CC 1 120 OG 1 120 CC 1 160 OG 1 CC 160 1 160 CC 180 OG X 1 OG 180 1 180 CC CC 180 1  1350 1250 1050 1000 875 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1  1 1 1 1 1 1 X  1 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  760 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  640 610 590 510 490 400 350 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 0 0 0 1 1 1 1 1 X 0 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 X 1 1 1  1 1 1 1 1 1 1 1 X 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  96  APPENDIX II  Table A-II.1. Continued. Loci (bp)  Primer 352 Distance Sample DNA TR (m) # # 0 36 OG 1 0 37 OG 2 0 38 OG 3 0 31 CC 4 CC 0 32 5 0 CC 33 6 20 40 OG 7 20 46 OG 8 20 47 OG 9 20 192 CC 10 20 193 CC 11 20 194 CC 12 40 50 OG 13 40 141 OG 14 40 142 OG 15 40 202 CC 16 40 203 CC 17 40 204 CC 18 60 147 OG 19 60 148 OG 20 60 149 OG 21 60 207 CC 22 60 208 CC 23 60 209 CC 24 120 157 OG 25 120 158 OG 26 120 159 OG 27 CC 120 237 28 120 238 CC 29 120 239 CC 30 140 167 OG 31 140 168 OG 32 140 169 OG 33 140 247 CC 34 140 248 CC 35 140 249 CC 36 160 174 OG 37 160 175 OG 38 160 176 OG 39 160 250 CC 40 160 251 CC 41 160 252 CC 42 180 11 OG 43 180 12 OG 44 180 13 OG 45 0 39 OG 46 0 34 CC 47 0 35 CC 48 0 189 CC 49 20 48 OG 50  1375  1350  1180  925  1 0 0 0 0 X X 1 1 0 0 0 1 1 1 1 1 1 1 1  1 1 1 0 0 X X 1 1 1 1 1 1 1 1 1 1 1 1 1  1 0 0 0 1 X X X 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 0 X 0 X 0 1 1 0 0 1 1 1 1 1 0 1 1 1 X  1 1 1 1 1  1 1 1 1 1 1 1 X  1 1 1 1 1 1 1 1 1 1 1 1 1 X  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1  875 650 480 450 1 0 0 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 0 1 0 0 X X 1 X 1 1 1 0 0 1 1 1 1 0 1 X 1 1 1 0 0 1 0 0 0 1 0 X 1 X 1 1 1 1 1 1 1 1 0 X 0 1 1 1 X  1 0 0 0 1 X X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X  97  APPENDIX II  Table A-II.1. Continued. Loci (bp)  Primer 352 Sample # 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101  DNA # 49 195 196 197 143 144 145 205 206 150 151 152 210 211 212 154 155 217 218 219 156 226 227 228 160 161 162 240 241 242 170 171 172 260 177 178 179 253 254 255 190 198 199 200 153 213 214 215 220 221 222  TR OG CC CC CC OG OG OG CC CC OG OG OG CC CC CC OG OG CC CC CC OG CC CC CC OG OG OG CC CC CC OG OG OG CC OG OG OG CC CC CC CC CC CC CC OG CC CC CC CC CC CC  Distance (m) 20 20 20 20 40 40 40 40 40 60 60 60 60 60 60 80 80 80 80 80 100 100 100 100 120 120 120 120 120 120 140 140 140 140 160 160 160 160 160 160 0 20 20 20 60 60 60 60 80 80 80  1375 X 1 0 1 1 1 • 1 1 0 0 0 0 0 1 1 1 1 0 1 X 0 0 1 0 1 0 1 0 1 1 0 1 0 0 0 0 0 1 1  1350  1180  1 1 1 1 1. 1  X 1 1 1 0 1 0 1 1 0 0 0 1 1 1 1  1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 X 1 1 0 1 0 0  1 1 X 1 1 1 1 1 0  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1 1  925 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1 1  875 650 480 450 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  X 1 1 1 0 1 0 1 1 0 0 0 1 1 1 1 X 1 1 1 0 0 1 1 1 1 0 1 0 1 0 0 0 1 0 0 0 1 1 0 1 1 1 1 1 1 1 0 1 1 0  1 1 1 1 1 1 1 1 1 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  APPENDIX II  98  Table A-11.1. Continued.  P r i m e r 352 Sample  DNA  #  #  102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150  223 224 225 229 230 231 232 233 235 163 164 165 243 244 245 173 180 181 182 256 257 14 15 184 185 186 16 17 18 19 20 261 262 263 191 201 146 216 234 236 166 246 183 258 259 187 188 264 265  TR  CC CC cc cc cc cc cc cc cc OG OG OG CC CC CC OG OG OG OG CC CC OG OG OG OG OG CC CC CC CC CC CC CC CC CC CC OG CC CC CC OG CC OG CC CC OG OG CC CC  L o c i (bp)  Distance (m)  80 80 80 100 100 100 ' 100 100 100 120 120 120 120 120 120 140 160 160 160 160 160 180 180 180 180 180 180 180 180 180 180 180 180 180 0 20 40 60 80 100 120 120 160 160 160 180 180 180 180  1375  1350  1180  925  875  650  480  450  0 1 1 0 1 1 0 1 1 1 1 0 0 0 1 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 1 0 1 1 X 0 0 0 0 1 0 1 1 1 0 0 1  1 1 1 1 X 0 1 1 1 1 1 1 1 0 0 1 0 1 1 0 1 0 1 1 1 1 1 1 0 1 0 1 1 1 1 1 X 0 0 0 1 1 1 1 1 1 0 0 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 T 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 X 1 1 1 1 0 0 0 1 0 0 1 0 0 0 1 1 0 1 0 1 0 0 0 0 0 1 1 1 1 1 1 X 0 0 0 0 1 1 1 1 0 0 1 1  1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1 1 1  1 1  APPENDIX II  Table A-11.1. Continued. Primer 400 Samp # 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50  DNA # 36 37 38 31 32 33 40 46  TR  Dist (m)  OG OG OG CC CC CC OG OG 47 OG 192 CC 193 CC 194 CC 50 OG 141 OG 142 OG 202 CC 203 CC 204 CC 147 OG 148 OG 149 OG 207 CC 208 CC 209 CC 157 OG 158 OG 159 OG 237 CC 238 CC 239 CC 167 OG 168 OG 169 OG 247 CC 248 CC 249 CC 174 OG 175 OG 176 OG 250 CC 251 CC 252 CC 11 OG 12 OG 13 OG 39 OG 34 CC 35 CC 189 CC 48 OG  0 0 0 0 0 0 20 20 20 20 20 20 40 40 40 40 40 40 60 60 60 60 60 60 120 120 120 120 120 120 140 140 140 140 140 140 160 160 160 160 160 160 180 180 180 0 0 0 0 20  Loci (bp) 1180  1020  980  900  780  760  675  390  350  1 1  1 1 0 1 1 X 0 0 1 1 1 1 X 1 1 1 1 1 X 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 X 1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 0 1 1 X 0 0 1 1 1 1 X 0 0 1 1 1 X 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1  1 0 1 1 0 1 0 0 1 1 1 1 1 1 1 0 1 1 X 1 0 0 1 1 1 0 1 1 1 1 1 1 1 1 1 1. 1 1 1 1 1 0 1 1 1 0 1 1 1 0  1 1 X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0  1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1  1 1 1 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1  1  1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  100  APPENDIX II Table A-II.1 Continued.  Primer 400  Loci (bp)  Dist Samp DNA 1180 1020 980 900 780 760 675 390 350 TR (m) # # 1 1 1 1 1 0 0 0 0 49 OG 20 51 1 1 1 1 1 1 1 0 1 195 CC 20 52 1 1 1 1 1 1 1 0 1 20 196 CC 53 1 1 1 1 1 1 1 0 1 20 197 CC 54 1 1 1 0 1 1 0 OG 40 143 55 1 1 1 1 1 1 0 1 0 40 144 OG 56 1 1 1 1 1 0 1 0 145 OG 40 57 1 1 1 1 0 1 0 1 1 205 CC 40 58 1 1 1 1 0 1 1 1 1 206 CC 40 59 1 1 1 1 1 1 1 1 0 150 OG 60 60 1 1 1 1 1 0 151 OG 60 61 1 1 1 1 1 1 X 152 OG 60 62 1 1 1 1 1 1 1 1 0 210 CC 60 63 1 1 1 1 1 1 1 0 211 CC 60 64 1 1 1 1 1 1 1 1 0 212 CC 60 65 1 1 1 1 1 1 1 1 0 154 OG 80 66 1 1 1 1 0 1 1 1 1 155 OG 80 67 1 1 1 1 1 1 1 1 217 CC 80 68 1 1 1 1 1 1 1 1 1 218 CC 80 69 1 1 1 1 1 1 1 1 0 219 CC 80 70 1 1 1 1 1 1 1 1 1 156 OG 100 71 1 1 1 1 0 1 1 1 1 226 CC 100 72 1 1 1 1 0 1 1 1 1 227 CC 100 73 1 1 1 1 1 1 1 0 228 CC 100 74 1 1 1 1 1 1 1 1 0 160 OG 120 75 1 1 1 1 1 1 1 1 0 161 OG 120 76 1 1 1 1 1 1 1 1 0 162 OG 120 77 1 1 1 1 1 1 1 0 240 CC 120 78 1 1 1 1 1 1 1 1 0 241 CC 120 79 1 1 1 1 1 1 1 1 0 242 CC 120 80 1 1 1 1 1 1 1 0 170 OG 140 81 1 1 1 1 1 1 1 1 0 171 OG 140 82 1 1 1 1 1 1 1 0 172 OG 140 83 1 1 1 1 1 1 0 260 CC 140 84 1 1 1 1 0 1 1 1 1 177 OG 160 85 1 1 1 1 1 1 1 1 0 178 OG 160 86 1 1 1 1 1 1 1 1 0 179 OG 160 87 1 1 1 1 1 1 1 1 0 253 CC 160 88 1 1 1 1 1 1 1 1 0 254 CC 160 89 1 1 1 1 1 1 1 1 0 255 CC 160 90 1 1 1 1 1 1 1 1 1 0 190 CC 91 1 1 1 1 1 1 1 1 1 198 CC 20 92 1 1 1 1 1 1 1 1 1 199 CC 20 93 1 1 1 1 1 1 1 1 1 200 CC 20 94 1 1 1 1 1 X 153 OG 60 95 1 1 1 1 1 1 1 1 213 CC 60 96 1 1 1 1 1 1 1 1 1 214 CC 60 97 1 1 • 1 1 1 1 1 •1 1 215 CC .60 98 1 1 1 1 1 1 1 1 1 220 CC 80 99 1 1 1 1 1 1 1 1 1 221 CC 80 100 80 222 c c 1 1 1 1 1 1 1 1 1 101  APPENDIX II  Table A-II.1. Continued. Loci (bp)  Primer 400 Samp # 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117  DNA # 223 224 225 229 230 231 232 233 235  118  163 164 165 243 244 245 173 180  119 120  181 182  121 122  256 257 14  123 124 125 126 127  15 184 185 186  128 129 130  16 17  131  19  18  132  20  133 134  261 262  135  263  136 137  201  191  139  146 216  140  234  141 142  236 166  143 144 145  246 183 258  146  259  147  187  138  148  188  149  264  150  265  TR  Dist (m)  CC CC CC CC CC CC CC CC CC OG OG OG CC CC CC OG OG OG OG CC CC OG OG OG OG OG CC CC CC CC CC CC CC CC CC CC OG CC CC CC OG CC OG CC CC OG OG CC CC  80 80 80 100 100 100 100 100 100 120 120 120 120 120 120 140 160 160 160 160 160 180 180 180 180 180 180 180 180 180 180 180 180 180 0 20 40 60 80 100 120 120 160 160 160 180 180 180 180  1180  1020  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  1 1  1 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1  1 1  980  900  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  780  1 0 1 1 1 1 1 1 1 1 1 1 0 1 1 1 1 1 1 1 1 1 1 1 0 0 1 1 0 1 0 0 1 1 1 1 X 0 1 1 0 1 1 1 1 0 0 0 0  760  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  675  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 . 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  390  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  350  0 1 1 1 1 1 1 1 1 1 1 1  1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1  APPENDIX 1I  102  CO  CM  Je  .Q. to  OT CM  00 CN  _J  00 CM  st  f-  CD  CO CM  LO st  OT  r- ro O  CN  CM  0.  CM •SI-  CD o O CD m CD O CN C N O C D o CD O CN C N O C D CO C D O O CN T— O CN OD O O CN T— O s t NO o to  CNJ CN  CN  T—  CN O CN  o CO  OT  IO  T—  00  st  f~ CO  St  CO  CD o O st CD o O st CD o O  st  CO  CN  <0 CD  Q.  CD o 00  00 CM  sf st  s t CO CM s f CO CN  CM •sf  CM CN  St  T -  CN  O sf  CD o 00 o T— CD o O 00 O o D CJ CT— CJ CoO CJ CJ o CJ C D CD o O CD CD oC D  CD CO  CO  CD CO  o  O CN  CD o O CD o (O co O in C N CD o O •st  s t  T—  CO T—  CN T—  sf CO  CO CO  CN CO  O  CD O CD O  in  cu n o m  5  CO  LL  •sf  CN CD  o ° I o Pi  m  CD  •Sf CN  O CD  CO CN  00  CN CN  00 OD  TCN  fCO  O CM  CD CO  OT  CN CD  OT  OO  •<CD  00  T~~  1-  T -  CD  o CD  CD  CD  O •sf  o  sf  O •sf  LO  "Sf  00  O  st  CN  00 CO OO  O °  o  CD  in i-  CD IO  St  CO LO  T -  co r- O 1-  LO  CN  CD LO  T -  o  i-  CM  CO CD  T -  DO OT C O CN T— Do 00 0 0 C CN O CN CD OC N r- rCN O o co CC ND CJ CJ C D O in LC NO O O CD st  CJ  .Q. Jc to .Q. Jc co c  CU  .2  a.  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OT CN  CD o O m oo CJ o CN C D CJ C D o s t r-~ O CN C D CJ C D CO C D CJ o CN C D O co eo  O  m  O  hCM  o  JC CO  CN  o  T—  O CN  to CJ CJ o  to  cu Q Q  -9-  o o  o CD  s  OT  -J  T— o T— o  St  _l  to  CD O CD O CN OD CD O O CN o r- CJ O O OT C O O O o  st  CM  T—  CM  f-  .§-  o o o CN  o  00  in  f-  _ l  T—  tn  st  o  CO  skip  o  skip  o  o  •st  o o  r--  T -  f~-  o o  o P  8 O O O CJ CJ O CD O  in  E re £o  CO LL  o I co | oI oo I oI co | ol 0 0  oo CJ •sf Z .a Jc CO  Jc  to  .Q. Jc co  Jc  to  0)  4—'  1  Q . CO  o  ro E £ *->  ro o  CO  LL  £  tn  a  CD  o  c Q. CO ro  E £ ro o  CO LL  in  b  103  A P P E N D I X II  o  .&  •§•  0  .3. 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